Cord blood-derived biologics lead to robust axonal regeneration in benzalkonium chloride-injured mouse corneas by modulating the Il-17 pathway and neuropeptide Y.

BACKGROUND: Umbilical cord blood-derived therapeutics, such as serum (UCS) and platelet-rich plasma (UCPRP), are popular treatment options in clinical trials and can potentially be utilized to address a clinically unmet need caused by preservatives, specifically benzalkonium chloride (BAK), present in ophthalmic formulations. As current clinical interventions for secondary injuries caused by BAK are suboptimal, this study will explore the feasibility of utilizing UCS and UCPRP for cornea treatment and investigate the underlying mechanisms associated with this approach. METHODS: Mice's corneas were administered BAK to induce damage. UCS and UCPRP were then utilized to attempt to treat the injuries. Ocular tests were performed on the animals to evaluate recovery, while immunostaining, RNA-seq, and subsequent bioinformatics analysis were conducted to investigate the treatment mechanism. RESULTS: BAK administration led to widespread inflammatory responses in the cornea. Subsequent treatment with UCS and UCPRP led to the downregulation of immune-related 'interactions between cytokine receptors' and 'IL-17 signaling' pathways. Although axonal enhancers such as Ngf, Rac2, Robo2, Srgap1, and Rock2 were found to be present in the injured group, robust axonal regeneration was observed only in the UCS and UCPRP treatment groups. Further analysis revealed that, as compared to normal corneas, inflammation was not restored to pre-injury levels post-treatment. Importantly, Neuropeptide Y (Npy) was also involved in regulating immune responses, indicating neuroimmune axis interactions. CONCLUSIONS: Cord blood-derived therapeutics are feasible options for overcoming the sustained injuries induced by BAK in the cornea. They also have potential applications in areas where axonal regeneration is required.

Cell spray printing combined with Lycium barbarum glycopeptide promotes repair of corneal epithelial injury.

The corneal epithelium, located as the outermost layer of the cornea, is inherently susceptible to injuries that may lead to corneal opacities and compromise visual acuity. Rapid restoration of corneal epithelial injury is crucial for maintaining the transparency and integrity of the cornea. Cell spray treatment emerges as an innovative and effective approach in the field of regenerative medicine. In our study, a cell spray printing platform was established, and the optimal printing parameters were determined to be a printing air pressure of 5 PSI (34.47 kPa) and a liquid flow rate of 30 ml/h. Under these conditions, the viability and phenotype of spray-printed corneal epithelial cells were preserved. Moreover, Lycium barbarum glycopeptide (LBGP), a glycoprotein purified from wolfberry, enhanced proliferation while simultaneously inhibiting apoptosis of the spray-printed corneal epithelial cells. We found that the combination of cell spray printing and LBGP facilitated the rapid construction of multilayered cell sheets on flat and curved collagen membranes in vitro. Furthermore, the combined cell spray printing and LBGP accelerated the recovery of the rat corneal epithelium in the mechanical injury model. Our findings offer a therapeutic avenue for addressing corneal epithelial injuries and regeneration.

Sodium butyrate ameliorates diabetic retinopathy in mice via the regulation of gut microbiota and related short-chain fatty acids.

