Advertisement

Molecular and Cellular Biochemistry

, Volume 401, Issue 1–2, pp 155–164 | Cite as

The effect of extended passaging on the phenotype and osteogenic potential of human umbilical cord mesenchymal stem cells

  • Zhe Shi
  • Liang ZhaoEmail author
  • Gengtao Qiu
  • Ruixuan He
  • Michael S. DetamoreEmail author
Article

Abstract

Retaining biological characteristics in the extended passaging is crucial for human umbilical cord mesenchymal stem cells (hUCMSCs) in tissue engineering. We aimed to assess morphology, viability, MSC marker expression, and osteogenic activity of hUCSMCs after extended passaging. Passages 4 (P4) and 16 (P16) hUCMSCs displayed similar morphology and viability. The flow cytometry results showed that CD73, CD90, and CD105 were highly expressed at P1–P16. CD166 expression decreased progressively from 90 % at P2 to 61.5 % at P5 (p < 0.05), followed by stable expression through P16. Results from calcium deposition alkaline phosphatase activity and RT-PCR assay showed that both P4 and P16 hUCMSCs differentiated down an osteogenic lineage, with no significant difference in osteogenic capacity (p < 0.05). High-passage UMCSCs maintained stable expression of MSC CD markers as well as stable osteogenic activity. hUCMSCs may thus be suitable for tissue engineering and regenerative medicine applications.

Keywords

Mesenchymal stem cell Human umbilical cord Proliferation Osteogenic differentiation CD166 

Notes

Acknowledgments

We gratefully acknowledge Prof. Zhengliang Chen at the Southern Medical University for FCM assistance and useful discussions. This study was supported by National Natural Science Foundation of China 31328008 (LZ), 31100695(LZ) Natural Science Foundation of Guangdong s20130010014253 (LZ), Guangdong Provincial Science and Technology Project 2012B010200024 (LZ) and Guangzhou Science and Technology Project 2012027(LZ).

Conflict of Interest

None declared.

