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.

Tissue engineering RPE sheet derived from hiPSC-RPE cell spheroids supplemented with Y-27632 and RepSox.

BACKGROUND: Retinal pigment epithelium (RPE) cell therapy is a promising way to treat many retinal diseases. However, obtaining transplantable RPE cells is time-consuming and less effective. This study aimed to develop novel strategies for generating engineered RPE patches with physiological characteristics. RESULTS: Our findings revealed that RPE cells derived from human induced pluripotent stem cells (hiPSCs) successfully self-assembled into spheroids. The RPE spheroids treated with Y27632 and Repsox had increased expression of epithelial markers and RPE-specific genes, along with improved cell viability and barrier function. Transcriptome analysis indicated enhanced cell adhesion and extracellular matrix (ECM) organization in RPE spheroids. These RPE spheroids could be seeded and bioprinted on collagen vitrigel (CV) membranes to construct engineered RPE sheets. Circular RPE patches, obtained by trephining a specific section of the RPE sheet, exhibited abundant microvilli and pigment particles, as well as reduced proliferative capacity and enhanced maturation. CONCLUSIONS: Our study suggests that the supplementation of small molecules and 3D spheroid culture, as well as the bioprinting technique, can be effective methods to promote RPE cultivation and construct engineered RPE sheets, which may support future clinical RPE cell therapy and the development of RPE models for research applications.

mRNA sequencing reveals the distinct gene expression and biological functions in cardiac fibroblasts regulated by recombinant fibroblast growth factor 2.

After myocardial injury, cardiac fibroblasts (CFs) differentiate into myofibroblasts, which express and secrete extracellular matrix (ECM) components for myocardial repair, but also promote myocardial fibrosis. Recombinant fibroblast growth factor 2 (FGF2) protein drug with low molecular weight can promote cell survival and angiogenesis, and it was found that FGF2 could inhibit the activation of CFs, suggesting FGF2 has great potential in myocardial repair. However, the regulatory role of FGF2 on CFs has not been fully elucidated. Here, we found that recombinant FGF2 significantly suppressed the expression of alpha smooth muscle actin (α-SMA) in CFs. Through RNA sequencing, we analyzed mRNA expression in CFs and the differently expressed genes regulated by FGF2, including 430 up-regulated genes and 391 down-regulated genes. Gene ontology analysis revealed that the differentially expressed genes were strongly enriched in multiple biological functions, including ECM organization, cell adhesion, actin filament organization and axon guidance. The results of gene set enrichment analysis (GSEA) show that ECM organization and actin filament organization are down-regulated, while axon guidance is up-regulated. Further cellular experiments indicate that the regulatory functions of FGF2 are consistent with the findings of the gene enrichment analysis. This study provides valuable insights into the potential therapeutic role of FGF2 in treating cardiac fibrosis and establishes a foundation for further research to uncover the underlying mechanisms of CFs gene expression regulated by FGF2.

Fibroblast growth factor 2 acts as an upstream regulator of inhibition of pulmonary fibroblast activation.

Fibroblast growth factor (FGF) signaling plays a crucial role in lung development and repair. Fibroblast growth factor 2 (FGF2) can inhibit fibrotic gene expression and suppress the differentiation of pulmonary fibroblasts (PFs) into myofibroblasts in vitro, suggesting that FGF2 is a potential target for inhibiting pulmonary fibrosis. To gain deeper insights into the molecular mechanism underlying FGF2-mediated regulation of PFs, we performed mRNA sequencing analysis to systematically and globally uncover the regulated genes and biological functions of FGF2 in PFs. Gene Ontology analysis revealed that the differentially expressed genes regulated by FGF2 were enriched in multiple cellular functions including extracellular matrix (ECM) organization, cytoskeleton formation, ß-catenin-independent Wnt signaling pathway, supramolecular fiber organization, epithelial cell proliferation, and cell adhesion. Gene Set Enrichment Analysis and cellular experiments confirmed that FGF2 can suppress ECM and actin filament organization and increase PFs proliferation. Taken together, these findings indicate that FGF2 acts as an upstream regulator of the inhibition of PFs activation and may play a regulatory role in pulmonary fibrosis.

Establishment of iPS cell line (KLRMMEi003-A) from a patient with Usher syndrome due to USH2A mutation.

We generated an induced pluripotent stem (iPS) cell line by reprogramming peripheral blood mononuclear cells of a patient with Usher syndrome type II carrying USH2A gene mutation (c.8559-2A > G). The iPS cell line with confirmed patient-specific point mutation exhibited typical iPS cell characteristics and maintained a normal karyotype. It can be used as 2D and 3D models to investigate the underlying pathogenic mechanism and lay a solid foundation for future personalized therapy.

Regulation of the Keratocyte Phenotype and Cell Behavior Derived from Human Induced Pluripotent Stem Cells by Substrate Stiffness.