BACKGROUND: Diabetic retinopathy (DR) development is associated with disturbances in the gut microbiota and related metabolites. Butyric acid is one of the short-chain fatty acids (SCFAs), which has been found to possess a potential antidiabetic effect. However, whether butyrate has a role in DR remains elusive. This study aimed to investigate the effect and mechanism of sodium butyrate supplementation on DR. METHODS: C57BL/6J mice were divided into three groups: Control group, diabetic group, and diabetic with butyrate supplementation group. Type 1 diabetic mouse model was induced by streptozotocin. Sodium butyrate was administered by gavage to the experimental group daily for 12 weeks. Optic coherence tomography, hematoxylin-eosin, and immunostaining of whole-mount retina were used to value the changes in retinal structure. Electroretinography was performed to assess the retinal visual function. The tight junction proteins in intestinal tissue were evaluated using immunohistochemistry. 16S rRNA sequencing and LC-MS/MS were performed to determine the alteration and correlation of the gut microbiota and systemic SCFAs. RESULTS: Butyrate decreased blood glucose, food, and water consumption. Meanwhile, it alleviated retinal thinning and activated microglial cells but improved electroretinography visual function. Additionally, butyrate effectively enhanced the expression of ZO-1 and Occludin proteins in the small intestine. Crucially, only butyric acid, 4-methylvaleric acid, and caproic acid were significantly decreased in the plasma of diabetic mice and improved after butyrate supplementation. The deeper correlation analysis revealed nine genera strongly positively or negatively correlated with the above three SCFAs. Of note, all three positively correlated genera, including norank_f_Muribaculaceae, Ileibacterium, and Dubosiella, were significantly decreased in the diabetic mice with or without butyrate treatment. Interestingly, among the six negatively correlated genera, Escherichia-Shigella and Enterococcus were increased, while Lactobacillus, Bifidobacterium, Lachnospiraceae_NK4A136_group, and unclassified_f_Lachnospiraceae were decreased after butyrate supplementation. CONCLUSION: Together, these findings demonstrate the microbiota regulating and diabetic therapeutic effects of butyrate, which can be used as a potential food supplement alternative to DR medicine.

Probucol attenuates high glucose-induced Müller cell damage through enhancing the Nrf2/p62 signaling pathway.

PURPOSE: This study investigated the protective effect of probucol on Müller cells exposed to high glucose conditions and examined potential mechanisms of action. METHODS: Primary human retinal Müller cells were incubated with high glucose (HG, 35 mM) in the present or absence of different concentrations of probucol for 24 h. Cell viability was determined using the CCK-8 method. Mitochondrial membrane potential (MMP) was measured using JC-1 staining and cell cycle by flow cytometry. The expression of nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit, and p62 was quantified using quantitative polymerase chain reaction and western blot. RESULTS: We found that HG inhibited cell proliferation, arrested cell cycle, and increased MMP in human Müller cells. Probucol activated the Nrf2/p62 pathway and upregulated the anti-apoptotic protein, Bcl2, and attenuated HG-mediated damage in Müller cells. CONCLUSIONS: Our results suggest that probucol may protect Müller cells from HG-induced damage through enhancing the Nrf2/p62 signaling pathway.

COL10A1 is a novel factor in the development of choroidal neovascularization.

With the dramatic rise in the aging population, researching age-related macular degeneration (AMD), especially the severe form neovascular AMD (nAMD), has become more important than ever. In this study, we found that collagen type X was increased in retina-choroid tissue of mice with laser-induced choroidal neovascularization (CNV) based on immunohistofluorescence. RNA sequencing and bioinformatic analyses were performed to compare the retina-choroid tissue complex of the CNV mouse model to normal controls. Collagen type X alpha 1 chain (Col10a1) was among the most significantly upregulated genes, and the results were validated with an animal model at the mRNA and protein levels by quantitative real-time polymerase chain reaction (qPCR) and western blotting, respectively. COL10A1 was also upregulated in human retinal microvascular endothelial cells (HRMECs), human umbilical vein endothelial cells (HUVECs), RPE19 cells and RF/6A cells under hypoxic conditions. Next, in vitro and in vivo experiments were performed to study the effect of COL10A1 on neovascularization. siRNA knockdown of COL10A1 suppressed the proliferation and tube formation ability of HRMECs under hypoxic conditions. Snail family transcriptional repressor 1 (SNAIL1) and angiopoietin-2 (ANGPT2) were downregulated in COL10A1 knockdown HRMECs under hypoxic conditions and thus were potential downstream genes. Significant decreases in CNV leakage and CNV lesion area, as assessed by fundus fluorescein angiography (FFA) and immunofluorescence of choroidal flat mounts, respectively, were observed in a mouse model intravitreally injected with anti-collagen X monoclonal antibody (mAb) compared to the controls. In conclusion, COL10A1 promotes CNV formation and may represent a new candidate target for the treatment and diagnosis of nAMD and other neovascular diseases.

