Project description:Expression data from undifferentiated human embryonic stem cells (hESC) and Day 3.5 mesodermal progenitor (CD326neg CD56+) population
Project description:Our understanding of how mesodermal tissue is formed, has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESC) is initiated by Epithelial to Mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that CD326negCD56+ cells, generated from hESC in the presence of activin A, BMP4, VEGF and FGF2, represent a novel, multi-potent mesoderm-committed progenitor population. CD326negCD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle and cardiomyocytes, while lacking the pluripotency of hESC. CD326negCD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a novel approach to study how germ layer specification is regulated, and offer a unique target for tissue engineering. We used microarrays to compare gene expression profile of early mesodermal progenitors with undifferentiated hESC (H9 line). Mesoderm induction from hESC was initiatiated with combination of morphogens and growth factors including activin A, bone morphogenic protein 4, basic fibroblast growth factor and vascular endothelial growth factor. Proposed mesodermal progenitor population was isolated by FACS on day 3.5 of culture based on the presence of CD56 expression and the absenbce of CD326 expression.
Project description:Our understanding of how mesodermal tissue is formed, has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESC) is initiated by Epithelial to Mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that CD326negCD56+ cells, generated from hESC in the presence of activin A, BMP4, VEGF and FGF2, represent a novel, multi-potent mesoderm-committed progenitor population. CD326negCD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle and cardiomyocytes, while lacking the pluripotency of hESC. CD326negCD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a novel approach to study how germ layer specification is regulated, and offer a unique target for tissue engineering. We used microarrays to compare gene expression profile of early mesodermal progenitors with undifferentiated hESC (H9 line).
Project description:We performed time-dependent, genome-wide analysis to explore gene expression profiles during in vitro cardiogenesis from embryonic stem cells to cardiac progenitor cells. Total RNA was extracted from undifferentiated stem cells, early mesodermal cells, cardiac mesodermal cells and cardiac progenitor cells. As a result of cluster analysis, Cited gene family was considered as candidate genes in cardiogenesis. Cited4 gene expression was specific for early cardiogenesis and Cited4 functioned as a cell cycle controling factor for early cardiac progenitor cells. Embryoid body was formed as in vitro cardiogenesis. Total RNA was extracted from Rex1-positive undifferentiated stem cells at day 0, Bra-positive early mesodermal cells at day 4.5, Flk1-positive cardiac mesodermal cells at day 4.5 and Nkx2.5-positive cardiac progenitor cells at day 7.5.
Project description:We analyzed the global transcriptome signature over the time course of the cardiac differentiation from hESC by RNA-seq. We characterized the genome-wide transcriptome profile of 5 distinct stages; undifferentiated hESC (day 0), mesodermal precursor stage (hMP, day 2), cardiac progenitor stage (hCP, day 5), immature cardiomyocyte (hCM14) and hESC-CMS differentiated for 14 additional days (hCM28). While the stem cell signature decreases over the five stages, the signatures associated with heart and smooth muscle development increase, indicating the efficient cardiac differentiation of our protocol.
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.
Project description:RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes
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.
Project description:Whole proteome profiling and quantification was performed on an isogenic Huntington disease (IsoHD) human embryonic stem cell (hESC) allelic panel. The IsoHD hESCs harbour 30, 45, 65 and 81 CAG repeats in the first exon of HTT. Whole proteome quantification was also performed on neural progenitor cells derived from the IsoHD hESC panel.
Project description:Despite the progress in safety and efficacy of cell therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells (NPCs) with rostral identity has remained a major challenge. Here we reported the generation of an LMX1A knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further proteomic analysis of enriched LMX1A+ cells identified several membrane associated proteins including CNTN2, enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of hPSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson’s disease symptoms in animal models. Our study establishes an efficient approach for purification of large numbers of hPSC-derived dopaminergic progenitors for therapeutic applications.