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

doi:10.1186/s13287-015-0137-7

. 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 Zhou³, Shuang Tan^{4

5}, Guangqian Zhou⁶, Lars Aagaard⁷, Lin Xie⁸, Cody Bünger⁹, Lars Bolund¹⁰, Yonglun Luo¹¹

Affiliations

¹ Orthopedic Research Lab, Aarhus University, 8000, Aarhus C, Denmark. KANG.RAN@CLIN.AU.DK.
² Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing, 210028, China. KANG.RAN@CLIN.AU.DK.
³ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. YAZH@BIOMED.AU.DK.
⁴ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. SHUANG.TAN@HUM-GEN.AU.DK.
⁵ Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, 518060, Shenzhen, China. SHUANG.TAN@HUM-GEN.AU.DK.
⁶ Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, 518060, Shenzhen, China. GQZHOU@SZU.EDU.CN.
⁷ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. AAGAARD@BIOMED.AU.DK.
⁸ Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing, 210028, China. XIELIN117@126.COM.
⁹ Orthopedic Research Lab, Aarhus University, 8000, Aarhus C, Denmark. CODYBUNG@RM.DK.
¹⁰ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. BOLUND@BIOMED.AU.DK.
¹¹ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. ALUN@BIOMED.AU.DK.

PMID: 26282538
PMCID: PMC4539932
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 et al. Stem Cell Res Ther. 2015.

. 2015 Aug 18;6(1):144.

doi: 10.1186/s13287-015-0137-7.

Authors

Ran Kang^{1

2}, Yan Zhou³, Shuang Tan^{4

5}, Guangqian Zhou⁶, Lars Aagaard⁷, Lin Xie⁸, Cody Bünger⁹, Lars Bolund¹⁰, Yonglun Luo¹¹

Affiliations

¹ Orthopedic Research Lab, Aarhus University, 8000, Aarhus C, Denmark. KANG.RAN@CLIN.AU.DK.
² Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing, 210028, China. KANG.RAN@CLIN.AU.DK.
³ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. YAZH@BIOMED.AU.DK.
⁴ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. SHUANG.TAN@HUM-GEN.AU.DK.
⁵ Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, 518060, Shenzhen, China. SHUANG.TAN@HUM-GEN.AU.DK.
⁶ Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, 518060, Shenzhen, China. GQZHOU@SZU.EDU.CN.
⁷ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. AAGAARD@BIOMED.AU.DK.
⁸ Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing, 210028, China. XIELIN117@126.COM.
⁹ Orthopedic Research Lab, Aarhus University, 8000, Aarhus C, Denmark. CODYBUNG@RM.DK.
¹⁰ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. BOLUND@BIOMED.AU.DK.
¹¹ Department of Biomedicine, the Health Faculty, Aarhus University, 8000, Aarhus C, Denmark. ALUN@BIOMED.AU.DK.

PMID: 26282538
PMCID: PMC4539932
DOI: 10.1186/s13287-015-0137-7

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**
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**
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**
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**
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**
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**
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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References

1. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41–9. doi: 10.1038/nature00870. - DOI - PubMed
1. Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008;180:2581–7. doi: 10.4049/jimmunol.180.4.2581. - DOI - PubMed
1. Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials. 2005;26:5158–66. doi: 10.1016/j.biomaterials.2005.01.002. - DOI - PubMed
1. Kang R, Luo Y, Zou L, Xie L, Lysdahl H, Jiang X, et al. Osteogenesis of human induced pluripotent stem cells derived mesenchymal stem cells on hydroxyapatite contained nanofibers. RSC Adv. 2014;4:5734–9. doi: 10.1039/c3ra44181d. - DOI
1. Meinel L, Karageorgiou V, Fajardo R, Snyder B, Shinde-Patil V, Zichner L, et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng. 2004;32:112–22. doi: 10.1023/B:ABME.0000007796.48329.b4. - DOI - PubMed

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[1] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41–9. doi: 10.1038/nature00870. - DOI - PubMed

[2] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41–9. doi: 10.1038/nature00870. - DOI - PubMed

[3] Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008;180:2581–7. doi: 10.4049/jimmunol.180.4.2581. - DOI - PubMed

[4] Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008;180:2581–7. doi: 10.4049/jimmunol.180.4.2581. - DOI - PubMed

[5] Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials. 2005;26:5158–66. doi: 10.1016/j.biomaterials.2005.01.002. - DOI - PubMed

[6] Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials. 2005;26:5158–66. doi: 10.1016/j.biomaterials.2005.01.002. - DOI - PubMed

[7] Kang R, Luo Y, Zou L, Xie L, Lysdahl H, Jiang X, et al. Osteogenesis of human induced pluripotent stem cells derived mesenchymal stem cells on hydroxyapatite contained nanofibers. RSC Adv. 2014;4:5734–9. doi: 10.1039/c3ra44181d. - DOI

[8] Kang R, Luo Y, Zou L, Xie L, Lysdahl H, Jiang X, et al. Osteogenesis of human induced pluripotent stem cells derived mesenchymal stem cells on hydroxyapatite contained nanofibers. RSC Adv. 2014;4:5734–9. doi: 10.1039/c3ra44181d. - DOI

[9] Meinel L, Karageorgiou V, Fajardo R, Snyder B, Shinde-Patil V, Zichner L, et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng. 2004;32:112–22. doi: 10.1023/B:ABME.0000007796.48329.b4. - DOI - PubMed

[10] Meinel L, Karageorgiou V, Fajardo R, Snyder B, Shinde-Patil V, Zichner L, et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng. 2004;32:112–22. doi: 10.1023/B:ABME.0000007796.48329.b4. - DOI - PubMed

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

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

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