Suppression of EZH2 inhibits TGF-ß1-induced EMT in human retinal pigment epithelial cells.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is critically involved in the occurrence of subretinal fibrosis. This study aimed to investigate the role of enhancer of zeste homolog 2 (EZH2) in EMT of human primary RPE cells and the underlying mechanisms of the anti-fibrotic effect of EZH2 suppression. Primary cultures of human RPE cells were treated with TGF-ß1 for EMT induction. EZH2 was silenced by siRNA to assess the expression levels of epithelial and fibrotic markers using qRT-PCR, Western blot, and immunofluorescence staining assay. Furthermore, the cellular migration, proliferation and barrier function of RPE cells were evaluated. RNA-sequencing analyses were performed to investigate the underlying mechanisms of EZH2 inhibition. Herein, EZH2 silencing up-regulated epithelial marker ZO-1 and downregulated fibrotic ones including α-SMA, fibronectin, and collagen 1, alleviating EMT induced by TGF-ß1 in RPE cells. Moreover, silencing EZH2 inhibited cellular migration and proliferation, but didn't affect cell apoptosis. Additionally, EZH2 suppression contributed to improved barrier functions after TGF-ß1 stimulation. The results from RNA sequencing suggested that the anti-fibrotic effect of EZH2 inhibition was associated with the MAPK signaling pathway, cytokine-cytokine receptor interaction, and the TGF-beta signaling pathway. Our findings provide evidence that the suppression of EZH2 might reverse EMT and maintain the functions of RPE cells. EZH2 could be a potential therapeutic avenue for subretinal fibrosis.

Tissue-Specific Gamma-Flicker Light Noninvasively Ameliorates Retinal Aging.

Aging is a risk factor for multiple retinal degeneration diseases. Entraining brain gamma oscillations with gamma-flicker light (γFL) has been confirmed to coordinate pathological changes in several Alzheimer's disease mouse models and aged mice. However, the direct effect of γFL on retinal aging remains unknown. We assessed retinal senescence-associated beta-galactosidase (ß-gal) and autofluorescence in 20-month-old mice and found reduced ß-gal-positive cells in the inner retina and diminished lipofuscin accumulation around retinal vessels after 6 days of γFL. In immunofluorescence, γFL was further demonstrated to ameliorate aging-related retinal changes, including a decline in microtubule-associated protein 1 light chain 3 beta expression, an increase in complement C3 activity, and an imbalance between the anti-oxidant factor catalase and pro-oxidant factor carboxymethyl lysine. Moreover, we found that γFL can increase the expression of activating transcription factor 4 (ATF4) in the inner retina, while revealing a decrease of ATF4 expression in the inner retina and positive expression in the outer segment of photoreceptor and RPE layer for aged mice. Western blotting was then used to confirm the immunofluorescence results. After mRNA sequencing (NCBI Sequence Read Archive database: PRJNA748184), we found several main mechanistic clues, including mitochondrial function and chaperone-mediated protein folding. Furthermore, we extended γFL to aged Apoe-/- mice and showed that 1-m γFL treatment even improved the structures of retinal-pigment-epithelium basal infolding and Bruch's membrane. Overall, γFL can orchestrate various pathological characteristics of retinal aging in mice and might be a noninvasive, convenient, and tissue-specific therapeutic strategy for retinal aging.

Aberrant Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem Cells of a Retinitis Pigmentosa Patient with the PRPF6 Mutation.

