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

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

Yen Shun Chen et al. Stem Cells Transl Med.

Abstract

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.

Figures

Figure 1.
Figure 1.
Morphology of iPSC-derived MSCs by the SB method. Representative phase contrast images of iPSCs incubated for 10 days in mTeSR medium (A), mTeSR medium + SB (B), KOSR medium (C), or KOSR medium + SB (D) (magnification, ×4 and ×20 for each). Left panels show cells at 10 days in the designated conditions, and right panels show the same cells 2 days after passage into MSC media. Qualitative survival of cells to MP1 based on the images is indicated on the far right, with − indicating no cells attaching and surviving, + indicating a few cells, and ++++ indicating many cells. (E): Regions of SB-induced differentiation were evident as cells developing an enlarged, flattened epithelial-like morphology either on the periphery of colonies (left images) or at the center of a colony (right images, ES4CL1 cells shown). Arrows mark the boundary between undifferentiated cells and epithelial-like cells observed after 10 days SB treatment. (F): ES cell- and iPSC-derived MSCs from the SB method (MP2 and MP1, respectively) showed a fibroblast-like morphology similar to that of primary adult and fetal bone marrow MSCs (magnification, ×10). Abbreviations: ES, embryonic stem; inhib, inhibitor; iPS, induced pluripotent stem; KOSR, knockout serum replacement; MP, mesenchymal passage; MSC, mesenchymal stem/stromal cell; SB, SB431542.
Figure 2.
Figure 2.
Morphology of ESC- and iPSC-derived mesenchymal stem/stromal cells (MSCs) by the embryoid body (EB) method. Shown is the morphology of the ESCs (A) and iPSCs (B) grown for 10 days in EB suspension culture (magnification, ×4). EBs were then transferred into tissue culture flasks with MSC medium, where they rapidly attached to the vessel ([C] and [D]; images are 4 days after attachment), after which the center of undifferentiated cells was removed by aspiration, and outgrowth cells were allowed to become confluent before further passaging. (E, F): ESC- and iPSC-derived MSCs form the EB method (mesenchymal passage 2) showed a standard MSC-like fibroblastic morphology. Abbreviations: ESC, embryonic stem cell; iPSC, induced pluripotent stem cell.
Figure 3.
Figure 3.
Cell surface phenotype of iPS-derived MSCs. CD and other immunological surface markers used to define human MSCs were applied here to fetal MSCs, iPS-MSCs (inhib.), iPS-MSCs (EB), and undifferentiated iPSCs. Abbreviations: EB, embryoid body; fMSC, fetal mesenchymal stem/stromal cell; inhib, inhibitor; iPS, induced pluripotent stem; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem/stromal cell.
Figure 4.
Figure 4.
Mesodermal differentiation of ES cell- and iPSC-derived MSCs. Shown is in vitro mesodermal differentiation of fetal MSC (fMSC), iPS-MSCs (inhibitor), iPS-MSCs (EB), ES-MSCs (inhibitor), ES-MSCs (EB), and fMSC under osteogenic conditions with von Kossa staining, adipogenic conditions with Oil Red O staining, and chondrogenic pellet culture conditions with PAS staining. Abbreviations: EB, embryoid body; ES, embryonic stem; iPS, induced pluripotent stem; MSC, mesenchymal stem/stromal cell; PAS, periodic acid-Schiff.
Figure 5.
Figure 5.
Defining the mechanism of SB431542-induced differentiation and characterization of iPS-MSC markers, growth potential, and telomerase activity. (A): Short-term cumulative population doublings calculated for iPS-MSCs (inhibitor) in blue, iPS-MSCs (EB) in green, aMSCs in black, and fMSCs in red. (B): Long-term cumulative doublings showing convergence of the growth curves for iPS-MSCs (inhibitor) in blue and iPS-MSCs (EB) in green. (C): Cumulative cell number of iPS-MSCs (inhibitor) in blue, iPS-MSCs (EB) in green. (D–G): After 4 days of SB431542 treatment, differentiation was observed spreading from both the center of the pluripotent cell colonies (D, F) and the periphery of colonies (E, G) (HES3 cells shown). Arrows denote boundary of undifferentiated cells and point toward the differentiated cells. This morphological change coincided with a loss of OCT4 nuclear expression (D, E) (red) and plasma membrane ECAD expression (F, G) (red), with both remaining in the undifferentiated cells. However, there was