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Engineered Extracellular Vesicles from Antler Blastema Progenitor Cells: A Therapeutic Choice for Spinal Cord Injury
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  • Shijie Yang
    Shijie Yang
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Borui Xue
    Borui Xue
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    Air Force 986(th) Hospital, The Fourth Military Medical University, Xi’an 710001, P.R. China
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  • Yongfeng Zhang
    Yongfeng Zhang
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Yongfeng Zhang
  • Haining Wu
    Haining Wu
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Haining Wu
  • Beibei Yu
    Beibei Yu
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Beibei Yu
  • Shengyou Li
    Shengyou Li
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Shengyou Li
  • Teng Ma
    Teng Ma
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Xue Gao
    Xue Gao
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Yiming Hao
    Yiming Hao
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Lingli Guo
    Lingli Guo
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Qi Liu
    Qi Liu
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
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  • Xueli Gao
    Xueli Gao
    School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, P.R. China
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  • Yujie Yang
    Yujie Yang
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Yujie Yang
  • Zhenguo Wang
    Zhenguo Wang
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Zhenguo Wang
  • Mingze Qin
    Mingze Qin
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    More by Mingze Qin
  • Yunze Tian
    Yunze Tian
    Department of Thoracic Surgery, Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    More by Yunze Tian
  • Longhui Fu
    Longhui Fu
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
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  • Bisheng Zhou
    Bisheng Zhou
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
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  • Luyao Li
    Luyao Li
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    More by Luyao Li
  • Jianzhong Li*
    Jianzhong Li
    Department of Thoracic Surgery, Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    *Email: [email protected]
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  • Shouping Gong*
    Shouping Gong
    Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    Xi’an Medical University, Xi’an 710021, P.R. China
    *Email: [email protected]
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  • Bing Xia*
    Bing Xia
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    *Email: [email protected]
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  • Jinghui Huang*
    Jinghui Huang
    Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-0991-0591
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ACS Nano

Cite this: ACS Nano 2025, 19, 6, 5995–6013
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https://doi.org/10.1021/acsnano.4c10298
Published January 22, 2025

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Abstract

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Deer antler blastema progenitor cells (ABPCs) are promising for regenerative medicine due to their role in annual antler regeneration, the only case of complete organ regeneration in mammals. ABPC-derived signals show great potential for promoting regeneration in tissues with limited natural regenerative ability. Our findings demonstrate the capability of extracellular vesicles from ABPCs (EVsABPC) to repair spinal cord injury (SCI), a condition with low regenerative capacity. EVsABPC significantly enhanced the proliferation of neural stem cells (NSCs) and activated neuronal regenerative potential, resulting in a 5.2-fold increase in axonal length. Additionally, EVsABPC exhibited immunomodulatory effects, shifting macrophages from M1 to M2. Engineered with activated cell-penetrating peptides (ACPPs), EVsABPC significantly outperformed EVs from rat bone marrow stem cells (EVsBMSC) and neural stem cells (EVsNSC), promoting a 1.3-fold increase in axonal growth, a 30.6% reduction in neuronal apoptosis, and a 2.6-fold improvement in motor function recovery. These findings support ABPC-derived EVs as a promising therapeutic candidate for SCI repair.

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Spinal cord injury (SCI) is a debilitating condition that results in irreversible harm and permanent impairment of sensory, autonomic, and motor function. (1−3) Epidemiological data from the World Health Organization (WHO) have revealed more than 250,000 SCI patients every year, with late mortality from acute SCI ranging from 4.4% to 16.7% worldwide. (4−6) Despite advancements in SCI management, current treatments─such as surgery, corticosteroids, and rehabilitation─offer only limited benefits. These approaches help reduce inflammation or provide supportive care but fall short of restoring lost neural function or promoting meaningful nerve regeneration. (7) Half of the affected individuals remain paralyzed, with life expectancies of decades with permanent disabilities. Due to the limited ability of the central nervous system to repair itself following injury, re-establishment of the neural network is essential for functional recovery. (8) Nevertheless, axonal growth after SCI is limited by the poor intrinsic regenerative capacity of neurons and the hostile extrinsic injury environment that compromises inflammation, myelin-associated inhibitors, and glial scars. (9,10) Therefore, it is crucial to target the diminished intrinsic regenerative ability of neurons and relieve extrinsic inhibitory cues within the environment. (10,11) Hence, developing a strategy that tackles both challenges may enhance the current state of SCI treatment.
Studies on SCI treatment have shown promising results with stem cell-based therapies. (12) Mesenchymal stem cells (MSCs) are particularly noteworthy among existing stem cell options for SCI treatment because they are typically obtained from postnatal tissues and have fewer safety and ethical concerns. (13) To date, MSCs sourced from diverse origins, such as adipose tissue, bone marrow, and umbilical cord, have been shown to improve the outcome of SCI. (14,15) Pro-regenerative factors from MSCs can enhance the intrinsic neuronal regenerative effects of insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF). (16,17) In addition, MSCs help establish a beneficial microenvironment that facilitates axonal regrowth by ameliorating neuroinflammation and inhibiting neuron apoptosis. (18) Nevertheless, MSCs typically undergo dynamic cellular senescence during multipassage cultivation in vitro. (19,20) Additionally, MSCs are generally obtained from mammalian postnatal tissues, which show relatively constrained regenerative ability and typically contribute to partial tissue regeneration in mammals, resulting in a ceiling effect in the promotion of tissue regeneration. (21) Therefore, the discovery of novel MSCs with robust regenerative potential in mammals may revolutionize stem cell studies within the domain of regenerative medicine.
In a recent study, we discovered a new type of MSCs in the growing deer antler, denoted antler blastema progenitor cells (ABPCs). (22) ABPCs and their progeny drive bony antler growth at an astonishing rate. The growth rate of antlers is up to 2.75 cm per day, allowing them to achieve a mass of up to 15 kg and a length of 120 cm within three months, which is the fastest organ growth recorded in mammals. (23) Intriguingly, ABPCs exhibit negligible signs of senescence and retain robust proliferative and regenerative capacities after 50 passages in vitro. (22) This is in contrast to commonly used MSCs, such as those from the umbilical cord, adipose tissue, and bone marrow, which generally undergo obvious cellular senescence after 10–15 passages of in vitro culture. (24) To our knowledge, ABPCs are the only MSCs from postnatal mammalian tissue that support complete organ regeneration in mammals, highlighting their robust pro-regenerative ability in promoting regeneration. (25,26) In addition, ABPCs have immunomodulatory, antiapoptotic, and anti-inflammatory properties consistent with the inherent nature of MSCs. (27)
These unique characteristics make ABPCs appealing for promoting tissue regeneration, particularly for poorly regenerated tissues such as the spinal cord. However, direct injection of ABPCs into the human body is not clinically acceptable because of ethical and safety concerns. (27) Extracellular vesicles (EVs) are nanosized membranous vesicles that transport different informative cargoes (including lipids, proteins, metabolites, and nucleic acids) from their parent cells. (28) Preclinical and clinical research has suggested that stem cell-derived EVs provide clinical benefits similar to those of their donor cells without the biosafety concerns inherent to stem cell therapy. (29) Therefore, EV-based therapy is an ideal alternative to stem cell therapy in medical applications. (30,31) In this study, we addressed the gap in knowledge regarding the role of EVs derived from ABPCs (EVsABPC) in SCI repair. EVsABPC are uniquely advantageous as they retain potent regenerative ABPC signals, which are not present in EVs from more commonly used stem cells like bone marrow stem cells (BMSCs) and neural stem cells (NSCs). Additionally, to overcome the challenges of targeted delivery in SCI, we engineered EVsABPC with activated cell-penetrating peptides (ACPP), enhancing their localization to the injury site. By comparing EVsABPC to BMSC- and NSC-derived EVs (EVsBMSC; EVsNSC), our study demonstrates how this innovative approach can more effectively promote neural regeneration, modulate the immune response, and improve functional recovery in SCI patients. The workflow of this study is illustrated in Scheme 1.

Scheme 1

Scheme 1. Schematic Illustration of Antler Blastema Progenitor Cell-Derived Extracellular Vesicles and Their Therapeutic Role in Spinal Cord Injury (SCI)a
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a(A) Isolation and modification of EVsABPC, derived from antler blastema progenitor cells (ABPCs) isolated from deer antler, were modified with DSPE-PEG and activatable cell-penetrating peptides (ACPP) to enhance their targeting ability at the SCI lesion site. (B) Intravenous injection of ACPP@EVsABPC in spinal cord injury (SCI) rats enables them to cross the blood-spinal cord barrier (BSCB) and selectively accumulate at the injury site. (C) SCI is characterized by an acute inflammatory response, axonal disruption and neuronal damage, and glial scar formation, which collectively inhibit neural repair and functional recovery. At the lesion site, ACPP@EVsABPC promote neural stem cell proliferation by enhancing cell cycle progression through the S phase, facilitate axonal growth, and reduce neuronal apoptosis via modulation of the Wnt and Hippo signaling pathways. Additionally, EVsABPC regulate inflammation by promoting macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype through the NF-κB signaling pathway, thereby supporting tissue repair and functional recovery.

Results

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Isolation and Characterization of EVsABPC

The periodic rapid growth of antlers is a meticulous process involving osteogenesis, neurogenesis, and angiogenesis, partly attributable to bioactive cues from ABPCs. (22,32) To avoid ethical and safety concerns of xenogeneic ABPCs, EVs were purified from ABPCs (EVsABPC), which inherit a multitude of biological signals from their originating cells. (27) Their features were characterized and compared with those of EVs from the most widely used NSCs (EVsNSC) and BMSCs (EVsBMSC) extracted from Sprague–Dawley (SD) rats (Figure 1A). EVsABPC exhibited analogous morphology, surface markers, and size distribution to EVsNSC and EVsBMSC (Figure 1B–D). Transmission electron microscopy (TEM) revealed the characteristic cup-shaped or spheroidal morphology of all EVs (Figure 1B). Nanoparticle tracking analysis (NTA) revealed that the overall particle size distribution profiles of EVs from the three different sources were in a similar range, with a primary peak at an approximate diameter of 154.4 nm (Figure 1C). Protein content-based characterization of the three types of EVs using Western blotting validated the elevated expression of EVs marker proteins CD63 and HSP90 while showing negative expression of the microvesicle-specific marker ANNEXIN A1 (ANXA1; Figure 1D). To ensure precise measurements and mitigate non-EV contaminants, we used the ExoView platform to characterize single EVs in a multiplex manner. (33) The captured EVs were labeled with fluorescent antibodies targeting CD9, CD81, and CD63. All EVs expressed the tetraspanin markers CD81, CD9, and CD63 (Figure 1E), confirming the uniformity of surface markers across the EVs.

Figure 1

Figure 1. Characterization and proteomic analysis of EVsABPC, EVsNSC, and EVsBMSC. (A) Schematic depicting the extraction process of EVs from ABPCs, NSCs, and BMSCs. The supernatants from the cultured cells were collected and subjected to ultracentrifugation to isolate the EVs. (B) TEM images validated the morphology of EVsNSC, EVsBMSC, and EVsABPC. Scale bar, 200 nm. (C) Quantification and size analyses of EVsABPC, EVsNSC, and EVsBMSC using NTA. (D) Western blot analysis of EVs marker proteins (CD63, HSP90, and ANXA1). An equal amount (2 μg) of protein in EVsNSC, EVsBMSC, and EVsABPC was loaded in each lane, using NSC, BMSC, and ABPC lysates as controls for surface markers. (E) ExoView analysis of EVsABPC, EVsNSC, and EVsBMSC. Representative composite images show EV markers CD9 (blue), CD81 (green), and CD63 (red). Scale bar, 5 μm. (F) Schematic illustration of the proteomic analysis workflow. EVsNSC, EVsBMSC, and EVsABPC were subjected to trypsin digestion and analyzed using a label-free peptide quantification method, followed by bioinformatic analysis. (G) Venn diagram showing the overlap of identified proteins among EVsABPC, EVsNSC, and EVsBMSC. (H) Volcano map indicating DEPs between EVsABPC and EVsNSC. Upregulated proteins are denoted using red dots, while downregulated proteins are indicated using blue dots; nonsignificant proteins are represented using gray dots. (I) GO term enrichment analyses depicting enriched biological processes upregulated between EVsABPC and EVsNSC. (J) Volcano plot showing DEPs between EVsABPC and EVsBMSC. (K) GO term enrichment analyses depicting enriched biological processes upregulated between EVsABPC and EVsBMSC.

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Further proteomic analysis was performed to characterize the composition of the different EVs using liquid chromatography-tandem mass spectrometry (LC-MS/MS; Figure 1F). Principal component analysis (PCA) indicated that the three biological replicates of each EV were homogeneous, confirming the repeatability and reliability of the data (Figure S1A). Venn diagrams demonstrated that among the 1432 proteins identified, 257 overlapped with all EV types (Figure 1G). A unique proteomic profile was identified in EVsABPCvia Gene Ontology (GO) analysis compared to EVsNSC, in which 79 upregulated proteins were involved in wound healing (including PPIA, ANXA5, and TIMP1), synaptic vesicle maturation (including PICALM, ATP6 V1B2, and ATP6 V1A), immune response (including B2M, MASP1, and CORO1A), neuronal cellular homeostasis (including APP, ATP6 V1B2, and ATP6 V1A), and response to growth factors (including SERPINE1, APP, and CORO1A; Figure 1H–I; |log2(FC)| > 1 and P < 0.05). Furthermore, compared to EVsBMSC, 139 upregulated proteins were identified in EVsABPC, which were related to synaptic signaling (including CACNA1C, CTBP2, and GRIK3), acute-phase response (including F8, SAA1, and SERPINA1A), and developmental maturation (including RHOA, THBS3, and PEBP1; Figure 1J–K, |log2(FC)| > 1, and P < 0.05). Selected proteins, including RHOA, PICALM, and B2M, identified as upregulated and associated with wound healing pathway proteins were further validated using Western blot to strengthen the credibility of the proteomic findings (Figure S1B,C). Subsequent Rectome pathway knowledgebase enrichment analysis indicated that upregulated differentially expressed proteins (DEPs) participated in axon guidance and nervous system development compared to EVsNSC and EVsBMSC (Figure S1D,E). Collectively, EVsABPC contained a variety of unique biological factors that favor neurogenesis, highlighting their therapeutic potential in addressing neurological injuries and disorders.

EVsABPC Enhanced NSC Proliferation In Vitro

Proteomic analysis revealed marked enrichment of proteins associated with nervous system development (including RHOA, DSCAM, and GFRA1) within EVsABPC compared to that within EVsBMSC (Figure 2A–B). Given the crucial role of NSCs in nervous system development and repair, (34) we investigated the impact of EVsABPC on NSCs, with a particular focus on NSC proliferation and differentiation. We first extracted NSCs from the cerebral cortex of E14 SD rats and characterized them using the specific markers Nestin and SOX2 (Figure S2A). EVs labeled with PKH26 were observed within the soma and processes of NSCs, as shown by the colocalization of PKH26 and Nestin staining in Figure S2B. This indicates successful internalization and distribution of EVs within the NSCs. Subsequently, NSCs were treated with either EVsABPC or EVsBMSC, using basal culture medium as vehicle control (Figure 2C). Upon treatment with EVsABPC, the proportion of Ki67-positive cells significantly increased (19.5 ± 0.7%). Compared to the EVsBMSC and vehicle groups, 1.3-fold and 2.1-fold increases were observed, respectively, suggesting a stronger ability of EVsABPC to promote the proliferation of NSCs (Figure 2D–E). Western blot analysis confirmed these results, demonstrating markedly elevated levels of Ki67 and Nestin in the EVsABPC group compared to those in the other groups (Figure 2F–G).

Figure 2

Figure 2. EVsABPC enhanced NSC proliferation in vitro. (A) Circular heatmap showing DEPs related to nervous system development between EVsABPC and EVsBMSC. (B) Violin plot depicting proteins related to nervous system development regulation in EVsABPC and EVsBMSC. Central box in each violin represents the interquartile range (IQR). Statistical significance between groups was determined using an unpaired Student’s t-test. ***P < 0.001. (C) Schematic of EVsABPC and EVsBMSC effects on NSCs proliferation. (D–E) Ki67 immunofluorescence staining (D) and quantification (E) in primary NSCs treated with vehicle, EVsBMSC, or EVsABPC (n = 3). Scale bar, 25 μm. (F) Western blot detection of Nestin, Ki67, and GAPDH protein levels in the vehicle, EVsBMSC, and EVsABPC groups. (G) Quantification of Nestin and Ki67 protein levels after different treatments (n = 3). (H) Schematic representation of FUCCI-based cell cycle evaluation. (I) Representative cell cycle images. Circle indicates tracked cell transitioning from G1 to G2/M within 24 h. Scale bar, 25 μm. (J) Bar graphs depicting the proportion of cells in different cell cycle phases over time under different treatments. (K) Line graphs showing dynamic variations in the proportion of cells in G1, S, and G2-M phases over time under different treatments (n = 3). Solid lines represent mean values for each phase, while shaded areas indicate standard deviation (SD). (L) Comparison of dynamic changes in the proportion of NSCs in S phase at different time points over 24 h under different treatments (n = 3). (M) Schematic illustration of evaluating effects of EVsABPC on NSC differentiation. (N) Representative immunofluorescence images showing expression of GFAP and TUJ1 in NSCs treated with vehicle, EVsBMSC, and EVsABPC for 2 d. Scale bar, 100 μm. (O) Measurement of proportion of TUJ1+ cells in each treatment group (n = 4). (P) Measurement of longest neurite length in TUJ1+ cells from each treatment group (n = 4). (E, G, O, and P) Statistical data are presented as mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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We used a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to observe single-cell behaviors with precise spatiotemporal resolution. This system employs genes linked to fluorescent reporters that emit different colors specific to each cell cycle phase, allowing real-time imaging of cell cycle phases (Figure 2H). (35) We induced the expression of FUCCI reporters in NSCs via lentiviral transduction. Cell populations with red, yellow, or green light correspond to cells in the quiescent G1 phase, early S-phase, or proliferating late-S/G2-M phases, respectively (Figure 2I). A representative example of a cell transitioning from the G1 (red) to the G2-M phase (green) is shown in Figure 2I. In each group, we observed synchronized oscillations in the proportions of cells in the G1 (red), S (yellow), and G2-M (green) phases as the overall cell population cycled over time, reflecting typical cell cycle dynamics (Figure 2J–K). (36) The proportion of cells in the S phase (yellow) accumulated over 24 h in the EVsABPC group, demonstrating a marked increase compared to the other groups (Figure 2L).
Next, we explored the effects of EVsABPC on NSC differentiation (Figure 2M). Immunostaining was performed for specific markers of neurons and astrocytes using βIII-tubulin (TUJ1) and glial fibrillary acidic protein (GFAP), respectively, to examine the differentiation ratio of NSCs into neurons and astrocytes (Figure 2N). No significant differences were observed in the proportions of TUJ1+ and GFAP+ cells between the groups (Figure 2N–O and S2C; P > 0.05), indicating a negligible effect of EVsABPC on the differentiation of NSCs. Nevertheless, we observed a marked rise in the length of Tuj1+ cells in the EVsABPC group (106.5 ± 8.13 μm), which was 2.6-fold greater than that in the vehicle group and 1.4-fold that in the EVsBMSC group (Figure 2P, S2D). This finding suggested that EVsABPC could promote the outgrowth of neurite neurons upon their differentiation from NSCs rather than affecting the lineage specification of NSCs.

EVsABPC Promoted Neurite Outgrowth and Branching

EVsABPC contained abundant proteins associated with axon guidance (including RPS2, ACTG1, and RHOA) in contrast to EVsNSC, highlighting their potential to drive axon growth (Figure 3A–B). Therefore, dorsal root ganglion (DRG) neurons were utilized to evaluate the potential impacts of EVsABPC on axonal outgrowth (Figure 3C). Intriguingly, different EVs increased the complexity of neurite branching, which was associated with neural circuit development, including the establishment of synapses and guidance of axons. (37) Sholl analysis demonstrated that in DRG neurons, the branching complexity in the EVsABPC group was approximately 1.5-fold, 2.7-fold, and 4.1-fold higher than that in the EVsNSC, EVsBMSC, and vehicle groups, respectively (Figure 3D). Upon treatment with EVsABPC for 48 h, the axon length of neurons significantly increased, reaching a maximum of 5.2-fold that of the vehicle, 1.3-fold that of EVsNSC, and 2.2-fold that of the EVsBMSC (Figure S3A) groups. In DRG explants, EVs from different sources showed a notable elevation in the mean length of the five longest axons in comparison to the vehicle group. The average length of DRG explants was the highest in the EVsABPC group (2,017.5 ± 95.6 μm), followed by EVsNSC (1,445.8 ± 164.9 μm), EVsBMSC (1,205.6 ± 46.9 μm) groups, and the lowest in the vehicle group (969.3 ± 40.0 μm; Figure 3E). Furthermore, in DRG explants, EVsABPC significantly increased branching complexity, which was 2.5-fold, 2.3-fold, and over 3-fold higher than that in the EVsNSC, EVsBMSC, and vehicle groups, respectively (Figure S3B). Collectively, the vehicle group had the shortest neurite outgrowth and the lowest number of branches in both the DRG neurons and explants. The EVsNSC and EVsBMSC groups exhibited moderate increases in neurite outgrowth and branching. Notably, the EVsABPC group exhibited the strongest effects, with the most extended neurites and branches. These results demonstrated that EVsABPC outperformed the other EVs in establishing neuronal branching complexity, highlighting their potential for establishing synaptic connections.