Substrate stiffness has been indicated as an important factor to control stem cell fate, including proliferation and differentiation. To optimize the stiffness for the differentiation process from h-iPSCs (human induced pluripotent stem cells) into h-iCSCs (human corneal stromal cells derived from h-iPSCs) and the phenotypic maintenance of h-iCSCs in vitro, h-iPSCs were cultured on matrigel-coated tissue culture plate (TCP) (106 kPa), matrigel-coated polydimethylsiloxane (PDMS) 184 (1250 kPa), and matrigel-coated PDMS 527 (4 kPa) before they were differentiated to h-iCSCs. Immunofluorescence staining, quantitative real-time polymerase chain reaction (RT-qPCR), and western blot demonstrated that the stiffer substrate TCP promoted the h-iCSCs' differentiation from h-iPSCs. On the contrary, softer PDMS 527 was more effective to maintain the phenotype of h-iCSCs cultured in vitro. Finally, we cultured h-iCSCs on PDMS 527 until P3 and seeded them on a biomimetic collagen membrane to form the single-layer and multiple-layer bioengineered corneal stroma with high transparency properties and cell survival rate. In conclusion, the study is helpful for differentiating h-iPSCs to h-iCSCs and corneal tissue engineering by manipulating stiffness mechanobiology.

Prevalence, Characteristics, and Risk Factors of Retinal Hemorrhage among Full-Term Neonates in Southern China.

Neonatal retinal hemorrhage (RH) is the most common ocular fundus disease among newborns. Early detection and timely intervention are vital for reducing the risk of visual impairment caused by RH. However, little is known about the prevalence, characteristics, and risk factors of RH in southern China. Full-term infants born in Qingyuan City during the first 10 days of each month in 2021 were included in this study. All infants underwent RetCam III retinal examinations. Detailed information on retinal hemorrhage, including involved eyes, bleeding severity, and affected area (extrafoveal macula, fovea, or optic disc), and clinical information on the neonates and their mothers was collected. The results showed that among the 1072 eligible neonates, 266 (24.8%) had neonatal retinal hemorrhage. Consistent bilateral retinal hemorrhage severity was observed in 83.2% of the cases. The prevalence of optic disc involved RH, extrafoveal macular involved RH and foveal involved RH were 23.7%, 81.2% and 2.63%, respectively. Multivariate logistic regression analysis showed that lower birth weight (OR, 0.63; 95% CI, 0.40-0.99; p < 0.05) and vaginal delivery (OR, 20.6; 95% CI, 9.10-46.5; p < 0.001) were risk factors of neonatal RH. The area under the ROC curve of vaginal delivery, combined with birth weight, as predictors of neonatal RH was 0.73, with 85.3% sensitivity and 23.9% specificity. The birth weight cutoff was 3460 g. Our results suggested that neonatal RH is common in full-term neonates in southern China. It usually has the same severity in both eyes and mostly involves the extrafoveal macular region. Vaginal delivery and low birth weight are risk factors for neonatal RH.

LM22B-10 promotes corneal nerve regeneration through in vitro 3D co-culture model and in vivo corneal injury model.

Corneal nerve wounding often causes abnormalities in the cornea and even blindness in severe cases. In this study, we construct a dorsal root ganglion-corneal stromal cell (DRG-CSC, DS) co-culture 3D model to explore the mechanism of corneal nerve regeneration. Firstly, this model consists of DRG collagen grafts sandwiched by orthogonally stacked and orderly arranged CSC-laden plastic compressed collagen. Nerve bundles extend into the entire corneal stroma within 14 days, and they also have orthogonal patterns. This nerve prevents CSCs from apoptosis in the serum withdrawal medium. The conditioned medium (CM) for CSCs in collagen scaffolds contains NT-3, IL-6, and other factors. Among them, NT-3 notably promotes the activation of ERK-CREB in the DRG, leading to the growth of nerve bundles, and IL-6 induces the upregulation of anti-apoptotic genes. Then, LM22B-10, an activator of the NT-3 receptor TrkB/TrkC, can also activate ERK-CREB to enhance nerve growth. After administering LM22B-10 eye drops to regular and diabetic mice with corneal wounding, LM22B-10 significantly improves the healing speed of the corneal epithelium, corneal sensitivity, and corneal nerve density. Overall, the DS co-culture model provides a promising platform and tools for the exploration of corneal physiological and pathological mechanisms, as well as the verification of drug effects in vitro. Meanwhile, we confirm that LM22B-10, as a non-peptide small molecule, has future potential in nerve wound repair. STATEMENT OF SIGNIFICANCE: The cornea accounts for most of the refractive power of the eye. Corneal nerves play an important role in maintaining corneal homeostasis. Once the corneal nerves are damaged, the corneal epithelium and stroma develop lesions. However, the mechanism of the interaction between corneal nerves and corneal cells is still not fully understood. Here, we construct a corneal stroma-nerve co-culture in vitro model and reveal that NT-3 expressed by stromal cells promotes nerve growth by activating the ERK-CREB pathway in nerves. LM22B-10, an activator of NT-3 receptors, can also induce nerve growth in vitro. Moreover, it is used as eye drops to enhance corneal epithelial wound healing, corneal nerve sensitivity and density of nerve plexus in corneal nerve wounding model in vivo.

