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

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 b (TGF-b)/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), a-smooth muscle actin, Nkx2.5, cTNT]. Continuous inhibition of TGF-b signaling in EB outgrowth cultures (SB-OG) enriched for myocyte progenitor cells; markers were PAX7 þ (25%), MYOD1 þ (52%), and NCAM þ (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 þ (100%), CD73 þ (98%), CD146 þ (96%), and CD166 þ (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 (À167.9-fold), ACTA1 (À161-fold), MYBPH (À139-fold), ACTC (À100.3-fold), MYH8 (À45.5-fold), and MYOT (À41.8-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-b signaling pathway. ß

Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts

“…In mouse trophoblast stem cells, activin promotes the acquisition of a labyrinth cell fate (60). Interestingly, the occurrence of EMT upon passage of SB431542-treated hESCs has been reported but in dissimilar culture conditions (61,62).…”

Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

“…SB431542 treatment has previously been used to generate mesodermal progenitors from hESCs [10,14]. Sanchez et al [14], however, found that hESCs, but not iPSCs, underwent differentiation into CD90 ϩ CD73 ϩ MSCs after treatment with SB431542.…”

“…Mahmood et al treated hESCs with SB431542 during EB formation to upregulate paraxial mesodermal and myogenic markers and then derived MSC-like cells from EB outgrowth cultures [10]. Sanchez et al exposed hESCs to SB431542 for 28 days to induce differentiation into multipotent progenitors [14].…”

Direct Differentiation of Human Embryonic Stem Cells toward Osteoblasts and Chondrocytes through an Intermediate Mesenchyme Progenitor Lineage

“…Successive outgrowth cultures of the MCP, in the presence of 10% fetal bovine serum (FBS), demonstrated a step-wise progression from a heterogeneous EB outgrowth cell population, through an MCP population, to 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%). Lastly these mesenchymal progenitors verified their ability to differentiate into osteoblasts in vitro and could form ectopic bone upon subcutaneous implantation with hydroxyl-apatite/tricalcium phosphate ceramic powder (HA/TCP) in immune deficient mice [27].…”

“…Detailed cellular and molecular analysis revealed the differentiation of SB-treated hEBs into muscle progenitor cells (MPC) [27]. Successive outgrowth cultures of the MCP, in the presence of 10% fetal bovine serum (FBS), demonstrated a step-wise progression from a heterogeneous EB outgrowth cell population, through an MCP population, to 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%).…”