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ROCK Inhibition extends passage of human embryonic stem cell derived retinal pigmented epithelium

ABSTRACT: Human embryonic stem cell derived retinal pigmented epithelial cells (hESC-RPE) are in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only five to six passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We describe a novel passaging protocol and show that inhibition of Rho-associated, coiled coil containing protein kinases (ROCK1 and ROCK2) using Y-27632, allows extended passage of hESC-RPE in serum-free culture with maintenance of the RPE phenotype. After 30 population doublings, hESC-RPE at passage 13 maintain normal karyotype, display typical, polarized epithelial morphology, and continue to express RPE-specific genes. Passage 13 hESC-RPE show protein localization patterns similar to passage 2 cells, and display similar levels of growth factor secretion and phagocytosis of photoreceptor outer segments. Microarray analysis from day 2 cells shows several key pathways are altered by ROCK inhibition, including stimulation of the cell cycle and suppression of TGFβ and Wnt signaling. These findings describe a means to greatly increase the yield of functional hESC-RPE for use in research and clinical trials.

ORGANISM(S): Homo sapiens

PROVIDER: GSE56618 | GEO | 2014/06/30

SECONDARY ACCESSION(S): PRJNA244089

REPOSITORIES: GEO

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ROCK Inhibition extends passage of human embryonic stem cell derived retinal pigmented epithelium

Project description:Human embryonic stem cell derived retinal pigmented epithelial cells (hESC-RPE) are in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only five to six passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We describe a novel passaging protocol and show that inhibition of Rho-associated, coiled coil containing protein kinases (ROCK1 and ROCK2) using Y-27632, allows extended passage of hESC-RPE in serum-free culture with maintenance of the RPE phenotype. After 30 population doublings, hESC-RPE at passage 13 maintain normal karyotype, display typical, polarized epithelial morphology, and continue to express RPE-specific genes. Passage 13 hESC-RPE show protein localization patterns similar to passage 2 cells, and display similar levels of growth factor secretion and phagocytosis of photoreceptor outer segments. Microarray analysis from day 2 cells shows several key pathways are altered by ROCK inhibition, including stimulation of the cell cycle and suppression of TGFM-NM-2 and Wnt signaling. These findings describe a means to greatly increase the yield of functional hESC-RPE for use in research and clinical trials. Two-condition experiment, Control vs Y-27632 treated hESC-RPE passage 5. Biological replicates: 4 control, 4 Y-27632. The experiments were technically carried out as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but processed as though they are single channel (Cy3 and Cy5 signals are calculated; Cy3/Cy5 ratios are not calculated). Therefore, there are 4 raw data files for total 8 samples.

2014-06-30 | E-GEOD-56618 | biostudies-arrayexpress

Characterization of hES derived Retinal Pigment Epithelial (RPE). RPE proliferation involves a non-classical Epithelial-Mesenchymal Transition (EMT).

Project description:Retinal Pigment Epithelial (RPE) cells are located behind the retina and are critical for photoreceptor survival. Loss of RPE is associated with several pathogenic conditions such as Age Related Macular Degeneration and Retinitis Pigmentosa. RPE derived from human embryonic stem cells (hESC) offer a potential source for producing these cells for therapy. Here we report the molecular and cellular characterization of RPE differentiated from hESC. hESC derived RPE are capable of proliferation and lose their epithelial characteristics before becoming confluent and re-differentiating back into their typical pigmented, cobblestoned appearance. During the proliferative phase, they adopt a mesenchymal morphology and express mesenchymal markers. Our results demonstrate that this apparent Epithelial-Mesenchymal Transition is not regulated by the classical EMT transcription factors SNAIL and SLUG. Furthermore, it is possible to regulate RPE de-differentiation and re-differentiation by modulating the Wnt and BMP pathway respectively. These findings further our understanding of the genesis and expansion of RPE which is essential for their therapeutic use.

2012-10-24 | E-MTAB-854 | biostudies-arrayexpress

RPE Redifferentiation (miRNA)

2013-01-17 | E-MTAB-960 | biostudies-arrayexpress

Effect of cAMP and seeding density on RPE redifferentiation

Project description:Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESC) differentiated into RPE progeny have potential to provide an unlimited supply of cells for transplantation but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield following differentiation. Our data show that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of TGFβ signalling. This in turn results in enhanced uptake of epithelial identity. In line with these findings, targeted manipulation of the TGFβ pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell derived RPE. Modulation of these pathways has potential to favorably impact upon scalability and clinical translation of hESC-derived RPE as a cell therapy. A sample of Gene Pool™ cDNA, from human fetal normal brain tissue (Invitrogen D8830-01) is included for reference.

