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. 2015 Aug 18;6(1):144.
doi: 10.1186/s13287-015-0137-7.

Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity

Ran Kang  1   2 , Yan Zhou  3 , Shuang Tan  4   5 , Guangqian Zhou  6 , Lars Aagaard  7 , Lin Xie  8 , Cody Bünger  9 , Lars Bolund  10 , Yonglun Luo  11
Affiliations

Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity

Ran Kang et al. Stem Cell Res Ther. .

Abstract

Introduction: Previously, we established a simple method for deriving mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSC-MSCs). These iPSC-MSCs were capable of forming osteogenic structures in scaffolds and nanofibers. The objective of this study is to systematically characterize the mesenchymal characteristics of the iPSC-MSCs by comparing them to bone marrow-derived MSCs (BM-MSCs).

Methods: Two iPSC-MSC lines (named as mRNA-iPSC-MSC-YL001 and lenti-iPSC-MSC-A001) and one BM-MSC line were used for the study. Cell proliferation, presence of mesenchymal surface markers, tri-lineage differentiation capability (osteogenesis, chondrogenesis, adipogenesis), and expression of "stemness" genes were analyzed in these MSC lines.

Results: The iPSC-MSCs were similar to BM-MSCs in terms of cell morphology (fibroblast-like) and surface antigen profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD11b-, CD14-, CD31-, CD34-, CD45- and HLA-DR-. A faster proliferative capability was seen in both iPSC-MSCs lines compared to the BM-MSCs. The iPSC-MSCs showed adequate capacity of osteogenesis and chondrogenesis compared to the BM-MSCs, while less adipogenic potential was found in the iPSC-MSCs. The iPSC-MSCs and the tri-lineage differentiated cells (osteoblasts, chondrocytes, adipocytes) all lack expression of "stemness" genes: OCT4, SOX2, GDF3, CRIPTO, UTF1, DPPA4, DNMT3B, LIN28a, and SAL4.

Conclusions: The MSCs derived from human iPSCs with our method have advanced proliferation capability and adequate osteogenic and chondrogenic properties compared to BM-MSCs. However, the iPSC-MSCs were less efficient in their adipogenicity, suggesting that further modifications should be applied to our method to derive iPSC-MSCs more closely resembling the naïve BM-MSCs if necessary.

