Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.
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ABSTRACT:
SUBMITTER: Ali M
PROVIDER: S-EPMC8452521 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in mammals (rabbits) and non-human primates (monkeys). We injected cryopreserved hESC-derived CECs in rabbits and monkeys either immediately after removing 8 mm of the central portion of the CE or a few days later when corneal edema developed. All clinical models developed deturgesced and clear corneas 2-3 weeks following the CEC injection and remained comparable to the CE of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues examined for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet’s membrane.
2021-09-29 | GSE173692 | GEO
Project description:Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty
| PRJNA726726 | ENA
Project description:Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.
| S-EPMC9821383 | biostudies-literature
Project description:Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic approaches. A stem cell-based regenerative medicine approach using induced pluripotent stem cells (iPSCs) offers a promising solution, as they possess self-renewal capabilities, can be derived from adult somatic cells, and can be differentiated into all cell types including corneal endothelial cells (CECs). This review discusses the progress and challenges in developing protocols to induce iPSCs into CECs, focusing on the different media formulations used to differentiate iPSCs to neural crest cells (NCCs) and subsequently to CECs, as well as the characterization methods and markers that define iPSC-derived CECs. The hurdles and solutions for the clinical application of iPSC-derived cell therapy are also addressed, including the establishment of protocols that adhere to good manufacturing practice (GMP) guidelines. The potential risks of genetic mutations in iPSC-derived CECs associated with long-term in vitro culture and the danger of potential tumorigenicity following transplantation are evaluated. In all, this review provides insights into the advancement and obstacles of using iPSC in the treatment of corneal endothelial dysfunction.
| S-EPMC10418878 | biostudies-literature
Project description:Vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. Human genotype-phenotype studies of endothelium are limited by the unavailability of patient-specific endothelial cells. To establish a cellular platform for studying endothelial biology, we have generated vascular endothelium from human induced pluripotent stem cells (iPSCs) exhibiting the rich functional phenotypic plasticity of mature primary vascular endothelium. These endothelial cells respond to diverse proinflammatory stimuli, adopting an activated phenotype including leukocyte adhesion molecule expression, cytokine production, and support for leukocyte transmigration. They maintain dynamic barrier properties responsive to multiple vascular permeability factors. Importantly, biomechanical or pharmacological stimuli can induce pathophysiologically relevant atheroprotective or atheroprone phenotypes. Our results demonstrate that iPSC-derived endothelium possesses a repertoire of functional phenotypic plasticity and is amenable to cell-based assays probing endothelial contributions to inflammatory and cardiovascular diseases.
| S-EPMC3757754 | biostudies-literature
Project description:BackgroundHuman corneal endothelial cells lack regenerative capacity through cell division in vivo. Consequently, in the case of trauma or dystrophy, the only available treatment modality is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor which faces a high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink.MethodsCorneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas.ResultsThe bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10 days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer.ConclusionsOur results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.
| S-EPMC10941625 | biostudies-literature
Project description:Purpose:To establish Myh11 as a marker of a subset of corneal endothelial cells (CECs), and to demonstrate the feasibility of restoring the corneal endothelium with Myh11-lineage (Myh11-Lin[+]) adipose-derived stromal cells (ASCs). Methods:Intraperitoneal administration of tamoxifen and (Z)-4-hydroxytamoxifen eyedrops were used to trace the lineage of Myh11-expressing cells with the Myh11-Cre-ERT2-flox-tdTomato mouse model. Immunostaining and Western blot characterized marker expression and spatial distribution of Myh11-Lin(+) cells in the cornea, and administration of 5-ethynyl-2'-deoxyuridine labeled proliferating cells. ASCs were isolated from epididymal adipose Myh11+ mural cells and treated with cornea differentiation media to evaluate corneal endothelial differentiation potential. Differentiated ASCs were injected into the anterior chamber to test for incorporation into corneal endothelium following scratch injury. Results:A subset of CECs express Myh11, a marker previously thought restricted to only mural cells. Myh11-Lin(+) CECs marked a stable subpopulation of cells in the cornea endothelium. Myh11-Lin(+) ASCs undergo CEC differentiation in vitro and incorporate into injured corneal endothelium. Conclusions:Dystrophy and dysfunction of the corneal endothelium accounts for almost half of all corneal transplants, the maintenance of the cornea endothelium is poorly understood, and there are a lack of mouse models to study specific CEC populations. We establish a mouse model that can trace the cell fate of a subpopulation of CECs based on Myh11 expression. A subset of ASCs that share this Myh11 transcriptional lineage are capable of differentiating into CECs that can incorporate into injured corneal endothelium, revealing a potential cell source for creating engineered transplant material.
| S-EPMC6905658 | biostudies-literature
Project description:The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.
| S-EPMC5992532 | biostudies-literature
Project description:Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.
| S-EPMC3195418 | biostudies-literature
Project description:BackgroundCorneal transplantation is the most effective clinical treatment for irreversible corneal endothelial decompensation. However, while visual rehabilitation can be achieved by corneal transplantation, transplant rejection, poor postoperative visual acuity, and lack of suitable donor tissue are currently the greatest obstacles to corneal transplantation. As a result, endothelial cell-based therapy has emerged as an alternative to corneal transplantation treatment. Human induced pluripotent stem cells (hiPSCs) were induced to differentiate into human corneal endothelial cell (hCEC)-like cells in our study, which aimed to provide an experimental basis for studying the clinical translation and application of induced PSC (iPSC) to corneal endothelial cell (CEC) differentiation.MethodsA two-step method to convert hiPSCs into hCEC-like cells was applied in the study. First, the transforming growth factor beta (TGF-β) and Wnt signaling pathways were regulated by adding SB4315542 and CHIR99021, and hiPSCs were induced to differentiate into neural crest cells (NCCs) by a chemically defined and serum-free in vitro induction method. Subsequently, NCCs were induced to differentiate into hCEC-like cells by adding B27, platelet-derived growth factor (PDGF)-BB, and XAV939 (Wnt pathway inhibitor) to CEC culture medium.ResultsIPSCs were directionally differentiated into NCCs. During the process of differentiation, iPSCs gradually lost the monoclonal morphology of PSCs. Moreover, β-catenin and SRY-related HMG-box gene 10 (SOX10) proteins were immunohistochemically expressed on the 7th day of differentiation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results demonstrated that the cells expressed SOX9, SOX10, nerve growth factor receptor (NGFR), human natural killer-1 (HNK-1), and β-catenin, indicating that they were successfully directionally differentiated from iPSCs to NCCs. After 5-7 days of differentiation, the cells demonstrated a hexagonal morphology of monolayer tight junctions. Immunofluorescence results demonstrated that the cells expressed CEC indicator zonula occludens-1 (ZO-1) protein. QRT-PCR results demonstrated that the cells expressed collagen type IV alpha 1 (COL4A1), COL8A2, COL8A1, and ZO-1, which indicated that they were successfully directionally differentiated from NCC to CEC.ConclusionsOur study successfully provided a simple and efficient method with clear chemical composition and serum-free media to directionally differentiate hiPSCs into hCEC-like cells, thereby advancing the results of previous studies on directional transformation into epithelial cells.
| S-EPMC9096426 | biostudies-literature