Pre-mRNA processing factors (PRPFs) are vital components of the spliceosome and are involved in the physiological process necessary for pre-mRNA splicing to mature mRNA. As an important member, PRPF6 mutation resulting in autosomal dominant retinitis pigmentosa (adRP) is not common. Recently, we reported the establishment of an induced pluripotent stem cells (iPSCs; CSUASOi004-A) model by reprogramming the peripheral blood mononuclear cells of a PRPF6-related adRP patient, which could recapitulate a consistent disease-specific genotype. In this study, a disease model of retinal pigment epithelial (RPE) cells was generated from the iPSCs of this patient to further investigate the underlying molecular and pathological mechanisms. The results showed the irregular morphology, disorganized apical microvilli and reduced expressions of RPE-specific genes in the patient's iPSC-derived RPE cells. In addition, RPE cells carrying the PRPF6 mutation displayed a decrease in the phagocytosis of fluorescein isothiocyanate-labeled photoreceptor outer segments and exhibited impaired cell polarity and barrier function. This study will benefit the understanding of PRPF6-related RPE cells and future cell therapy.

Dry eye syndrome: comprehensive etiologies and recent clinical trials.

Dry eye syndrome (DES) is multifactorial and likely to be a cause of concern more so than ever given the rapid pace of modernization, which is directly associated with many of the extrinsic causative factors. Additionally, recent studies have also postulated novel etiologies that may provide the basis for alternative treatment methods clinically. Such insights are especially important given that current approaches to tackle DES remains suboptimal. This review will primarily cover a comprehensive list of causes that lead to DES, summarize all the upcoming and ongoing clinical trials that focuses on treating this disease as well as discuss future potential treatments that can improve inclusivity.

Effect of porcine corneal stromal extract on keratocytes from SMILE-derived lenticules.

Propagating large amounts of human corneal stromal cells (hCSCs) in vitro while maintaining the physiological quality of their phenotypes is necessary for their application in cell therapy. Here, a novel medium to propagate hCSCs obtained from small incision lenticule extraction (SMILE)-derived lenticules was investigated and the feasibility of intrastromal injection of these hCSCs was assessed. Primary hCSCs were cultured in porcine corneal stroma extract (pCSE) with RIFA medium including ROCK inhibitor Y27632, insulin-transferrin-selenium, fibroblast growth factor 2, L-ascorbate 2-phosphate and 0.5% FBS (RIFA medium + pCSE). Protein profiling of the pCSE was identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). After subculturing in RIFA medium + pCSE or 10% FBS normal medium (NM), hCSCs at P4 were transplanted into mouse corneal stroma. Compared with NM, ALDH3A1, keratocan and lumican were significantly more expressed in the RIFA medium + pCSE. ALDH3A1 was also more expressed in the RIFA medium + pCSE than in the RIFA medium. Fibronectin and α-SMA were less expressed in the RIFA medium + pCSE than in the NM. Using Metascape analysis, the pCSE with its anti-fibrosis, pro-proliferation and anti-apoptosis activities, was beneficial for hCSC cultivation. The intrastromally implanted hCSCs in the RIFA medium + pCSE had positive CD34 expression but negative CD45 expression 35 days after injection. We provide a valuable new medium that is advantageous for the proliferation of hCSCs with the properties of physiological keratocytes. Intrastromal injection of hCSCs in RIFA medium + pCSE has the potential for clinical cell therapy.

The daily gene transcription cycle in mouse retina.

Many physiological retinal processes, such as outer segment disk shedding and visual sensitivity, exhibit a daily rhythm. However, the detailed transcriptome dynamics and related biological processes of the retina are not fully understood. Retinal tissues were collected from C57BL/6J male mice housed in a 12h light/12h dark (LD) cycle for 4 weeks, at Zeitgeber time (ZT) 0, 4, 8, 12, 16, and 20. Total RNA was extracted from the tissues and used for unique identifier RNA sequencing experiments. The rhythmicity of gene expression was determined using the MetaCycle R package. We found that 1741 genes (10.26%) were rhythmically expressed in the retina. According to the expression patterns, the rhythmically expressed genes were assigned to four clusters, each with about 361-492 genes, using the Mfuzz R package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were conducted to identify pathways and biological processes of the profiled genes. Genes in Clusters 1 and 4 were associated with glycolysis and energy production, showed higher activity at night (from ZT16 to ZT20), and were enriched in the Hif-1α signaling pathway and low-oxygen-related terms. Genes in Cluster 2 were predominantly involved in cilium assembly and organization and were relatively upregulated during the day. Genes in Cluster 3 were associated with ribosome biosynthesis and were highly expressed during the day-night transition period. Taken together, these results demonstrate that a large proportion of retinal genes are expressed rhythmically. Genes involved in energy production and glycolysis are highly expressed at night, leading to relative hypoxia and activation of the Hif-1α signaling pathway. Genes associated with the formation of photoreceptor cilia are expressed during the day.

