To study the biological functions and applications of human amniotic epithelial cell-derived extracellular vesicles (hAEC-EVs), the cargos of hAEC-EVs were analyzed using miRNA sequencing and proteomics analysis. The hAECs and hAEC-EVs in this study had specific characteristics. Multi-omics analyses showed that extracellular matrix (ECM) reorganization, inhibition of excessive myofibroblasts, and promotion of target cell adhesion to the ECM were their primary functions. We evaluated the application of hAEC-EVs for corneal alkali burn healing in rabbits and elucidated the fundamental mechanisms. Slit-lamp images revealed that corneal alkali burns induced central epithelial loss, stromal haze, iris, and pupil obscurity in rabbits. Slit-lamp examination and histological findings indicated that hAEC-EVs facilitated re-epithelialization of the cornea after alkali burns, reduced scar formation and promoted the restoration of corneal tissue transparency. Significantly fewer α-SMA-positive myofibroblasts were observed in the hAEC-EV-treated group than the PBS group. HAEC-EVs effectively promoted the proliferation and migration of hCECs and hCSCs in vitro and activated the focal adhesion signaling pathway. We demonstrated that hAEC-EVs were excellent cell-free candidates for the treatment of ECM lesion-based diseases, including corneal alkali burns. HAEC-EVs promoted ECM reorganization and cell adhesion of target tissues or cells via orderly activation of the focal adhesion signaling pathway.

Background: Systemic sclerosis (SSc) is a disease that features severe fibrosis of the skin and lacks effective therapy. Bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) are potential stem cell-based tools for the treatment of SSc. Methods: BMSCs were isolated from the bone marrow of mice and identified with surface markers according to multilineage differentiation. EVs were isolated from the BMSC culture medium by ultracentrifugation and identified with a Nanosight NS300 particle size analyzer, transmission electron microscopy (TEM), and western blot. The microRNAs (miRNAs) of BMSC-derived EVs (BMSC-EVs) were studied via miRNA sequencing (miRNA-seq) and bioinformatic analysis. An SSc mouse model was established via subcutaneous bleomycin (BLM) injection, and the mice were treated with BMSCs or BMSC-derived EVs. Skin tissues were dissociated and analyzed with H&E staining, RNA sequencing (RNA-seq), western blot, and immunohistochemical staining. Results: Evident pathological changes, like fibrosis and inflammation, were induced in the skin of BLM-treated mice. BMSCs and BMSC-EVs effectively intervened such pathological manifestations and disease processes in a very similar way. The effects of the BMSC-EVs were found to be caused by the miRNAs they carried, which were proven to be involved in regulating the proliferation and differentiation of multiple cell types and in multiple EV-related biological processes. Furthermore, TGF-β1-positive cells and α-SMA-positive myofibroblasts were significantly increased in the scleroderma skin of BLM-treated mice but evidently reduced in the scleroderma skin of the EV-treated SSc group. In addition, the numbers of mast cells and infiltrating macrophages and lymphocytes were evidently increased in the skin of BLM-treated mice but significantly reduced by EV treatment. In line with these observations, there were significantly higher mRNA levels of the inflammatory cytokines Il6, Il10, and Tnf-α in SSc mice than in control mice, but the levels decreased following EV treatment. Through bioinformatics analysis, the TGFβ and WNT signaling pathways were revealed to be closely involved in the pathogenic changes seen in mouse SSc, and these pathways could be therapeutic targets for treating the disease. Conclusions: BMSC-derived EVs could be developed as a potential therapy for treating skin dysfunction in SSc, especially considering that they show similar efficacy to BMSCs but have fewer developmental regulatory requirements than cell therapy. The effects of EVs are generated by the miRNAs they carry, which alleviate SSc pathogenic changes by regulating the WNT and TGFβ signaling pathways.

Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their limited differentiation capacity in vivo attenuates their therapeutic function. Therefore, the differentiation of MSCs into retinal pigment epithelial (RPE) cells in vitro and their subsequent transplantation into the subretinal space is expected to improve the outcome of cell therapy. Here, we transdifferentiated human umbilical cord MSCs (hUCMSCs) into induced RPE (iRPE) cells using a cocktail of five transcription factors (TFs): CRX, NR2E1, C-MYC, LHX2, and SIX6. iRPE cells exhibited RPE specific properties, including phagocytic ability, epithelial polarity, and gene expression profile. In addition, high expression of PTPN13 in iRPE cells endows them with an epithelial-to-mesenchymal transition (EMT)-resistant capacity through dephosphorylating syntenin1, and subsequently promoting the internalization and degradation of transforming growth factor-β receptors. After grafting into the subretinal space of the sodium iodate-induced rat AMD model, iRPE cells demonstrated a better therapeutic function than hUCMSCs. These results suggest that hUCMSC-derived iRPE cells may be promising candidates to reverse AMD pathophysiology.

Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-β. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-β1 stimulation and the PKA pathway. The effect of H89 on the TGF-β-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-β-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

Millions of people are affected with retinal diseases that eventually cause blindness, and retinal progenitor cell (RPC) transplantation is a promising therapeutic avenue. However, RPC expansion and the underlying regulation mechanisms remain elusive. Adult mouse neural RPCs (mNRPCs) were isolated and amplified with the combination of basic fibroblast growth factor (bFGF) and glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021. The progenitor characteristics were evaluated with RT-PCR, immunocytochemistry (ICC), western blot, flow cytometry, and transcriptome analysis prior to transplantation. By treating cells with or without bFGF and CHIR99021 at different time points, the mechanism for mNRPCs’ self-renewal was investigated by transcriptome analysis and western blot assay. mNRPCs were self-renewing in the presence of bFGF and CHIR99021 and showed prominent RPC characteristics. bFGF was essential in promoting cell cycle by facilitating G1/S and G2/M transitions. bFGF combined with CHIR99021 activated the non-canonical Wnt5A/Ca2+ pathway and form a calcium homeostasis. In addition, the self-renewing mNRPCs could differentiate into rod photoreceptor-like cells and retinal pigment epithelium (RPE)-like cells by in vitro induction. When green fluorescent protein (GFP)-labeled cells were transplanted into the subretinal space (SRS) of Pde6b (rd1) mice (also known as RD1 mice, or rodless mice), the cells survived for more than 12 weeks and migrated into the retina. Parts of the recipient retina showed positive expression of photoreceptor marker rhodopsin. Transplanted cells can migrate into the retina, mainly into the inner cell layer (INL) and ganglion cell layer (GCL). Some cells can differentiate into astrocytes and amacrine cells. Cultured mNRPCs did not form tumors after transplanted into NOD/SCID mice for 6 months. Present study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.