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Figures

Fig. 1
Fig. 1
Derivation of iPSC-MSCs from iPSCs produced by lentiviral-mediated programming of normal human dermal fibroblasts. a One lenti-iPSC line characterized by live cell imaging of TRA-1-60+ and CD44–; immunofluorescence staining of NANOG+ and OCT3/4+, and phase-contrast image of the iPSC clone used for MSC differentiation. b Cell cycle analysis of the lenti-iPSC clone. c Phase-contrast morphology of the BM-MSC, mRNA-iPSC-MSC-YL001 and the lenti-iPSC-MSC-A001 derived from the lenti-iPSCs. Magnification 200×. d A representative DAPI banded karyotype result for BM-MSC (P12), mRNA-iPSC-MSC-YL001 (P13) and the lenti-iPSC-MSC-A001 (P10). BM bone marrow, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell, NHDF normal human dermal fibroblast
Fig. 2
Fig. 2
Flow cytometric analysis of mesenchymal markers CD11b–, CD14–, CD29+, CD31–, CD34–, CD44+, CD45–, CD73+, CD90+, CD105+, and HLA-DR– in BM-MSCs (P13), mRNA-iPSC-MSC-YL001 (P14), lenti-iPSC-MSC-A001 (P10). Unstained cells were used as flow cytometry control; 10,000 events were recorded for each sample. Bottom panel: percentage of cells positive for each marker is calculated by normalizing to the unstained cells. A minus value was replaced with “0”. BM bone marrow, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell
Fig. 3
Fig. 3
Characterization of cell proliferation and growth. Analysis of the cell proliferation rate with a Click IT cell proliferation assay in a control cell line (K562, chronic myeloid leukemia-derived cell line), b lenti-iPSC-MSC-YL001 (P7), c mRNA-iPSC-MSC-A001 (P7) and d BM-MSCs (P8). Blue plots represents negative control without EdU incubation. Pink plots represents cells treated with EdU. e Cumulative population doubling assay of the BM-MSCs (P11), miPSC-MSC-YL001 (P11) and lenti-iPSC-MSC-A001 (P8) for a period of 2 weeks. BM bone marrow, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell
Fig. 4
Fig. 4
Comparative analyses of the osteogenic capacity of the iPSC-MSCs and BM-MSCs. a Alizarin Red staining of calcium in the BM-MSCs, the mRNA-iPSC-MSC-YL001, and the lenti-iPSC-MSC-A001 with growth medium or osteogenic medium for 3 weeks. Images are representative of six repeats. In the left corner there is an image from one well of the 96-well plate. Scale bar for the microscopic images is 300 μm. b Quantitative calcium deposition of the BM-MSC, mRNA-iPSC-MSC-YL001, and lenti-iPSC-MSC-A001 cells after 3 weeks in the MSC growth medium or osteogenic medium (n = 8). c Quantitative PCR analysis of the expression of osteogenic genes RUNX2, ALP, COL1A1, and OC in the BM-MSC, mRNA-iPSC-MSC-YL001, lenti-iPSC-MSC-A001 after 3 weeks in the MSC growth medium or osteogenic medium (n = 6). *P < 0.05. BM bone marrow, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell
Fig. 5
Fig. 5
Comparative analysis of the chondrogenic and adipogenic capacities. Toluidine blue staining of cartilaginous extracellular matrix of the BM-MSCs (a and d), the mRNA-iPSC-MSC-YL001 (b and e), and the lenti-iPSC-MSC-A001 (c and f) after 3 weeks in MSC growth medium (a–c) or chondrogenic medium (d–f). Oil-Red O staining of lipids in the BM-MSCs (g and j), the mRNA-iPSC-MSC-YL001 (h and k), and the lenti-iPSC-MSC-A001 (i and l) after 3 weeks in MSC growth medium (g–i) or adipogenic medium (j–l). Arrow heads indicate the cartilaginous extracellular matrix (d–f) and lipids (j–l). Scale bar: a–f, 150 μm; g–l, 300 μm
Fig. 6
Fig. 6
Gene expression analysis of “stemness” genes in the NHDFs, iPSCs (two lines: mRNA-iPSC and lenti-iPSC), BM-MSCs, mRNA-iPSC-MSC-YL001, lenti-iPSC-MSC-A001 and the three derived lineages (osteogenic, chondrogenic, adipogenic). Expression of eleven stemness genes (POU class 5 homeobox 1 (OCT4/ POU5F1), Kruppel-like factor 4 (KLF4), v-myc avian myelocytomatosis viral oncogene homolog (C-MYC), SRY (sex determining region Y)-box 2 (SOX2), growth differentiation factor 3 (GDF3), teratocarcinoma-derived growth factor 1 (CRIPTO/ TDGF1), undifferentiated embryonic cell transcription factor 1 (UTF1), developmental pluripotency associated 4 (DPPA4), DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B), lin-28 homolog A (LIN28A) and spalt-like transcription factor 4 (SALL4)) was analyzed by PCR. a BM-MSCs in growth medium, and after 3 weeks in osteogenic, chondrogenic and adipogenic medium; b NHDF, mRNA-iPSCs, mRNA-iPSC-MSC-YL001 in growth medium, and mRNA-iPSC-MSC-YL001 after 3 weeks in osteogenic, chondrogenic and adipogenic medium; c NHDF, lenti-iPSCs, lenti-iPSC-MSC-A001 in growth medium, and lviPSC-MSC-A001 after 3 weeks in osteogenic, chondrogenic and adipogenic medium. BM bone marrow, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell, NHDF normal human dermal fibroblast
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