Inhibition of enhancer of zeste homolog 2 prevents corneal myofibroblast transformation in vitro.

PURPOSE: Corneal fibroblast can be transformed into corneal myofibroblasts by TGF-ß1. Enhancer of zeste homolog 2 (EZH2) upregulation has been observed in the occurrence of other fibrotic disorders. We investigated the role of EZH2 in the progression of corneal fibrosis and the antifibrotic effect of EZH2 inhibition in corneal fibroblasts (CFs). METHODS: Primary CFs were isolated from corneal limbi and the CFs were treated with TGF-ß1 to induce fibrosis. EPZ-6438 and EZH2 siRNA were used to inhibit EZH2 expression. Myofibroblast activation and extracellular matrix (ECM) protein synthesis was detected by quantitative real-time PCR, western blotting, and immunofluorescence staining assay. The functions of myofibroblast were evaluated by cell migration and collagen gel contraction assays. Molecular mechanisms involved in EZH2 inhibition were investigated by RNA sequencing. RESULTS: TGF-ß1 activated EZH2 expression in CFs. Treatment with EPZ-6438 (5 µM) and EZH2 siRNA considerably suppressed corneal myofibroblast activation and ECM protein synthesis in CFs induced by TGF-ß1 when compared to the control group. EPZ-6438 (5 µM) suppressed cell migration and gel contraction in CFs. RNA sequencing results revealed that antifibrotic genes were activated after EZH2 inhibition to suppress corneal myofibroblast activation. CONCLUSION: Inhibition of EZH2 suppresses corneal myofibroblast activation and ECM protein synthesis, and could serve as a novel therapeutic target for preventing corneal scarring.

WRN inhibits oxidative stress-induced apoptosis of human lensepithelial cells through ATM/p53 signaling pathway and its expression is downregulated by DNA methylation.

BACKGROUND: Apoptosis and oxidative stress are the main etiology of age related cataract (ARC). This article aims to investigate the role of WRN in lens epithelial cells (LECs). METHODS: We estimated the methylation level of WRN in anterior lens capsule tissues of ARC patients. SRA01/04 (LECs) cells were treated with H2O2 or combined with 5-aza-2-deoxycytidine (5-Aza-CdR) or chloroquine. CCK8 and flow cytometry were performed to explore proliferation and apoptosis. The content of ROS was detected by fluorescent probe DCFH-DA. The gene and protein expression was assessed by quantitative real-time PCR or western blot. RESULTS: WRN was down-regulated and the methylation level of WRN was increased in the anterior lens capsule tissues. WRN overexpression and 5-Aza-CdR enhanced proliferation and repressed apoptosis and oxidative stress of SRA01/04 cells. 5-Aza-CdR enhanced WRN expression. WRN knockdown inhibited proliferation and promoted apoptosis and oxidative stress of SRA01/04 cells, which was rescued by 5-Aza-CdR. WRN overexpression and 5-Aza-CdR repressed ATM/p53 signaling pathway. Furthermore, chloroquine inhibited proliferation and promoted apoptosis and oxidative stress of SRA01/04 cells by activating ATM/p53 signaling pathway. The influence conferred by chloroquine was abolished by WRN overexpression. CONCLUSION: Our study reveals that DNA methylation mediated WRN inhibits apoptosis and oxidative stress of human LECs through ATM/p53 signaling pathway.