Establishment of iPS cell line (KLRMMEi002-A) by reprogramming peripheral blood mononuclear cells from a patient with USH2A-associated Usher syndrome.

USH type 2 (USH2) is an autosomal recessive disorder that is characterized by inherited retinopathies and sensorineural hearing loss. USH type 2 (USH2) is frequently caused by USH2A mutations, which account for 74-90% of USH2 cases. We used peripheral blood mononuclear cells (PBMCs) from a USH2 patient with a USH2A gene mutation (c.8559-2A > G) to create an induced pluripotent stem (iPS) cell line. The patient-specific iPS cell line with the specific point mutation exhibited typical iPS cell characteristics, and it can be used as a model to investigate the pathogenic mechanisms underlying USH2A-associated retinal degeneration and sensorineural hearing loss.

Retinal organoids and microfluidic chip-based approaches to explore the retinitis pigmentosa with USH2A mutations.

Retinitis pigmentosa (RP) is a leading cause of vision impairment and blindness worldwide, with limited medical treatment options. USH2A mutations are one of the most common causes of non-syndromic RP. In this study, we developed retinal organoids (ROs) and retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs) of RP patient to establish a sustainable in vitro RP disease model. RT-qPCR, western blot, and immunofluorescent staining assessments showed that USH2A mutations induced apoptosis of iPSCs and ROs, and deficiency of the extracellular matrix (ECM) components. Transcriptomics and proteomics findings suggested that abnormal ECM-receptor interactions could result in apoptosis of ROs with USH2A mutations via the PI3K-Akt pathway. To optimize the culture conditions of ROs, we fabricated a microfluidic chip to co-culture the ROs with RPE cells. Our results showed that this perfusion system could efficiently improve the survival rate of ROs. Further, ECM components such as laminin and collagen IV of ROs in the RP group were upregulated compared with those maintained in static culture. These findings illustrate the potential of microfluidic chip combined with ROs technology in disease modelling for RP.

Mechanotransduction Regulates Reprogramming Enhancement in Adherent 3D Keratocyte Cultures.

Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFß/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture.

In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion (DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates (T-DRG) versus three-dimensional collagen hydrogels (C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis- and myelination-related genes were highly expressed in C-DRG, while epithelial-mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B (S100B) and lower α-smooth muscle actin (α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China (approval No. 2020-IRB16), on March 15, 2020.

Protective effects of human iPS-derived retinal pigmented epithelial cells on retinal degenerative disease.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited retinal disease characterized by progressive loss of photoreceptor cells. This study aim at exploring the effect of retinal pigment epithelium (RPE) derived from human-induced pluripotent stem cell (hiPSC-RPE) on the retina of retinal degeneration 10 (rd10) mice, which are characterized with progressive photoreceptor death. METHODS: We generated RPE from hiPSCs by sequential supplementation with retinal-inducing factors and RPE specification signaling factors. The three-dimensional (3D) spheroid culture method was used to obtain optimal injectable hiPSC-RPE cells. Subretinal space transplantation was conducted to deliver hiPSC-RPE cells into the retina of rd10 mice. Neurotrophic factor secretion from transplanted hiPSC-RPE cells was detected by enzyme-linked immunosorbent assay (ELISA). Immunostaining, Western blotting, electroretinography (ERG), and visual behavior testing were performed to determine the effects of hiPSC-RPE on the retinal visual function in rd10 mice. RESULTS: Our data demonstrated that hiPSC-RPE cells exhibited classic RPE properties and phenotype after the sequential RPE induction from hiPSCs. hiPSC-RPE cells co-cultured with mouse retinal explants or retinal ganglion cells 5 (RGC5) exhibited decreased apoptosis. The viability and functional properties of hiPSC-RPE cells were enhanced by 3D spheroid culture. Transplanted hiPSC-derived RPE cells were identified by immunostaining with human nuclear antigen staining in the retina of rd10 14 days after subretinal space injection. The pigment epithelium-derived factor level was increased significantly. The expression of CD68, microglial activation marker, reduced after transplantation. The light avoidance behavior and ERG visual function in rd10 mice improved by the transplantation of hiPSC-RPE cells. CONCLUSION: Our findings suggest that injectable hiPSC-RPE cells after 3D spheroid culture can rescue the structure and function of photoreceptors by sub-retinal transplantation, which lay the foundation for future clinical cell therapy to treat RP and other retinal degeneration diseases.