Figure 3

Figure 3. EVsABPC enhanced neurite outgrowth and reduce neuronal apoptosis. (A) Heatmap of DEPs related to axon guidance in EVsABPC and EVsNSC. (B) Violin plot of axon guidance proteins in EVsABPC and EVsNSC. Central box in each violin represents IQR. Statistical significance between groups was determined using an unpaired Student’s t-test. ***P < 0.001. (C) Schematic of EVsNSC, EVsBMSC, or EVsABPC effects on DRG neurite outgrowth. (D) Representative images (left) showing TUJ1-stained DRG neurons treated with vehicle, EVsNSC, EVsBMSC, or EVsABPC for 48 h. Scale bar, 50 μm. Sholl analysis (right) demonstrates the effects of different treatments on neurite branching complexity in DRG neurons. Representative Sholl plots were shown as insets, which were normalized against the vehicle group (n = 3). (E) Images depicting TUJ1 (green) /S100 (red) staining (left) in DRG explants treated with vehicle, EVsNSC, EVsBMSC, or EVsABPC for 48 h. Scale bar, 500 μm. Quantification of the mean length of the five longest axons in DRG explants (right) under different treatments (n = 5). (F) Workflow of RNA sequencing (RNA-seq) analysis. (G) RNA-seq analyses of regulated pathways in Neurons_Vehicle vs Neurons_EVsABPC. Left: Up- and downregulated genes. Middle: DEG expression heatmap. Right: Functional enrichment. (H) GSEA of biological pathways including Wnt signaling, axon guidance, Hippo signaling, and mast cell activation. (I) Western blot of key proteins in Wnt pathways in neurons treated with EVsABPC and the Wnt inhibitor XAV939. (J) Representative images of TUNEL staining in neuron cultures. Red indicates TUNEL-positive apoptotic cells, and green indicates NeuN-positive neurons. Scale bar, 50 μm. (K) Quantification of apoptosis in neuron culture. All statistical data are represented as mean ± SD. (E and K) Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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Functional deficits following SCI primarily stem from the depletion of mature neurons, a consequence directly linked to apoptotic processes in neural cells. (38) We examined the effects of different EVs on Ventral spinal cord (VSC4.1) neurons. Neuronal apoptosis was induced using lipopolysaccharide (LPS), a well-established cell model of neuronal apoptosis in SCI. (39) We found that EVs from different stem cells rescued neuronal apoptosis induced by LPS (67.34 ± 9.8%), with the strongest ability observed in EVsABPC, which decreased apoptosis to 15.33 ± 2.9%. EVsNSC and EVsBMSC also demonstrated significant effects, although to a lesser extent, reducing apoptosis to approximately 35.12 ± 3.3% and 29.14 ± 8.8%, respectively (Figure S3C–D). This aligns with previous findings that stem cell-derived EVs can suppress neuronal apoptosis following SCI. (40)
We further examined the underlying mechanisms involved in the positive effects of EVsABPC on neurite outgrowth and branching using RNA sequencing (RNA-Seq) (Figure 3F). The PCA plot showed the clustering of neurons treated with EVsABPC compared to the vehicle group, confirming the repeatability and reliability of the data (Figure S4A). The heatmap and volcano plot (Figure 3G and S4B) revealed that 799 genes exhibited significant differential expression following EVsABPC treatment. Among these, 671 genes showed increased expression, whereas 128 genes exhibited decreased expression (|log2(FC)| > 0.58 and P < 0.05). GO enrichment analysis revealed that the upregulated differentially expressed genes (DEGs) were associated with neuron projection development (including Bcl2, Cd2ap, and Dst), axonogenesis (including Apc, Bcl2, and Cd2ap), and axon guidance (including Egr2, Gli3, and Hoxa2), which are pivotal for nerve regeneration (Figure 3G).
Gene set enrichment analysis (GSEA) in EVsABPC-treated neurons revealed significant enrichment in multiple biological pathways, including the Hippo signaling pathway, canonical Wnt signaling pathway, axon guidance pathway, and mast cell activation pathway related to immune response (Figure 3H). Among the enriched pathways identified, the Hippo and Wnt signaling pathways were of particular interest due to their established roles in promoting cell survival and regeneration. (41,42) Since treatment with EVsABPC alone significantly reduced neuronal apoptosis, to further investigate this hypothesis, neurons were pretreated with the Wnt pathway inhibitor XAV939 to suppress Wnt signaling activity (Figure 3I). (43) XAV939 treatment significantly suppressed the expression of beta-catenin (β-catenin) and increased the levels of GSK-3β, indicating inhibited Wnt signaling activity (Figure S4C–E). Further analysis showed that, compared to the EVsABPC group, the levels of the antiapoptotic protein BCL-2 were significantly decreased, while the levels of the pro-apoptotic protein BAX were markedly increased in the XAV939 group. However, subsequent treatment with EVsABPC in XAV939-pretreated neurons (XAV939 + EVsABPC group) partially restored the expression of BCL-2 and significantly reduced the levels of BAX compared to the XAV939 group alone (Figure S4F–G). Additionally, TUNEL and NeuN immunofluorescence staining showed that the TUNEL/NeuN double-positive cell ratio was significantly elevated in the XAV939-pretreated group. Although this ratio decreased following subsequent EVsABPC treatment, it remained higher than that in the group treated with EVsABPC alone, further supporting the involvement of the Wnt pathway in the antiapoptotic effects of EVsABPC (Figure 3J–K).
To explore the role of the Hippo signaling pathway in the effect of EVs on neural apoptosis, we used the Hippo pathway activator Verteporfin (VTP) to enhance the activity of the Hippo pathway. (44) VTP treatment alone resulted in a marked increase in phosphorylated YAP (p-YAP), accompanied by a decrease in BCL-2 levels and an increase in BAX levels (Figure S4H), indicating enhanced neuronal apoptosis due to Hippo pathway activation. Subsequent treatment with EVsABPC in VTP-treated neurons led to a reduction in p-YAP levels, an increase in BCL-2 levels, and a decrease in BAX levels (Figure S4H–K), suggesting that EVsABPC counteracted the pro-apoptotic effects induced by Hippo pathway activation. Collectively, these findings indicated that EVsABPC supported neuronal survival by regulating both the Wnt and Hippo signaling pathways.

EVsABPC Exhibited Robust Immunomodulatory Properties

EVs derived from MSCs exhibit immunomodulatory properties. (45) Given that ABPCs are a unique type of MSCs, a variety of molecules related to the modulation of inflammatory responses, such as VWF, TIMP, and COL6A1, were identified in EVsABPC (Figure S5A). In addition, the score for the gene set of the innate immune system was significantly higher in EVsABPC than in EVsBMSC and EVsNSC (Figure S5B), suggesting a potentially stronger immunomodulatory ability of EVsABPC.
Next, we investigated the immunomodulatory properties of EVsABPC, focusing on the shift of macrophages from a proinflammatory (M1) to a tissue-repairing (M2) phenotype, given their pivotal role in immunoregulation and nerve regeneration (Figure 4A). Upon treatment with lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ), we observed a marked elevation in proinflammatory factors in RAW264.7 macrophages, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). This finding suggested that a combination of LPS and IFN-γ was sufficient to switch M0 macrophages into the proinflammatory M1 phenotype (Figure 4B). Notably, the expression of these proinflammatory factors was significantly reduced following EV application, with EVsABPC showing the most pronounced effect (Figure 4C). Immunofluorescence staining showed that PKH26-labeled EVsABPC were internalized by macrophages and distributed within the soma of M1 macrophages (Figure S5C). In addition, the fluorescence intensity of iNOS+ cells (M1) significantly decreased after EVsABPC treatment, showing a reduction to approximately 14.1% of the level observed in the LPS + IFN-γ group, 35.4% of the level observed in the EVsNSC group, and 39.1% of the level observed in the EVsBMSC group. Conversely, the EVsABPC-treated group exhibited a remarkable increase in the fluorescence intensity of Arg1+ cells (M2), which was approximately 2.7-fold greater than that in the LPS + IFN-γ group (Figure 4D–E). An identical trend was observed using fluorescence-activated cell sorting (FACS). Upon treatment with LPS + IFN-γ, the CD206+/F4/80+ polarization ratio (M2/M0) increased from 1.90% (vehicle) to 5.87% (EVsNSC), 7.99% (EVsBMSC), and further to 12.53% (EVsABPC; Figure 4F–G). Additionally, the CD86+/F4/80+ polarization ratio (M1/M0) was 73.25% after treatment with LPS + IFN-γ. Following treatment with EVsNSC, EVsBMSC, and EVsABPC, the proportion of M1 cells was reduced to 32.75%, 30.32%, and 27.74%, respectively (Figure S5D). These findings further confirmed that EVsABPC possessed a robust capability to switch from M1 to M2. To investigate the possible mechanism by which EVsABPC repopulated macrophages, we assessed the relative levels of proteins involved in the inflammatory Nuclear Factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway, which is critical for regulating the M1/M2 switch. (46) We found that EVsABPC inhibited the NF-κB pathway, as evidenced by the decreased expression of phosphorylated IκBα (p-IκBα) and phosphorylated P65 (p-P65). Collectively, these results demonstrate that EVsABPC inhibited M1 macrophages and promoted M2 macrophages, probably by regulating the NF-κB pathway (Figure 4H).

Figure 4

Figure 4. EVsABPC exhibited robust immunomodulatory ability in vitro and in vivo. (A) Schematic of evaluating the immunomodulatory effect of EVs on macrophage polarization in RAW264.7 cells. (B) Western blot showing proinflammatory factors iNOS and TNF-α in RAW 264.7 cells treated with LPS + IFN-γ, with or without different EVs. (C) Quantification of the relative iNOS and TNF-α expression levels in macrophages (n = 3). (D) Immunofluorescence images showing effects of EVs on macrophage polarization. Top: iNOS (red), F4/80 (green), DAPI (blue). Bottom: Arg1 (red), F4/80 (green), DAPI (blue). Scale bar, 50 μm. (E) Quantification of the fluorescence intensity of iNOS+ cells (M1) and Arg1+ cells (M2) (n = 4). (F) Flow cytometric analysis showing the percentage of CD86+ and CD206+ cells in F4/80+ gated macrophages after different treatments. (G) Quantification of the percentage of CD206+ cells (M2) in F4/80+ macrophages (primarily in Q2). (H) Western blot showing Arg1, IκBα, p-IκBα, p-P65, and P65 in macrophages treated with LPS + IFN-γ, with or without EVsABPC. (I) Immunofluorescence images showing effects of EVs on microglial polarization in the spinal cord at 14 dpi. Left: iNOS (red), iba1 (green), DAPI (blue). Right: Arg1 (red), iba1 (green), DAPI (blue). Scale bar, 200 μm. Insets show higher magnification of boxed areas. Scale bar, 50 μm. (J) Quantification of the fluorescence intensity of iNOS+ and Arg1+ microglia in the spinal cord (n = 4). (K) ELISA assay assessing spinal proinflammatory cytokine IL-1β, IL-6, and TNF-α levels following different treatments. (n = 3). (C, E, J, and K) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant. a.u: arbitrary unit.

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Next, we investigated the immunomodulatory ability of EVsABPCin vivo. Fourteen days following treatment with EVs, the abundance of Arg1+ cells was the highest in the EVsABPC group, followed by the EVsNSC and EVsBMSC groups, and was lowest in the vehicle group. The opposite trend was observed for iNOS+ intensity, with the most significant value in the vehicle group and the lowest value in the EVsABPC group (Figure 4I–J). These findings confirmed the ability of EVsABPC to switch M1 macrophages to the M2 phenotype in vivo. In addition, EVs treatment significantly attenuated the upregulation of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in the focal areas after SCI, with EVsABPC showing the most pronounced inhibition of proinflammatory cytokines (Figure 4K). Thus, EVsABPC possessed a robust ability to ameliorate inflammatory responses and switch to the M1/M2 transition, which was beneficial for cell survival and tissue regeneration.

Delivery System for EVs to Target SCI Lesions

Current therapeutic applications of EVs involve nontissue-specific targeting, which results in their nonselective distribution across diverse tissues upon systematic administration. (47) To address this issue, the surface of EVs was modified with activatable cell-penetrating peptides (ACPPs), which comprised a sequence of polyanionic inhibitory domain, polycationic CPP, and peptide linker matrix that exhibited sensitivity to metalloproteinases (MMPs; Figure 5A). (48,49) In SCI lesions rich in MMPs, the linker undergoes selective cleavage, causing the dissociation of the inhibitory acidic peptide based on its off-rate, thereby exposing the cationic CPP for the delivery of EVs into adjacent cells. (49) Docking analysis revealed specific interactions between MMP2 and ACPP peptides, including multiple hydrogen bonds and salt bridges (Figure 5B). The close-up view highlights critical amino acid residues such as GLU-33, ASN-45, and ARG-67 (colored purple), alongside key interactions like GLU-345 with ARG-15 and ASP-210 with ARG-16, which contribute to stabilizing the binding pocket (Figure 5B, Table 1). Subsequently, we performed mass spectrometry on the ACPP-engineered EVs. Figure 5C shows an example of a DIA spectrum containing the target peptide (EEEEEEEEEEPLGLAGR) predicted using DeepNovo-DIA. Each panel highlights fragment ions that corroborate the respective peptide: red denotes y ions, and blue denotes b ions. The error plot matches the experimentally obtained and theoretical mass peaks, indicating high measurement accuracy (within ±0.05 Da). Collectively, these results confirmed the peptide sequence and successful construction of ACPP@EVs.

Figure 5

Figure 5. ACPP-modified EVsABPC enhanced targeted delivery to SCI sites. (A) Schematic diagram illustrating the modification of EVs with ACPPs to form ACPP@EVs. (B) Docking simulation between MMP2 and ACPP peptides showing specific interactions, including hydrogen bonds and salt bridges, with key amino acid residues highlighted in purple. (C) Mass spectrometry analysis confirming the peptide sequence (EEEEEEEEEEPLGLAGR) in ACPP@EVsABPC, as indicated by the DIA spectrum with fragment ions (red, y ion; blue, b ion) and an error plot showing the measurement accuracy. (D) ACPP@EVs labeled with a red fluorescent protein (RFP) were administered to SCI rats via tail vein injection, and their distribution was monitored 12 h postinjection by in vivo and ex vivo imaging. (E) Representative in vivo images showing the distribution of Dir@EVsABPC and ACPP@EVsABPC at the injury site 12 h postinjection. (F) Quantification of radiant efficiency at the injury site (n = 3). (G–H) Ex vivo imaging of the major organs showing the distribution of Dir@EVsABPC (G) and ACPP@EVsABPC (H). (I–J) Quantification of radiant efficiency in the spinal cord (I), spleen, lungs, kidneys, and liver (J) (n = 3). (K) Two-photon imaging of the dynamics of EVs from the vasculature to the spinal cord 4 h postinjection, with blood vessels (green) and EVs (red). Scale bar (left), 200 μm; Scale bar (right), 50 μm. (L) Quantification of fluorescence intensity of EVs in the spinal cord, as visualized by two-photon imaging (n = 5). (M) Quantification of the fluorescence intensity of Dir@EVsABPC and ACPP@ EVsABPC in spinal cord histology (n = 4). (N) Representative images of EV distribution in the spinal cord, with DAPI (blue) staining nuclei and Dir@EVs and ACPP@EVs (red). Scale bar, 25 μm. (F, I, J, L, and M) All statistical data are represented as mean ± SD. Statistical significance between groups was determined using an unpaired Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant. a.u: arbitrary unit.

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We further labeled ACPP@EVsABPC with a red fluorescent protein (RFP) to examine its in vivo distribution, using Dir-labeled EVs as controls. EVs were then administered to SCI rats via tail vein injection, and their distribution was monitored by in vivo imaging of small animals 12 h postinjection (Figure 5D). The fluorescence signals in rats injected with Dir@EVsABPC were predominantly distributed outside the SCI site. In contrast, in the ACPP@EVsABPC group, the fluorescence signals were mainly localized at the site of SCI, which was 1.8 times greater than that in the Dir@EVsABPC group (Figure 5E–F). The major organs of rats were dissected and arranged accordingly, as shown in Figure 5D. In the ACPP@EVsABPC group, a notable increase in fluorescence signals was observed in the spinal cord, measuring 2.3-fold higher compared to that in the Dir@EVsABPC group. (Figure 5G–I). In contrast, the kidneys, liver, spleen, and lungs showed a decrease in fluorescence to approximately 47.6, 35.7, 45.5, and 29.4% of the levels observed in the DiR@EVsABPC group, respectively (Figure 5J). These findings indicated that engineering EVsABPC with ACPP increased their targeting of the spinal cord after injury. Figure S6A–C shows the ex vivo distribution of ACPP-loaded EVsNSC and EVsBMSC.
The transport of EVs from the vasculature to the adjacent spinal cord was further visualized using two-photon imaging, with blood vessels marked with FITC (green) and EVs labeled with RFP or Dir (red). Four hours postinjection, high-intensity red fluorescence signals were observed in the infarct zone of spinal cord lesions in the ACPP@EVsABPC group (Figure 5K). The fluorescence intensity of EVs in the ACPP@EVsABPC group was nearly 3.6-fold higher than that in the Dir@EVsABPC group (Figure 5L). Similarly, a significantly higher intensity of red fluorescence was confirmed in the ACPP@EVsABPC group by histological analysis of local spinal cord lesions, which was 9.4-fold higher than that in the Dir@EVsABPC group (Figure 5M–N). Thus, using a combination of in vivo imaging and ex vivo two-photon imaging techniques, we provide direct evidence that ACPP@EVsABPC selectively accumulates at the SCI sites.
To evaluate the anti-inflammatory properties of ACPP further, we measured the expression levels of proinflammatory cytokines TNF-α and IL-1β in LPS + IFN-γ pretreated macrophages across three groups: vehicle control (LPS + IFN-γ alone), unmodified EVsABPC, and ACPP@EVsABPC. Both the unmodified EVsABPC and ACPP@EVsABPC groups significantly reduced TNF-α and IL-6 levels compared to the vehicle control group (Figure S6D). However, no significant difference was detected between the unmodified EVsABPC and ACPP@EVsABPC groups, suggesting that the observed anti-inflammatory effect is primarily due to the inherent properties of EVsABPC.

EVsABPC Facilitated Neural Network Reconstruction after SCI

Next, we examined the potential therapeutic effects of ACPP@EVsABPC on neural regeneration in the rat traumatic SCI model (Figure 6A and Figure S7A). After SCI, the rats were administered different ACPP@EVs three times weekly via tail vein injection until sacrifice, after which they were successfully transferred to the spinal cord lesions in vivo. Fourteen days postinjury (dpi), sagittal sections of the injured spinal cord were stained with GFAP to visualize astrocytes and with TUJ1 to mark newborn neurons. In the vehicle group, there was a restricted presence of TUJ1+ cells (9.85 ± 2.17%) observed from the rostral to the caudal of the lesion site, especially the epicenter. This was accompanied by overactivity and accumulation of astrocytes (Figure 6B). In contrast, TUJ1 positive cells were more evenly distributed within the lesion site in the ACPP@EV-treated groups (Figure 6C–E). Among the three types of EVs, ACPP@EVsABPC exhibited the greatest neuroprotective effect, with 81.59 ± 5.82% of neurons, which was 1.2 times the proportion found in the ACPP@EVsNSC group (67.38 ± 7.92%) and more than 1.4 times that in the ACPP@EVsBMSC group (58.07 ± 1.80%; Figure 6F). Additionally, ACPP@EVsABPC was significantly more effective than the vehicle in limiting the overactivity and accumulation of astrocytes during secondary injury (Figure 6G). At 7 dpi, we found a significantly higher density of neurons in the ACPP@EVsABPC group than in the other groups via Nissl staining, which represented a 1.9-fold higher density in the vehicle group, 1.2-fold higher density in the ACPP@EVsNSC group, and 1.5-fold higher density in the ACPP@EVsBMSC group (Figure 6H–I). These findings suggest that EVsABPC have the ability to optimize the proportion of neurons and astrocytes for the promotion of the spinal cord and have a greater neuroprotective effect than EVs from NSCs and BMSCs.

Figure 6

Figure 6. ACPP@EVsABPC promoted neural regeneration and reduce neuronal apoptosis post-SCI in vivo. (A) Schedule of SCI rats treated with ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC. Treatment included intravenous injections (i.v.) three times weekly, followed by assessments at various time points to evaluate neural regeneration and motor function recovery. (B–E) Representative immunofluorescence images of sagittal sections of the injured spinal cord stained for GFAP (green) and TUJ1 (red) to visualize astrocytes and newborn neurons at 14 dpi treated with ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC, respectively. Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas (b1-b3, c1-c3, d1-d3, e1-e3). Scale bar, 50 μm. (F–G) Quantification of the areas of TUJ1+ neurons (F) and GFAP+ astrocytes (G) in the lesion site (n = 3). (H–I) Representative Nissl staining images and quantification of the average optical density of neurons at 7 dpi (n = 5). Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas. Scale bar, 50 μm. (J–K) Quantification of the areas of NF+ axons and GAP43+ axons at 28 dpi (n = 3). (L) Representative immunofluorescence images of sagittal sections of the injured spinal cord stained for NF (white) and GAP43 (purple) to visualize nerve fibers and regenerating axons. Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas (L1–L4). Scale bar, 50 μm. (M–N) Representative transmission electron micrographs (M) and scatterplots (N) comparing the myelin thickness and the correlation and linear regression between axon diameter and G-ratio in the vehicle, ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC treatment groups. Axons were selected from 4 rats per group for measurement (n = 60 measured axons for each group). Scale bars, 4 μm. Correlation analyses were performed using Pearson’s correlation. (F, G, I, J, and K) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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The primary cause of functional impairments after SCI is the depletion of mature neurons, directly caused by the initiation of programmed cell death in neural cells. (50) Thus, we examined the antiapoptotic effects of ACPP@EVsABPCin vivo using TUNEL staining (Figure S7B–C). At 7 dpi, the lowest apoptosis in neurons was found in the ACPP@EVsABPC (53.7 ± 3.2%) group, followed by the ACPP@EVsBMSC (71.1 ± 2.4%) and ACPP@EVsNSC (70.7 ± 3.5%) groups, and the highest in the vehicle group (84.3 ± 1.2%). These findings suggest a strong antiapoptotic effect of ACPP@EVsABPC, which may contribute to greater neuronal preservation and facilitate neural network reconstruction after SCI.
At 28 dpi, we immunostained growth-associated protein-43 (GAP43) to visualize regenerated budding axons and neurofilament 200 (NF) to observe mature nerve fibers. Compared with the other groups, GAP43 positive axons in the EVsABPC group were arranged in a relatively uniform and oriented pattern (Figure 6J–L), indicating better neural network reconstruction after SCI. Additionally, we detected robust neurite regrowth in the ACPP@EVsABPC group, with significantly larger areas of GAP43 and NF than those in the other groups (Figure 6J–K). Dotted NF+ fibers were found in the vehicle group and were mostly limited to injured areas. In contrast, nerve fibers in the different EV groups were distributed across the lesion regions, with a length more than twice that of the vehicle group. Notably, ACPP@EVsABPC induced the greatest increase in nerve fiber length, which was 1.3-fold longer than that in the ACPP@EVsNSC group and 1.5-fold longer than that in the ACPP@EVsBMSC group (Figure S8A). This finding suggests that EVsABPC have a greater ability to promote the regrowth of new nerve fibers than EVs from NSCs and BMSCs.
Demyelination leaves axons exposed, making them prone to atrophy and degeneration, which worsens dysfunction. Promoting myelin repair could be a potential strategy for restoring function after SCI. (14) To explore this, we examined the effects of EVsABPC on axonal remyelination. Using transmission electron microscopy, we observed extensive demyelination in the vehicle group, characterized by vacuole-like structures, thinner myelin sheaths, and pronounced delamination (Figure 6M). While there were no significant differences in axonal diameter among groups (Figure S8B), we noted a significant reduction in the G-ratio (axon-to-fiber ratio) in the ACPP@EVsABPC group (0.75 [95% CI: 0.74–0.76]) compared to that in the other groups (Figure S8C). This suggests that ACPP@EVsABPC treatment led to a high degree of remyelination. Additionally, although the G-ratio showed a weak correlation with axon diameter, the myelin thickness in rats treated with ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC was more proportional to axon diameter compared to the vehicle group. Notably, the ACPP@EVsABPC group exhibited the thickest myelin sheaths, further emphasizing a hypermyelination phenotype (Figure 6N).
Further, we used immunostaining for myelin basic protein (MBP) to visualize remyelinated axons. In contrast to the vehicle group, where few myelinated axons were found at the caudal ends or lesion center, MBP-positive cells and myelinated axons were significantly more abundant in the ACPP@EVsABPC group after 6 weeks (Figure S8D–E). Together, these findings indicated that ACPP@EVsABPC treatment accelerated myelination in the central nervous system, suggesting it may be a promising approach for promoting recovery in SCI.

EVsABPC Improved Motor Function Recovery after SCI

We assessed the neurological function recovery after 8 weeks of EVsABPC treatment via comprehensive assessments of behavior, motor function, and neurophysiology. Images of the right claw captured during the climbing test are shown in Figure 7A. The rats in the vehicle group dragged hind limbs in a contractured state two months postsurgery. In contrast, after treatment with different EVs, rats exhibited slow and coordinated movements of both hind limbs, with the most significant improvement observed in the EVsABPC group.

Figure 7

Figure 7. ACPP@EVsABPC improved motor function recovery post-SCI. (A) Representative images of the right hindlimb of rats during the climbing test showing the effects of treatment with ACPP@EVsNSC, ACPP@EVsBMSC, or ACPP@EVsABPC. (B) Representative 3D stress diagrams of the right hindlimb at 8 wpi. (C) The representative footprints recorded by the Catwalk system (cyan for right front, RF; yellow for left front, LF; green for left hind, LH; and magenta for right hind, RH). (D–E) Quantification of the base of support (D) and stride length (E) in different treatment groups based on footprint patterns (n = 3). (F–G) Quantification of CMAP amplitude (F) and latency (G) in the different treatment groups (n = 4). (H) Representative MEP waveforms in the different groups. (I) BBB locomotor rating scale of hindlimbs in different groups during the 8-week treatment period (n = 5). All statistical data are represented as mean ± SD (D, E, F, and J) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. (I) Statistical differences were determined using two-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the Vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to ACPP@EVsBMSC group; @P < 0.05, @@P < 0.01, @@@P < 0.001 compared to ACPP@EVsNSC group. ns: not significant.