no accompanying loss of NANOG expression (F, G) (green) in either the differentiated or undifferentiated cells during SB431542 treatment. (H): Undifferentiated iPSCs (upper row) and iPS-MSCs (inhibitor) at mesenchymal passage 2 (lower row) were analyzed for expression of pluripotency markers, including OCT4 (left panels, red), Tra 1–81 (center panels, red), and NANOG (right panels, green) by immunofluorescence microscopy. (I): Cells were further analyzed for the expression of ECAD and NCAD (left panels, green and red, respectively), VIM (center panels, red), and HuNu (right panels, red). In all images, nuclei were counterstained with DAPI (blue). Abbreviations: aMSC, adult mesenchymal stem/stromal cell; DAPI, 4′,6-diamidino-2-phenylindole; EB, embryoid body; ECAD, E-cadherin; fMSC, fetal mesenchymal stem/stromal cell; HuNu, human nuclear antigen; iPS, induced pluripotent stem; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem/stromal cell; NCAD, N-cadherin; Pop., population; VIM, vimentin.
Figure 6.
Figure 6.
Gene expression profile of SB-induced differentiation of ESCs and iPSCs. (A): The expression of 53 genes selected from the quantitative reverse transcription-polymerase chain reaction array in ESC-derived and iPSC-derived MSCs. ESCs/iPSCs were cultured in mTeSR, KOSR medium in the presence of 10 μM SB for 10 days (KOSR+SB), and then after KOSR+SB, cells were subcultured in MSC medium and RNA extracted after one or two passages. Data are categorized into markers of pluripotency, germ layers (ectoderm, mesoderm, endoderm), hematopoietic cells, MSCs, and EMT. Expression levels were normalized to GAPDH and compared with those in mTeSR culture conditions. The heat map profile is presented as follows: red, high expression; white, medium expression; blue, low expression relative to overall expression over the three samples (time points) for a given probe. (B–E): Scatter plot comparisons of the gene expression profiles of the undifferentiated iPSC mTeSR condition with iPSC KOSR+SB (B) and iPSC-derived MSCs (iPS-MSCs) (D) at mesenchymal passage 1, and similarly of gene expression profiles of undifferentiated ESCs in mTeSR with ESC KOSR+SB (C) and ESC-derived MSCs (ES-MSCs) (E) at mesenchymal passage 2. Pink lines indicate boundaries of fourfold difference in gene expression, and highly expressed marker genes are indicated. Gene expression levels are depicted on a log10 scale. The scatter plots show the fold changes calculated using the 2−ΔCt formula. (F): iPSCs and ESCs were cultured on a Matrigel-coated plate in mTeSR medium (lanes 1 and 4) and treated with 10 μM SB in either mTeSR medium (lanes 2 and 5) or KOSR medium (lanes 3 and 6). After 10 days of treatment, the proteins in the cell lysates were subjected to Western blot analysis for SMAD2 (upper panel) and pSMAD2 (middle panel), and equal loading was assessed by Western blotting of the cell lysates for actin (bottom panel). Abbreviations: EMT, epithelial-to-mesenchymal transition; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; KOSR, knockout serum replacement; MSC, mesenchymal stem/stromal cell; SB, SB431542.
Figure 7.
Figure 7.
Gene expression analysis of iPSC-derived mesenchymal stem/stromal cell (MSCs) induced by the SB method. The iPSCs were cultured in mTeSR or KOSR medium with 10 μM SB treatment for 10 days and then subcultured in MSC medium. The 10-day SB-treated cells and iPS-MSCs at MP1 were analyzed for gene expression. (A–C): OCT4, SOX2, and ECAD genes associated with pluripotency. (D, E): PAX6 and CDX2 genes expressed by ectoderm. (F–H): NCAM, BMP4, and MSX2 genes expressed by mesodermal lineages. (I, J): SOX7 and SOX17 expressed by cell lineages from the endoderm. (K, L): The expression of the LEFTY1 and LEFTY2 genes was regulated by TGF-β signaling pathway. (M-P): Genes expressed by cell lineages from MSCs, positive for CD29, CD73, CD105, and CD44. (Q–T): Genes associated with the epithelial-to-mesenchymal transition pathway, NCAD, ZEB1, SNAI1, and TWIST2. Gene expression was normalized to GAPDH. Samples were in duplicate. Abbreviations: ECAD, E-cadherin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iPS, induced pluripotent stem; iPSC, induced pluripotent stem cell; KOSR, knockout serum replacement; NCAD, N-cadherin; SB, SB431542.

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