Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/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 beta (TGF-beta)/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), alpha-smooth muscle actin, Nkx2.5, cTNT]. Continuous inhibition of TGF-beta 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-beta signaling pathway.
Mahmood
Amer
A
Medical Biotechnology Centre, Department of Endocrinology, University Hospital of Odense, University of South Denmark, Odense, Denmark.
Harkness
Linda
L
Schrøder
Henrik Daa
HD
Abdallah
Basem M
BM
Kassem
Moustapha
M
D028341:Activins / Q000378:metabolism*; D050156:Adipogenesis / Q000187:drug effects; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D004149:Dioxoles / Q000494:pharmacology*; D004475:Ectoderm / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009132:Muscles / Q000166:cytology; D055457:Nodal Protein / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010012:Osteogenesis / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D033581:Stem Cell Transplantation; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D015415:Biomarkers; D004149:Dioxoles; C526007:NODAL protein, human; D055457:Nodal Protein; D016212:Transforming Growth Factor beta; D028341:Activins