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Following this, we conducted a thorough quantitative assessment of motor function using the CatWalk system at 8 weeks postinjury (wpi). (51) Gait analysis of SCI rats was limited to forepaw data because of hindlimb paralysis. A typical 3D image showed that the hind toes of EV-treated rats supported their weight, with the most significant improvement observed in the EVsABPC-treated group (Figure 7B). In addition, the footprints of rats in the vehicle group exhibited a linear pattern, indicating continuous hindlimb dragging. In contrast, EVsABPC group rats were able to lift their hindlimbs off the ground, exhibiting frequent plantar stepping and occasional forelimb-hindlimb coordination, demonstrating recovery of motor function (Figure 7C). Additionally, ACPP@EVsABPC significantly decreased the support base in SCI rats while increasing stride length, demonstrating a substantial improvement in locomotor coordination between the fore and hind limbs (Figure 7D–E).
Motor-evoked potential (MEP) testing revealed that compound muscle action potential (CMAP) signals from the right hind limbs in the vehicle group decreased to approximately 31.25% of those in the sham group 8 weeks post-SCI. In contrast, the CMAPs signal amplitude was 2.3 ± 0.2 mV in the ACPP@EVsABPC group, which was approximately 1.6-fold higher than that in the ACPP@EVsNSC group, 2.1-fold of that in the ACPP@EVsBMSC group, and 2.6-fold of that in the vehicle group (Figure 7F–H). Additionally, the Basso-Beattie-Bresnahan (BBB) assay was utilized to evaluate locomotor function recovery, conducted 1 day prior to injury and again until sacrifice. At 56 dpi, the rats in vehicle group continued to drag their hind limbs. In contrast, we observed the best motor function improvement in the ACPP@EVsABPC group, whereas rats in the ACPP@EVsNSC and ACPP@EVsBMSC groups displayed plantar placement with some degree of support; however, their stepping lacked coordination (Figure 7I). Collectively, these results demonstrate that ACPP@EVsABPC is superior to ACPP@EVsNSC and ACPP@EVsBMSC in enhancing neurological function after SCI.
We also assessed the biomedical safety of ACPP@EVsABPC. As shown in Figure S9A, histological analysis of major organs was conducted at 28 dpi. Consistent with expectations, Hematoxylin and Eosin (H&E) staining revealed no significant tissue damage in cardiomyocytes, hepatic cells, splenic tissues, alveolar cells, or renal glomeruli (Figure S9A). Additionally, we performed a quantitative analysis of inflammatory cells (including monocytes and lymphocytes), which showed that the number of inflammatory cells remained within the normal range (Figure S9B). (52) To further verify the biosafety of ACPP@EVsABPC, we evaluated a complete blood count (Figure S9C), along with ALT, AST, BUN, and cholesterol levels (Figure S9D) at 28 dpi, assessing liver function, kidney function, lipid metabolism, and immune status. The results showed no significant abnormalities across different treatment groups, indicating that ACPP@EVsABPC had no adverse effects on major physiological functions.

Discussion

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This study identified ABPCs as a viable source of EVs that contain an abundance of unique pro-regenerative factors, including proneurogenic factors (RPS2, ACTG1, and RHOA) and immunomodulatory factors (Timp1, B2M, and VWF). In this study, we found that EVsABPC facilitated the proliferation of NSCs and promoted the extension of axons in differentiated neurons. In addition, EVsABPC showed a stronger neuroprotective effect in neurons than EVs from NSCs and BMSCs by enhancing neurite outgrowth and mitigating apoptosis. Moreover, EVsABPC exhibited strong immunomodulatory ability by switching the activation of macrophages from the inflammatory M1 phenotype toward the reparative M2 phenotype. In the therapeutic context, EVsABPC achieved better outcomes at both the histological and functional levels in rats with SCI than the most extensively studied EVs (EVsNSC and EVsBMSC). These findings suggest that ABPCs are a novel and practical source of EVs that contain a wealth of systemic pro-regenerative factors and have considerable translational value in the treatment of SCI.
The therapeutic potential of NSCs and BMSCs in regenerative medicine is widely recognized. (53,54) Numerous studies have increasingly noted that the advantageous effects of stem cells are primarily attributed to their paracrine activity rather than the persistent engraftment of transplanted stem cells. (15) Over the past decade, stem cell-derived EVs have been recognized as crucial mediators of stem cell paracrine action. (12) In addition, both preclinical and clinical studies indicate that EVs derived from stem cells can provide comparable therapeutic benefits to those of the donor cells themselves while circumventing the biosafety concerns associated with stem cell therapy. (29) Therefore, to avoid ethical and safety concerns regarding the direct application of xenogeneic and allogenic cells, EVs from different stem cells were used as they lack the ability to self-replicate, thereby addressing concerns about the potential risk of tumor formation following stem cell transplantation. (55) In this study, we found the potent ability of EVsABPC to promote nerve regeneration and modulate inflammatory responses, which was stronger than that of EVs from NSCs and BMSCs. The benefits of EVsABPC are likely due to their unique stemness and renewability of ABPCs. ABPCs can be expanded in vitro for at least 50 passages without significant senescence, whereas MSCs exhibit signs of senescence after 10–15 rounds of in vitro culture. (22,25) These characteristics make ABPCs ideal for producing stable phenotypic EVs in large quantities to support EV-based therapies.
The mechanisms through which ABPCs enable regeneration are complex and diverse. An intriguing observation was the rapid growth of nerve fibers toward the antler tip, where ABPCs reside. (22,32) Therefore, chemotaxis cues within ABPCs are likely to play a pivotal role in guiding the directional growth of nerve fibers, of which EVs may be a major mediator for their communication. In addition, the natural process of antler shedding in deer, which occurs without inflammation, highlights the intrinsic anti-inflammatory properties of ABPCs, which are critical for tissue healing and regeneration. Additionally, ABPCs exhibit high expression levels of genes associated with many pathways, notably the Hippo and Wnt signaling pathways. These pathways are fundamental for enhancing the regenerative functions of ABPCs and supporting cellular processes that are crucial for recovery. The Hippo signaling pathway is a conserved mechanism crucial for regulating cell proliferation and apoptosis. (42) YAP functions as a mechanically activated downstream effector of this pathway. (56) In addition, the Wnt signaling pathway promotes the survival and regeneration of neural cells by activating downstream effectors such as β-catenin. (41,57) The careful modulation of these pathways by ABPCs underlines their potential in therapeutic applications, bridging critical gaps in current regenerative medicine approaches and offering novel avenues for treatment.
EVsABPC offer several advantages in translational research. First, EVsABPC carry unique regenerative signals closely associated with the distinctive regenerative properties of ABPCs. Unlike BMSCs and NSCs, ABPCs can be consistently sourced from the annual regeneration cycle of sika deer antlers, resulting in higher EV yields and greater potential for neural regeneration. (22) Additionally, in our study, EVsABPC were modified with activated cell-penetrating peptides (ACPP) to enhance targeted delivery to the injury site. (49) Previous studies have shown that unmodified EVs are rapidly cleared in vivo and exhibit low retention rates. In contrast, the ACPP modification in our study significantly improved the local retention and therapeutic efficacy of EVsABPC, demonstrating superior neural regeneration and motor function recovery in the SCI model. Finally, the utilization of EVsABPC presents a viable strategy to overcome the limitations of stem cell therapies, including low retention and survival rates due to immune rejection. Furthermore, EVs are stable during long-term frozen storage or storage at room temperature after lyophilization, which is important for translational purposes. (58,59)
With the development of novel treatments, several concerns must be addressed before advancing current findings for clinical applications. First, a limitation of this study is the selection of the SCI model. Although the clip compression model offers certain advantages in simulating chronic compression and ischemic environments, it does not fully capture the complex pathology of human SCI. To further assess the clinical translational potential of our findings, future studies could incorporate multiple SCI models, such as those created using the IH Impactor or MASCIS Impactor, which may better simulate the complex pathology resulting from acute contusive injuries. (60,61) Furthermore, to increase clinical relevance, these findings should be replicated across different animal models. Conducting experiments in rats, mice, pigs, and even nonhuman primates would provide additional validation of EVsABPC’s safety and efficacy to enhance understanding of its therapeutic potential. Second, although EVs are safe for use in rodents and nonhuman primates because of their cell-free properties, a comprehensive study, including long-term safety monitoring (such as 3 or 6 months), is necessary to exclude any potential pro-tumorigenic effects. (62) An additional safety concern regarding EVs from different species is possible adverse immunological reactions, although this phenomenon was not observed in the current study. (62) Third, given the profound effect of EVsABPC on NSC proliferation and neurite outgrowth, a tissue-specific targeting strategy for EVs may allow neuroprotective treatment in specific tissues. In addition, while the current extraction techniques have provided promising results, future studies could incorporate advanced techniques for more precise and scalable EV isolation, especially microfluidic technologies. Incorporating these innovations in EVs extraction can minimize contamination risks and reduce time and labor, addressing scalability challenges associated with traditional methods. (63) Finally, EVsABPC-associated factors critical for dictating function need to be thoroughly assessed, which may reveal new factors that do not exist in other stem cells and potentially provide alternative translational avenues for specific tissue injury and repair.

Conclusions

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ABPCs are a unique type of mesenchymal stem cell that drives complete organ regeneration in mammals annually. EVsABPC contain pro-regenerative signals that differ from those in EVs derived from other stem cells. EVsABPC boost neural stem cell proliferation, activate neuronal regenerative potential, modulate immune response, and switch the M1/M2 polarization of macrophages. Engineered EVsABPC delivered intravenously significantly improve axonal growth, reduce astrogliosis, and greatly enhance motor functional recovery after SCI, outperforming EVs from other stem cell sources. These findings suggest that ABPCs are a practical source of EVs containing pro-regenerative factors and offer a promising therapeutic strategy for SCI repair.

Experimental Section

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Primary Extraction and Culture of Antler Blastema Progenitor Cells (ABPCs)

ABPCs were isolated according to a previously established protocol detailed by Qin et al., published in Science. (22) Briefly, the hard antlers of a two-year-old male sika deer were used to extract ABPCs. Five days after the antlers were cast, cells within the blastema were sorted using flow cytometry to identify CX43+FGFR2+ cells as ABPCs. Single-cell suspensions were prepared in Dulbecco’s modified Eagle Medium (DMEM; Gibco, 11965092) with 1% penicillin-streptomycin (Gibco, 15140122), 0.1% Mycoplasma Removal Agent, and fetal bovine serum (FBS; Gibco, 10099141C). Subsequently, these cells were plated in 10 cm culture dishes at a density of 2 × 105 cells/cm2. After 24 h, nonadherent cells were removed, and adherent cells were cultured. Upon reaching 80–90% confluence, the cells were harvested and reseeded at the same density for further expansion. ABPCs at passages 6–8 (P6–P8) were used in the experiments. This adjusted protocol ensures the effective isolation and maintenance of ABPCs with strong regenerative capabilities that are ready for further experimental use.

Primary Extraction and Culture of Neural Stem Cells

NSCs were extracted from the cerebral cortex of E14 SD rat embryos. Under sterile conditions, the cerebral cortex was carefully dissected from the embryos and mechanically dissociated into a single-cell suspension using fine forceps and gentle pipetting. The resulting cell suspension was cultured in Neurobasal medium (Gibco, Carlsbad, CA, USA) supplemented with 20 μL/mL B27 (Gibco, Carlsbad, CA, USA), 20 ng/mL epidermal growth factor (EGF, PeproTech), 20 ng/mL basic fibroblast growth factor (bFGF, Invitrogen, Carlsbad, CA, USA), and 1% penicillin-streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2, and the culture medium was changed every 2–3 d to ensure optimal growth conditions. When the cells aggregated into neurospheres and reached 80–90% confluence (P0), they were passaged using TrypLE Express (Thermo Fisher Scientific). Throughout the culture period, cell morphology and proliferation were regularly observed under a microscope to monitor cell health and growth. The presence of NSCs was confirmed using immunofluorescence staining for specific markers such as Nestin and SOX2, ensuring the effective isolation and culture of NSCs for subsequent experimental procedures. The NSCs were divided into three groups: vehicle Group, NSCs treated with the standard culture medium; EVsBMSC Group, NSCs treated with EVsBMSC (2 × 106 particles/mL) for 24 h; and EVsABPC Group, NSCs treated with EVsABPC (2 × 106 particles/mL) for 24 h.

Primary Extraction and Culture of DRG Neurons and Explants

SD rat pups aged 1–3 days were used for this procedure. The heads of the pups were removed, and the skin and bones along the spine and thoracic and cervical vertebrae were incised with microscissors to fully expose the DRG on both sides of the spinal canal. Each small bulbous dorsal root ganglion was removed using fine ophthalmic forceps. The outer membrane surrounding the DRG neurons was carefully peeled off, and the ganglia were cut into small pieces. (64) Collagenase type IV and 0.25% trypsin were added to the dish to digest DRG neurons, and digestion was terminated after 1 h by adding 4 mL of DF-12 medium containing FBS. The mixture was centrifuged at 1000 rpm for 5 min. The pellet was resuspended in Neurobasal complete medium, filtered through a 100 μm cell strainer, and inoculated onto cell culture plates that had been coated with matrix gel overnight. The same procedure was followed for DRG explants, but the ganglia were not cut into pieces before digestion, and the digestion time was reduced to 5 min. The DRG neurons and explants were treated with PBS, EVsNSC, EVsBMSC, or EVsABPC (2 × 106 particles/mL) for 48 h. The effects of the different treatments on DRG neurons and explant health and growth were observed and analyzed. Sholl analysis was conducted using ImageJ with the Sholl Analysis plugin, where concentric circles were drawn around the soma, and the number of dendritic intersections at each radius was quantified. Statistical analyses were conducted to compare the differences across groups.

Culture of RAW 264.7 Cells

RAW 264.7 cells (Procell, Wuhan, China, Cat# CL-0190) were divided into several groups. In the vehicle Group, the medium was replaced with fresh growth medium, with no additional treatment applied. In the LPS + IFN-γ group, RAW 264.7 macrophages were incubated with LPS (100 ng/mL, #L3129, Sigma-Aldrich) and IFN-γ (20 ng/mL, #L17001, Sigma-Aldrich) for 24 h, followed by a medium change to the normal growth medium. In the LPS + IFN-γ + EVsNSC group, macrophages were treated with LPS and IFN-γ for 24 h, followed by a medium change to normal growth medium and the addition of EVsNSC (2 × 106 particles/mL) for 24 h. In the LPS + IFN-γ + EVsBMSC group, macrophages were treated with LPS and IFN-γ for 24 h, followed by a medium change and the addition of EVsBMSC (2 × 106 particles/mL) for 24 h. In the LPS + IFN-γ + EVsABPC group, macrophages were treated with LPS and IFN-γ for 24 h, followed by a medium change and the addition of EVsABPC (2 × 106 particles/mL) for 24 h. All groups were incubated under the same conditions. After the treatment period, the cells were further processed to evaluate the effects of EVs on macrophage activation and polarization.

Culture of VSC 4.1 Motoneurons

VSC 4.1 neurons (EditGene #EDWT0066) were grown in a fully humidified incubator at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. In the LPS group, VSC 4.1 neurons were incubated with LPS (1000 ng/mL, #L3129, Sigma-Aldrich) for 24 h. For the LPS + EVsNSC group, VSC 4.1 motoneurons were incubated with LPS for 24 h, followed by a medium change to normal growth medium and the addition of EVsNSC (2 × 106 particles/mL) for 24 h. In the LPS + EVsBMSC group, VSC 4.1 motoneurons were incubated with LPS for 24 h, followed by a medium change to normal growth medium and the addition of EVsBMSC (2 × 106 particles/mL) for 24 h. In the LPS + EVsABPC group, VSC 4.1 motoneurons were incubated with LPS for 24 h, followed by a medium change to normal growth medium and the addition of EVsABPC (2 × 106 particles/mL) for 24 h.

EVs Isolation and Characterization

EVs with a diameter greater than 100 nm were isolated and purified using gradient centrifugation, following a previously described protocol. We first cultured ABPCs using a serum-free medium to avoid contamination with extracellular vesicles. Cell viability was assessed using the CCK-8 assay, ensuring that only culture supernatants from cells with over 90% viability were used for EV collection and purification. The collected supernatant was initially centrifuged at 750 × g for 20 min to remove cells, followed by a second centrifugation at 2,000 × g for 30 min to further eliminate cell debris and apoptotic bodies. The supernatant was then centrifuged at 16,000 × g for 70 min to precipitate the EV particles. The obtained pellet was resuspended in prechilled PBS and centrifuged again at 16,000 × g for 70 min to ensure purity. All centrifugation steps were performed at 4 °C to maintain EV integrity. The final EV pellet was resuspended in 100 μL of prechilled PBS and stored at −80 °C for subsequent analysis. EVs were then characterized using transmission electron microscopy (TEM; TECNAI Spirit, FEI, USA), nanoparticle tracking analysis (NTA; ZetaView, Particle Metrix, Germany), and Western blotting. EVs were quantified using both NTA and bicinchoninic acid (BCA) analysis (Beyotime Biotechnology, Shanghai, China). The concentration and size distribution of the EVs were assessed using NTA, while BCA analysis was employed for quantification. For the Western blotting process to identify EV markers, 2 μg of lysates from NSCs, BMSCs, and ABPCs, and 2 μg of EVsNSC, EVsBMSC, and EVsABPC, were loaded into each lane. For the experiments in vitro, we used extracellular vesicles (EVs) at a concentration of 2 × 106 particles/mL, corresponding to a protein concentration of 50–70 μg/mL as determined by the BCA assay. For in vivo experiments, the administered dosage was 1 × 109 particles per rat, with the protein concentration ranging from 500 to 700 μg/mL.

Single Particle Interferometric Reflectance Imaging Sensor with ExoView

The characterization of EVs was performed using the ExoView platform (ExoView, Boston, MA, USA), which allows for a detailed analysis of EV size, concentration, and surface marker expression to further validate the purity of our EV samples. (65) First, the isolated EVs were resuspended in precooled PBS at a concentration of 1× 106 particles/mL, which is suitable for ExoView analysis. The prepared EV samples were then loaded onto ExoView chips (EV-TM-FLEX-CAR), which are specifically designed to capture EVs based on their surface markers. The ExoView platform uses fluorescently labeled antibodies to specifically detect surface markers on the EVs, providing detailed information on the presence and abundance of these markers. The ExoView system captures high-resolution images of the EVs, enabling the precise measurement of their size and concentration. The data obtained from the ExoView system were analyzed using its software that generated comprehensive reports on the size distribution, concentration, and surface marker profiles of the EVs.

Liquid Chromatography–Mass Spectrometry (LC–MS/MS)

Protein (100 μg) from each sample was digested using a filter-aided sample preparation (FASP) method for proteomic analysis. The resulting peptides were desalted using C18 spin columns (Thermo Fisher Scientific) and dried via vacuum centrifugation. The mass spectrometer was operated in data-dependent acquisition mode by selecting the top 20 most abundant precursor ions for HCD fragmentation.

Proteomics Analysis

Raw MS data were processed using the MaxQuant software (version 1.6.1.0). The MS/MS spectra were searched against the UniProt human protein database. Carbamidomethylation of cysteine was designated as a fixed modification, while oxidation of methionine and acetylation of protein N-termini were set as variable modifications. A maximum of two missed cleavages was allowed. Peptide and protein identifications were filtered using a 1% false discovery rate (FDR). Label-free quantification (LFQ) was performed using the MaxLFQ algorithm. Quantified proteins were analyzed using Perseus software (version 1.6.2.3). Only proteins identified in at least two out of the three biological replicates with LFQ intensity values were considered. Regarding the analysis methods for differentially expressed proteins, we employed the limma package in R (version 4.3.2) to perform the analysis of gene expression. Initially, standard preprocessing steps, including quality control, normalization, and filtering, were conducted to ensure data integrity. A linear model was then fitted to log2-transformed expression values using the lmFit function to assess the effects of experimental conditions. Following this, the eBayes function was applied to compute moderated t-statistics, log-fold changes, and associated p-values for each protein. To identify differentially expressed proteins, thresholds of absolute log-fold change (|LogFC|) > 1.5 and P < 0.05 were used. Functional enrichment analysis was performed using Metascape (https://metascape.org) (P < 0.05).

Fluorescent Ubiquitination-Based Cell Cycle Indicator (FUCCl) System

A lentiviral vector containing an A FUCCI reporter gene (EFLA-mKO2-T2A-mAzami-Green) was developed by Genechem, China. (36) This vector is designed to be selectively expressed at specific cell cycle stages. The utilization of reporter genes with different fluorescent colors allows for real-time imaging of the various phases of the cell cycle, where red, yellow, and green fluorescence correspond to cells in the G1, G1/S transition, and S/G2-M phases, respectively. The specific synthesis method was as follows: the target gene was PCR-amplified, followed by digestion of the vector with PacI and EcoRI. The PCR product was then purified. Next, the purified PCR fragment was ligated into the digested vector to obtain the recombinant plasmid. This recombinant plasmid was subsequently transfected into suitable packaging cells along with helper plasmids to produce the lentivirus. Finally, we collected the viral supernatant, concentrated it, and used it to infect and label the NSCs. Subsequently, the labeled NSCs were treated with PBS, EVsBMSC (2 × 106 particles/mL), and EVsABPC (2 × 106 particles/mL) for 24 h. Following treatment, the cells were observed and analyzed statistically analysis using the High Content Imaging System (ImagePress Micro, Molecular Devices).

Preparation of ACPP@EVs

First, a Tris·HCl buffer solution was prepared by dissolving 0.44 g of Tris·HCl in 45 mL of deionized water, and the pH was adjusted to 6.8–7.4 using a 1% ammonia solution. Next, 5 mg of DSPE was dissolved in 200 μL of anhydrous ethanol. The peptide, EEEEEEEE(E8)-6-aminohexanoyl-PLGLAG-RRRRRRRR(R8), (49) was dissolved in 1 mL of Tris·HCl buffer and added to the DSPE solution, followed by rinsing the peptide container twice with Tris·HCl buffer and adding the rinses to achieve a total volume of 3.2 mL. Then, 1.8 mL of Tris·HCl buffer was added to bring the final volume to 5 mL. The reaction mixture was protected from light by wrapping the tube in aluminum foil and mixing overnight on a shaker. Subsequently, EVsNSC, EVsBMSC, and EVsABPC (1 × 1011 particles/mL) were added to the reaction mixture and incubated for an additional period to allow conjugation. The solution was transferred to a dialysis membrane and dialyzed against 2 L of deionized water with an appropriate amount of anhydrous ethanol while stirring overnight. The water was changed every 12 h to ensure thorough purification. Finally, the number of purified EV particles number was measured using NTA at a concentration of approximately 1 × 1010 particles/mL.

Validation of Targeted EV Construction Using Mass Spectrometry

We performed a mass spectrometry (MS) analysis. The engineered EVs were lysed, and the proteins were digested with trypsin. The resulting peptides were then purified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The raw data were processed and analyzed using PEAKS Studio X+ software (Bioinformatics Solutions Inc.) to identify specific marker proteins and peptides that indicated successful targeting and EV construction. (66)

Animals

We used adult female Sprague–Dawley (SD) rats, aged 8–10 weeks and weighing approximately 220–250 g. These rats were obtained from the Animal Experiment Centre of The Fourth Military Medical University, China (License No. IACUC-20241365), and were housed in clean cages at a controlled temperature of 22–25 °C, with a 12-h light/dark cycle and ad libitum access to food and water. All experimental procedures were approved by the Ethics Committee of The Fourth Military Medical University, adhering to the 3Rs principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and reduce suffering. The rats were randomly divided into five experimental groups, with at least five rats in each group: 1) Sham group, which received surgery without SCI induction; 2) Vehicle group, untreated SCI rats; 3) ACPP@EVsNSC group, SCI rats treated with intravenous ACPP@EVsNSC at a dose of 1 × 109 particles per injection three times weekly; 4) ACPP@EVsBMSC group, SCI rats treated with intravenous ACPP@EVsBMSC at a dose of 1 × 109 particles per injection three times weekly; and 5) ACPP@EVsABPC group, SCI rats treated with intravenous ACPP@EVsABPC at a dose of 1 × 109 particles per injection three times weekly. Sampling time points were set at 3, 7, 14, 28, and 56 dpi to evaluate the repair outcomes at different stages. At each time point, the BBB score was used to assess motor function recovery. Specific analyses included Nissl staining at 7 dpi to observe neuronal density, GFAP and TUJ1 staining at 14 dpi to evaluate the distribution of astrocytes and newly formed neurons, quantification of nerve fiber length and electrophysiological analysis at 28 dpi, HE staining and blood biochemistry at 28 dpi, and MBP staining and TEM analysis of myelin structure at 56 dpi.

