Tissue Engineering and Regenerative Medicine

, Volume 15, Issue 5, pp 649–659 | Cite as

Characterization of Human Fetal Cartilage Progenitor Cells During Long-Term Expansion in a Xeno-Free Medium

  • Hwal Ran Kim
  • Jiyoung Kim
  • So Ra Park
  • Byoung-Hyun Min
  • Byung Hyune Choi
Original Article



Stem cell therapy requires a serum-free and/or chemically-defined medium for commercialization, but it is difficult to find one that supports long-term expansion of cells without compromising their stemness, particularly for novel stem cells.


In this study, we tested the efficiency of StemPro® MSC SFM Xeno Free (SFM-XF), a serum-free medium, for the long-term expansion of human fetal cartilage-derived progenitor cells (hFCPCs) from three donors in comparison to that of the conventional α-Modified Eagle’s Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS).


We found that SFM-XF supported the expansion of hFCPCs for up to 28–30 passages without significant changes in the doubling time, while α-MEM with 10% FBS showed a rapid increase in doubling time at 10–18 passages depending on the donor. Senescence of hFCPCs was not observed until passage 10 in both media but was induced in approximately 15 and 25% of cells at passage 20 in SFM-XF and α-MEM with 10% FBS, respectively. The colony forming ability of hFCPCs in SFX-XF was also comparable to that in α-MEM with 10% FBS. hFCPCs expressed pluripotency genes like Oct-4, Sox-2, Nanog, SCF, and SSEA4 at passage 20 and 31 in SFM-XF; however, this was not observed when cells were cultured in α-MEM with 10% FBS. The ability of hFCPCs to differentiate into three mesodermal lineages decreased gradually in both media but it was less significant in SFM-XF. Finally we found no chromosomal abnormality after long-term culture of hFCPCs until passage 17 by karyotype analysis.


These results suggest that SFM-XF supports the long-term expansion of hFCPCs without significant phenotypic and chromosomal changes. This study have also shown that hFCPCs can be mass-produced in vitro, proving their commercial value as a novel source for developing cell therapies.


Human fetal cartilage progenitor cells Serum-free medium Cell therapy Pluripotency 



This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI17C2191).

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare regarding this study.

Ethical statement

This study was approved by the institutional review board (IRB) of the Ajou University Medical Center (IRB No.: AJIRB-CRO-16-139).


