Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-β/activin/nodal signaling using SB-431542
Open Access
- 27 May 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Bone and Mineral Research
- Vol. 25 (6), 1216-1233
- https://doi.org/10.1002/jbmr.34
Abstract
Directing differentiation of human embryonic stem cells (hESCs) into specific cell types using an easy and reproducible protocol is a prerequisite for the clinical use of hESCs in regenerative-medicine procedures. Here, we report a protocol for directing the differentiation of hESCs into mesenchymal progenitor cells. We demonstrate that inhibition of transforming growth factor β (TGF-β)/activin/nodal signaling during embryoid body (EB) formation using SB-431542 (SB) in serum-free medium markedly upregulated paraxial mesodermal markers (TBX6, TBX5) and several myogenic developmental markers, including early myogenic transcriptional factors (Myf5, Pax7), as well as myocyte-committed markers [NCAM, CD34, desmin, MHC (fast), α-smooth muscle actin, Nkx2.5, cTNT]. Continuous inhibition of TGF-β signaling in EB outgrowth cultures (SB-OG) enriched for myocyte progenitor cells; markers were PAX7+ (25%), MYOD1+ (52%), and NCAM+ (CD56) (73%). DNA microarray analysis revealed differential upregulation of 117 genes (>2-fold compared with control cells) annotated to myogenic development and function. Moreover, these cells showed the ability to contract (80% of the population) and formed myofibers when implanted intramuscularly in vivo. Interestingly, SB-OG cells cultured in 10% fetal bovine serum (FBS) developed into a homogeneous population of mesenchymal progenitors that expressed CD markers characteristic of mesenchymal stem cells (MSCs): CD44+ (100%), CD73+ (98%), CD146+ (96%), and CD166+ (88%) with the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Furthermore, microarray analysis of these cells revealed downregulation of genes related to myogenesis: MYH3 (−167.9-fold), ACTA1 (−161-fold), MYBPH (−139-fold), ACTC (−100.3-fold), MYH8 (−45.5-fold), and MYOT (−41.8-fold) and marked upregulation of genes related to mesoderm-derived cell lineages. In conclusion, our data provides a simple and versatile protocol for directing the differentiation of hESCs into a myogenic lineage and then further into mesenchymal progenitors by blocking the TGF-β signaling pathway. © 2010 American Society for Bone and Mineral ResearchKeywords
This publication has 78 references indexed in Scilit:
- Derivation of cranial neural crest-like cells from human embryonic stem cellsBiochemical and Biophysical Research Communications, 2008
- Transcriptional Regulation of OsteoblastsCells Tissues Organs, 2008
- Osteogenic properties of human myogenic progenitor cellsMechanisms of Development, 2008
- Two Cell Lineages, myf5 and myf5-Independent, Participate in Mouse Skeletal MyogenesisDevelopmental Cell, 2008
- Megf10 regulates the progression of the satellite cell myogenic programThe Journal of cell biology, 2007
- Alternative Splicing Events Identified in Human Embryonic Stem Cells and Neural ProgenitorsPLoS Computational Biology, 2007
- Human embryonic stem cell transplantation to repair the infarcted myocardiumHeart, 2007
- Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cellsNature Biotechnology, 2006
- Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cellsNature Biotechnology, 2002
- Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based TherapiesTissue Engineering, 2001