ABSTRACT: Pigment Epithelium Derived Factor (PEDF) secreted from iPSC derived-RPE facilitates apoptotic causes cell death, not the differentiation, of iPSCs
Project description:We show that Retinal pigment epithelium (RPE) secreted-factor, pigment epithelium derived factor (PEDF) secreted/derived from primary or iPSC-derived retinal pigment epithelium (RPE)RPE, dramatically inhibitsed the cell growth of iPSCs. PEDF was detected abundantly in culture supernatant media of primary and iPSC-derived RPE. We examined the gene expression in primary RPE and iPS-derived RPE. Two samples: RPE derived from 253G1 iPSC, Primary RPE.
Project description:We show that Retinal pigment epithelium (RPE) secreted-factor, pigment epithelium derived factor (PEDF) secreted/derived from primary or iPSC-derived retinal pigment epithelium (RPE)RPE, dramatically inhibitsed the cell growth of iPSCs. PEDF was detected abundantly in culture supernatant media of primary and iPSC-derived RPE. We examined the gene expression in primary RPE and iPS-derived RPE.
Project description:Compare transcriptomes of control and USH1B patient iPSC-derived retinal pigment epithelium (RPE) to elucidate disease mechanisms of Usher syndrome type IB (USH1B). USH1B patient fibroblasts were collected at Great Ormond Street Hospital (GOSH) and reprogrammed to iPSCs. Control and patient iPSCs differentiated in vitro to generate retinal pigment epithelium (RPE) and collected for RNA-seq at 24 week. Sequencing was performed at University College London (UCL) Genomics on a NovaSeq 6000 system. Data aligned to the human genome UCSC hg38 using RNA-STAR 2.5.2b.
Project description:Age-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells. We studied the chemistry of retinal progenitor cells derived from iPSC through our patented unified differentiation protocol with the aim to take the cells for clincal benefits to needly patients. RPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host's outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye.
Project description:Pigment Epithelium-Derived Factor (PEDF) has recently been identified as a factor that is significantly upregulated in late-stage osteoarthritic cartilage in which chondrocytes are confronted with terminal differentiation and cell death. Since PEDF is known to induce cell death of endothelial cells, it may also be responsible for terminal differentiation and cell death in cartilage.
Project description:Pigment Epithelium-Derived Factor (PEDF) has recently been identified as a factor that is significantly upregulated in late-stage osteoarthritic cartilage in which chondrocytes are confronted with terminal differentiation and cell death. Since PEDF is known to induce cell death of endothelial cells, it may also be responsible for terminal differentiation and cell death in cartilage. Using cDNA microarray analysis, we found PEDF among the factors with the strongest differential expression and significant higher levels (118.5-fold) in osteophytic cartilage compared with articular cartilage. This study explored if PEDF interferes with the stable chondrocyte phenotype by promoting terminal differentiation or cell death.
Project description:To evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), we performed poly-A RNA sequencing on nuclear and cytoplasmic fractions from induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide, as well as untreated controls.
Project description:Comparing fibroblasts and derived -iPSC and - RPE cells from human AMD and non-AMD donors Retinal pigment epithelium (RPE) generated from skin biopsies of donors with age-related macular degeneration (AMD) exhibit a disease phenotype and a distinct transcriptome compared to age-matched controls. We investigated whether similar differences existed in the skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from them. Hierarchical cluster and principal component analyses revealed significant overlap in the transcriptome of fibroblasts of AMD and non-AMD donors. After reprogramming, iPSCs exhibited slight differences. In contrast, the transcriptome of RPE derived from AMD and normal donors segregated into two distinct clusters. Differences in the expression of specific genes that were evident between normal and AMD-derived RPE were not observed in fibroblasts or iPSCs. Mitochondrial respiration was reduced in RPE from AMD patients but not in fibroblast or iPSCs. RPE derived from AMD patients have a distinct transcriptome and phenotype compared to controls that is not observed in their corresponding skin fibroblasts or iPSCs.
Project description:PURPOSE: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. METHODS: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. RESULTS: ZSCAN10 and Lin28a were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and Lin28a were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and Lin28a in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10 and Lin28a expression in iPSCs was generally uniform. The LOD for ZSCAN10 and Lin28a in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of DDCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC / 10,000 RPE cells in both iPSC lines. CONCLUSIONS: qPCR for ZSCAN10 and Lin28a detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.
Project description:We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients.