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.

FIG. 1.

hESC monolayer differentiation. hESC were differentiated in EGM2-MV for 20–30 days. (A) Within 3–5 days, hESC (arrowhead) began forming epithelial foci (arrow), (B) expanded in a circular pattern (arrow) until (C) the entire culture presented an epithelial phenotype. (D) The epithelial phenotype underwent EMT with passaging. (E) Time line for differentiation of hESC to epithelium and EMT. (10 × magnification, A–D).

FIG. 2.

hESC-derived epithelial cells express mesodermal markers. RNA was acquired from WA09 on d0, 5, 10, 15, 20, 25, and 30 and examined by RT-qPCR with respect to 18S, normalized to d0 and the transformed data [ln(RQ)] analyzed for significance (n = 4; *p < 0.05). The RQ value (y-axis) is plotted versus time in days (x-axis) and is shown as the average ± standard error.

FIG. 3.

Flow cytometry indicates hESC to epithelial to mesenchymal changes. (A) WA09 samples were examined by flow cytometry at p0 (passage 0 or pluripotence) and p1 (passage 1 at ∼30 days). (B) Protein expression for WA09 (n = 3 or 4) and BG01 (n = 2) was compared between passages 1 (p1) and 7 (p7) (*p < 0.05). Color images available online at www.liebertonline.com/ten.

FIG. 4.

hES-MC are osteogenic and chondrogenic, but not adipogenic. hES-MC and bone marrow–derived human MSC were subject to MSC three-lineage differentiation protocols. For negative controls, both cell types were cultured in normal growth media. (A) Osteogenic conditions and von Kossa staining. (B) Chondrogenic conditions and Alcian blue staining. (C) Adipogenic conditions and Oil Red-O staining. Images are representative of WA09 (n = 3) and BG01 (n = 2). (10 × magnification). Color images available online at www.liebertonline.com/ten.

FIG. 5.

hES-MC contract and remodel collagen I lattice. KF and derived hES-MC were seeded into rat tail collagen I lattices floating for 7 days. (A) Bright field images show the degree of contraction and remodeling compared to the original lattice size (B4 and E22h from WA09; E21b from BG01; NC, No Cells). (B) Contraction quantification (n = 3). *p < 0.05. Color images available online at www.liebertonline.com/ten.

FIG. 5.

hES-MC contract and remodel collagen I lattice. KF and derived hES-MC were seeded into rat tail collagen I lattices floating for 7 days. (A) Bright field images show the degree of contraction and remodeling compared to the original lattice size (B4 and E22h from WA09; E21b from BG01; NC, No Cells). (B) Contraction quantification (n = 3). *p < 0.05. Color images available online at www.liebertonline.com/ten.

FIG. 6.

TGF-β1, but not PDGF-B, induce αSMA expression in hES-MC. hES-MC of passage 5 or 6 were plated in 10 ng/mL of PDGF-B or TGF-β1 for 12 days and then immunostained for αSMA, F-actin, and DAPI. 40 ×, scale bars = 10 μm. Images are representative of two independent experiments with two WA09-derived cell lines (B4 and E28h) and one BG01-derived line (E21b). Color images available online at www.liebertonline.com/ten.