Muse cell spheroids have therapeutic effect on corneal scarring wound in mice and tree shrews.

Stem cell therapy holds promises for treating corneal scarring. Here, we use multilineage-differentiating stress-enduring (Muse) cells to study their differentiation and therapeutic potential for treating corneal injury. Muse cells were isolated from lipoaspirate, which presented biphenotype properties of both pluripotent stem cells and some mesenchymal stem cells. Muse cells expanded by about 100-fold from the initial seeding cell number to Muse spheroids with the maintenance of the Muse cell phenotype and high cell viability at 33 days by static spheroid culture. We revealed that Muse spheroids were activated by the dynamic rotary cell culture system (RCCS), as characterized by increased stemness, improved activity, and enhanced adherence. Gene and protein expression of the pluripotent markers OCT3/4, SOX2, and NANOG and of the proliferation marker KI67 in Muse spheroids cultured under RCCS were higher than those in the static group. These activated Muse spheroids enabled ready differentiation into corneal stromal cells (CSCs) expressing characteristic marker genes and proteins. Furthermore, implantation of Muse cells-differentiated CSCs (Muse-CSCs) laden assembled with two orthogonally stacked stretched compressed collagen (cell-SCC) in mouse and tree shrew wounded corneas prevented the formation of corneal scarring, increased corneal re-epithelialization and nerve regrowth, and reduced the severity of corneal inflammation and neovascularization. cell-SCC retained the capacity to suppress corneal scarring after long-distance cryopreserved transport. Thus, Muse cell therapy is a promising avenue for developing therapeutics for treating corneal scarring.

Establishment of a human induced pluripotent stem cell line (CSUASOi005-A), from peripheral blood mononuclear cells of a patient with X-linked juvenile retinoschisis carrying a novel mutation in RS1 gene.

X-linked retinoschisis (XLRS) is a one of most common retinal genetic diseases of juvenile progressive vitreoretinal degeneration in males, which caused by the mutation of RS1 gene. In this study, an induced pluripotent stem cell (iPSC) line was generated from human peripheral blood mononuclear cells (PBMC) of a 13-year-old male patient with X-linked juvenile retinoschisis carrying a novel mutation in RS1 gene. The iPSCs exhibited iPSC morphology, expression of the pluripotency markers and in vitro differentiation potential, and the CSUASOi005-A iPSC line retained the original mutation (c.527T > A) of RS1 with a normal karyotype.

Transcriptomic Analysis of the Developmental Similarities and Differences Between the Native Retina and Retinal Organoids.

Purpose: We performed a bioinformatic transcriptome analysis to determine the alteration of gene expression between the native retina and retinal organoids in both mice and humans. Methods: The datasets of mouse native retina (GSE101986), mouse retinal organoids (GSE102794), human native retina (GSE104827), and human retinal organoids (GSE119320) were obtained from Gene Expression Omnibus. After normalization, a principal component analysis was performed to categorize the samples. The genes were clustered to classify them. A functional analysis was performed using the bioinformatics tool Gene ontology enrichment to analyze the biological processes of selected genes and cellular components. Results: The development of retinal organoids is slower than that in the native retina. In the early stage, cell proliferation predominates. Subsequently, neural differentiation is dominant. In the later stage, the dominant differentiated cells are photoreceptors. Additionally, the fatty acid metabolic process and mitochondria-related genes are upregulated over time, and the glycogen catabolic process and activin receptors are gradually downregulated in human retinal organoids. However, these trends are opposite in mouse retinal organoids. There are two peaks in mitochondria-related genes, one in the early development period and another during the photoreceptor development period. It takes about five times longer for human retinal development to achieve similar levels of mouse retinal development. Conclusions: Our study reveals the similarities and differences in the developmental features of retinal organoids as well as the corresponding relationship between mouse and human retinal development.

Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells.

Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.

Establishment of a non-integrate iPS cell line CSUASOi002-A, from urine-derived cells of a female patient with macular corneal dystrophy carrying compound heterozygous CHST6 mutations.

We report the human induced pluripotent stem cell line (iPSC) CSUASOi002-A, generated from urine-derived cells (UCs) from a 51-year-old female patient carrying compound heterozygous mutations (c.62_63delTinsGA and c.C892T) in the carbohydrate sulfotransferase 6 gene (CHST6). This patient was from a Chinese family of three siblings with macular corneal dystrophy (MCD). Patient UCs were reprogrammed by electroporation using the episomal plasmids (OCT4, SOX2, KLF4, l-MYC, LIN28 and shP53). The human MCD-UiPS cell line CSUASOi002-A retained the disease-associated genotype, while expressed pluripotent stem cell markers and could be differentiated into cells of all three germ layers.