Skip to main content

Advertisement

SpringerLink
Log in

Low-density expansion protects human synovium-derived stem cells from replicative senescence: a preliminary study

  • Research Article
  • Published:
Drug Delivery and Translational Research Aims and scope Submit manuscript

Abstract

Our hypothesis in this study is that low seeding density expansion could retain human synovium-derived stem cell (hSDSC) “stemness”, defined as higher proliferation and multi-differentiation capacity; retention of “stemness” probably occurs through the mitogen-activated protein kinase (MAPK) signaling pathway. hSDSCs were expanded in conventional plastic flasks for two consecutive passages at either low or high density (30 or 3,000 cells/cm2). Expanded cells were assessed for the effect of seeding density on their morphology, proliferation, apoptosis, stem cell surface markers, and multi-lineage differentiation capacity (chondrogenic, adipogenic, and osteogenic differentiation) using flow cytometry, biochemical analysis, histology, immunostaining, and real-time polymerase chain reaction. The MAPK signaling pathway (Erk1/2, p38, and JNK) and senescence-associated markers (p21 and caveolin) were also evaluated for their role in cell density-based monolayer expansion using western blot. Our data suggested that low seeding density expansion yielded hSDSCs with enhanced proliferation and multi-differentiation capacity compared to those grown at high seeding density, despite the fact that the cells expanded at both high and low density had lower osteogenic capacity. Low seeding density also down-regulated Erk1/2 and JNK expression and up-regulated p38 expression, which might be responsible for the retained “stemness” in the cells expanded at low density. Low seeding density expansion could retain hSDSC proliferation and multi-differentiation capacity and protect cells from replicative senescence.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Harris JD, Siston RA, Pan X, Flanigan DC. Autologous chondrocyte implantation: a systematic review. J Bone Joint Surg Am. 2010;92(12):2220–33.

    Article  PubMed  Google Scholar 

  2. Csaki C, Schneider PR, Shakibaei M. Mesenchymal stem cells as a potential pool for cartilage tissue engineering. Ann Anat. 2008;190(5):395–412.

    Article  PubMed  CAS  Google Scholar 

  3. De Bari C, Dell’Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum. 2001;44(8):1928–42.

    Article  PubMed  Google Scholar 

  4. Estes BT, Diekman BO, Guilak F. Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells. Biotechnol Bioeng. 2008;99(4):986–95.

    Article  PubMed  CAS  Google Scholar 

  5. Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I. Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol. 2008;36(9):1176–85.

    Article  PubMed  Google Scholar 

  6. Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 2000;97(7):3213–8.

    Article  PubMed  CAS  Google Scholar 

  7. Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA. 2001;98(14):7841–5.

    Article  PubMed  CAS  Google Scholar 

  8. Jones B, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage tissue engineering and regeneration. Tissue Eng Part B Rev. 2012;18(4):301–11.

    Google Scholar 

  9. Kurth TB, Dell’accio F, Crouch V, Augello A, Sharpe PT, De Bari C. Functional mesenchymal stem cell niches in the adult knee joint synovium in vivo. Arthritis Rheum. 2011;63(5):1289–300.

    Article  PubMed  Google Scholar 

  10. Pei M, He F, Boyce BM, Kish VL. Repair of full-thickness femoral condyle cartilage defects using allogeneic synovial cell-engineered tissue constructs. Osteoarthritis Cartilage. 2009;17(6):714–22.

    Article  PubMed  CAS  Google Scholar 

  11. Pei M, He F, Vunjak-Novakovic G. Synovium-derived stem cell-based chondrogenesis. Differentiation. 2008;76(10):1044–56.

    Article  PubMed  CAS  Google Scholar 

  12. Pei M, He F, Kish V, Vunjak-Novakovic G. Engineering of functional cartilage tissue using stem cells from synovial lining: a preliminary study. Clin Orthop Relat Res. 2008;466(8):1880–9.

    Article  PubMed  Google Scholar 

  13. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005;52(8):2521–9.

    Article  PubMed  Google Scholar 

  14. Segawa Y, Muneta T, Makino H, Nimura A, Mochizuki T, Ju YJ, et al. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res. 2009;27(4):435–41.

    Article  PubMed  CAS  Google Scholar 

  15. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001;22(2):153–83.

    Article  PubMed  CAS  Google Scholar 

  16. Li J, He F, Pei M. Creation of an in vitro microenvironment to enhance human fetal synovium-derived stem cell chondrogenesis. Cell Tissue Res. 2011;345(3):357–65.

    Article  PubMed  Google Scholar 

  17. Pei M, He F, Kish VL. Expansion on extracellular matrix deposited by human bone marrow stromal cells facilitates stem cell proliferation and tissue-specific lineage potential. Tissue Eng Part A. 2011;17(23–24):3067–76.

    Article  PubMed  CAS  Google Scholar 

  18. Li JT, Pei M. Cell senescence: a challenge in cartilage engineering and regeneration. Tissue Eng Part B. 2012;18(4):270–87.

    Google Scholar 

  19. Csaszar E, Kirouac DC, Yu M, Wang W, Qiao W, Cooke MP, et al. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell. 2012;10(2):218–29.

