Functional comparison of human-induced pluripotent stem cell-derived mesenchymal cells and bone marrow-derived mesenchymal stromal cells from the same donor

Abstract

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases, and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are, thus, of interest, as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However, characterization of such MSC-like cells is currently inadequate, especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study, we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs), and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile, and they were capable of differentiation in vitro along the osteogenic, chondrogenic, and adipogenic lineages. However, compared with the parental BMSCs, iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We, therefore, conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics.

FIG. 1.

Pluripotency and normal karyotype of M-iPSCs. (A) Immunofluorescence staining for Oct3/4, TRA-1-81 (red), and Sox2, SSEA4, TRA-1-60 (green) overlaid with DAPI staining of the nuclei (blue) of M-iPSCs in comparison to AE-iPSCs and BMSCs. Scale bar: 100 μm. (B) Comparison of levels of pluripotency-associated genes OCT3/4, SOX2, NANOG, GDF3, TDGF, and DPPA5 in M-iPSCs in comparison to AE-iPSCs and BMSCs analyzed by qRT-PCR. Values of fold changes are mean±standard deviation of three independent replicates relative to housekeeping genes. (C) Teratoma formation by M-iPSCs after injection into mouse testes containing tissues of all three germ layers. Hematoxylin- and Eosin-stained sections showed retinal epithelium (a) and neural rosettes (d), cartilage (b) and connective tissue (e), intestinal (c), and glandular (f) structures. Scale bars: 100 μm. (D) Normal karyotype in (male) passage 13 M-iPSCs. BMSC, bone marrow stromal cell; iPSCs, induced pluripotent stem cells. Color images available online at www.liebertpub.com/scd

FIG. 2.

Morphology and growth rates of iMPCs. (A) Typical cell morphology of M-iMPCs. Scale bar: 200 μm. Insets are fourfold magnifications. A-iMPC morphology was not markedly different from M-iMPCs. (B) Growth rates of BMSCs (black line) in comparison to M-iMPCs (solid gray line) and A-iMPCs (dashed gray line) derived using different procedures (EB, SD, CC, and GM). Values are mean±standard deviation of four to six replicate cell lines for iMPCs and three replicates from the same donor for BMSCs. Results are presented up to passage 6. Analysis was carried out up to passage 18, with all iMPCs ceasing proliferation after passage 9 (data not shown). CC, coculture; EB, embryoid body; GM, growth medium; iMPCs, iPSC-derived mesenchymal stem cell-like progenitor cells; SD, spontaneous differentiation.

FIG. 3.

Flow cytometric analysis of surface marker expression in M-iMPC-EBs and M-iMPC-SDs (A) as well as A-iMPC-CCs and A-iMPC-EBs (B) in comparison to BMSCs and iPSCs. Assessed surface markers included those typically present on BMSCs (CD73, CD105, and CD44), typically absent on BMSCs (CD45, HLA DR), and associated with pluripotency (TRA-1-60). Analysis of iMPC surface markers was performed with pools of four to six replicate cell lines.

FIG. 4.

Mesenchymal gene expression in M-iMPCs compared with BMSCs and M-iPSCs. (A) Heat map of PCR array results for each of the three BMSC, M-iPSC, and M-iMPC-SD, -EB, -CC, and GM replicates. (B) Expression of selected pluriptency-associated genes (top) and MSC-associated genes (bottom) by BMSCs, iPSCs, and iMPCs. Values are the mean of three replicates, and error bars represent 95% confidence intervals. (C) Volcano plots of differentially expressed genes between M-iPSCs and BMSCs (top), M-iMPCs and M-iPSCs (middle), as well as M-iMPCs and BMSCs (bottom). Genes with a>10-fold change (>4-fold for iMPC vs. BMSC) are colored red (over-expressed) and green (under-expressed). The vertical gray lines represent P=0.05, and the vertical blue lines represent significance at the Bonferroni-adjusted P value (P<5.6×10⁻⁴). Fold changes and P values of the individual genes are itemized in Table 2. All 12 iMPC lines that were investigated in the PCR array were grouped together (n=12) for this analysis. (D) Hierarchical cluster analysis (Pearson) of the average linkage between groups. MSC, mesenchymal stem cell. Color images available online at www.liebertpub.com/scd

FIG. 5.

Quality of in vitro trilineage differentiation capacity of iMPCs assessed via histological staining. (A) Alizarin Red staining of matrix calcification in BMSCs and M-iMPCs after 3 weeks under osteogenic conditions. Best and worst results are shown for each derivation method. (B) Quantitative analysis of Alizarin Red incorporation for BMSCs versus M-iMPCs and A-iMPCs. (C) Best and worst results of Oil Red O staining of lipid vesicles after 3 weeks in adipogenic medium for BMSCs and M-iMPCs. (D) Quantitative analysis of dye incorporation for BMSCs versus M-iMPCs and A-iMPCs. (E) Alcian blue staining of sGAG deposition (left) and collagen type II immunostaining (right) of BMSCs and M-iMPCs after 3 weeks in chondrogenic pellet culture. Best results are shown for M-iMPCs. (F) Quantitative sGAG assay of chondrogenic BMSC, M-iMPC, and A-iMPC pellets. Scale bars: 100 μm for (A); 50 μm for (C, E). Median values are presented (bars), with the boxes representing first and third quartile and whiskers representing maximal and minimal values. Outliers are depicted as circles. Group sizes were n=3 for BMSCs (independent replicates from one donor) and n=4–6 for iMPCs (replicate cell lines). Significant differences (P<0.05) between iMPCs and BMSCs are designated by an asterisk. Significant differences (P<0.05) between M-iMPCs and A-iMPCs are designated by §. sGAG, sulfated glycosaminoglycan. Color images available online at www.liebertpub.com/scd

FIG. 6.

Quantitative gene expression rates of osteogenic (A–C), adipogenic (D–F), and chondrogenic (G–I) lineage markers. Gene abbreviations are according to NCBI gene database. Values are median (bars), with the boxes representing first and third quartile and whiskers representing maximal and minimal values. Outliers (1.5 to 3-fold IQR) are depicted as circles. Group sizes were n=3 for BMSCs (independent replicates from one donor) and n=4–6 for iMPCs (replicate cell lines). Significant differences (P<0.05) between iMPCs and BMSCs are designated by an asterisk. Significant differences (P<0.05) between M-iMPCs and A-iMPCs are designated by §.