In vitro pigmentation of human iPSC-derived retinal pigment epithelium cells is not indicative of their quality for cell transplantation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE242184 | GEO | 2024/05/04
REPOSITORIES: GEO
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Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma. 60 pooled eyes from 36 hpf wild type or vsx2:Gal4/dsRed:14xUAS:YapS87A embryos were pooled for one sample. Three wild type and three vsx2:Gal4/dsRed:14xUAS:YapS87A pools were analyzed for RNA.
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Project description:We report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.
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Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma.
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Project description:In vitro pigmentation of human iPSC-derived retinal pigment epithelium cells is not indicative of their quality for cell transplantation
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Project description:Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that span spatiotemporal scales. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. While pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. By designing a pigmentation oscillator, in this study we delineate the role of interferon gamma (IFN-γ) signaling in skin pigmentation homeostasis. IFN-γ signaling reversibly controls maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. We show that this temporal effect is mediated through interferon regulatory factor-1 (IRF-1) independent of the central regulator microphthalmia-associated transcription factor (MITF). Chronic expression of IFN-γ in mouse and human skin shows hypopigmentation phenotype, further providing a strong support to the under-appreciated function of IFN-γ in skin pigmentation. Our studies thus reveal the hitherto missing link between immune system and pigmentation, which may have implications in depigmentary and malignant cutaneous disorders. there are 12 samples from two consecutive cycles of pigmentation oscillator, termed set 1 and set 2 analysis is performed wrt to day 0 of first cycle
2014-02-03 | E-GEOD-54359 | biostudies-arrayexpress
Project description:The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNAseq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd). were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67≥FC≥1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, andtight junctions were down regulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNAseq results. Thus, the RPE quickly down-regulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space and the opsonization of orphaned outer segments following RD.
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Project description:Retinal Pigment Epithelium (RPE) derived from two human embryonic stem cell lines, H1 and H9, were compared with human fetal RPE (hfRPE) using RNA-seq. Nominally, the transcriptome of H1-derived RPE showed greater overlap with hfRPE. For cells maintained in the medium used to differentiate RPE, 6.2% (H1-RPE) and 4.2% (H9-RPE) of the transcripts were expressed in amounts that were statistically different from hfRPE (false discovery rate: 5%). After adaptation to the serum-free medium, SFM-1, only 1.0 % (H1-RPE) and 1.9% (H9-RPE) were expressed in amounts that were statistically different. For RPE signature genes, statistical differences were observed for 1.0 % (H1-RPE) and 1.9% (H9-RPE) of the transcripts. For some barrier-function related mRNAs the statistical differences were greater than these small differences would predict. For adhesion proteins and plasma membrane transporters, the differences were as great as 6.9% and 4.3%, respectively. After adaptation to SFM-1, the statistical differences between H1- and H9-RPE were only 0.4% for all transcripts, 1.4% for signature genes, and 0.7% for membrane transporters. No statistical differences were observed for the transcripts related to tight junctions, adhesion junctions or ion channels. In summary, SFM-1 promoted the maturation of stem cell-derived RPE, and the statistical difference between two stem cell lines was minimal. RNA sequencing of hfRPE from three fetuses (reference sample) and three independent isolates of RPE derived from the H1, and three from the H9, human embryonic stem cell (hESC) lines. The cultures were maintained in a serum-free medium, SFM-1. Comparisons were also made to cultures maintained in the medium used to differentiate the cells, KSR.
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