SCI Model

Rats were anesthetized via intraperitoneal injection of sodium pentobarbital and placed in a prone position. The dorsal area was shaved, and the skin was disinfected with iodophor. After disinfection, the skin, subcutaneous tissue, and paravertebral muscles were carefully dissected to expose the T9 vertebra. The T9 spinous process and vertebral plate were removed to expose the spinal cord without causing injury, using fine forceps. The spinal cord was then compressed vertically using microvascular clips (RWD Life Science, R31005–10) paired with a clip applicator (RWD Life Science, R34001–14) for 5 s. Successful compression was confirmed by observing limb extension and tail twitching. Postcompression, muscles, and skin were sutured using sterile techniques, and cefuroxime was administered intraperitoneally. The rats were allowed to recover at room temperature. Postoperatively, rats in the ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC groups received their respective EVs via intravenous injections three times weekly until sacrifice. Manual bladder expression was also performed twice daily until reflex bladder emptying was restored.
Rats were humanely euthanized with an overdose of isoflurane at 3, 7-, 14-, 28-, and 56-days post-SCI. A median thoracoabdominal incision was made, followed by perfusion with 10 mL of cold saline via the left ventricle and then 100 mL of cold 4% paraformaldehyde (Beyotime, Shanghai, China, Cat# P0099–100 mL). The spinal cord segments (0.5 cm) above and below the lesion site were harvested and fixed overnight in 4% paraformaldehyde.

Electrophysiological Analysis

Electrophysiological tests were performed at 28 dpi to evaluate the functional status of sensorimotor signal conduction. The sciatic nerves of the rats were exposed under anesthesia. For motor-evoked potential (MEP) measurements, a stimulating electrode was inserted into the spinal motor cortex (SMC), and a recording electrode was placed into the sciatic nerve. The waveforms, amplitudes, and latencies of the MEPs were recorded and analyzed.

Gait Analysis

The Catwalk XT (Noldus) system was employed to identify neurological gait disorders. One week before modeling, the rats were trained to learn the procedure. Gait analysis was conducted in the fourth week after EV treatment to assess the recovery of motor function. The system recorded the footprints of the rats as they voluntarily crossed a glass plate toward a goal box. For the analysis, a minimum of three successful runs were required for each group. Additionally, based on the measurement data, the Catwalk XT system was used to calculate the BBB score.

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 9.2.0 software (GraphPad Software, San Diego, CA, USA). We used the Shapiro–Wilk test to assess normality for each group’s data distribution. The homogeneity of variances was evaluated using the F-test for two-group comparisons and the Brown–Forsythe test for comparisons involving more than two groups. When data met the assumptions of normality and homogeneity of variances, they were reported as mean ± standard deviation (SD), and we performed an unpaired t test for two-group comparisons or one-way ANOVA followed by Bonferroni’s multiple comparisons test for comparisons among multiple groups. For data with two variables (group and time), two-way ANOVA was applied. For data deviating from a normal distribution, we applied the nonparametric Mann–Whitney U test for two-group comparisons and the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test for multigroup comparisons. These data were reported as median with interquartile range (IQR). Correlation analyses were performed using Pearson’s correlation. Detailed statistical methods are provided in each figure legend. All differences among and between groups were considered statistically significant at P < 0.05. Significance levels were defined as follows: *P < 0.05, ** P < 0.01, *** P < 0.001.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c10298.

  • Detailed experimental procedures including descriptions of primary extraction and culture of BMSCs, ELISA for cytokine quantification, H&E and Nissl staining procedures, two-photon microscopy for EV transport dynamics, RNA sequencing analysis, transmission electron microscopy, molecular mechanism analysis, and flow cytometry for macrophage analysis; comparative proteomic analysis of EVsABPC, EVsNSC, and EVsBMSC (Figure S1); identification of NSCs and analysis of the effects of EVsBMSC and EVsABPC on NSCs proliferation and differentiation (Figure S2); enhancement of neurite outgrowth and reduction of neuronal apoptosis by EVsABPC (Figure S3); bioinformatics analysis and validation of key signaling pathways in neurons treated with EVsABPC (Figure S4); evaluation of inflammatory response and macrophage polarization induced by different EVs treatments (Figure S5); ex vivo distribution of ACPP@ EVs (Figure S6); evaluation of neuronal apoptosis and treatment effects post SCI (Figure S7); ACPP@EVsABPC promoted neural regeneration and axonal remyelination in SCI (Figure S8); safety evaluation of EVsABPC treatment on major organs and physiological parameters (Figure S9); and analysis of the protein interaction between MMP2 and ACPP peptide segments (Table 1) (PDF)

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Author Information

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  • Corresponding Authors
    • Jianzhong Li - Department of Thoracic Surgery, Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China;  Email: [email protected]
    • Shouping Gong - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China;  Xi’an Medical University, Xi’an 710021, P.R. China;  Email: [email protected]
    • Bing Xia - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China;  Email: [email protected]
    • Jinghui Huang - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China;  Orcidhttps://orcid.org/0000-0002-0991-0591;  Email: [email protected]
  • Authors
    • Shijie Yang - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China;  Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Borui Xue - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China;  Air Force 986(th) Hospital, The Fourth Military Medical University, Xi’an 710001, P.R. China
    • Yongfeng Zhang - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China;  Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Haining Wu - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Beibei Yu - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China;  Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Shengyou Li - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Teng Ma - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Xue Gao - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Yiming Hao - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Lingli Guo - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Qi Liu - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Xueli Gao - School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, P.R. China
    • Yujie Yang - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Zhenguo Wang - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Mingze Qin - Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, P.R. China
    • Yunze Tian - Department of Thoracic Surgery, Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    • Longhui Fu - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    • Bisheng Zhou - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
    • Luyao Li - Department of Neurosurgery, The Second Affiliated Hospital of Xi’an Jiao Tong University, Xi’an 710004, P.R. China
  • Author Contributions

    S.Y., B.X., Y.Z, and H.W. contributed equally to this work. The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript.

  • Funding

    This work was financially supported by the National Key R&D Program of China (Grant 2022YFB3808000), the National Natural Science Foundation of China (Grants 82122043, 82201537, and 82372404), and the Key Research and Development Project of Shaanxi Province (2022ZDLSF04-01).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We are grateful to Zhuanzhuan Wang (Biomedical Experimental Center of Xi’an Jiaotong University) for the technical support.

