Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF‐β/activin/nodal signaling using SB‐431542

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 Research