Local Group 2 Innate Lymphoid Cells Promote Corneal Regeneration after Epithelial Abrasion.

Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.

PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. However, little is known about its presence or effects in human adipose-derived stem cells (hADSCs). In this study, the expression of PACAP type I receptor (PAC1R) was first confirmed in hADSCs. Maxadilan, a specific agonist of PAC1R, could increase hADSC proliferation as determined by Cell Counting Kit-8 and cell cycle analysis and promote migration as shown in wound-healing assays. Maxadilan also showed anti-apoptotic activity in hADSCs against serum withdrawal-induced apoptosis based on Annexin V/propidium iodide analysis and mitochondrial membrane potential assays. The anti-apoptotic effects of maxadilan correlated with the down-regulation of Cleaved Caspase 3 and Caspase 9 as well as up-regulation of Bcl-2. The chemical neural differentiation potential could be enhanced by maxadilan as indicated through quantitative PCR, Western blot and cell morphology analysis. Moreover, cytokine neural redifferentiation of hADSCs treated with maxadilan acquired stronger neuron-like functions with higher voltage-dependent tetrodotoxin-sensitive sodium currents, higher outward potassium currents and partial electrical impulses as determined using whole-cell patch clamp recordings. Maxadilan up-regulated the Wnt/ß-catenin signalling pathway associated with dimer-dependent activity of PAC1R, promoting cell viability that was inhibited by XAV939, and it also activated the protein kinase A (PKA) signalling pathway associated with ligand-dependent activity of PAC1R, enhancing cell viability and neural differentiation potential that was inhibited by H-89. In summary, these results demonstrated that PAC1R is present in hADSCs, and maxadilan could enhance hADSC viability and neural differentiation potential in neural differentiation medium.

Matrigel and Activin A promote cell-cell contact and anti-apoptotic activity in cultured human retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured in vitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (â–³Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured in vitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased â–³Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of ß-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, ß-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/ß-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/ß-catenin pathway. This study will benefit the understanding of hRPE cells and future cell therapy.

Effects of induced pluripotent stem cells-derived conditioned medium on the proliferation and anti-apoptosis of human adipose-derived stem cells.

Human adipose-derived stem cells (hASCs) become an appealing source for regenerative medicine. However, with the multi-passage or cryopreservation for large-scale growth procedures in terms of preclinical and clinical purposes, hASCs often reveal defective cell viability, which is a major obstacle for cell therapy. In our study, the effects of induced pluripotent stem cells-derived conditioned medium (iPS-CM) on the proliferation and anti-apoptosis in hASCs were investigated. hASCs at passage 1 were identified by the analysis of typical surface antigens with flow cytometry assay and adipogenic and osteogenic differentiation. The effect of iPS-CM on the proliferation in hASCs was analyzed by cell cycle assay and Ki67/P27 quantitative polymerase chain reaction analysis. The effect of iPS-CM on the anti-apoptosis of hASCs irradiated by 468 J/m(2) of ultraviolet C was investigated by annexin v/propidium iodide analysis, mitochondrial membrane potential assay, intracellular reactive oxygen species assay, Western blotting and caspase activity assays. The effect of iPS-CM on the surface antigen expressions of hASCs was analyzed using flow cytometry assay. The levels of Activin A and bFGF in culture supernatant of hASCs with different treatments were also detected by enzyme-linked immunosorbent assay. iPS-CM promoted proliferation and inhibited apoptosis of hASCs. This discovery demonstrates that iPS-CM might be used as one of the available means to overcome the propagation obstacle for hASCs and make for large-scale growth procedures in terms of preclinical and clinical purposes.

Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids.

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.

Retinal organoids with X-linked retinoschisis RS1 (E72K) mutation exhibit a photoreceptor developmental delay and are rescued by gene augmentation therapy.

BACKGROUND: X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. METHODS: hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. RESULTS: ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. CONCLUSIONS: The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.