ABBREVIATIONS

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SCI

spinal cord injury

MSC

mesenchymal stem cell

NGF

nerve growth factor

IGF-1

insulin-like growth factor-1

ABPCs

antler blastema progenitor cells

EV

extracellular vesicle

ACPP

activated cell-penetrating peptides

TEM

transmission electron microscopy

LC–MS/MS

liquid chromatography–tandem mass spectrometry

PCA

principal component analysis

GO

gene ontology

DEP

differentially expressed protein

FUCCI

fluorescent ubiquitination-based cell cycle indicator

LPS

lipopolysaccharide

IFN-γ

interferon-gamma

DEG

differentially expressed gene

GSEA

gene set enrichment analysis

iNOS

inducible nitric oxide synthase

TNF-α

tumor necrosis factor-alpha

MBP

myelin basic protein

FACS

fluorescence-activated cell sorting

PBS

phosphate-buffered saline

MEP

metalloproteinase (MMP); Motor-evoked potential

CMAP

compound muscle action potential

BBB

Basso–Beattie–Bresnahan

dpi

days postinjury

BMSC

bone marrow stem cell

NSC

neural stem cell

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    Nature Reviews Neuroscience (2020), 21 (7), 366-383CODEN: NRNAAN; ISSN:1471-003X. (Nature Research)
    Abstr.: Spinal cord injury remains a scientific and therapeutic challenge with great cost to individuals and society. The goal of research in this field is to find a means of restoring lost function. Recently we have seen considerable progress in understanding the injury process and the capacity of CNS neurons to regenerate, as well as innovations in stem cell biol. This presents an opportunity to develop effective transplantation strategies to provide new neural cells to promote the formation of new neuronal networks and functional connectivity. Past and ongoing clin. studies have demonstrated the safety of cell therapy, and preclin. research has used models of spinal cord injury to better elucidate the underlying mechanisms through which donor cells interact with the host and thus increase long-term efficacy. While a variety of cell therapies have been explored, we focus here on the use of neural progenitor cells obtained or derived from different sources to promote connectivity in sensory, motor and autonomic systems.
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  11. 11
    Liu, H.; Zhang, J.; Xu, X.; Lu, S.; Yang, D.; Xie, C. SARM1 Promotes Neuroinflammation and Inhibits Neural Regeneration after Spinal Cord Injury through NF-κB Signaling. Theranostics 2021, 11 (9), 4187– 4206,  DOI: 10.7150/thno.49054
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    11
    SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-ΚB signaling
    Liu, Huitao; Zhang, Jingjing; Xu, Xingxing; Lu, Sheng; Yang, Danlu; Xie, Changnan; Jia, Mengxian; Zhang, Wenbin; Jin, Lingting; Wang, Xiwu; Shen, Xiya; Li, Fayi; Wang, Wangfei; Bao, Xiaomei; Li, Sijia; Zhu, Minyu; Wang, Wei; Wang, Ying; Huang, Zhihui; Teng, Honglin
    Theranostics (2021), 11 (9), 4187-4206CODEN: THERDS; ISSN:1838-7640. (Ivyspring International Publisher)
    Axonal degeneration is a common pathol. feature in many acute and chronic neurol. diseases such as spinal cord injury (SCI). SARM1 (sterile alpha and TIR motif-contg. 1), the fifth TLR (Toll-like receptor) adaptor, has diverse functions in the immune and nervous systems, and recently has been identified as a key mediator of Wallerian degeneration (WD). However, the detailed functions of SARM1 after SCI still remain unclear. Modified Allen's method was used to establish a contusion model of SCI in mice. Furthermore, to address the function of SARM1 after SCI, conditional knockout (CKO) mice in the central nervous system (CNS), SARM1Nestin-CKO mice, and SARM1GFAP-CKO mice were successfully generated by Nestin-Cre and GFAP-Cre transgenic mice crossed with SARM1flox/flox mice, resp. Immunostaining, Hematoxylin-Eosin (HE) staining, Nissl staining and behavioral test assays such as footprint and Basso Mouse Scale (BMS) scoring were used to examine the roles of SARM1 pathway in SCI based on these conditional knockout mice. Drugs such as FK866, an inhibitor of SARM1, and apoptozole, an inhibitor of heat shock protein 70 (HSP70), were used to further explore the mol. mechanism of SARM1 in neural regeneration after SCI. We found that SARM1 was upregulated in neurons and astrocytes at early stage after SCI. SARM1Nestin-CKO and SARM1GFAP-CKO mice displayed normal development of the spinal cords and motor function. Interestingly, conditional deletion of SARM1 in neurons and astrocytes promoted the functional recovery of behavior performance after SCI. Mechanistically, conditional deletion of SARM1 in neurons and astrocytes promoted neuronal regeneration at intermediate phase after SCI, and reduced neuroinflammation at SCI early phase through downregulation of NF-ΚB signaling after SCI, which may be due to upregulation of HSP70. Finally, FK866, an inhibitor of SARM1, reduced the neuroinflammation and promoted the neuronal regeneration after SCI. Our results indicate that SARM1-mediated prodegenerative pathway and neuroinflammation promotes the pathol. progress of SCI and anti-SARM1 therapeutics are viable and promising approaches for preserving neuronal function after SCI.
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  12. 12
    Zipser, C. M.; Cragg, J. J.; Guest, J. D.; Fehlings, M. G.; Jutzeler, C. R.; Anderson, A. J. Cell-Based and Stem-Cell-Based Treatments for Spinal Cord Injury: Evidence from Clinical Trials. Lancet Neurol. 2022, 21 (7), 659– 670,  DOI: 10.1016/S1474-4422(21)00464-6
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    Li, L.; Mu, J.; Zhang, Y.; Zhang, C.; Ma, T.; Chen, L. Stimulation by Exosomes from Hypoxia Preconditioned Human Umbilical Vein Endothelial Cells Facilitates Mesenchymal Stem Cells Angiogenic Function for Spinal Cord Repair. ACS Nano 2022, 16 (7), 10811– 10823,  DOI: 10.1021/acsnano.2c02898
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    13
    Stimulation by Exosomes from Hypoxia Preconditioned Human Umbilical Vein Endothelial Cells Facilitates Mesenchymal Stem Cells Angiogenic Function for Spinal Cord Repair
    Li, Liming; Mu, Jiafu; Zhang, Yu; Zhang, Chenyang; Ma, Teng; Chen, Lu; Huang, Tianchen; Wu, Jiahe; Cao, Jian; Feng, Shiqing; Cai, Youzhi; Han, Min; Gao, Jianqing
    ACS Nano (2022), 16 (7), 10811-10823CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Revascularization treatment is a crit. measure for tissue engineering therapies like spinal cord repair. As multipotent stem cells, mesenchymal stem cells (MSCs) have proven to regulate the lesion microenvironment through feedback to the microenvironment signals. The angiogenic capacities of MSCs have been reported to be facilitated by vein endothelial cells in the niche. As emerging evidence demonstrated the roles of exosomes in cell-cell and cell-microenvironment communications, to cope with the ischemia complication for treatment of traumatic spinal cord injury, the study exts. the microenvironment factors to stimulate angiogenic MSCs through using exosomes (EX) derived from hypoxic preconditioned (HPC) human umbilical vein endothelial cells (HUVEC). The HPC treatment with a hypoxia time segment of only 15 min efficiently enhanced the function of EX in facilitating MSCs angiogenesis activity. MSCs stimulated by HPC-EX showed significant tube formation within 2 h, and the in vivo transplantation of the stimulated MSCs elicited effective nerve tissue repair after rat spinal cord transection, which could be attributed to the pro-angiogenic and anti-inflammatory impacts of the MSCs. Through the simulation of MSCs using HPC-tailored HUVEC exosomes, the results proposed an efficient angiogenic nerve tissue repair strategy for spinal cord injury treatment and could provide inspiration for therapies based on stem cells and exosomes.
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  14. 14
    Fan, L.; Liu, C.; Chen, X.; Zheng, L.; Zou, Y.; Wen, H. Exosomes-Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth. Adv. Sci. 2022, 9 (13), 2105586  DOI: 10.1002/advs.202105586
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    14
    Exosomes-Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth
    Fan, Lei; Liu, Can; Chen, Xiuxing; Zheng, Lei; Zou, Yan; Wen, Huiquan; Guan, Pengfei; Lu, Fang; Luo, Yian; Tan, Guoxin; Yu, Peng; Chen, Dafu; Deng, Chunlin; Sun, Yongjian; Zhou, Lei; Ning, Chengyun
    Advanced Science (Weinheim, Germany) (2022), 9 (13), 2105586CODEN: ASDCCF; ISSN:2198-3844. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Electroconductive hydrogels are very attractive candidates for accelerated spinal cord injury (SCI) repair because they match the elec. and mech. properties of neural tissue. However, electroconductive hydrogel implantation can potentially aggravate inflammation, and hinder its repair efficacy. Bone marrow stem cell-derived exosomes (BMSC-exosomes) have shown immunomodulatory and tissue regeneration effects, therefore, neural tissue-like electroconductive hydrogels loaded with BMSC-exosomes are developed for the synergistic treatment of SCI. These exosomes-loaded electroconductive hydrogels modulate microglial M2 polarization via the NF-κB pathway, and synergistically enhance neuronal and oligodendrocyte differentiation of neural stem cells (NSCs) while inhibiting astrocyte differentiation, and also increase axon outgrowth via the PTEN/PI3K/AKT/mTOR pathway. Furthermore, exosomes combined electroconductive hydrogels significantly decrease the no. of CD68-pos. microglia, enhance local NSCs recruitment, and promote neuronal and axonal regeneration, resulting in significant functional recovery at the early stage in an SCI mouse model. Hence, the findings of this study demonstrate that the combination of electroconductive hydrogels and BMSC-exosomes is a promising therapeutic strategy for SCI repair.
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    Huang, L.; Fu, C.; Xiong, F.; He, C.; Wei, Q. Stem Cell Therapy for Spinal Cord Injury. Cell Transplant. 2021, 30, 0963689721989266  DOI: 10.1177/0963689721989266
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    Zha, K.; Yang, Y.; Tian, G.; Sun, Z.; Yang, Z.; Li, X. Nerve Growth Factor (NGF) and NGF Receptors in Mesenchymal Stem/Stromal Cells: Impact on Potential Therapies. Stem Cells Transl. Med. 2021, 10 (7), 1008– 1020,  DOI: 10.1002/sctm.20-0290
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    16
    Nerve growth factor (NGF) and NGF receptors in mesenchymal stem/stromal cells: Impact on potential therapies
    Zha, Kangkang; Yang, Yu; Tian, Guangzhao; Sun, Zhiqiang; Yang, Zhen; Li, Xu; Sui, Xiang; Liu, Shuyun; Zhao, Jinmin; Guo, Quanyi
    Stem Cells Translational Medicine (2021), 10 (7), 1008-1020CODEN: SCTMC7; ISSN:2157-6580. (AlphaMed Press)
    A review. Mesenchymal stem/stromal cells (MSCs) are promising for the treatment of degenerative diseases and traumatic injuries. However, MSC engraftment is not always successful and requires a strong comprehension of the cytokines and their receptors that mediate the biol. behaviors of MSCs. The effects of nerve growth factor (NGF) and its two receptors, TrkA and p75NTR, on neural cells are well studied. Increasing evidence shows that NGF, TrkA, and p75NTR are also involved in various aspects of MSC function, including their survival, growth, differentiation, and angiogenesis. The regulatory effect of NGF on MSCs is thought to be achieved mainly through its binding to TrkA. p75NTR, another receptor of NGF, is regarded as a novel surface marker of MSCs. This review provides an overview of advances in understanding the roles of NGF and its receptors in MSCs as well as the effects of MSC-derived NGF on other cell types, which will provide new insight for the optimization of MSC-based therapy.
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  17. 17
    Shen, H.; Gu, X.; Wei, Z. Z.; Wu, A.; Liu, X.; Wei, L. Combinatorial Intranasal Delivery of Bone Marrow Mesenchymal Stem Cells and Insulin-like Growth Factor-1 Improves Neurovascularization and Functional Outcomes Following Focal Cerebral Ischemia in Mice. Exp. Neurol. 2021, 337, 113542  DOI: 10.1016/j.expneurol.2020.113542
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    Andrzejewska, A.; Dabrowska, S.; Lukomska, B.; Janowski, M. Mesenchymal Stem Cells for Neurological Disorders. Adv. Sci. 2021, 8 (7), 2002944  DOI: 10.1002/advs.202002944
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    Mesenchymal Stem Cells for Neurological Disorders
    Andrzejewska, Anna; Dabrowska, Sylwia; Lukomska, Barbara; Janowski, Miroslaw
    Advanced Science (Weinheim, Germany) (2021), 8 (7), 2002944CODEN: ASDCCF; ISSN:2198-3844. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Neurol. disorders are becoming a growing burden as society ages, and there is a compelling need to address this spiraling problem. Stem cell-based regenerative medicine is becoming an increasingly attractive approach to designing therapies for such disorders. The unique characteristics of mesenchymal stem cells (MSCs) make them among the most sought after cell sources. Researchers have extensively studied the modulatory properties of MSCs and their engineering, labeling, and delivery methods to the brain. The first part of this review provides an overview of studies on the application of MSCs to various neurol. diseases, including stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, and other less frequently studied clin. entities. In the second part, stem cell delivery to the brain is focused. This fundamental but still understudied problem needs to be overcome to apply stem cells to brain diseases successfully. Here the value of cell engineering is also emphasized to facilitate MSC diapedesis, migration, and homing to brain areas affected by the disease to implement precision medicine paradigms into stem cell-based therapies.
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  19. 19
    Wang, C.-Z.; Chen, S.-M.; Chen, C.-H.; Wang, C.-K.; Wang, G.-J.; Chang, J.-K. The Effect of the Local Delivery of Alendronate on Human Adipose-Derived Stem Cell-Based Bone Regeneration. Biomaterials 2010, 31 (33), 8674– 8683,  DOI: 10.1016/j.biomaterials.2010.07.096
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    The effect of the local delivery of alendronate on human adipose-derived stem cell-based bone regeneration
    Wang, Chau-Zen; Chen, Shih-Mao; Chen, Chung-Hwan; Wang, Chih-Kuang; Wang, Gwo-Jaw; Chang, Je-Ken; Ho, Mei-Ling
    Biomaterials (2010), 31 (33), 8674-8683CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Recent studies have shown that alendronate (Aln) enhances the osteogenesis of osteoblasts and bone marrow mesenchymal stem cells. In this study, the authors hypothesize that Aln may act as an osteo-inductive factor to stimulate the osteogenic differentiation of human adipose-derived stem cells (hADSCs) for bone regeneration. The in vitro effect of Aln (1-10 μM) on the osteogenic ability of hADSCs was evaluated by examg. mineralization and alk. phosphatase (ALP) activity. Bone morphogenetic protein 2 (BMP2) expression was measured using a real-time polymerase chain reaction and western blot anal. The authors' results indicated that 5 μM Aln was sufficient to enhance BMP2 expression, ALP activity and mineralization in hADSCs. The in vivo effect of locally administered Aln on bone repair was examd. in a rat crit.-sized (7-mm) calvarial defect that was implanted with a hADSC-seeded poly(lactic-co-glycolic acid) (PLGA) scaffold. Aln (5 μM/100 μl/day) was injected locally into the defect site for one week. New bone formation was evaluated by radiog. and histol. analyses at 8 and 12 wk post-implantation. The expression levels of human BMP2 (hBMP2) and hADSC localization in defect sites were examd. using immunohistochem. anal. and fluorescent in situ hybridization, resp. Results showed that local treatment of Aln on hADSC-seeded PLGA scaffolds at week 12 had a maximal effect on bone regeneration, enhancing mineralization and bone matrix formation. In addn., hADSCs and hBMP2 were also detected at the defect sites. These results demonstrated that local delivery of Aln, a potent osteo-inductive factor, enhances hADSC osteogenesis and bone regeneration.
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    Religa, P. The Future Application of Induced Pluripotent Stem Cells in Vascular Regenerative Medicine. Cardiovasc. Res. 2012, 96 (3), 348– 349,  DOI: 10.1093/cvr/cvs319
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    Nombela-Arrieta, C.; Ritz, J.; Silberstein, L. E. The Elusive Nature and Function of Mesenchymal Stem Cells. Nat. Rev. Mol. Cell Biol. 2011, 12 (2), 126– 131,  DOI: 10.1038/nrm3049
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    The elusive nature and function of mesenchymal stem cells
    Nombela-Arrieta, Cesar; Ritz, Jerome; Silberstein, Leslie E.
    Nature Reviews Molecular Cell Biology (2011), 12 (2), 126-131CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    Mesenchymal stem cells (MSCs) are a diverse subset of multipotent precursors present in the stromal fraction of many adult tissues and have drawn intense interest from translational and basic investigators. MSCs have been operationally defined by their ability to differentiate into osteoblasts, adipocytes and chondrocytes after in vitro expansion. Nevertheless, their identity in vivo, heterogeneity, anatomical localization and functional roles in adult tissue homeostasis have remained enigmatic and are only just starting to be uncovered.
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    Qin, T.; Zhang, G.; Zheng, Y.; Li, S.; Yuan, Y.; Li, Q. A Population of Stem Cells with Strong Regenerative Potential Discovered in Deer Antlers. Science 2023, 379 (6634), 840– 847,  DOI: 10.1126/science.add0488
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    Yao, B.; Wang, C.; Zhou, Z.; Zhang, M.; Zhao, D.; Bai, X. Comparative Transcriptome Analysis of the Main Beam and Brow Tine of Sika Deer Antler Provides Insights into the Molecular Control of Rapid Antler Growth. Cell. Mol. Biol. Lett. 2020, 25 (1), 42,  DOI: 10.1186/s11658-020-00234-9
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    Bajpai, V. K.; Mistriotis, P.; Loh, Y.-H.; Daley, G. Q.; Andreadis, S. T. Functional Vascular Smooth Muscle Cells Derived from Human Induced Pluripotent Stem Cells via Mesenchymal Stem Cell Intermediates. Cardiovasc. Res. 2012, 96 (3), 391– 400,  DOI: 10.1093/cvr/cvs253
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    Wang, D.; Berg, D.; Ba, H.; Sun, H.; Wang, Z.; Li, C. Deer Antler Stem Cells Are a Novel Type of Cells That Sustain Full Regeneration of a Mammalian Organ─Deer Antler. Cell Death Dis. 2019, 10 (6), 443,  DOI: 10.1038/s41419-019-1686-y
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    Rong, X.; Chu, W.; Zhang, H.; Wang, Y.; Qi, X.; Zhang, G. Antler Stem Cell-Conditioned Medium Stimulates Regenerative Wound Healing in Rats. Stem Cell Res. Ther. 2019, 10 (1), 326,  DOI: 10.1186/s13287-019-1457-9
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    Lei, J.; Jiang, X.; Li, W.; Ren, J.; Wang, D.; Ji, Z.; Wu, Z.; Cheng, F.; Cai, Y.; Yu, Z.-R.; Belmonte, J. C. I.; Li, C.; Liu, G.-H.; Zhang, W.; Qu, J.; Wang, S. Exosomes from Antler Stem Cells Alleviate Mesenchymal Stem Cell Senescence and Osteoarthritis. Protein Cell 2022, 13 (3), 220– 226,  DOI: 10.1007/s13238-021-00860-9
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    Jin, S.; Wang, Y.; Wu, X.; Li, Z.; Zhu, L.; Niu, Y. Young Exosome Bio-Nanoparticles Restore Aging-Impaired Tendon Stem/Progenitor Cell Function and Reparative Capacity. Adv. Mater. 2023, 35 (18), 2211602  DOI: 10.1002/adma.202211602
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    Tan, F.; Li, X.; Wang, Z.; Li, J.; Shahzad, K.; Zheng, J. Clinical Applications of Stem Cell-Derived Exosomes. Signal Transduction Targeted Ther. 2024, 9 (1), 17,  DOI: 10.1038/s41392-023-01704-0
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    Xia, B.; Gao, X.; Qian, J.; Li, S.; Yu, B.; Hao, Y. A Novel Superparamagnetic Multifunctional Nerve Scaffold: A Remote Actuation Strategy to Boost In Situ Extracellular Vesicles Production for Enhanced Peripheral Nerve Repair. Adv. Mater. 2024, 36 (3), 2305374  DOI: 10.1002/adma.202305374
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    Han, M.; Yang, H.; Lu, X.; Li, Y.; Liu, Z.; Li, F. Three-Dimensional-Cultured MSC-Derived Exosome-Hydrogel Hybrid Microneedle Array Patch for Spinal Cord Repair. Nano Lett. 2022, 22 (15), 6391– 6401,  DOI: 10.1021/acs.nanolett.2c02259
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    Three-dimensional-cultured mesenchymal stem cells-derived exosome-hydrogel hybrid microneedle array patch for spinal cord repair
    Han, Min; Yang, Hongru; Lu, Xiangdong; Li, Yuming; Liu, Zihao; Li, Feng; Shang, Zehan; Wang, Xiaofeng; Li, Xuze; Li, Junliang; Liu, Hong; Xin, Tao
    Nano Letters (2022), 22 (15), 6391-6401CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Exosomes derived from mesenchymal stem cells (MSCs) have been proven to exhibit great potentials in spinal cord injury (SCI) therapy. However, conventional two-dimensional (2D) culture will inevitably lead to the loss of stemness of MSCs, which substantially limits the therapeutic potency of MSCs exosomes (2D-Exo). Exosomes derived from three-dimensional culture (3D-Exo) possess higher therapeutic efficiency which have wide applications in spinal cord therapy. Typically, conventional exosome therapy that relies on local repeated injection results in secondary injury and low efficiency. It is urgent to develop a more reliable, convenient, and effective exosome delivery method to achieve const. in situ exosomes release. Herein, we proposed a controlled 3D-exohydrogel hybrid microneedle array patch to achieve SCI repair in situ. Our studies suggested that MSCs with 3D-culturing could maintain their stemness, and consequently, 3D-Exo effectively reduced SCI-induced inflammation and glial scarring. Thus, it is a promising therapeutic strategy for the treatment of SCI.
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    Nieto-Diaz, M. Deer Antler Innervation and Regeneration. Front. Biosci. 2012, 17 (1), 1389,  DOI: 10.2741/3993
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    S, S.; Camardo, A.; Dahal, S.; Ramamurthi, A. Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair. Mol. Pharmaceutics 2023, 20 (6), 2801– 2813,  DOI: 10.1021/acs.molpharmaceut.2c00769
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    33
    Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair
    S, Sajeesh; Camardo, Andrew; Dahal, Shataakshi; Ramamurthi, Anand
    Molecular Pharmaceutics (2023), 20 (6), 2801-2813CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Extracellular vesicles (EVs) are nanosized vesicles that carry cell-specific biomol. information. Our previous studies showed that adult human bone marrow mesenchymal stem cell (BM-MSC)-derived EVs provide antiproteolytic and proregenerative effects in cultures of smooth muscle cells (SMCs) derived from an elastase-infused rat abdominal aortic aneurysm (AAA) model, and this is promising toward their use as a therapeutic platform for naturally irreversible elastic matrix aberrations in the aortic wall. Since systemically administered EVs poorly home into sites of tissue injury, disease strategies to improve their affinity toward target tissues are of great significance for EV-based treatment strategies. Toward this goal, in this work, we developed a postisolation surface modification strategy to target MSC-derived EVs to the AAA wall. The EVs were surface-conjugated with a short, synthetic, azide-modified peptide sequence for targeted binding to cathepsin K (CatK), a cysteine protease overexpressed in the AAA wall. Conjugation was performed using a copper-free click chem. method. We detd. that such conjugation improved EV uptake into cultured aneurysmal SMCs in culture and their binding to the wall of matrix injured vessels ex vivo. The proregenerative and antiproteolytic effects of MSC-EVs on cultured rat aneurysmal SMCs were also unaffected following peptide conjugation. From this study, it appears that modification with short synthetic peptide sequences seems to be an effective strategy for improving the cell-specific uptake of EVs and may be effective in facilitating AAA-targeted therapy.
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    Wu, W.; Jia, S.; Xu, H.; Gao, Z.; Wang, Z.; Lu, B. Supramolecular Hydrogel Microspheres of Platelet-Derived Growth Factor Mimetic Peptide Promote Recovery from Spinal Cord Injury. ACS Nano 2023, 17 (4), 3818– 3837,  DOI: 10.1021/acsnano.2c12017
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    34
    Supramolecular Hydrogel Microspheres of Platelet-Derived Growth Factor Mimetic Peptide Promote Recovery from Spinal Cord Injury
    Wu, Weidong; Jia, Shuaijun; Xu, Hailiang; Gao, Ziheng; Wang, Zhiyuan; Lu, Botao; Ai, Yixiang; Liu, Youjun; Liu, Renfeng; Yang, Tong; Luo, Rongjin; Hu, Chunping; Kong, Lingbo; Huang, Dageng; Yan, Liang; Yang, Zhimou; Zhu, Lei; Hao, Dingjun
    ACS Nano (2023), 17 (4), 3818-3837CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Neural stem cells (NSCs) are considered to be prospective replacements for neuronal cell loss as a result of spinal cord injury (SCI). However, the survival and neuronal differentiation of NSCs are strongly affected by the unfavorable microenvironment induced by SCI, which critically impairs their therapeutic ability to treat SCI. Herein, a strategy to fabricate PDGF-MP hydrogel (PDGF-MPH) microspheres (PDGF-MPHM) instead of bulk hydrogels is proposed to dramatically enhance the efficiency of platelet-derived growth factor mimetic peptide (PDGF-MP) in activating its receptor. PDGF-MPHM were fabricated by a piezoelec. ceramic-driven thermal electrospray device, had an av. size of 9μm, and also had the ability to activate the PDGFRβ of NSCs more effectively than PDGF-MPH. In vitro, PDGF-MPHM exerted strong neuroprotective effects by maintaining the proliferation and inhibiting the apoptosis of NSCs in the presence of myelin exts. In vivo, PDGF-MPHM inhibited M1 macrophage infiltration and extrinsic or intrinsic cells apoptosis on the seventh day after SCI. Eight weeks after SCI, the T10 SCI treatment results showed that PDGF-MPHM + NSCs significantly promoted the survival of NSCs and neuronal differentiation, reduced lesion size, and considerably improved motor function recovery in SCI rats by stimulating axonal regeneration, synapse formation, and angiogenesis in comparison with the NSCs graft group. Therefore, our findings provide insights into the ability of PDGF-MPHM to be a promising therapeutic agent for SCI repair.
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    Yano, S.; Tazawa, H.; Kagawa, S.; Fujiwara, T.; Hoffman, R. M. FUCCI Real-Time Cell-Cycle Imaging as a Guide for Designing Improved Cancer Therapy: A Review of Innovative Strategies to Target Quiescent Chemo-Resistant Cancer Cells. Cancers 2020, 12 (9), 2655,  DOI: 10.3390/cancers12092655
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    Koh, S.-B.; Mascalchi, P.; Rodriguez, E.; Lin, Y.; Jodrell, D. I.; Richards, F. M. A Quantitative FastFUCCI Assay Defines Cell Cycle Dynamics at a Single-Cell Level. J. Cell Sci. 2017, 130 (2), 512– 520,  DOI: 10.1242/jcs.195164
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    Geng, H.; Li, Z.; Li, Z.; Zhang, Y.; Gao, Z.; Sun, L. Restoring Neuronal Iron Homeostasis Revitalizes Neurogenesis after Spinal Cord Injury. Proc. Natl. Acad. Sci. U. S. A. 2023, 120 (46), e2220300120  DOI: 10.1073/pnas.2220300120
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    Tran, A. P.; Warren, P. M.; Silver, J. The Biology of Regeneration Failure and Success After Spinal Cord Injury. Physiol. Rev. 2018, 98 (2), 881– 917,  DOI: 10.1152/physrev.00017.2017
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    The biology of regeneration failure and success after spinal cord injury
    Tran, Amanda Phuong; Warren, Philippa Mary; Silver, Jerry
    Physiological Reviews (2018), 98 (2), 881-917CODEN: PHREA7; ISSN:1522-1210. (American Physiological Society)
    A review. Since no approved therapies to restore mobility and sensation following spinal cord injury (SCI) currently exist, a better understanding of the cellular and mol. mechanisms following SCI that compromise regeneration or neuroplasticity is needed to develop new strategies to promote axonal regrowth and restore function. Phys. trauma to the spinal cord results in vascular disruption that, in turn, causes blood-spinal cord barrier rupture leading to hemorrhage and ischemia, followed by rampant local cell death. As subsequent edema and inflammation occur, neuronal and glial necrosis and apoptosis spread well beyond the initial site of impact, ultimately resolving into a cavity surrounded by glial/fibrotic scarring. Understanding the formative events in glial scarring helps guide strategies towards the development of potential therapies to enhance axon regeneration and functional recovery at both acute and chronic stages following SCI. This review will also discuss the perineuronal net and how chondroitin sulfate proteoglycans (CSPGs) deposited in both the glial scar and net impede axonal outgrowth at the level of the growth cone. We will end the review with a summary of current CSPG-targeting strategies that help to foster axonal regeneration, neuroplasticity/sprouting, and functional recovery following SCI.
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  39. 39
    Li, S.; Wang, P.; Ye, M.; Yang, K.; Cheng, D.; Mao, Z. Cysteine-Activatable Near-Infrared Fluorescent Probe for Dual-Channel Tracking Lipid Droplets and Mitochondria in Epilepsy. Anal. Chem. 2023, 95 (11), 5133– 5141,  DOI: 10.1021/acs.analchem.3c00226
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    Mathew, B.; Ravindran, S.; Liu, X.; Torres, L.; Chennakesavalu, M.; Huang, C.-C. Mesenchymal Stem Cell-Derived Extracellular Vesicles and Retinal Ischemia-Reperfusion. Biomaterials 2019, 197, 146– 160,  DOI: 10.1016/j.biomaterials.2019.01.016
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    Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion
    Mathew, Biji; Ravindran, Sriram; Liu, Xiaorong; Torres, Leianne; Chennakesavalu, Mohansrinivas; Huang, Chun-Chieh; Feng, Liang; Zelka, Ruth; Lopez, Jasmine; Sharma, Monica; Roth, Steven
    Biomaterials (2019), 197 (), 146-160CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Retinal ischemia is a major cause of vision loss and impairment and a common underlying mechanism assocd. with diseases such as glaucoma, diabetic retinopathy, and central retinal artery occlusion. The regenerative capacity of the diseased human retina is limited. Our previous studies have shown the neuroprotective effects of intravitreal injection of mesenchymal stem cells (MSC) and MSC-conditioned medium in retinal ischemia in rats. Based upon the hypothesis that the neuroprotective effects of MSCs and conditioned medium are largely mediated by extracellular vesicles (EVs), MSC derived EVs were tested in an in-vitro oxygen-glucose deprivation (OGD) model of retinal ischemia. Treatment of R28 retinal cells with MSC-derived EVs significantly reduced cell death and attenuated loss of cell proliferation. Mechanistic studies on the mode of EV endocytosis by retinal cells were performed in vitro. EV endocytosis was dose- and temp.-dependent, saturable, and occurred via cell surface heparin sulfate proteoglycans mediated by the caveolar endocytic pathway. The administration of MSC-EVs into the vitreous humor 24 h after retinal ischemia in a rat model significantly enhanced functional recovery, and decreased neuro-inflammation and apoptosis. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia. They were present in the vitreous humor for four weeks after intravitreal administration, with saturable binding to vitreous humor components. Overall, this study highlights the potential of MSC-EV as biomaterials for neuroprotective and regenerative therapy in retinal disorders.
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    Ciani, L.; Salinas, P. C. WNTS in the Vertebrate Nervous System: From Patterning to Neuronal Connectivity. Nat. Rev. Neurosci. 2005, 6 (5), 351– 362,  DOI: 10.1038/nrn1665
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    Signaling in neural development: WNTS in the vertebrate nervous system: from patterning to neuronal connectivity
    Ciani, Lorenza; Salinas, Patricia C.
    Nature Reviews Neuroscience (2005), 6 (5), 351-362CODEN: NRNAAN; ISSN:1471-003X. (Nature Publishing Group)
    A review. WNT signaling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity and cell movements. In the nervous system, WNT signaling also regulates neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation. Studies, from invertebrates to mammals, have led to a considerable understanding of WNT signal transduction pathways. This knowledge provides a framework for the study of the mechanisms by which WNTs regulate diverse neuronal functions. Manipulation of the WNT pathways could provide new strategies for nerve regeneration and neuronal circuit modulation.
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    Feltri, M. L.; Poitelon, Y. HIPPO Stampede in Nerve Sheath Tumors. Cancer Cell 2018, 33 (2), 160– 161,  DOI: 10.1016/j.ccell.2018.01.016
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    Tian, X.; Hou, W.; Bai, S.; Fan, J.; Tong, H.; Bai, Y. XAV939 Promotes Apoptosis in a Neuroblastoma Cell Line via Telomere Shortening. Oncol. Rep. 2014, 32 (5), 1999– 2006,  DOI: 10.3892/or.2014.3460
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    Barrette, A. M.; Ronk, H.; Joshi, T.; Mussa, Z.; Mehrotra, M.; Bouras, A. Anti-Invasive Efficacy and Survival Benefit of the YAP-TEAD Inhibitor Verteporfin in Preclinical Glioblastoma Models. Neuro-Oncol. 2022, 24 (5), 694– 707,  DOI: 10.1093/neuonc/noab244
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    Liu, H.; Li, R.; Liu, T.; Yang, L.; Yin, G.; Xie, Q. Immunomodulatory Effects of Mesenchymal Stem Cells and Mesenchymal Stem Cell-Derived Extracellular Vesicles in Rheumatoid Arthritis. Front. Immunol. 2020, 11, 1912,  DOI: 10.3389/fimmu.2020.01912
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    Xiong, T.; Yang, K.; Zhao, T.; Zhao, H.; Gao, X.; You, Z. Multifunctional Integrated Nanozymes Facilitate Spinal Cord Regeneration by Remodeling the Extrinsic Neural Environment. Adv. Sci. 2023, 10 (7), 2205997  DOI: 10.1002/advs.202205997
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    Multifunctional Integrated Nanozymes Facilitate Spinal Cord Regeneration by Remodeling the Extrinsic Neural Environment
    Xiong, Tiandi; Yang, Keni; Zhao, Tongtong; Zhao, Haitao; Gao, Xu; You, Zhifeng; Fan, Caixia; Kang, Xinyi; Yang, Wen; Zhuang, Yan; Chen, Yanyan; Dai, Jianwu
    Advanced Science (Weinheim, Germany) (2023), 10 (7), 2205997CODEN: ASDCCF; ISSN:2198-3844. (Wiley-VCH Verlag GmbH & Co. KGaA)
    High levels of reactive oxygen species (ROS) and inflammation create a complicated extrinsic neural environment that dominates the initial post-injury period after spinal cord injury (SCI). The compensatory pathways between ROS and inflammation limited the efficacy of modulating the above single treatment regimen after SCI. Here, novel "nanoflower" Mn3O4 integrated with "pollen" IRF-5SiRNA was designed as a combination antioxidant and anti-inflammatory treatment after SCI. The "nanoflower" and "pollen" structure was encapsulated with a neutrophil membrane for protective and targeted delivery. Furthermore, valence-engineered nanozyme Mn3O4 imitated the cascade response of antioxidant enzymes with a higher substrate affinity compared to natural antioxidant enzymes. Nanozymes effectively catalyzed ROS to generate O2, which is advantageous for reducing oxidative stress and promoting angiogenesis. The screened "pollen" IRF-5SiRNA could reverse the inflammatory phenotype by reducing interferon regulatory factors-5 (IRF-5) expression (protein level: 73.08% and mRNA level: 63.10%). The decreased expression of pro-inflammatory factors reduced the infiltration of inflammatory cells, resulting in less neural scarring. In SCI rats, multifunctional nanozymes enhanced the proliferation of various neuronal subtypes (motor neurons, interneurons, and sensory neurons) and the recovery of locomotor function, demonstrating that the remodeling of the extrinsic neural environment is a promising strategy to facilitate nerve regeneration.
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    Wang, L.; Wang, D.; Ye, Z.; Xu, J. Engineering Extracellular Vesicles as Delivery Systems in Therapeutic Applications. Adv. Sci. 2023, 10 (17), 2300552  DOI: 10.1002/advs.202300552
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    Miranpuri, G. S.; Schomberg, D. T.; Alrfaei, B.; King, K. C.; Rynearson, B.; Wesley, V. S. Role of Matrix Metalloproteinases 2 in Spinal Cord Injury-Induced Neuropathic Pain. Ann. Neurosci. 2016, 23 (1), 25– 32,  DOI: 10.1159/000443553
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    Shen, K.; Sun, G.; Chan, L.; He, L.; Li, X.; Yang, S. Anti-Inflammatory Nanotherapeutics by Targeting Matrix Metalloproteinases for Immunotherapy of Spinal Cord Injury. Small 2021, 17 (41), 2102102  DOI: 10.1002/smll.202102102
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    Anti-Inflammatory Nanotherapeutics by Targeting Matrix Metalloproteinases for Immunotherapy of Spinal Cord Injury
    Shen, Kui; Sun, Guodong; Chan, Leung; He, Lizhen; Li, Xiaowei; Yang, Shuxian; Wang, Baocheng; Zhang, Hua; Huang, Jiarun; Chang, Minmin; Li, Zhizhong; Chen, Tianfeng
    Small (2021), 17 (41), 2102102CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Neuroinflammation is critically involved in the repair of spinal cord injury (SCI), and macrophages assocd. with inflammation propel the degeneration or recovery in the pathol. process. Currently, efforts have been focused on obtaining efficient therapeutic anti-inflammatory drugs to treat SCI. However, these drugs are still unable to penetrate the blood spinal cord barrier and lack the ability to target lesion areas, resulting in unsatisfactory clin. efficacy. Herein, a polymer-based nanodrug delivery system is constructed to enhance the targeting ability. Because of increased expression of matrix metalloproteinases (MMPs) in injured site after SCI, MMP-responsive mol., activated cell-penetrating peptides (ACPP), is introduced into the biocompatible polymer PLGA-PEI-mPEG (PPP) to endow the nanoparticles with the ability for diseased tissue-targeting. Meanwhile, etanercept (ET), a clin. anti-inflammation treatment medicine, is loaded on the polymer to regulate the polarization of macrophages, and promote locomotor recovery. The results show that PPP-ACPP nanoparticles possess satisfactory lesion targeting effects. Through inhibited consequential prodn. of proinflammation cytokines and promoted anti-inflammation cytokines, ET@PPP-ACPP could decrease the percentage of M1 macrophages and increase M2 macrophages. As expected, ET@PPP-ACPP accumulates in lesion area and achieves effective treatment of SCI; this confirmed the potential of nano-drug loading systems in SCI immunotherapy.
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    Zhu, B.; Gu, G.; Ren, J.; Song, X.; Li, J.; Wang, C. Schwann Cell-Derived Exosomes and Methylprednisolone Composite Patch for Spinal Cord Injury Repair. ACS Nano 2023, 17 (22), 22928– 22943,  DOI: 10.1021/acsnano.3c08046
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    Yang, J.; Yang, K.; Man, W.; Zheng, J.; Cao, Z.; Yang, C.-Y. 3D Bio-Printed Living Nerve-like Fibers Refine the Ecological Niche for Long-Distance Spinal Cord Injury Regeneration. Bioact. Mater. 2023, 25, 160– 175,  DOI: 10.1016/j.bioactmat.2023.01.023
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    3D bio-printed living nerve-like fibers refine the ecological niche for long-distance spinal cord injury regeneration
    Yang, Jia; Yang, Kaiyuan; Man, Weitao; Zheng, Jingchuan; Cao, Zheng; Yang, Chun-Yi; Kim, Kunkoo; Yang, Shuhui; Hou, Zhaohui; Wang, Guihuai; Wang, Xiumei
    Bioactive Materials (2023), 25 (), 160-175CODEN: BMIAD4; ISSN:2452-199X. (KeAi Communications Co., Ltd.)
    3D bioprinting holds great promise toward fabricating biomimetic living constructs in a bottom-up assembly manner. To date, various emergences of living constructs have been bioprinted for in vitro applications, while the conspicuous potential serving for in vivo implantable therapies in spinal cord injury (SCI) has been relatively overlooked. Herein, living nerve-like fibers are prepd. via extrusion-based 3D bioprinting for SCI therapy. The living nerve-like fibers are comprised of neural stem cells (NSCs) embedded within a designed hydrogel that mimics the extracellular matrix (ECM), assembled into a highly spatial ordered architecture, similar to densely arranged bundles of the nerve fibers. The pro-neurogenesis ability of these living nerve-like fibers is tested in a 4 mm-long complete transected SCI rat model. Evidence shows that living nerve-like fibers refine the ecol. niche of the defect site by immune modulation, angiogenesis, neurogenesis, neural relay formations, and neural circuit remodeling, leading to outstanding functional reconstruction, revealing an evolution process of this living construct after implantation. This effective strategy, based on biomimetic living constructs, opens a new perspective on SCI therapies.
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    Goodman, Z. D. Grading and Staging Systems for Inflammation and Fibrosis in Chronic Liver Diseases. J. Hepatol. 2007, 47 (4), 598– 607,  DOI: 10.1016/j.jhep.2007.07.006
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    Qin, T.; Li, C.; Xu, Y.; Qin, Y.; Jin, Y.; He, R. Local Delivery of EGFR+NSCs-Derived Exosomes Promotes Neural Regeneration Post Spinal Cord Injury via miR-34a-5p/HDAC6 Pathway. Bioact. Mater. 2024, 33, 424– 443,  DOI: 10.1016/j.bioactmat.2023.11.013
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    Nie, H.; Jiang, Z. Bone Mesenchymal Stem Cell-Derived Extracellular Vesicles Deliver microRNA-23b to Alleviate Spinal Cord Injury by Targeting Toll-like Receptor TLR4 and Inhibiting NF-κB Pathway Activation. Bioengineered 2021, 12 (1), 8157– 8172,  DOI: 10.1080/21655979.2021.1977562
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    Welsh, J. A.; Goberdhan, D. C. I.; O’Driscoll, L.; Buzas, E. I.; Blenkiron, C.; Bussolati, B. Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches. J. Extracell. Vesicles 2024, 13 (2), e12404  DOI: 10.1002/jev2.12451
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    Piccolo, S.; Dupont, S.; Cordenonsi, M. The Biology of YAP/TAZ: Hippo Signaling and Beyond. Physiol. Rev. 2014, 94 (4), 1287– 1312,  DOI: 10.1152/physrev.00005.2014
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    The biology of YAP/TAZ: Hippo signaling and beyond
    Piccolo, Stefano; Dupont, Sirio; Cordenonsi, Michelangelo
    Physiological Reviews (2014), 94 (4), 1287-1312CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
    A review. The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biol. properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a mol. entry point into the mysteries of tissue biol., their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's phys. nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mech. signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metab. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context.
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    Cassani, M.; Fernandes, S.; Oliver-De La Cruz, J.; Durikova, H.; Vrbsky, J.; Patočka, M. YAP Signaling Regulates the Cellular Uptake and Therapeutic Effect of Nanoparticles. Adv. Sci. 2024, 11 (2), 2302965  DOI: 10.1002/advs.202302965
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    Pham, T. C.; Jayasinghe, M. K.; Pham, T. T.; Yang, Y.; Wei, L.; Usman, W. M. Covalent Conjugation of Extracellular Vesicles with Peptides and Nanobodies for Targeted Therapeutic Delivery. J. Extracell. Vesicles 2021, 10 (4), e12057  DOI: 10.1002/jev2.12057
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    Covalent conjugation of extracellular vesicles with peptides and nanobodies for targeted therapeutic delivery
    Pham, Tin Chanh; Jayasinghe, Migara Kavishka; Pham, Thach Tuan; Yang, Yuqi; Wei, Likun; Usman, Waqas Muhammad; Chen, Huan; Pirisinu, Marco; Gong, Jinhua; Kim, Seongkyeol; Peng, Boya; Wang, Weixi; Chan, Charlene; Ma, Victor; Nguyen, Nhung T. H.; Kappei, Dennis; Nguyen, Xuan-Hung; Cho, William C.; Shi, Jiahai; Le, Minh T. N.
    Journal of Extracellular Vesicles (2021), 10 (4), e12057CODEN: JEVOA4; ISSN:2001-3078. (John Wiley & Sons, Inc.)
    Natural extracellular vesicles (EVs) are ideal drug carriers due to their remarkable biocompatibility. Their delivery specificity can be achieved by the conjugation of targeting ligands. However, existing methods to engineer target-specific EVs are tedious or inefficient, having to compromise between harsh chem. treatments and transient interactions. Here, we describe a novel method for the covalent conjugation of EVs with high copy nos. of targeting moieties using protein ligases. Conjugation of EVs with either an epidermal growth factor receptor (EGFR)-targeting peptide or anti-EGFR nanobody facilitates their accumulation in EGFR-pos. cancer cells, both in vitro and in vivo. Systemic delivery of paclitaxel by EGFR-targeting EVs at a low dose significantly increases drug efficacy in a xenografted mouse model of EGFR-pos. lung cancer. The method is also applicable to the conjugation of EVs with peptides and nanobodies targeting other receptors, such as HER2 and SIRP alpha, and the conjugated EVs can deliver RNA in addn. to small mols., supporting the versatile application of EVs in cancer therapies. This simple, yet efficient and versatile method for the stable surface modification of EVs bypasses the need for genetic and chem. modifications, thus facilitating safe and specific delivery of therapeutic payloads to target cells.
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    Théry, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines. J. Extracell. Vesicles 2018, 7 (1), 1535750  DOI: 10.1080/20013078.2018.1535750
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    Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
    Thery Clotilde; Lavieu Gregory; Martin-Jaular Lorena; Mathieu Mathilde; Tkach Mercedes; Witwer Kenneth W; Huang Yiyao; Muth Dillon C; Powell Bonita H; Schoyen Tine Hiorth; Zhao Zezhou; Witwer Kenneth W; Datta Chaudhuri Amrita; Aikawa Elena; Aikawa Elena; Alcaraz Maria Jose; Anderson Johnathon D; Andriantsitohaina Ramaroson; Le Lay Soazig; Martinez M Carmen; Antoniou Anna; Antoniou Anna; Arab Tanina; Archer Fabienne; Atkin-Smith Georgia K; Baxter Amy A; Caruso Sarah; Cheng Lesley; Greening David W; Hill Andrew F; Jiang Lanzhou; Mathivanan Suresh; Poon Ivan Kh; Tixeira Rochelle; Ayre D Craig; Ayre D Craig; Bach Jean-Marie; Bosch Steffi; Bachurski Daniel; Baharvand Hossein; Shekari Faezeh; Baharvand Hossein; Balaj Leonora; Baldacchino Shawn; Bauer Natalie N; Bebawy Mary; Beckham Carla; Bedina Zavec Apolonija; Benmoussa Abderrahim; Boilard Eric; Gilbert Caroline; Berardi Anna C; Bergese Paolo; Radeghieri Annalisa; Bergese Paolo; Bergese Paolo; Busatto Sara; Radeghieri Annalisa; Bielska Ewa; Blenkiron Cherie; Bobis-Wozowicz Sylwia; Zuba-Surma Ewa K; Boireau Wilfrid; Elie-Caille Celine; Frelet-Barrand Annie; Bongiovanni Antonella; Borras Francesc E; Gamez-Valero Ana; Borras Francesc E; Borras Francesc E; Boulanger Chantal M; Loyer Xavier; Boulanger Chantal M; Loyer Xavier; Breakefield Xandra; Breglio Andrew M; Breglio Andrew M; Brennan Meadhbh A; Brennan Meadhbh A; Brennan Meadhbh A; Brigstock David R; Brigstock David R; Brisson Alain; Broekman Marike Ld; Broekman Marike Ld; Broekman Marike Ld; Bromberg Jacqueline F; Bromberg Jacqueline F; Bryl-Gorecka Paulina; Buch Shilpa; Hu Guoku; Liao Ke; Buck Amy H; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Busatto Sara; Buschmann Dominik; Mussack Veronika; Pfaffl Michael W; Bussolati Benedetta; Buzas Edit I; Buzas Edit I; Forsonits Andras; Hegyesi Hargita; Khamari Delaram; Kovacs Arpad Ferenc; Sodar Barbara W; Visnovitz Tamas; Vukman Krisztina V; Wiener Zoltan; Byrd James Bryan; Camussi Giovanni; Carter David Rf; Pink Ryan C; Chamley Lawrence W; Chang Yu-Ting; Chen Chihchen; Chen Chihchen; Chen Shuai; Chin Andrew R; Di Vizio Dolores; Mariscal Javier; Clayton Aled; Cocks Alex; Webber Jason P; Clerici Stefano P; Cocucci Emanuele; Cocucci Emanuele; Coffey Robert J; Cordeiro-da-Silva Anabela; Couch Yvonne; Coumans Frank Aw; Nieuwland Rienk; Coyle Beth; Jackson Hannah K; Crescitelli Rossella; Lasser Cecilia; Lotvall Jan; Shelke Ganesh Vilas; Criado Miria Ferreira; D'Souza-Schorey Crislyn; Das Saumya; de Candia Paola; De Santana Eliezer F; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; Del Portillo Hernando A; Del Portillo Hernando A; Del Portillo Hernando A; Demaret Tanguy; Lombard Catherine A; Deville Sarah; Deville Sarah; Mertens Inge; Devitt Andrew; Dhondt Bert; Dieterich Lothar C; Dolo Vincenza; Giusti Ilaria; Dominguez Rubio Ana Paula; Dominici Massimo; Dominici Massimo; Dourado Mauricio R; Dourado Mauricio R; Driedonks Tom Ap; Nolte-'t Hoen Esther Nm; Stoorvogel Willem; van der Grein Susanne G; van Herwijnen Martijn Jc; Wauben Marca Hm; Duarte Filipe V; Rodrigues Silvia C; Duncan Heather M; Duncan Heather M; Eichenberger Ramon M; Sotillo Javier; Ekstrom Karin; El Andaloussi Samir; Gorgens Andre; El Andaloussi Samir; Erdbrugger Uta; Musante Luca; Falcon-Perez Juan M; Falcon-Perez Juan M; Fatima Farah; Fish Jason E; Fish Jason E; Gustafson Dakota; Schneider Raphael; Flores-Bellver Miguel; Fricke Fabia; Fricke Fabia; Fuhrmann Gregor; Fuhrmann Gregor; Fuhrmann Gregor; Gabrielsson Susanne; Gamez-Valero Ana; Gardiner Chris; Gartner Kathrin; Gaudin Raphael; Gaudin Raphael; Gho Yong Song; Park Jaesung; Giebel Bernd; Gorgens Andre; Gimona Mario; Rohde Eva; Goberdhan Deborah Ci; Gorgens Andre; Gorski Sharon M; Xu Jing; Gorski Sharon M; Xu Jing; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gualerzi Alice; Gupta Gopal N; Handberg Aase; Handberg Aase; 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  • Abstract