  1. 1.
    Cooper K, Viswanathan C. Establishment of a mesenchymal stem cell bank. Stem Cells Int. 2011;2011:905621.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zhao K, Liu Q. The clinical application of mesenchymal stromal cells in hematopoietic stem cell transplantation. J Hematol Oncol. 2016;9:46.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Estrada JC, Torres Y, Benguria A, Dopazo A, Roche E, Carrera-Quintanar L, et al. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis. 2013;4:e691.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Turinetto V, Vitale E, Giachino C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy. Int J Mol Sci. 2016. Scholar
  5. 5.
    Rabbani M, Tafazzoli-Shadpour M, Shokrgozar MA, Janmaleki M, Teymoori M. Cyclic stretch effects on adipose-derived stem cell stiffness, morphology and smooth muscle cell gene expression. Tissue Eng Regen Med. 2017;14:279–86.CrossRefGoogle Scholar
  6. 6.
    Osipova EY, Shamanskaya TV, Kurakina OA, Nikitina VA, Purbueva BB, Ustugov AY, et al. Biological characteristics of mesenchymal stem cells during ex vivo expansion. Br J Med Med Res. 2011;1:85–95.CrossRefGoogle Scholar
  7. 7.
    Chun SY, Park GB, Kwon TG, Choi SH. Analysis of stability of human urine derived stem cells during serial subcultures. Tissue Eng Regen Med. 2015;12 Suppl 2:122–31.CrossRefGoogle Scholar
  8. 8.
    Son Y. Recent advances in stem cell researches and their future perspectives in regenerative medicine. Tissue Eng Regen Med. 2017;14:641–2.CrossRefGoogle Scholar
  9. 9.
    Oikonomopoulos A, van Deen WK, Manansala AR, Lacey PN, Tomakili TA, Ziman A, et al. Optimization of human mesenchymal stem cell manufacturing: the effects of animal/xeno-free media. Sci Rep. 2015;13:16570.CrossRefGoogle Scholar
  10. 10.
    Agata H, Watanabe N, Ishii Y, Kubo N, Ohshima S, Yamazaki M, et al. Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions. Biochem Biophys Res Commun. 2009;382:353–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Patrikoski M, Juntunen M, Boucher S, Campbell A, Vemuri MC, Mannerström B, et al. Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy-compliant human adipose stem cells. Stem Cell Res Ther. 2013;4:27.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Usta SN, Scharer CD, Xu J, Frey TK, Nash RJ. Chemically defined serum-free and xeno-free media for multiple cell lineages. Ann Transl Med. 2014;2:97.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Heiskanen A, Satomaa T, Tiitinen S, Laitinen A, Mannelin S, Impola U, et al. N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells. 2007;25:197–202.CrossRefPubMedGoogle Scholar
  14. 14.
    Choi WH, Kim HR, Lee SJ, Jeong N, Park SR, Choi BH, et al. Fetal cartilage-derived cells have stem cell properties and are a highly potent cell source for cartilage regeneration. Cell Transplant. 2016;25:449–61.CrossRefPubMedGoogle Scholar
  15. 15.
    Lee SJ, Oh HJ, Truong MD, Lee KB, Kim J, Kim YJ, et al. Therapeutic possibility of human fetal cartilage-derived progenitor cells in rat arthritis model. Tissue Eng Regen Med. 2015;12 Suppl 2:147–54.CrossRefGoogle Scholar
  16. 16.
    Santos Fd, Andrade PZ, Abecasis MM, Gimble JM, Chase LG, Campbell AM, et al. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods. 2011;17:1201–10.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chase LG, Yang S, Zachar V, Yang Z, Lakshmipathy U, Bradford J, et al. Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells. Stem Cells Transl Med. 2012;1:750–8.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Samsonraj RM, Rai B, Sathiyanathan P, Puan KJ, Rötzschke O, Hui JH, et al. Establishing criteria for human mesenchymal stem cell potency. Stem Cells. 2015;33:1878–91.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mafi P, Hindocha S, Mafi R, Griffin M, Khan WS. Adult mesenchymal stem cells and cell surface characterization—a systematic review of the literature. Open Orthop J. 2011;5 Suppl 2:253–60.CrossRefGoogle Scholar
  20. 20.
    Schrobback K, Wrobel J, Hutmacher DW, Woodfield TB, Klein TJ. Stage-specific embryonic antigen-4 is not a marker for chondrogenic and osteogenic potential in cultured chondrocytes and mesenchymal progenitor cells. Tissue Eng Part A. 2013;19:1316–26.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Amini S, Fathi F, Mobalegi J, Sofimajidpour H, Ghadimi T. The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines. Anat Cell Biol. 2014;47:1–11.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, et al. Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem. 2005;280:24731–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Miyazawa K, Tanaka T, Nakai D, Morita N, Suzuki K. Immunohistochemical expression of four different stem cell markers in prostate cancer: high expression of NANOG in conjunction with hypoxia-inducible factor-1alpha expression is involved in prostate epithelial malignancy. Oncol Lett. 2014;8:985–92.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhang W, Sui Y, Ni J, Yang T. Insights into the Nanog gene: a propeller for stemness in primitive stem cells. Int J Biol Sci. 2016;12:1372–81.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ma Y, Liang D, Liu J, Axcrona K, Kvalheim G, Giercksky KE, et al. Synergistic effect of SCF and G-CSF on stem-like properties in prostate cancer cell lines. Tumour Biol. 2012;33:967–78.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.CrossRefPubMedGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular Science and TechnologyAjou UniversitySuwonKorea
  2. 2.Cell Therapy CenterAjou University Medical CenterSuwonKorea
  3. 3.Department of Physiology and BiophysicsInha University College of MedicineIncheonKorea
  4. 4.Department of Orthopedic Surgery, School of MedicineAjou UniversitySuwonKorea
  5. 5.Department of Biomedical SciencesInha University College of MedicineIncheonKorea

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