Human Embryonic Stem Cell–Derived Mesoderm-like Epithelium Transitions to Mesenchymal Progenitor Cells

Abstract

Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They are able to self-renew indefinitely, potentially making them a source for large-scale production of therapeutic cell lines. Here, we developed a monolayer differentiation culture that induces hESC (WA09 and BG01) to form epithelial sheets with mesodermal gene expression patterns (BMP4, RUNX1, and GATA4). These E-cadherin+ CD90low cells then undergo apparent epithelial-mesenchymal transition for the derivation of mesenchymal progenitor cells (hESC-derived mesenchymal cells [hES-MC]) that by flow cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) markers, but positive for markers associated with mesenchymal stem cells (CD73, CD90, CD105, and CD166). To determine their functionality, we tested their capacity to produce the three lineages associated with mesenchymal stem cells and found they could form osteogenic and chondrogenic, but not adipogenic lineages. The derived hES-MC were able to remodel and contract collagen I lattice constructs to an equivalent degree as keloid fibroblasts and were induced to express alpha-smooth muscle actin when exposed to transforming growth factor (TGF)-beta1, but not platelet derived growth factor-B (PDGF-B). These data suggest that the derived hES-MC are multipotent cells with potential uses in tissue engineering and regenerative medicine and for providing a highly reproducible cell source for adult-like progenitor cells.

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

“…4). We conclude that iPS-and ES-MSCs displayed a greater propensity to undergo osteogenic and chondrogenic differentiation than adipogenic differentiation, and in this respect, our results resemble findings reported in ESC-derived MSCs [13,19,33], fetal, and other gestational product-derived MSCs [2, 5, 34 -38].…”

“…3) [16]. This ECAD-NCAD switching observed in our MSC differentiation protocol was not observed by Boyd et al [19] during the generation of MSCs from hESCs and iPS cells with an EMT differentiation method, suggesting either that cadherin switching is not required for in vitro type 1 EMT or that the two protocols generate two distinct MSC-like cell populations. These data and the strong induction of recognized inducers of EMT, such as BMP4, ZEB1, and ZEB2, suggests that SB431542 triggers both intrinsic and autocrine mechanisms in iPSCs that collectively prime a subset of cells for a mesenchymal stromal cell fate.…”

“…Our one-step, small molecule-based method allows for a substantially shorter culture period (20 days) to generate MSClike cells compared with ϳ30 days of differentiation required in our EB-based protocol and 40ϩ days with other monolayerbased protocols [19]. A further advantage is that it avoids the initial heterogeneity of cell populations seen with the EB method when placed in MSC media and thus an apparent lower propensity for unwanted cell types remaining in the MSC culture.…”

“…Interestingly, the reverse process of mesenchymal-to-epithelial transition has been implicated in the reprogramming of fibroblasts to iPSCs [17,18]. Boyd et al used a 30-day epithelial culture to differentiate manually dissociated hESCs, which after subsequent passaging underwent an apparent spontaneous EMT, generating cells with decreased pluripotency and increased mesodermal/MSC marker expression and bilineage mesenchymal differentiation potential [19].…”

Neurotrophic Effects of Leukemia Inhibitory Factor on Neural Cells Derived from Human Embryonic Stem Cells

“…Semiquantitative Reverse Transcription Polymerase Chain Reaction and Quantitative Real-Time PCR RNA was isolated from NP cells and neurons using RNeasy plus kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) [21]. One microgram RNA was reverse transcribed using Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA, http://www.clontech.com).…”