References

  1. 1.
    Mravic M, Peault B, James AW (2014) Current trends in bone tissue engineering. BioMed Res Int 2014:865270PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Shrivats AR, McDermott MC, Hollinger JO (2014) Bone tissue engineering: state of the union. Drug Discov Today 19:781–786PubMedCrossRefGoogle Scholar
  3. 3.
    Stappenbeck TS, Miyoshi H (2009) The role of stromal stem cells in tissue regeneration and wound repair. Science 324:1666–1669PubMedCrossRefGoogle Scholar
  4. 4.
    Liechty KW, MacKenzie TC, Shaaban AF et al (2000) Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6:1282–1286PubMedCrossRefGoogle Scholar
  5. 5.
    Knight MN, Hankenson KD (2013) Mesenchymal stem cells in bone regeneration. Adv Wound Care 2:306–316CrossRefGoogle Scholar
  6. 6.
    Baba K, Yamazaki Y, Ikemoto S, Aoyagi K, Takeda A, Uchinuma E (2012) Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum. J Cranio-Maxillo-facial Surg 40:768–772CrossRefGoogle Scholar
  7. 7.
    Jiang Y, Jahagirdar BN, Reinhardt RL et al (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41–49PubMedCrossRefGoogle Scholar
  8. 8.
    Roberts SJ, Owen HC, Tam WL et al (2014) Humanized culture of periosteal progenitors in allogeneic serum enhances osteogenic differentiation and in vivo bone formation. Stem Cells Transl Med 3:218–228PubMedCrossRefGoogle Scholar
  9. 9.
    Koga H, Muneta T, Ju YJ et al (2007) Synovial stem cells are regionally specified according to local microenvironments after implantation for cartilage regeneration. Stem Cells 25:689–696PubMedCrossRefGoogle Scholar
  10. 10.
    Romagnoli C, Brandi ML (2014) Adipose mesenchymal stem cells in the field of bone tissue engineering. World J Stem Cells 6:144–152PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    de Villiers JA, Houreld N, Abrahamse H (2009) Adipose derived stem cells and smooth muscle cells: implications for regenerative medicine. Stem Cell Rev 5:256–265PubMedCrossRefGoogle Scholar
  12. 12.
    Derubeis AR, Cancedda R (2004) Bone marrow stromal cells (BMSCs) in bone engineering: limitations and recent advances. Ann Biomed Eng 32:160–165PubMedCrossRefGoogle Scholar
  13. 13.
    Mueller SM, Glowacki J (2001) Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem 82:583–590PubMedCrossRefGoogle Scholar
  14. 14.
    Katsara O, Mahaira LG, Iliopoulou EG et al (2011) Effects of donor age, gender, and in vitro cellular aging on the phenotypic, functional, and molecular characteristics of mouse bone marrow-derived mesenchymal stem cells. Stem Cells Dev 20:1549–1561PubMedCrossRefGoogle Scholar
  15. 15.
    Baksh D, Yao R, Tuan RS (2007) Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 25:1384–1392PubMedCrossRefGoogle Scholar
  16. 16.
    Can A, Karahuseyinoglu S (2007) Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells 25:2886–2895PubMedCrossRefGoogle Scholar
  17. 17.
    Friedman R, Betancur M, Boisse L, Tuncer H, Cetrulo C, Klingemann H (2007) Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation. Biol Blood Marrow Transpl 13:1477–1486CrossRefGoogle Scholar
  18. 18.
    Wang L, Seshareddy K, Weiss ML, Detamore MS (2009) Effect of initial seeding density on human umbilical cord mesenchymal stromal cells for fibrocartilage tissue engineering. Tissue Eng Part A 15:1009–1017PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Wang L, Tran I, Seshareddy K, Weiss ML, Detamore MS (2009) A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A 15:2259–2266PubMedCrossRefGoogle Scholar
  20. 20.
    Wang HS, Hung SC, Peng ST et al (2004) Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 22:1330–1337PubMedCrossRefGoogle Scholar
  21. 21.
    Weiss ML, Troyer DL (2006) Stem cells in the umbilical cord. Stem Cell Rev 2:155–162PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Chelluboina B, Klopfenstein JD, Pinson DM, Wang DZ, Veeravalli KK (2014) Stem cell treatment after cerebral ischemia regulates the gene expression of apoptotic molecules. Neurochem Res 39:1511–1521PubMedCrossRefGoogle Scholar
  23. 23.
    Bharathiraja C, Sukirtha R, Krishnan M, Achiraman S (2014) Interaction of Wharton’s Jelly derived fetal mesenchymal cells with tumor cells. Curr Stem Cell Res Ther 9:504–507PubMedCrossRefGoogle Scholar
  24. 24.
    Nagamura-Inoue T, He H (2014) Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility. World J Stem Cells 6:195–202PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE (2005) Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 23:220–229PubMedCrossRefGoogle Scholar
  26. 26.
    Fu YS, Cheng YC, Lin MY et al (2006) Conversion of human umbilical cord mesenchymal stem cells in Wharton’s jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism. Stem Cells 24:115–224PubMedCrossRefGoogle Scholar