    Article  PubMed  CAS  Google Scholar 

  20. Pei M, Li JT, Shoukry M, Zhang Y. A review of decellularized stem cell matrix: a novel cell expansion system for cartilage tissue engineering. Eur Cell Mater. 2011;22:333–43.

    PubMed  CAS  Google Scholar 

  21. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells. 2002;20(6):530–41.

    Article  PubMed  Google Scholar 

  22. Ben-Ze’ev A, Farmer SR, Penman S. Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts. Cell. 1980;21(2):365–72.

    Article  PubMed  Google Scholar 

  23. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell. 2004;6(4):483–95.

    Article  PubMed  CAS  Google Scholar 

  24. Chang TC, Chen YC, Yang MH, Chen CH, Hsing EW, Ko BS, et al. Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner. PLoS One. 2010;5(2):e9187.

    Article  PubMed  Google Scholar 

  25. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol. 1999;107(2):275–81.

    Article  PubMed  CAS  Google Scholar 

  26. Bobick BE, Kulyk WM. Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling. Birth Defects Res C Embryo Today. 2008;84(2):131–54.

    Article  PubMed  CAS  Google Scholar 

  27. Lim IK, Won Hong K, Kwak IH, Yoon G, Park SC. Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts. Mech Ageing Dev. 2000;119(3):113–30.

    Article  PubMed  CAS  Google Scholar 

  28. Oh CD, Chang SH, Yoon YM, Lee SJ, Lee YS, Kang SS, et al. Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes. J Biol Chem. 2000;275(8):5613–9.

    Article  PubMed  CAS  Google Scholar 

  29. Weston AD, Chandraratna RA, Torchia J, Underhill TM. Requirement for RAR-mediated gene repression in skeletal progenitor differentiation. J Cell Biol. 2002;158(1):39–51.

    Article  PubMed  CAS  Google Scholar 

  30. Bobick BE, Kulyk WM. MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis. Exp Cell Res. 2006;312(7):1079–92.

    Article  PubMed  CAS  Google Scholar 

  31. Fu L, Tang T, Miao Y, Zhang S, Qu Z, Dai K. Stimulation of osteogenic differentiation and inhibition of adipogenic differentiation in bone marrow stromal cells by alendronate via ERK and JNK activation. Bone. 2008;43(1):40–7.

    Article  PubMed  CAS  Google Scholar 

  32. Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem. 2000;275(13):9645–52.

    Article  PubMed  CAS  Google Scholar 

  33. Chiu LH, Yeh TS, Huang HM, Lu SJ, Yang CB, Tsai YH. Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells. J Cell Physiol. 2011;227(6):2412–20.

    Article  Google Scholar 

  34. Park JS, Kim HY, Kim HW, Chae GN, Oh HT, Park JY, et al. Increased caveolin-1, a cause for the declined adipogenic potential of senescent human mesenchymal stem cells. Mech Ageing Dev. 2005;126(5):551–9.

    Article  PubMed  CAS  Google Scholar 

  35. Park WY, Park JS, Cho KA, Kim DI, Ko YG, Seo JS, et al. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. J Biol Chem. 2000;275(27):20847–52.

    Article  PubMed  CAS  Google Scholar 

  36. Galbiati F, Volonte D, Liu J, Capozza F, Frank PG, Zhu L, et al. Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism. Mol Biol Cell. 2001;12(8):2229–44.

    PubMed  CAS  Google Scholar 

  37. Yew TL, Chiu FY, Tsai CC, Chen HL, Lee WP, Chen YJ, et al. Knockdown of p21(Cip1/Waf1) enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells. Aging Cell. 2011;10(2):349–61.

    Article  PubMed  CAS  Google Scholar 

  38. Plasilova M, Schonmeyr B, Fernandez J, Clavin N, Soares M, Mehrara BJ. Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21. Plast Reconstr Surg. 2009;123(2 Suppl):149S–57.

    Article  PubMed  CAS  Google Scholar 

  39. Li JT, Pei M. Optimization of an in vitro three-dimensional microenvironment to reprogram synovium-derived stem cells for cartilage tissue engineering. Tissue Eng Part A. 2011;17:703–12.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Suzanne Smith for editing the manuscript. This study was supported by a faculty start-up fund from West Virginia University.

Conflict of interest

We declare no conflict of interest in relation with this paper.

Declaration of ethical standards

The experiments in this work comply with the current laws of the USA.

Author information

Authors and Affiliations

  1. Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, P.O. Box 9196, One Medical Center Drive, Morgantown, WV, 26506-9196, USA

    Jingting Li, Brendan Jones, Ying Zhang & Ming Pei

  2. Division of Exercise Physiology, West Virginia University, Morgantown, WV, 26506, USA

    Jingting Li & Ming Pei

  3. Mechanical and Aerospace Engineering, West Virginia University, Morgantown, WV, 26506, USA

    Ying Zhang & Ming Pei

  4. School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

    Tatiana Vinardell

Authors
  1. Jingting Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brendan Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatiana Vinardell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ming Pei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ming Pei.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, J., Jones, B., Zhang, Y. et al. Low-density expansion protects human synovium-derived stem cells from replicative senescence: a preliminary study. Drug Deliv. and Transl. Res. 2, 363–374 (2012). https://doi.org/10.1007/s13346-012-0094-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13346-012-0094-y

Keywords

Search

Navigation