Unknown,Transcriptomics,Genomics,Proteomics

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Directing differentiation of pluripotent stem cells towards retinal pigment epithelium lineage

ABSTRACT: Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro models of disease, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of a robust and efficient method to derive RPE with high reproducibility in an adherent, monolayer system using sequential inhibition and activation of the Activin and BMP signalling pathways. We use whole genome transcript analysis to characterize cells at different stages of differentiation to gain further understanding of the developmental dynamics and fate specification of RPE.

ORGANISM(S): Homo sapiens

SUBMITTER: Alex Gutteridge

PROVIDER: E-MTAB-4259 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Directing differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a perquisite for the clinical use of hESC in regenerative medicine protocols. Here, we report the generation of mesodermal cells with differentiation potential to myocytes, osteoblasts, chondrocytes and adipocytes. We demonstrate that during hESC differentiation as embryoid bodies (EB), inhibition of TGF-b/Activin/Nodal signaling using SB-431542 (SB) markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), early myogenic transcriptional factors (Myf5, Pax7) as well as myocyte committed markers (NCAM, CD34, Desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT). Establishing EB outgrowth cultures (SB-OG) in the presence of SB (1 uM) led to further enrichment of cells expressing markers for myocyte progenitor cell: CD34+ (33%), NCAM+ (CD56) (73%), PAX7 (25%) and mature myocyte proteins (MYOD1, tropomyocin, fast MHC an; d SERCA1). Further analysis using DNA microarray revealed differential up-regulation of 117 genes (>2-fold compared to control cells) annotated to myogenic development and function. During ex vivo culture, contracting myocytes were observed (80% of the population) and the cells formed myofibres when implanted intramuscularly in vivo. Furthermore, in the presence of fetal bovine serum (10% FBS), SB-OG cells developed morphologically and phenotypically into a homogeneous stromal (mesenchymal) stem cell (MSC)-like population expressing characteristic MSC CD markers: CD44 (100%), CD73 (98%), CD146 (96%) and CD166 (88%). They were karyotypically normal and were able to differentiate ex vivo and in vivo into osteoblasts, adipocytes and chondrocytes. Experiment Overall Design: Human ESCs were differentiated as EBs for 10 days, where after EBs were plated onto fibronectin coated petri dishes. At confluency the outgrowth culture was passaged, at passage 5 3 samples were taken from both control-OG and SB-OG. the samples were mixed and frozen.

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Directing differentiation of pluripotent stem cells towards retinal pigment epithelium lineage

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