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    Scheme 1

    Scheme 1. Schematic Illustration of Antler Blastema Progenitor Cell-Derived Extracellular Vesicles and Their Therapeutic Role in Spinal Cord Injury (SCI)a
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    a(A) Isolation and modification of EVsABPC, derived from antler blastema progenitor cells (ABPCs) isolated from deer antler, were modified with DSPE-PEG and activatable cell-penetrating peptides (ACPP) to enhance their targeting ability at the SCI lesion site. (B) Intravenous injection of ACPP@EVsABPC in spinal cord injury (SCI) rats enables them to cross the blood-spinal cord barrier (BSCB) and selectively accumulate at the injury site. (C) SCI is characterized by an acute inflammatory response, axonal disruption and neuronal damage, and glial scar formation, which collectively inhibit neural repair and functional recovery. At the lesion site, ACPP@EVsABPC promote neural stem cell proliferation by enhancing cell cycle progression through the S phase, facilitate axonal growth, and reduce neuronal apoptosis via modulation of the Wnt and Hippo signaling pathways. Additionally, EVsABPC regulate inflammation by promoting macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype through the NF-κB signaling pathway, thereby supporting tissue repair and functional recovery.

    Figure 1

    Figure 1. Characterization and proteomic analysis of EVsABPC, EVsNSC, and EVsBMSC. (A) Schematic depicting the extraction process of EVs from ABPCs, NSCs, and BMSCs. The supernatants from the cultured cells were collected and subjected to ultracentrifugation to isolate the EVs. (B) TEM images validated the morphology of EVsNSC, EVsBMSC, and EVsABPC. Scale bar, 200 nm. (C) Quantification and size analyses of EVsABPC, EVsNSC, and EVsBMSC using NTA. (D) Western blot analysis of EVs marker proteins (CD63, HSP90, and ANXA1). An equal amount (2 μg) of protein in EVsNSC, EVsBMSC, and EVsABPC was loaded in each lane, using NSC, BMSC, and ABPC lysates as controls for surface markers. (E) ExoView analysis of EVsABPC, EVsNSC, and EVsBMSC. Representative composite images show EV markers CD9 (blue), CD81 (green), and CD63 (red). Scale bar, 5 μm. (F) Schematic illustration of the proteomic analysis workflow. EVsNSC, EVsBMSC, and EVsABPC were subjected to trypsin digestion and analyzed using a label-free peptide quantification method, followed by bioinformatic analysis. (G) Venn diagram showing the overlap of identified proteins among EVsABPC, EVsNSC, and EVsBMSC. (H) Volcano map indicating DEPs between EVsABPC and EVsNSC. Upregulated proteins are denoted using red dots, while downregulated proteins are indicated using blue dots; nonsignificant proteins are represented using gray dots. (I) GO term enrichment analyses depicting enriched biological processes upregulated between EVsABPC and EVsNSC. (J) Volcano plot showing DEPs between EVsABPC and EVsBMSC. (K) GO term enrichment analyses depicting enriched biological processes upregulated between EVsABPC and EVsBMSC.

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    Figure 2

    Figure 2. EVsABPC enhanced NSC proliferation in vitro. (A) Circular heatmap showing DEPs related to nervous system development between EVsABPC and EVsBMSC. (B) Violin plot depicting proteins related to nervous system development regulation in EVsABPC and EVsBMSC. Central box in each violin represents the interquartile range (IQR). Statistical significance between groups was determined using an unpaired Student’s t-test. ***P < 0.001. (C) Schematic of EVsABPC and EVsBMSC effects on NSCs proliferation. (D–E) Ki67 immunofluorescence staining (D) and quantification (E) in primary NSCs treated with vehicle, EVsBMSC, or EVsABPC (n = 3). Scale bar, 25 μm. (F) Western blot detection of Nestin, Ki67, and GAPDH protein levels in the vehicle, EVsBMSC, and EVsABPC groups. (G) Quantification of Nestin and Ki67 protein levels after different treatments (n = 3). (H) Schematic representation of FUCCI-based cell cycle evaluation. (I) Representative cell cycle images. Circle indicates tracked cell transitioning from G1 to G2/M within 24 h. Scale bar, 25 μm. (J) Bar graphs depicting the proportion of cells in different cell cycle phases over time under different treatments. (K) Line graphs showing dynamic variations in the proportion of cells in G1, S, and G2-M phases over time under different treatments (n = 3). Solid lines represent mean values for each phase, while shaded areas indicate standard deviation (SD). (L) Comparison of dynamic changes in the proportion of NSCs in S phase at different time points over 24 h under different treatments (n = 3). (M) Schematic illustration of evaluating effects of EVsABPC on NSC differentiation. (N) Representative immunofluorescence images showing expression of GFAP and TUJ1 in NSCs treated with vehicle, EVsBMSC, and EVsABPC for 2 d. Scale bar, 100 μm. (O) Measurement of proportion of TUJ1+ cells in each treatment group (n = 4). (P) Measurement of longest neurite length in TUJ1+ cells from each treatment group (n = 4). (E, G, O, and P) Statistical data are presented as mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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    Figure 3

    Figure 3. EVsABPC enhanced neurite outgrowth and reduce neuronal apoptosis. (A) Heatmap of DEPs related to axon guidance in EVsABPC and EVsNSC. (B) Violin plot of axon guidance proteins in EVsABPC and EVsNSC. Central box in each violin represents IQR. Statistical significance between groups was determined using an unpaired Student’s t-test. ***P < 0.001. (C) Schematic of EVsNSC, EVsBMSC, or EVsABPC effects on DRG neurite outgrowth. (D) Representative images (left) showing TUJ1-stained DRG neurons treated with vehicle, EVsNSC, EVsBMSC, or EVsABPC for 48 h. Scale bar, 50 μm. Sholl analysis (right) demonstrates the effects of different treatments on neurite branching complexity in DRG neurons. Representative Sholl plots were shown as insets, which were normalized against the vehicle group (n = 3). (E) Images depicting TUJ1 (green) /S100 (red) staining (left) in DRG explants treated with vehicle, EVsNSC, EVsBMSC, or EVsABPC for 48 h. Scale bar, 500 μm. Quantification of the mean length of the five longest axons in DRG explants (right) under different treatments (n = 5). (F) Workflow of RNA sequencing (RNA-seq) analysis. (G) RNA-seq analyses of regulated pathways in Neurons_Vehicle vs Neurons_EVsABPC. Left: Up- and downregulated genes. Middle: DEG expression heatmap. Right: Functional enrichment. (H) GSEA of biological pathways including Wnt signaling, axon guidance, Hippo signaling, and mast cell activation. (I) Western blot of key proteins in Wnt pathways in neurons treated with EVsABPC and the Wnt inhibitor XAV939. (J) Representative images of TUNEL staining in neuron cultures. Red indicates TUNEL-positive apoptotic cells, and green indicates NeuN-positive neurons. Scale bar, 50 μm. (K) Quantification of apoptosis in neuron culture. All statistical data are represented as mean ± SD. (E and K) Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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    Figure 4

    Figure 4. EVsABPC exhibited robust immunomodulatory ability in vitro and in vivo. (A) Schematic of evaluating the immunomodulatory effect of EVs on macrophage polarization in RAW264.7 cells. (B) Western blot showing proinflammatory factors iNOS and TNF-α in RAW 264.7 cells treated with LPS + IFN-γ, with or without different EVs. (C) Quantification of the relative iNOS and TNF-α expression levels in macrophages (n = 3). (D) Immunofluorescence images showing effects of EVs on macrophage polarization. Top: iNOS (red), F4/80 (green), DAPI (blue). Bottom: Arg1 (red), F4/80 (green), DAPI (blue). Scale bar, 50 μm. (E) Quantification of the fluorescence intensity of iNOS+ cells (M1) and Arg1+ cells (M2) (n = 4). (F) Flow cytometric analysis showing the percentage of CD86+ and CD206+ cells in F4/80+ gated macrophages after different treatments. (G) Quantification of the percentage of CD206+ cells (M2) in F4/80+ macrophages (primarily in Q2). (H) Western blot showing Arg1, IκBα, p-IκBα, p-P65, and P65 in macrophages treated with LPS + IFN-γ, with or without EVsABPC. (I) Immunofluorescence images showing effects of EVs on microglial polarization in the spinal cord at 14 dpi. Left: iNOS (red), iba1 (green), DAPI (blue). Right: Arg1 (red), iba1 (green), DAPI (blue). Scale bar, 200 μm. Insets show higher magnification of boxed areas. Scale bar, 50 μm. (J) Quantification of the fluorescence intensity of iNOS+ and Arg1+ microglia in the spinal cord (n = 4). (K) ELISA assay assessing spinal proinflammatory cytokine IL-1β, IL-6, and TNF-α levels following different treatments. (n = 3). (C, E, J, and K) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant. a.u: arbitrary unit.

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    Figure 5

    Figure 5. ACPP-modified EVsABPC enhanced targeted delivery to SCI sites. (A) Schematic diagram illustrating the modification of EVs with ACPPs to form ACPP@EVs. (B) Docking simulation between MMP2 and ACPP peptides showing specific interactions, including hydrogen bonds and salt bridges, with key amino acid residues highlighted in purple. (C) Mass spectrometry analysis confirming the peptide sequence (EEEEEEEEEEPLGLAGR) in ACPP@EVsABPC, as indicated by the DIA spectrum with fragment ions (red, y ion; blue, b ion) and an error plot showing the measurement accuracy. (D) ACPP@EVs labeled with a red fluorescent protein (RFP) were administered to SCI rats via tail vein injection, and their distribution was monitored 12 h postinjection by in vivo and ex vivo imaging. (E) Representative in vivo images showing the distribution of Dir@EVsABPC and ACPP@EVsABPC at the injury site 12 h postinjection. (F) Quantification of radiant efficiency at the injury site (n = 3). (G–H) Ex vivo imaging of the major organs showing the distribution of Dir@EVsABPC (G) and ACPP@EVsABPC (H). (I–J) Quantification of radiant efficiency in the spinal cord (I), spleen, lungs, kidneys, and liver (J) (n = 3). (K) Two-photon imaging of the dynamics of EVs from the vasculature to the spinal cord 4 h postinjection, with blood vessels (green) and EVs (red). Scale bar (left), 200 μm; Scale bar (right), 50 μm. (L) Quantification of fluorescence intensity of EVs in the spinal cord, as visualized by two-photon imaging (n = 5). (M) Quantification of the fluorescence intensity of Dir@EVsABPC and ACPP@ EVsABPC in spinal cord histology (n = 4). (N) Representative images of EV distribution in the spinal cord, with DAPI (blue) staining nuclei and Dir@EVs and ACPP@EVs (red). Scale bar, 25 μm. (F, I, J, L, and M) All statistical data are represented as mean ± SD. Statistical significance between groups was determined using an unpaired Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant. a.u: arbitrary unit.

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    Figure 6

    Figure 6. ACPP@EVsABPC promoted neural regeneration and reduce neuronal apoptosis post-SCI in vivo. (A) Schedule of SCI rats treated with ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC. Treatment included intravenous injections (i.v.) three times weekly, followed by assessments at various time points to evaluate neural regeneration and motor function recovery. (B–E) Representative immunofluorescence images of sagittal sections of the injured spinal cord stained for GFAP (green) and TUJ1 (red) to visualize astrocytes and newborn neurons at 14 dpi treated with ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC, respectively. Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas (b1-b3, c1-c3, d1-d3, e1-e3). Scale bar, 50 μm. (F–G) Quantification of the areas of TUJ1+ neurons (F) and GFAP+ astrocytes (G) in the lesion site (n = 3). (H–I) Representative Nissl staining images and quantification of the average optical density of neurons at 7 dpi (n = 5). Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas. Scale bar, 50 μm. (J–K) Quantification of the areas of NF+ axons and GAP43+ axons at 28 dpi (n = 3). (L) Representative immunofluorescence images of sagittal sections of the injured spinal cord stained for NF (white) and GAP43 (purple) to visualize nerve fibers and regenerating axons. Scale bar, 500 μm. Insets show higher-magnification images of the boxed areas (L1–L4). Scale bar, 50 μm. (M–N) Representative transmission electron micrographs (M) and scatterplots (N) comparing the myelin thickness and the correlation and linear regression between axon diameter and G-ratio in the vehicle, ACPP@EVsNSC, ACPP@EVsBMSC, and ACPP@EVsABPC treatment groups. Axons were selected from 4 rats per group for measurement (n = 60 measured axons for each group). Scale bars, 4 μm. Correlation analyses were performed using Pearson’s correlation. (F, G, I, J, and K) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

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    Figure 7

    Figure 7. ACPP@EVsABPC improved motor function recovery post-SCI. (A) Representative images of the right hindlimb of rats during the climbing test showing the effects of treatment with ACPP@EVsNSC, ACPP@EVsBMSC, or ACPP@EVsABPC. (B) Representative 3D stress diagrams of the right hindlimb at 8 wpi. (C) The representative footprints recorded by the Catwalk system (cyan for right front, RF; yellow for left front, LF; green for left hind, LH; and magenta for right hind, RH). (D–E) Quantification of the base of support (D) and stride length (E) in different treatment groups based on footprint patterns (n = 3). (F–G) Quantification of CMAP amplitude (F) and latency (G) in the different treatment groups (n = 4). (H) Representative MEP waveforms in the different groups. (I) BBB locomotor rating scale of hindlimbs in different groups during the 8-week treatment period (n = 5). All statistical data are represented as mean ± SD (D, E, F, and J) All statistical data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA with Bonferroni’s posthoc test. (I) Statistical differences were determined using two-way ANOVA with Bonferroni’s posthoc test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the Vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to ACPP@EVsBMSC group; @P < 0.05, @@P < 0.01, @@@P < 0.001 compared to ACPP@EVsNSC group. ns: not significant.

