isolation-small-extracellular-vesicles-derived-from-mesenchymal-stem

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

Third Military Medical University (Army Medical University)

Key Lab of Visual Damage and Regeneration & Restoration of Chongqing

Jinfeng Laboratory

Army Medical University

The stem cell factor receptor (SCFR) has been demonstrated to be expressed in the neural retina of mice, rat and human for decades. Previous reports indicated that the SCFR correlates with glia differentiation of late retinal progenitor cells (RPCs), retinal vasculogenesis and homeostasis of the blood-retinal barrier. However, the role of SCF/SCFR signaling in the growth and development of the neural retina (NR), especially in the early embryonic stage, remains poorly understood. Here, we show that SCF/SCFR signaling orchestrates invagination of the human embryonic stem cell (hESC)-derived NR via regulation of cell cycle progression, cytoskeleton dynamic and apical constriction of RPCs in the ciliary marginal zone (CMZ). Furthermore, activation of SCF/SCFR signaling promotes neurogenesis in the central-most NR via acceleration of the migration of immature ganglion cells and repressing apoptosis. Our study reveals an unreported role for SCF/SCFR signaling in controlling ciliary marginal cellular behaviors during early morphogenesis and neurogenesis of the human embryonic NR, providing a new potential therapeutic target for human congenital eye diseases such as anophthalmia, microphthalmia and congenital high myopia.

SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina.

Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation  and . RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1β, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia.

Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit/SSEA1 (C-Kit) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment.

Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

Retinal degeneration is a leading cause of irreversible vision impairment and blindness worldwide. Previous studies indicate that subretinal injection of human retinal progenitor cells (hRPCs) can delay the progression of retinal degeneration, preserve retinal function, and protect photoreceptor cells from death, albeit the mechanism is not well understood. In this study, small extracellular vesicles derived from hRPCs (hRPC-sEVs) were injected into the subretinal space of retinal dystrophic RCS rats. We find that hRPC-sEVs significantly preserve the function of retina and thickness of the outer nuclear layer (ONL), reduce the apoptosis of photoreceptors in the ONL, and suppress the inflammatory response in the retina of RCS rats. , we have shown that hRPC-sEV treatment could significantly reserve the low-glucose preconditioned apoptosis of photoreceptors and reduce the expression of pro-inflammatory cytokines in microglia Pathway analysis predicted the target genes of hRPC-sEV microRNAs involved in inflammation related biological processes and significantly enriched in processes autophagy, signal release, regulation of neuron death, and cell cycle. Collectively, our study suggests that hRPC-sEVs might be a favorable agent to delay retinal degeneration and highlights as a new paradigm for cell-free therapy.

Human Retinal Progenitor Cells Derived Small Extracellular Vesicles Delay Retinal Degeneration: A Paradigm for Cell-free Therapy.

Corneal alkali burn remains a clinical challenge in ocular emergency, necessitating the development of effective therapeutic drugs. Here, we observed the arachidonic acid metabolic disorders of corneas induced by alkali burns and aimed to explore the role of Prostaglandin E2 (PGE2), a critical metabolite of arachidonic acid, in the repair of alkali-burned corneas. We found a moderate dosage of PGE2 promoted the alkali-burned corneal epithelial repair, whereas a high dosage of PGE2 exhibited a contrary effect. This divergent effect is attributed to different dosages of PGE2 regulating ANXA1 expression differently. Mechanically, a high dosage of PGE2 induced higher GATA3 expression, followed by enhanced GATA3 binding to the  promoter to inhibit  expression. In contrast, a moderate dosage of PGE2 increased CREB1 phosphorylation and reduced GATA3 binding to the  promoter, promoting  expression. We believe PGE2 and its regulatory target ANXA1 could be potential drugs for alkali-burned corneas.

A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair.

Our previous study demonstrated that combined transplantation of bone marrow-derived mesenchymal stem cells and retinal progenitor cells in rats has therapeutic effects on retinal degeneration that are superior to transplantation of retinal progenitor cells alone. Bone marrow- derived mesenchymal stem cells regulate and interact with various cells in the retinal microenvironment by secreting neurotrophic factors and extracellular vesicles. Small extracellular vesicles derived from bone marrow-derived mesenchymal stem cells, which offer low immunogenicity, minimal tumorigenic risk, and ease of transportation, have been utilized in the treatment of various neurological diseases. These vesicles exhibit various activities, including anti-inflammatory actions, promotion of tissue repair, and immune regulation. Therefore, novel strategies using human retinal progenitor cells combined with BMSC-derived small extracellular vesicles may represent an innovation in stem cell therapy for retinal degeneration. In this study, we developed such an approach utilizing retinal progenitor cells combined with bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles to treat retinal degeneration in Royal College of Surgeons rats, a genetic model of retinal degeneration. Our findings revealed that the combination of bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles and retinal progenitor cells significantly improved visual function in these rats. The addition of bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles as adjuvants to stem cell transplantation with retinal progenitor cells enhanced the survival, migration, and differentiation of the exogenous retinal progenitor cells. Concurrently, these small extracellular vesicles inhibited the activation of regional microglia, promoted the migration of transplanted retinal progenitor cells to the inner nuclear layer of the retina, and facilitated their differentiation into photoreceptors and bipolar cells. These findings suggest that bone marrow-derived mesenchymal stem cell-derived small extracellular vesicles potentiate the therapeutic efficacy of retinal progenitor cells in retinal degeneration by promoting their survival and differentiation.

Mesenchymal stem cell-derived small extracellular vesicles enhance the therapeutic effect of retinal progenitor cells in retinal degenerative disease rats.

Previous studies have reported divergent sexual responses to aging; however, specific variations in gene expression between aging males and females and their potential association with age-related retinal diseases remain unclear. This study collected data from public databases and developed a comprehensive comparison of retina between aging females and males.

Article	Year
SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina. Development (Cambridge, England)\| PubMed ID: 31548215	2019
Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia. Journal of extracellular vesicles\| PubMed ID: 32373289	2020
Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells. Stem cell reports\| PubMed ID: 34214489	2021
Human Retinal Progenitor Cells Derived Small Extracellular Vesicles Delay Retinal Degeneration: A Paradigm for Cell-free Therapy. Frontiers in pharmacology\| PubMed ID: 34912217	2021
A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair. iScience\| PubMed ID: 38144456	2023
Mesenchymal stem cell-derived small extracellular vesicles enhance the therapeutic effect of retinal progenitor cells in retinal degenerative disease rats. Neural regeneration research\| PubMed ID: 39101643	2024
A Comprehensive Analysis of Sex-Biased Gene Expression in the Aging Human Retina Through a Combination of Single-Cell and Bulk RNA Sequencing. Investigative ophthalmology & visual science\| PubMed ID: 39804630	2025

Baishijiao Bian

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