2015-09-14 | E-MTAB-3878 | biostudies-arrayexpress

SHEF1.3-derived retinal pigment epithelium for cell therapy is free of stem cell contaminants

Project description:Safety is the principle consideration with any clinical program, for which hESC and their derived products hold specific challenges. Differentiated cell products derived from hESC must be free from pluripotent cells as these could potentially form teratomas. One relevant clinical program is transplantation of retinal pigment epithelial cells (RPE) derived from hESC. This has potential for halting visual decline in conditions where the RPE layer is damaged such as age-related macular degeneration (AMD). In this study we show that whole genome gene expression analysis of SHEF1.3 starting material and the P0 pigmented RPE foci shows that the two cell types are distinct.

2016-07-03 | E-MTAB-4154 | biostudies-arrayexpress

Genetic profiling of developing human embryonic stem cell derived Retinal pigmented epithelium

Project description:Age-related macular degeneration is a progressive disease resulting in impaired central vision. Degeneration of the retinal pigmented epithelial (RPE) monolayer is associated with the progression of the disease. To date no treatment is able to stop this progression, however new cell replacement studies using human embryonic stem cells (hESC) to generate RPE show a promising prospective. To improve cell replacement strategies a better understanding about the development of RPE cells is necessary. In the current study we therefore do extensive genetic profiling of hESC derived RPE cells at different stages during development based on their pigmentation.

2017-08-02 | GSE85907 | GEO

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation

Project description:Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here we conduct single-cell transcriptomic profiling of a hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, in which cells rapidly acquired a rostral embryo patterning signature, before converging towards the RPE lineage. At intermediate steps, we identified and examined the potency of a NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

2021-02-04 | GSE164092 | GEO

Reversal of persistent wound-induced retinal pigmented epithelial-to-mesenchymal transition by the TGFb pathway inhibitor, A-83-01.

Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD. Using a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we have shown that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40% of the transcriptome (see GEO:GSE62224). In contrast, at subconfluence fewer than 5% of expressed transcripts have 2-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFb pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and reverses the passage-dependent loss of epithelial potential. In this RNA-Seq based transcriptome analysis we show that the TGFb receptor kinase inhibitor, A-83-01, largely reverses the effects of passage and restores the transcriptome profile of Passage 4 RPE highly similar to that seen in differentiated Passage 0 RPE. Examination of mRNA expression in three different primary fetal RPE donor lines in 32 day old passage 0, passage 3, and passage 3 treated with 500 nM A-83-01 cultures

2015-07-06 | E-GEOD-67898 | biostudies-arrayexpress

Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

Project description:TGFbeta-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelial (RPE) differentiation after periods of sustained sub-confluent culture or repetitive passage. Inhibition of TGFbeta signaling using receptor kinase inhibitors forestalls the onset of passage-dependent EMT and can restore the capacity to differentiate to cells that previously underwent the mesenchymal switch [Radeke et al., 2015, Genome Med. 7:58]. However, even with the sustained inhibition of mesenchymal gene expression using TGFbeta signaling inhibitors the cells eventually lose the capacity to attain a characteristic phenotype. This suggests that there are additional mechanisms at play that contributeto the prevention of RPE differentiation after protracted periods of wound stimulus and mitosis. In this study we investigate the non-TGFbeta-mediated processes that contribute to the demise of the RPE phenotype after extended periods of proliferative wound response. Using comparative transcriptomics, we show that with increasing passage there is a downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential that cannot be prevented or reversed by inhibition of TGFbeta signaling using the TGFbeta receptor kinase inhibitor A-83-01. Importantly, among the RPE genes with decreased expression are several transcription factors known to be critical for RPE development. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 treatment restored the ability of passage 7 RPE to differentiate. Taken together, these results demonstrate that the loss of capacity to differentiate as a result of chronic wound stimulus is a product of both TGFbeta pathway-dependent increases in mesenchymal gene expression and a TGFbeta pathway-independent loss of RPE programming.

2016-12-01 | GSE78740 | GEO

Comparison of Trabecular Meshwork-Derived Cells (TMDCs) with other somatic Cell Types

Project description:Microarray study comparing trabecular meshwork-derived cells (TMDCs) from the iridocorneal angle of the human eye, with other human cell types such as scleral fibroblasts, corneal fibroblasts, retinal pigmented epithelial (RPE) cells, corneal stroma, human embryonic stem cells (hESC), neural precursors differentiated from hESC, human umbilical vein endothelial (HUVEC) cells and human adipose-tissue-derived mesenchymal stromal cells (MSC)

2012-05-01 | GSE28679 | GEO

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