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      21
      The elusive nature and function of mesenchymal stem cells
      Nombela-Arrieta, Cesar; Ritz, Jerome; Silberstein, Leslie E.
      Nature Reviews Molecular Cell Biology (2011), 12 (2), 126-131CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      Mesenchymal stem cells (MSCs) are a diverse subset of multipotent precursors present in the stromal fraction of many adult tissues and have drawn intense interest from translational and basic investigators. MSCs have been operationally defined by their ability to differentiate into osteoblasts, adipocytes and chondrocytes after in vitro expansion. Nevertheless, their identity in vivo, heterogeneity, anatomical localization and functional roles in adult tissue homeostasis have remained enigmatic and are only just starting to be uncovered.
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    22. 22
      Qin, T.; Zhang, G.; Zheng, Y.; Li, S.; Yuan, Y.; Li, Q. A Population of Stem Cells with Strong Regenerative Potential Discovered in Deer Antlers. Science 2023, 379 (6634), 840– 847,  DOI: 10.1126/science.add0488
      There is no corresponding record for this reference.
    23. 23
      Yao, B.; Wang, C.; Zhou, Z.; Zhang, M.; Zhao, D.; Bai, X. Comparative Transcriptome Analysis of the Main Beam and Brow Tine of Sika Deer Antler Provides Insights into the Molecular Control of Rapid Antler Growth. Cell. Mol. Biol. Lett. 2020, 25 (1), 42,  DOI: 10.1186/s11658-020-00234-9
      There is no corresponding record for this reference.
    24. 24
      Bajpai, V. K.; Mistriotis, P.; Loh, Y.-H.; Daley, G. Q.; Andreadis, S. T. Functional Vascular Smooth Muscle Cells Derived from Human Induced Pluripotent Stem Cells via Mesenchymal Stem Cell Intermediates. Cardiovasc. Res. 2012, 96 (3), 391– 400,  DOI: 10.1093/cvr/cvs253
      There is no corresponding record for this reference.
    25. 25
      Wang, D.; Berg, D.; Ba, H.; Sun, H.; Wang, Z.; Li, C. Deer Antler Stem Cells Are a Novel Type of Cells That Sustain Full Regeneration of a Mammalian Organ─Deer Antler. Cell Death Dis. 2019, 10 (6), 443,  DOI: 10.1038/s41419-019-1686-y
      There is no corresponding record for this reference.
    26. 26
      Rong, X.; Chu, W.; Zhang, H.; Wang, Y.; Qi, X.; Zhang, G. Antler Stem Cell-Conditioned Medium Stimulates Regenerative Wound Healing in Rats. Stem Cell Res. Ther. 2019, 10 (1), 326,  DOI: 10.1186/s13287-019-1457-9
      There is no corresponding record for this reference.
    27. 27
      Lei, J.; Jiang, X.; Li, W.; Ren, J.; Wang, D.; Ji, Z.; Wu, Z.; Cheng, F.; Cai, Y.; Yu, Z.-R.; Belmonte, J. C. I.; Li, C.; Liu, G.-H.; Zhang, W.; Qu, J.; Wang, S. Exosomes from Antler Stem Cells Alleviate Mesenchymal Stem Cell Senescence and Osteoarthritis. Protein Cell 2022, 13 (3), 220– 226,  DOI: 10.1007/s13238-021-00860-9
      There is no corresponding record for this reference.
    28. 28
      Jin, S.; Wang, Y.; Wu, X.; Li, Z.; Zhu, L.; Niu, Y. Young Exosome Bio-Nanoparticles Restore Aging-Impaired Tendon Stem/Progenitor Cell Function and Reparative Capacity. Adv. Mater. 2023, 35 (18), 2211602  DOI: 10.1002/adma.202211602
      There is no corresponding record for this reference.
    29. 29
      Tan, F.; Li, X.; Wang, Z.; Li, J.; Shahzad, K.; Zheng, J. Clinical Applications of Stem Cell-Derived Exosomes. Signal Transduction Targeted Ther. 2024, 9 (1), 17,  DOI: 10.1038/s41392-023-01704-0
      There is no corresponding record for this reference.
    30. 30
      Xia, B.; Gao, X.; Qian, J.; Li, S.; Yu, B.; Hao, Y. A Novel Superparamagnetic Multifunctional Nerve Scaffold: A Remote Actuation Strategy to Boost In Situ Extracellular Vesicles Production for Enhanced Peripheral Nerve Repair. Adv. Mater. 2024, 36 (3), 2305374  DOI: 10.1002/adma.202305374
      There is no corresponding record for this reference.
    31. 31
      Han, M.; Yang, H.; Lu, X.; Li, Y.; Liu, Z.; Li, F. Three-Dimensional-Cultured MSC-Derived Exosome-Hydrogel Hybrid Microneedle Array Patch for Spinal Cord Repair. Nano Lett. 2022, 22 (15), 6391– 6401,  DOI: 10.1021/acs.nanolett.2c02259
      31
      Three-dimensional-cultured mesenchymal stem cells-derived exosome-hydrogel hybrid microneedle array patch for spinal cord repair
      Han, Min; Yang, Hongru; Lu, Xiangdong; Li, Yuming; Liu, Zihao; Li, Feng; Shang, Zehan; Wang, Xiaofeng; Li, Xuze; Li, Junliang; Liu, Hong; Xin, Tao
      Nano Letters (2022), 22 (15), 6391-6401CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Exosomes derived from mesenchymal stem cells (MSCs) have been proven to exhibit great potentials in spinal cord injury (SCI) therapy. However, conventional two-dimensional (2D) culture will inevitably lead to the loss of stemness of MSCs, which substantially limits the therapeutic potency of MSCs exosomes (2D-Exo). Exosomes derived from three-dimensional culture (3D-Exo) possess higher therapeutic efficiency which have wide applications in spinal cord therapy. Typically, conventional exosome therapy that relies on local repeated injection results in secondary injury and low efficiency. It is urgent to develop a more reliable, convenient, and effective exosome delivery method to achieve const. in situ exosomes release. Herein, we proposed a controlled 3D-exohydrogel hybrid microneedle array patch to achieve SCI repair in situ. Our studies suggested that MSCs with 3D-culturing could maintain their stemness, and consequently, 3D-Exo effectively reduced SCI-induced inflammation and glial scarring. Thus, it is a promising therapeutic strategy for the treatment of SCI.
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    32. 32
      Nieto-Diaz, M. Deer Antler Innervation and Regeneration. Front. Biosci. 2012, 17 (1), 1389,  DOI: 10.2741/3993
      There is no corresponding record for this reference.
    33. 33
      S, S.; Camardo, A.; Dahal, S.; Ramamurthi, A. Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair. Mol. Pharmaceutics 2023, 20 (6), 2801– 2813,  DOI: 10.1021/acs.molpharmaceut.2c00769
      33
      Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair
      S, Sajeesh; Camardo, Andrew; Dahal, Shataakshi; Ramamurthi, Anand
      Molecular Pharmaceutics (2023), 20 (6), 2801-2813CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Extracellular vesicles (EVs) are nanosized vesicles that carry cell-specific biomol. information. Our previous studies showed that adult human bone marrow mesenchymal stem cell (BM-MSC)-derived EVs provide antiproteolytic and proregenerative effects in cultures of smooth muscle cells (SMCs) derived from an elastase-infused rat abdominal aortic aneurysm (AAA) model, and this is promising toward their use as a therapeutic platform for naturally irreversible elastic matrix aberrations in the aortic wall. Since systemically administered EVs poorly home into sites of tissue injury, disease strategies to improve their affinity toward target tissues are of great significance for EV-based treatment strategies. Toward this goal, in this work, we developed a postisolation surface modification strategy to target MSC-derived EVs to the AAA wall. The EVs were surface-conjugated with a short, synthetic, azide-modified peptide sequence for targeted binding to cathepsin K (CatK), a cysteine protease overexpressed in the AAA wall. Conjugation was performed using a copper-free click chem. method. We detd. that such conjugation improved EV uptake into cultured aneurysmal SMCs in culture and their binding to the wall of matrix injured vessels ex vivo. The proregenerative and antiproteolytic effects of MSC-EVs on cultured rat aneurysmal SMCs were also unaffected following peptide conjugation. From this study, it appears that modification with short synthetic peptide sequences seems to be an effective strategy for improving the cell-specific uptake of EVs and may be effective in facilitating AAA-targeted therapy.
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    34. 34
      Wu, W.; Jia, S.; Xu, H.; Gao, Z.; Wang, Z.; Lu, B. Supramolecular Hydrogel Microspheres of Platelet-Derived Growth Factor Mimetic Peptide Promote Recovery from Spinal Cord Injury. ACS Nano 2023, 17 (4), 3818– 3837,  DOI: 10.1021/acsnano.2c12017
      34
      Supramolecular Hydrogel Microspheres of Platelet-Derived Growth Factor Mimetic Peptide Promote Recovery from Spinal Cord Injury
      Wu, Weidong; Jia, Shuaijun; Xu, Hailiang; Gao, Ziheng; Wang, Zhiyuan; Lu, Botao; Ai, Yixiang; Liu, Youjun; Liu, Renfeng; Yang, Tong; Luo, Rongjin; Hu, Chunping; Kong, Lingbo; Huang, Dageng; Yan, Liang; Yang, Zhimou; Zhu, Lei; Hao, Dingjun
      ACS Nano (2023), 17 (4), 3818-3837CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Neural stem cells (NSCs) are considered to be prospective replacements for neuronal cell loss as a result of spinal cord injury (SCI). However, the survival and neuronal differentiation of NSCs are strongly affected by the unfavorable microenvironment induced by SCI, which critically impairs their therapeutic ability to treat SCI. Herein, a strategy to fabricate PDGF-MP hydrogel (PDGF-MPH) microspheres (PDGF-MPHM) instead of bulk hydrogels is proposed to dramatically enhance the efficiency of platelet-derived growth factor mimetic peptide (PDGF-MP) in activating its receptor. PDGF-MPHM were fabricated by a piezoelec. ceramic-driven thermal electrospray device, had an av. size of 9μm, and also had the ability to activate the PDGFRβ of NSCs more effectively than PDGF-MPH. In vitro, PDGF-MPHM exerted strong neuroprotective effects by maintaining the proliferation and inhibiting the apoptosis of NSCs in the presence of myelin exts. In vivo, PDGF-MPHM inhibited M1 macrophage infiltration and extrinsic or intrinsic cells apoptosis on the seventh day after SCI. Eight weeks after SCI, the T10 SCI treatment results showed that PDGF-MPHM + NSCs significantly promoted the survival of NSCs and neuronal differentiation, reduced lesion size, and considerably improved motor function recovery in SCI rats by stimulating axonal regeneration, synapse formation, and angiogenesis in comparison with the NSCs graft group. Therefore, our findings provide insights into the ability of PDGF-MPHM to be a promising therapeutic agent for SCI repair.
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    35. 35
      Yano, S.; Tazawa, H.; Kagawa, S.; Fujiwara, T.; Hoffman, R. M. FUCCI Real-Time Cell-Cycle Imaging as a Guide for Designing Improved Cancer Therapy: A Review of Innovative Strategies to Target Quiescent Chemo-Resistant Cancer Cells. Cancers 2020, 12 (9), 2655,  DOI: 10.3390/cancers12092655
      There is no corresponding record for this reference.
    36. 36
      Koh, S.-B.; Mascalchi, P.; Rodriguez, E.; Lin, Y.; Jodrell, D. I.; Richards, F. M. A Quantitative FastFUCCI Assay Defines Cell Cycle Dynamics at a Single-Cell Level. J. Cell Sci. 2017, 130 (2), 512– 520,  DOI: 10.1242/jcs.195164
      There is no corresponding record for this reference.
    37. 37
      Geng, H.; Li, Z.; Li, Z.; Zhang, Y.; Gao, Z.; Sun, L. Restoring Neuronal Iron Homeostasis Revitalizes Neurogenesis after Spinal Cord Injury. Proc. Natl. Acad. Sci. U. S. A. 2023, 120 (46), e2220300120  DOI: 10.1073/pnas.2220300120
      There is no corresponding record for this reference.
    38. 38
      Tran, A. P.; Warren, P. M.; Silver, J. The Biology of Regeneration Failure and Success After Spinal Cord Injury. Physiol. Rev. 2018, 98 (2), 881– 917,  DOI: 10.1152/physrev.00017.2017
      38
      The biology of regeneration failure and success after spinal cord injury
      Tran, Amanda Phuong; Warren, Philippa Mary; Silver, Jerry
      Physiological Reviews (2018), 98 (2), 881-917CODEN: PHREA7; ISSN:1522-1210. (American Physiological Society)
      A review. Since no approved therapies to restore mobility and sensation following spinal cord injury (SCI) currently exist, a better understanding of the cellular and mol. mechanisms following SCI that compromise regeneration or neuroplasticity is needed to develop new strategies to promote axonal regrowth and restore function. Phys. trauma to the spinal cord results in vascular disruption that, in turn, causes blood-spinal cord barrier rupture leading to hemorrhage and ischemia, followed by rampant local cell death. As subsequent edema and inflammation occur, neuronal and glial necrosis and apoptosis spread well beyond the initial site of impact, ultimately resolving into a cavity surrounded by glial/fibrotic scarring. Understanding the formative events in glial scarring helps guide strategies towards the development of potential therapies to enhance axon regeneration and functional recovery at both acute and chronic stages following SCI. This review will also discuss the perineuronal net and how chondroitin sulfate proteoglycans (CSPGs) deposited in both the glial scar and net impede axonal outgrowth at the level of the growth cone. We will end the review with a summary of current CSPG-targeting strategies that help to foster axonal regeneration, neuroplasticity/sprouting, and functional recovery following SCI.
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    39. 39
      Li, S.; Wang, P.; Ye, M.; Yang, K.; Cheng, D.; Mao, Z. Cysteine-Activatable Near-Infrared Fluorescent Probe for Dual-Channel Tracking Lipid Droplets and Mitochondria in Epilepsy. Anal. Chem. 2023, 95 (11), 5133– 5141,  DOI: 10.1021/acs.analchem.3c00226
      There is no corresponding record for this reference.
    40. 40
      Mathew, B.; Ravindran, S.; Liu, X.; Torres, L.; Chennakesavalu, M.; Huang, C.-C. Mesenchymal Stem Cell-Derived Extracellular Vesicles and Retinal Ischemia-Reperfusion. Biomaterials 2019, 197, 146– 160,  DOI: 10.1016/j.biomaterials.2019.01.016
      40
      Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion
      Mathew, Biji; Ravindran, Sriram; Liu, Xiaorong; Torres, Leianne; Chennakesavalu, Mohansrinivas; Huang, Chun-Chieh; Feng, Liang; Zelka, Ruth; Lopez, Jasmine; Sharma, Monica; Roth, Steven
      Biomaterials (2019), 197 (), 146-160CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      Retinal ischemia is a major cause of vision loss and impairment and a common underlying mechanism assocd. with diseases such as glaucoma, diabetic retinopathy, and central retinal artery occlusion. The regenerative capacity of the diseased human retina is limited. Our previous studies have shown the neuroprotective effects of intravitreal injection of mesenchymal stem cells (MSC) and MSC-conditioned medium in retinal ischemia in rats. Based upon the hypothesis that the neuroprotective effects of MSCs and conditioned medium are largely mediated by extracellular vesicles (EVs), MSC derived EVs were tested in an in-vitro oxygen-glucose deprivation (OGD) model of retinal ischemia. Treatment of R28 retinal cells with MSC-derived EVs significantly reduced cell death and attenuated loss of cell proliferation. Mechanistic studies on the mode of EV endocytosis by retinal cells were performed in vitro. EV endocytosis was dose- and temp.-dependent, saturable, and occurred via cell surface heparin sulfate proteoglycans mediated by the caveolar endocytic pathway. The administration of MSC-EVs into the vitreous humor 24 h after retinal ischemia in a rat model significantly enhanced functional recovery, and decreased neuro-inflammation and apoptosis. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia. They were present in the vitreous humor for four weeks after intravitreal administration, with saturable binding to vitreous humor components. Overall, this study highlights the potential of MSC-EV as biomaterials for neuroprotective and regenerative therapy in retinal disorders.
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    41. 41
      Ciani, L.; Salinas, P. C. WNTS in the Vertebrate Nervous System: From Patterning to Neuronal Connectivity. Nat. Rev. Neurosci. 2005, 6 (5), 351– 362,  DOI: 10.1038/nrn1665
      41
      Signaling in neural development: WNTS in the vertebrate nervous system: from patterning to neuronal connectivity
      Ciani, Lorenza; Salinas, Patricia C.
      Nature Reviews Neuroscience (2005), 6 (5), 351-362CODEN: NRNAAN; ISSN:1471-003X. (Nature Publishing Group)
      A review. WNT signaling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity and cell movements. In the nervous system, WNT signaling also regulates neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation. Studies, from invertebrates to mammals, have led to a considerable understanding of WNT signal transduction pathways. This knowledge provides a framework for the study of the mechanisms by which WNTs regulate diverse neuronal functions. Manipulation of the WNT pathways could provide new strategies for nerve regeneration and neuronal circuit modulation.
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    42. 42
      Feltri, M. L.; Poitelon, Y. HIPPO Stampede in Nerve Sheath Tumors. Cancer Cell 2018, 33 (2), 160– 161,  DOI: 10.1016/j.ccell.2018.01.016
      There is no corresponding record for this reference.
    43. 43
      Tian, X.; Hou, W.; Bai, S.; Fan, J.; Tong, H.; Bai, Y. XAV939 Promotes Apoptosis in a Neuroblastoma Cell Line via Telomere Shortening. Oncol. Rep. 2014, 32 (5), 1999– 2006,  DOI: 10.3892/or.2014.3460
      There is no corresponding record for this reference.
    44. 44
      Barrette, A. M.; Ronk, H.; Joshi, T.; Mussa, Z.; Mehrotra, M.; Bouras, A. Anti-Invasive Efficacy and Survival Benefit of the YAP-TEAD Inhibitor Verteporfin in Preclinical Glioblastoma Models. Neuro-Oncol. 2022, 24 (5), 694– 707,  DOI: 10.1093/neuonc/noab244
      There is no corresponding record for this reference.
    45. 45
      Liu, H.; Li, R.; Liu, T.; Yang, L.; Yin, G.; Xie, Q. Immunomodulatory Effects of Mesenchymal Stem Cells and Mesenchymal Stem Cell-Derived Extracellular Vesicles in Rheumatoid Arthritis. Front. Immunol. 2020, 11, 1912,  DOI: 10.3389/fimmu.2020.01912
      There is no corresponding record for this reference.
    46. 46
      Xiong, T.; Yang, K.; Zhao, T.; Zhao, H.; Gao, X.; You, Z. Multifunctional Integrated Nanozymes Facilitate Spinal Cord Regeneration by Remodeling the Extrinsic Neural Environment. Adv. Sci. 2023, 10 (7), 2205997  DOI: 10.1002/advs.202205997
      46
      Multifunctional Integrated Nanozymes Facilitate Spinal Cord Regeneration by Remodeling the Extrinsic Neural Environment
      Xiong, Tiandi; Yang, Keni; Zhao, Tongtong; Zhao, Haitao; Gao, Xu; You, Zhifeng; Fan, Caixia; Kang, Xinyi; Yang, Wen; Zhuang, Yan; Chen, Yanyan; Dai, Jianwu
      Advanced Science (Weinheim, Germany) (2023), 10 (7), 2205997CODEN: ASDCCF; ISSN:2198-3844. (Wiley-VCH Verlag GmbH & Co. KGaA)
      High levels of reactive oxygen species (ROS) and inflammation create a complicated extrinsic neural environment that dominates the initial post-injury period after spinal cord injury (SCI). The compensatory pathways between ROS and inflammation limited the efficacy of modulating the above single treatment regimen after SCI. Here, novel "nanoflower" Mn3O4 integrated with "pollen" IRF-5SiRNA was designed as a combination antioxidant and anti-inflammatory treatment after SCI. The "nanoflower" and "pollen" structure was encapsulated with a neutrophil membrane for protective and targeted delivery. Furthermore, valence-engineered nanozyme Mn3O4 imitated the cascade response of antioxidant enzymes with a higher substrate affinity compared to natural antioxidant enzymes. Nanozymes effectively catalyzed ROS to generate O2, which is advantageous for reducing oxidative stress and promoting angiogenesis. The screened "pollen" IRF-5SiRNA could reverse the inflammatory phenotype by reducing interferon regulatory factors-5 (IRF-5) expression (protein level: 73.08% and mRNA level: 63.10%). The decreased expression of pro-inflammatory factors reduced the infiltration of inflammatory cells, resulting in less neural scarring. In SCI rats, multifunctional nanozymes enhanced the proliferation of various neuronal subtypes (motor neurons, interneurons, and sensory neurons) and the recovery of locomotor function, demonstrating that the remodeling of the extrinsic neural environment is a promising strategy to facilitate nerve regeneration.
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    47. 47
      Wang, L.; Wang, D.; Ye, Z.; Xu, J. Engineering Extracellular Vesicles as Delivery Systems in Therapeutic Applications. Adv. Sci. 2023, 10 (17), 2300552  DOI: 10.1002/advs.202300552
      There is no corresponding record for this reference.
    48. 48
      Miranpuri, G. S.; Schomberg, D. T.; Alrfaei, B.; King, K. C.; Rynearson, B.; Wesley, V. S. Role of Matrix Metalloproteinases 2 in Spinal Cord Injury-Induced Neuropathic Pain. Ann. Neurosci. 2016, 23 (1), 25– 32,  DOI: 10.1159/000443553
      There is no corresponding record for this reference.
    49. 49
      Shen, K.; Sun, G.; Chan, L.; He, L.; Li, X.; Yang, S. Anti-Inflammatory Nanotherapeutics by Targeting Matrix Metalloproteinases for Immunotherapy of Spinal Cord Injury. Small 2021, 17 (41), 2102102  DOI: 10.1002/smll.202102102
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      Anti-Inflammatory Nanotherapeutics by Targeting Matrix Metalloproteinases for Immunotherapy of Spinal Cord Injury
      Shen, Kui; Sun, Guodong; Chan, Leung; He, Lizhen; Li, Xiaowei; Yang, Shuxian; Wang, Baocheng; Zhang, Hua; Huang, Jiarun; Chang, Minmin; Li, Zhizhong; Chen, Tianfeng
      Small (2021), 17 (41), 2102102CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Neuroinflammation is critically involved in the repair of spinal cord injury (SCI), and macrophages assocd. with inflammation propel the degeneration or recovery in the pathol. process. Currently, efforts have been focused on obtaining efficient therapeutic anti-inflammatory drugs to treat SCI. However, these drugs are still unable to penetrate the blood spinal cord barrier and lack the ability to target lesion areas, resulting in unsatisfactory clin. efficacy. Herein, a polymer-based nanodrug delivery system is constructed to enhance the targeting ability. Because of increased expression of matrix metalloproteinases (MMPs) in injured site after SCI, MMP-responsive mol., activated cell-penetrating peptides (ACPP), is introduced into the biocompatible polymer PLGA-PEI-mPEG (PPP) to endow the nanoparticles with the ability for diseased tissue-targeting. Meanwhile, etanercept (ET), a clin. anti-inflammation treatment medicine, is loaded on the polymer to regulate the polarization of macrophages, and promote locomotor recovery. The results show that PPP-ACPP nanoparticles possess satisfactory lesion targeting effects. Through inhibited consequential prodn. of proinflammation cytokines and promoted anti-inflammation cytokines, ET@PPP-ACPP could decrease the percentage of M1 macrophages and increase M2 macrophages. As expected, ET@PPP-ACPP accumulates in lesion area and achieves effective treatment of SCI; this confirmed the potential of nano-drug loading systems in SCI immunotherapy.
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    50. 50
      Zhu, B.; Gu, G.; Ren, J.; Song, X.; Li, J.; Wang, C. Schwann Cell-Derived Exosomes and Methylprednisolone Composite Patch for Spinal Cord Injury Repair. ACS Nano 2023, 17 (22), 22928– 22943,  DOI: 10.1021/acsnano.3c08046
      There is no corresponding record for this reference.
    51. 51
      Yang, J.; Yang, K.; Man, W.; Zheng, J.; Cao, Z.; Yang, C.-Y. 3D Bio-Printed Living Nerve-like Fibers Refine the Ecological Niche for Long-Distance Spinal Cord Injury Regeneration. Bioact. Mater. 2023, 25, 160– 175,  DOI: 10.1016/j.bioactmat.2023.01.023
      51
      3D bio-printed living nerve-like fibers refine the ecological niche for long-distance spinal cord injury regeneration
      Yang, Jia; Yang, Kaiyuan; Man, Weitao; Zheng, Jingchuan; Cao, Zheng; Yang, Chun-Yi; Kim, Kunkoo; Yang, Shuhui; Hou, Zhaohui; Wang, Guihuai; Wang, Xiumei
      Bioactive Materials (2023), 25 (), 160-175CODEN: BMIAD4; ISSN:2452-199X. (KeAi Communications Co., Ltd.)
      3D bioprinting holds great promise toward fabricating biomimetic living constructs in a bottom-up assembly manner. To date, various emergences of living constructs have been bioprinted for in vitro applications, while the conspicuous potential serving for in vivo implantable therapies in spinal cord injury (SCI) has been relatively overlooked. Herein, living nerve-like fibers are prepd. via extrusion-based 3D bioprinting for SCI therapy. The living nerve-like fibers are comprised of neural stem cells (NSCs) embedded within a designed hydrogel that mimics the extracellular matrix (ECM), assembled into a highly spatial ordered architecture, similar to densely arranged bundles of the nerve fibers. The pro-neurogenesis ability of these living nerve-like fibers is tested in a 4 mm-long complete transected SCI rat model. Evidence shows that living nerve-like fibers refine the ecol. niche of the defect site by immune modulation, angiogenesis, neurogenesis, neural relay formations, and neural circuit remodeling, leading to outstanding functional reconstruction, revealing an evolution process of this living construct after implantation. This effective strategy, based on biomimetic living constructs, opens a new perspective on SCI therapies.
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    52. 52
      Goodman, Z. D. Grading and Staging Systems for Inflammation and Fibrosis in Chronic Liver Diseases. J. Hepatol. 2007, 47 (4), 598– 607,  DOI: 10.1016/j.jhep.2007.07.006
      There is no corresponding record for this reference.
    53. 53
      Qin, T.; Li, C.; Xu, Y.; Qin, Y.; Jin, Y.; He, R. Local Delivery of EGFR+NSCs-Derived Exosomes Promotes Neural Regeneration Post Spinal Cord Injury via miR-34a-5p/HDAC6 Pathway. Bioact. Mater. 2024, 33, 424– 443,  DOI: 10.1016/j.bioactmat.2023.11.013
      There is no corresponding record for this reference.
    54. 54
      Nie, H.; Jiang, Z. Bone Mesenchymal Stem Cell-Derived Extracellular Vesicles Deliver microRNA-23b to Alleviate Spinal Cord Injury by Targeting Toll-like Receptor TLR4 and Inhibiting NF-κB Pathway Activation. Bioengineered 2021, 12 (1), 8157– 8172,  DOI: 10.1080/21655979.2021.1977562
      There is no corresponding record for this reference.
    55. 55
      Welsh, J. A.; Goberdhan, D. C. I.; O’Driscoll, L.; Buzas, E. I.; Blenkiron, C.; Bussolati, B. Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches. J. Extracell. Vesicles 2024, 13 (2), e12404  DOI: 10.1002/jev2.12451
      There is no corresponding record for this reference.
    56. 56
      Piccolo, S.; Dupont, S.; Cordenonsi, M. The Biology of YAP/TAZ: Hippo Signaling and Beyond. Physiol. Rev. 2014, 94 (4), 1287– 1312,  DOI: 10.1152/physrev.00005.2014
      56
      The biology of YAP/TAZ: Hippo signaling and beyond
      Piccolo, Stefano; Dupont, Sirio; Cordenonsi, Michelangelo
      Physiological Reviews (2014), 94 (4), 1287-1312CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
      A review. The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biol. properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a mol. entry point into the mysteries of tissue biol., their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's phys. nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mech. signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metab. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context.
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    57. 57
      Cassani, M.; Fernandes, S.; Oliver-De La Cruz, J.; Durikova, H.; Vrbsky, J.; Patočka, M. YAP Signaling Regulates the Cellular Uptake and Therapeutic Effect of Nanoparticles. Adv. Sci. 2024, 11 (2), 2302965  DOI: 10.1002/advs.202302965
      There is no corresponding record for this reference.
    58. 58
      Pham, T. C.; Jayasinghe, M. K.; Pham, T. T.; Yang, Y.; Wei, L.; Usman, W. M. Covalent Conjugation of Extracellular Vesicles with Peptides and Nanobodies for Targeted Therapeutic Delivery. J. Extracell. Vesicles 2021, 10 (4), e12057  DOI: 10.1002/jev2.12057
      58
      Covalent conjugation of extracellular vesicles with peptides and nanobodies for targeted therapeutic delivery
      Pham, Tin Chanh; Jayasinghe, Migara Kavishka; Pham, Thach Tuan; Yang, Yuqi; Wei, Likun; Usman, Waqas Muhammad; Chen, Huan; Pirisinu, Marco; Gong, Jinhua; Kim, Seongkyeol; Peng, Boya; Wang, Weixi; Chan, Charlene; Ma, Victor; Nguyen, Nhung T. H.; Kappei, Dennis; Nguyen, Xuan-Hung; Cho, William C.; Shi, Jiahai; Le, Minh T. N.
      Journal of Extracellular Vesicles (2021), 10 (4), e12057CODEN: JEVOA4; ISSN:2001-3078. (John Wiley & Sons, Inc.)
      Natural extracellular vesicles (EVs) are ideal drug carriers due to their remarkable biocompatibility. Their delivery specificity can be achieved by the conjugation of targeting ligands. However, existing methods to engineer target-specific EVs are tedious or inefficient, having to compromise between harsh chem. treatments and transient interactions. Here, we describe a novel method for the covalent conjugation of EVs with high copy nos. of targeting moieties using protein ligases. Conjugation of EVs with either an epidermal growth factor receptor (EGFR)-targeting peptide or anti-EGFR nanobody facilitates their accumulation in EGFR-pos. cancer cells, both in vitro and in vivo. Systemic delivery of paclitaxel by EGFR-targeting EVs at a low dose significantly increases drug efficacy in a xenografted mouse model of EGFR-pos. lung cancer. The method is also applicable to the conjugation of EVs with peptides and nanobodies targeting other receptors, such as HER2 and SIRP alpha, and the conjugated EVs can deliver RNA in addn. to small mols., supporting the versatile application of EVs in cancer therapies. This simple, yet efficient and versatile method for the stable surface modification of EVs bypasses the need for genetic and chem. modifications, thus facilitating safe and specific delivery of therapeutic payloads to target cells.
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    59. 59
      Théry, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines. J. Extracell. Vesicles 2018, 7 (1), 1535750  DOI: 10.1080/20013078.2018.1535750
      59
      Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
      Thery Clotilde; Lavieu Gregory; Martin-Jaular Lorena; Mathieu Mathilde; Tkach Mercedes; Witwer Kenneth W; Huang Yiyao; Muth Dillon C; Powell Bonita H; Schoyen Tine Hiorth; Zhao Zezhou; Witwer Kenneth W; Datta Chaudhuri Amrita; Aikawa Elena; Aikawa Elena; Alcaraz Maria Jose; Anderson Johnathon D; Andriantsitohaina Ramaroson; Le Lay Soazig; Martinez M Carmen; Antoniou Anna; Antoniou Anna; Arab Tanina; Archer Fabienne; Atkin-Smith Georgia K; Baxter Amy A; Caruso Sarah; Cheng Lesley; Greening David W; Hill Andrew F; Jiang Lanzhou; Mathivanan Suresh; Poon Ivan Kh; Tixeira Rochelle; Ayre D Craig; Ayre D Craig; Bach Jean-Marie; Bosch Steffi; Bachurski Daniel; Baharvand Hossein; Shekari Faezeh; Baharvand Hossein; Balaj Leonora; Baldacchino Shawn; Bauer Natalie N; Bebawy Mary; Beckham Carla; Bedina Zavec Apolonija; Benmoussa Abderrahim; Boilard Eric; Gilbert Caroline; Berardi Anna C; Bergese Paolo; Radeghieri Annalisa; Bergese Paolo; Bergese Paolo; Busatto Sara; Radeghieri Annalisa; Bielska Ewa; Blenkiron Cherie; Bobis-Wozowicz Sylwia; Zuba-Surma Ewa K; Boireau Wilfrid; Elie-Caille Celine; Frelet-Barrand Annie; Bongiovanni Antonella; Borras Francesc E; Gamez-Valero Ana; Borras Francesc E; Borras Francesc E; Boulanger Chantal M; Loyer Xavier; Boulanger Chantal M; Loyer Xavier; Breakefield Xandra; Breglio Andrew M; Breglio Andrew M; Brennan Meadhbh A; Brennan Meadhbh A; Brennan Meadhbh A; Brigstock David R; Brigstock David R; Brisson Alain; Broekman Marike Ld; Broekman Marike Ld; Broekman Marike Ld; Bromberg Jacqueline F; Bromberg Jacqueline F; Bryl-Gorecka Paulina; Buch Shilpa; Hu Guoku; Liao Ke; Buck Amy H; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Busatto Sara; Buschmann Dominik; Mussack Veronika; Pfaffl Michael W; Bussolati Benedetta; Buzas Edit I; Buzas Edit I; Forsonits Andras; Hegyesi Hargita; Khamari Delaram; Kovacs Arpad Ferenc; Sodar Barbara W; Visnovitz Tamas; Vukman Krisztina V; Wiener Zoltan; Byrd James Bryan; Camussi Giovanni; Carter David Rf; Pink Ryan C; Chamley Lawrence W; Chang Yu-Ting; Chen Chihchen; Chen Chihchen; Chen Shuai; Chin Andrew R; Di Vizio Dolores; Mariscal Javier; Clayton Aled; Cocks Alex; Webber Jason P; Clerici Stefano P; Cocucci Emanuele; Cocucci Emanuele; Coffey Robert J; Cordeiro-da-Silva Anabela; Couch Yvonne; Coumans Frank Aw; Nieuwland Rienk; Coyle Beth; Jackson Hannah K; Crescitelli Rossella; Lasser Cecilia; Lotvall Jan; Shelke Ganesh Vilas; Criado Miria Ferreira; D'Souza-Schorey Crislyn; Das Saumya; de Candia Paola; De Santana Eliezer F; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; Del Portillo Hernando A; Del Portillo Hernando A; Del Portillo Hernando A; Demaret Tanguy; Lombard Catherine A; Deville Sarah; Deville Sarah; Mertens Inge; Devitt Andrew; Dhondt Bert; Dieterich Lothar C; Dolo Vincenza; Giusti Ilaria; Dominguez Rubio Ana Paula; Dominici Massimo; Dominici Massimo; Dourado Mauricio R; Dourado Mauricio R; Driedonks Tom Ap; Nolte-'t Hoen Esther Nm; Stoorvogel Willem; van der Grein Susanne G; van Herwijnen Martijn Jc; Wauben Marca Hm; Duarte Filipe V; Rodrigues Silvia C; Duncan Heather M; Duncan Heather M; Eichenberger Ramon M; Sotillo Javier; Ekstrom Karin; El Andaloussi Samir; Gorgens Andre; El Andaloussi Samir; Erdbrugger Uta; Musante Luca; Falcon-Perez Juan M; Falcon-Perez Juan M; Fatima Farah; Fish Jason E; Fish Jason E; Gustafson Dakota; Schneider Raphael; Flores-Bellver Miguel; Fricke Fabia; Fricke Fabia; Fuhrmann Gregor; Fuhrmann Gregor; Fuhrmann Gregor; Gabrielsson Susanne; Gamez-Valero Ana; Gardiner Chris; Gartner Kathrin; Gaudin Raphael; Gaudin Raphael; Gho Yong Song; Park Jaesung; Giebel Bernd; Gorgens Andre; Gimona Mario; Rohde Eva; Goberdhan Deborah Ci; Gorgens Andre; Gorski Sharon M; Xu Jing; Gorski Sharon M; Xu Jing; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gualerzi Alice; Gupta Gopal N; Handberg Aase; Handberg Aase; Haraszti Reka A; Khvorova Anastasia; Harrison Paul; Hochberg Fred H; Nolan John P; Hochberg Fred H; Hoffmann Karl F; Holder Beth; Holder Beth; Holthofer Harry; Hosseinkhani Baharak; Huang Yiyao; Zheng Lei; Huber Veronica; Shahaj Eriomina; Hunt Stuart; Ibrahim Ahmed Gamal-Eldin; Ikezu Tsuneya; Inal Jameel M; Isin Mustafa; Ivanova Alena; Jacobsen Soren; Jacobsen Soren; Jay Steven M; Jayachandran Muthuvel; Jenster Guido; Martens-Uzunova Elena S; Johnson Suzanne M; Jones Jennifer C; Morales-Kastresana Aizea; Welsh Joshua A; Jong Ambrose; Jong Ambrose; Jovanovic-Talisman Tijana; Jung Stephanie; Kalluri Raghu; Kano Shin-Ichi; Kaur Sukhbir; Roberts David D; Kawamura Yumi; Kawamura Yumi; Keller Evan T; Tewari Muneesh; Keller Evan T; Khomyakova Elena; Khomyakova Elena; Kierulf Peter; Kim Kwang Pyo; Kislinger Thomas; Kislinger Thomas; Klingeborn Mikael; Klinke David J 2nd; Klinke David J 2nd; Kornek Miroslaw; Kornek Miroslaw; Kosanovic Maja M; Kramer-Albers Eva-Maria; Krasemann Susanne; Krause Mirja; Kusuma Gina D; Lim Rebecca; Kurochkin Igor V; Kusuma Gina D; Lim Rebecca; Kuypers Soren; Laitinen Saara; Langevin Scott M; Langevin Scott M; Languino Lucia R; Lannigan Joanne; Laurent Louise C; Lazaro-Ibanez Elisa; Shatnyeva Olga; Lee Myung-Shin; Lee Yi Xin Fiona; Lemos Debora S; Lenassi Metka; Leszczynska Aleksandra; Li Isaac Ts; Libregts Sten F; Ligeti Erzsebet; Lorincz Akos M; Lim Sai Kiang; Line Aija; Llorente Alicia; Lorenowicz Magdalena J; Lovett Jason; Myburgh Kathryn H; Lowry Michelle C; O'Driscoll Lorraine; Lu Quan; Lukomska Barbara; Lunavat Taral R; Maas Sybren Ln; Maas Sybren Ln; Malhi Harmeet; Marcilla Antonio; Marcilla Antonio; Mariani Jacopo; Martins Vilma Regina; Maugeri Marco; Nawaz Muhammad; McGinnis Lynda K; McVey Mark J; McVey Mark J; Meckes David G Jr; Meehan Katie L; Mertens Inge; Minciacchi Valentina R; Moller Andreas; Soekmadji Carolina; Moller Jorgensen Malene; Moller Jorgensen Malene; Morhayim Jess; Mullier Francois; Mullier Francois; Muraca Maurizio; Najrana Tanbir; Nazarenko Irina; Nazarenko Irina; Nejsum Peter; Neri Christian; Neri Tommaso; Nimrichter Leonardo; Noren Hooten Nicole; O'Grady Tina; O'Loghlen Ana; Ochiya Takahiro; Olivier Martin; Rak Janusz; Ortiz Alberto; Ortiz Alberto; Ortiz Alberto; Ortiz Luis A; Osteikoetxea Xabier; Ostergaard Ole; Ostergaard Ole; Ostrowski Matias; Pegtel D Michiel; Peinado Hector; Perut Francesca; Phinney Donald G; Pieters Bartijn Ch; Pisetsky David S; Pisetsky David S; Pogge von Strandmann Elke; Polakovicova Iva; Polakovicova Iva; Prada Ilaria; Pulliam Lynn; Raffai Robert L; Pulliam Lynn; Quesenberry Peter; Raffai Robert L; Raimondo Stefania; Rak Janusz; Ramirez Marcel I; Ramirez Marcel I; Raposo Graca; Rayyan Morsi S; Zheutlin Alexander R; Regev-Rudzki Neta; Ricklefs Franz L; Robbins Paul D; Rodrigues Silvia C; Rohde Eva; Rohde Eva; Rome Sophie; Rouschop Kasper Ma; Rughetti Aurelia; Russell Ashley E; Saa Paula; Sahoo Susmita; Salas-Huenuleo Edison; Salas-Huenuleo Edison; Sanchez Catherine; Saugstad Julie A; Saul Meike J; Schiffelers Raymond M; Vader Pieter; Schneider Raphael; Scott Aaron; Sharma Shivani; Sharma Shivani; Sharma Shivani; Shelke Ganesh Vilas; Shetty Ashok K; Shetty Ashok K; Shiba Kiyotaka; Siljander Pia R-M; Siljander Pia R-M; Silva Andreia M; Silva Andreia M; Vasconcelos M Helena; Silva Andreia M; Skowronek Agata; Snyder Orman L 2nd; Soares Rodrigo Pedro; Soekmadji Carolina; Stahl Philip D; Stott Shannon L; Stott Shannon L; Strasser Erwin F; Swift Simon; Tahara Hidetoshi; Tewari Muneesh; Tewari Muneesh; Timms Kate; Tiwari Swasti; Tiwari Swasti; Toh Wei Seong; Tomasini Richard; Torrecilhas Ana Claudia; Tosar Juan Pablo; Tosar Juan Pablo; Toxavidis Vasilis; Urbanelli Lorena; van Balkom Bas Wm; Van Keuren-Jensen Kendall; van Niel Guillaume; Verweij Frederik J; van Royen Martin E; van Wijnen Andre J; Vasconcelos M Helena; Vasconcelos M Helena; Vechetti Ivan J Jr; Veit Tiago D; Vella Laura J; Vella Laura J; Velot Emilie; Vestad Beate; Vestad Beate; Vestad Beate; Wahlgren Jessica; Watson Dionysios C; Watson Dionysios C; Weaver Alissa; Weber Viktoria; Wehman Ann M; Weiss Daniel J; Wendt Sebastian; Wheelock Asa M; Wolfram Joy; Wolfram Joy; Wolfram Joy; Xagorari Angeliki; Xander Patricia; Yan Xiaomei; Yanez-Mo Maria; Yanez-Mo Maria; Yin Hang; Yuana Yuana; Zappulli Valentina; Zarubova Jana; Zarubova Jana; Zarubova Jana; Zekas Vytautas; Zhang Jian-Ye; Zickler Antje M; Zimmermann Pascale; Zimmermann Pascale; Zivkovic Angela M; Zocco Davide
      Journal of extracellular vesicles (2018), 7 (1), 1535750 ISSN:2001-3078.
      The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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    60. 60
      Courtine, G.; Sofroniew, M. V. Spinal Cord Repair: Advances in Biology and Technology. Nat. Med. 2019, 25 (6), 898– 908,  DOI: 10.1038/s41591-019-0475-6
      60
      Spinal cord repair: advances in biology and technology
      Courtine, Gregoire; Sofroniew, Michael V.
      Nature Medicine (New York, NY, United States) (2019), 25 (6), 898-908CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)
      Individuals with spinal cord injury (SCI) can face decades with permanent disabilities. Advances in clin. management have decreased morbidity and improved outcomes, but no randomized clin. trial has demonstrated the efficacy of a repair strategy for improving recovery from SCI. Here, we summarize recent advances in biol. and engineering strategies to augment neuroplasticity and/or functional recovery in animal models of SCI that are pushing toward clin. translation.
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    61. 61
      Skinnider, M. A.; Gautier, M.; Teo, A. Y. Y.; Kathe, C.; Hutson, T. H.; Laskaratos, A. Single-Cell and Spatial Atlases of Spinal Cord Injury in the Tabulae Paralytica. Nature 2024, 631 (8019), 150– 163,  DOI: 10.1038/s41586-024-07504-y
      There is no corresponding record for this reference.
    62. 62
      Lener, T.; Gimona, M.; Aigner, L.; Börger, V.; Buzas, E.; Camussi, G. Applying Extracellular Vesicles Based Therapeutics in Clinical Trials – an ISEV Position Paper. J. Extracell. Vesicles 2015, 4 (1), 30087,  DOI: 10.3402/jev.v4.30087
      62
      Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper
      Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Boerger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A.; del Portillo, Hernando A.; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M.; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Goergens, Andre; Gupta, Ramesh C.; Hendrix, An; Hermann, Dirk M.; Hill, Andrew F.; Hochberg, Fred; Horn, Peter A.; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W.; Kraemer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J.; Lim, Sai Kiang; Loetvall, Jan; Maguire, Casey A.; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G.; Rivera, Francisco J.; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W. M.; Wauben, Marca; El Andaloussi, Samir; Thery, Clotilde; Rohde, Eva; Giebel, Bernd
      Journal of Extracellular Vesicles (2015), 4 (), 30087/1-30087/31CODEN: JEVOA4; ISSN:2001-3078. (Co-Action Publishing)
      Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiol. and pathophysiol. processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumor therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clin. therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufg., quality control and clin. investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clin. practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clin. scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technol. (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufg. and clin. application are highlighted. Prodn. and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clin. studies are addressed.
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    63. 63
      Elvira, K. S. Microfluidic Technologies for Drug Discovery and Development: Friend or Foe?. Trends Pharmacol. Sci. 2021, 42 (7), 518– 526,  DOI: 10.1016/j.tips.2021.04.009
      There is no corresponding record for this reference.
    64. 64
      Gao, X.; Li, S.; Yang, Y.; Yang, S.; Yu, B.; Zhu, Z. A Novel Magnetic Responsive miR-26a@SPIONs-OECs for Spinal Cord Injury: Triggering Neural Regeneration Program and Orienting Axon Guidance in Inhibitory Astrocytic Environment. Adv. Sci. 2023, 10 (32), 2304487  DOI: 10.1002/advs.202304487
      There is no corresponding record for this reference.
    65. 65
      Khan, N. Z.; Cao, T.; He, J.; Ritzel, R. M.; Li, Y.; Henry, R. J. Spinal Cord Injury Alters microRNA and CD81+ Exosome Levels in Plasma Extracellular Nanoparticles with Neuroinflammatory Potential. Brain. Behav. Immun. 2021, 92, 165– 183,  DOI: 10.1016/j.bbi.2020.12.007
      65
      Spinal cord injury alters microRNA and CD81+ exosome levels in plasma extracellular nanoparticles with neuroinflammatory potential
      Khan, Niaz Z.; Cao, Tuoxin; He, Junyun; Ritzel, Rodney M.; Li, Yun; Henry, Rebecca J.; Colson, Courtney; Stoica, Bogdan A.; Faden, Alan I.; Wu, Junfang
      Brain, Behavior, and Immunity (2021), 92 (), 165-183CODEN: BBIMEW; ISSN:0889-1591. (Elsevier Inc.)
      Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiol. of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, tech. limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examd. changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking anal. (NTA), and paralleled an overall redn. in total plasma extracellular nanoparticles. Western blot (WB) anal. of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView. Consistent with WB, we obsd. significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.
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    66. 66
      Tran, N. H.; Qiao, R.; Xin, L.; Chen, X.; Liu, C.; Zhang, X. Deep Learning Enables de Novo Peptide Sequencing from Data-Independent-Acquisition Mass Spectrometry. Nat. Methods 2019, 16 (1), 63– 66,  DOI: 10.1038/s41592-018-0260-3
      66
      Deep learning enables de novo peptide sequencing from data-independent-acquisition mass spectrometry
      Tran, Ngoc Hieu; Qiao, Rui; Xin, Lei; Chen, Xin; Liu, Chuyi; Zhang, Xianglilan; Shan, Baozhen; Ghodsi, Ali; Li, Ming
      Nature Methods (2019), 16 (1), 63-66CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      We present DeepNovo-DIA, a de novo peptide-sequencing method for data-independent acquisition (DIA) mass spectrometry data. We use neural networks to capture precursor and fragment ions across m/z, retention-time, and intensity dimensions. They are then further integrated with peptide sequence patterns to address the problem of highly multiplexed spectra. DIA coupled with de novo sequencing allowed us to identify novel peptides in human antibodies and antigens.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c10298.

    • Detailed experimental procedures including descriptions of primary extraction and culture of BMSCs, ELISA for cytokine quantification, H&E and Nissl staining procedures, two-photon microscopy for EV transport dynamics, RNA sequencing analysis, transmission electron microscopy, molecular mechanism analysis, and flow cytometry for macrophage analysis; comparative proteomic analysis of EVsABPC, EVsNSC, and EVsBMSC (Figure S1); identification of NSCs and analysis of the effects of EVsBMSC and EVsABPC on NSCs proliferation and differentiation (Figure S2); enhancement of neurite outgrowth and reduction of neuronal apoptosis by EVsABPC (Figure S3); bioinformatics analysis and validation of key signaling pathways in neurons treated with EVsABPC (Figure S4); evaluation of inflammatory response and macrophage polarization induced by different EVs treatments (Figure S5); ex vivo distribution of ACPP@ EVs (Figure S6); evaluation of neuronal apoptosis and treatment effects post SCI (Figure S7); ACPP@EVsABPC promoted neural regeneration and axonal remyelination in SCI (Figure S8); safety evaluation of EVsABPC treatment on major organs and physiological parameters (Figure S9); and analysis of the protein interaction between MMP2 and ACPP peptide segments (Table 1) (PDF)


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