  27. 27.
    Karahuseyinoglu S, Cinar O, Kilic E et al (2007) Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells 25:319–331PubMedCrossRefGoogle Scholar
  28. 28.
    Weiss ML, Anderson C, Medicetty S et al (2008) Immune properties of human umbilical cord Wharton’s jelly-derived cells. Stem Cells 26:2865–2874PubMedCrossRefGoogle Scholar
  29. 29.
    Mahaira LG, Katsara O, Pappou E et al (2014) IGF2BP1 expression in human mesenchymal stem cells significantly affects their proliferation and is under the epigenetic control of TET1/2 demethylases. Stem Cells Dev 23:2501–2512PubMedCrossRefGoogle Scholar
  30. 30.
    Vidal MA, Walker NJ, Napoli E, Borjesson DL (2012) Evaluation of senescence in mesenchymal stem cells isolated from equine bone marrow, adipose tissue, and umbilical cord tissue. Stem Cells Dev 21:273–283PubMedCrossRefGoogle Scholar
  31. 31.
    Scheers I, Lombard C, Paganelli M et al (2013) Human umbilical cord matrix stem cells maintain multilineage differentiation abilities and do not transform during long-term culture. PLoS ONE 8:e71374PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Corominas H, Clayburne G, Diaz-Lopez C, Schumacher HR (2007) Apatite crystal identification in dried smears and synovial fluid pellets with alizarin red staining. Clin Exp Rheumatol 25:935PubMedGoogle Scholar
  33. 33.
    Kawanishi M, Oura A, Furukawa K et al (2007) Redifferentiation of dedifferentiated bovine articular chondrocytes enhanced by cyclic hydrostatic pressure under a gas-controlled system. Tissue Eng 13:957–964PubMedCrossRefGoogle Scholar
  34. 34.
    Wang X, Phelan SA, Petros C et al (2004) Peroxiredoxin 6 deficiency and atherosclerosis susceptibility in mice: significance of genetic background for assessing atherosclerosis. Atherosclerosis 177:61–70PubMedCrossRefGoogle Scholar
  35. 35.
    Hao H, Chen G, Liu J et al (2013) Culturing on Wharton’s jelly extract delays mesenchymal stem cell senescence through p53 and p16INK4a/pRb pathways. PLoS ONE 8:e58314PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Otte A, Bucan V, Reimers K, Hass R (2013) Mesenchymal stem cells maintain long-term in vitro stemness during explant culture. Tissue Eng Part C Methods 19:937–948PubMedCrossRefGoogle Scholar
  37. 37.
    Gentleman E, Swain RJ, Evans ND et al (2009) Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation. Nat Mater 8:763–770PubMedCrossRefGoogle Scholar
  38. 38.
    Nagai A, Kim WK, Lee HJ et al (2007) Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow. PLoS ONE 2:e1272PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Pittenger M (2009) Sleuthing the source of regeneration by MSCs. Cell Stem Cell 5:8–10PubMedCrossRefGoogle Scholar
  40. 40.
    Sarugaser R, Hanoun L, Keating A, Stanford WL, Davies JE (2009) Human mesenchymal stem cells self-renew and differentiate according to a deterministic hierarchy. PLoS ONE 4:e6498PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Zucconi E, Vieira NM, Bueno DF et al (2010) Mesenchymal stem cells derived from canine umbilical cord vein–a novel source for cell therapy studies. Stem Cells Dev 19:395–402PubMedCrossRefGoogle Scholar
  42. 42.
    Troyer DL, Weiss ML (2008) Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 26:591–599PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    van Kempen LC, Nelissen JM, Degen WG et al (2001) Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction. J Biol Chem 276:25783–25790PubMedCrossRefGoogle Scholar
  44. 44.
    Bruder SP, Ricalton NS, Boynton RE et al (1998) Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res 13:655–663PubMedCrossRefGoogle Scholar
  45. 45.
    Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22:675–682PubMedCrossRefGoogle Scholar
  46. 46.
    Vacanti V, Kong E, Suzuki G, Sato K, Canty JM, Lee T (2005) Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture. J Cell Physiol 205:194–201PubMedCrossRefGoogle Scholar
  47. 47.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622PubMedCrossRefGoogle Scholar
  48. 48.
    Wang Y, Zhang Z, Chi Y et al (2013) Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death Dis 4:e950PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Orthopaedic Surgery, The Third Affilliated HospitalSouthern Medical UniversityGuangzhouChina
  2. 2.Department of Orthopaedic Surgery, Nanfang HospitalSouthern Medical UniversityGuangzhouChina
  3. 3.Department of Orthopaedic SurgeryShunDe First People HospitalFoshanChina
  4. 4.Department of Chemical & Petroleum EngineeringUniversity of KansasLawrenceUSA

Personalised recommendations