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Towards Optimal Growth Conditions for MSCs

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"Effects of Medium Supplements on Proliferation, Differentiation Potential, and In Vitro Expansion of Mesenchymal Stem Cells"

Mesenchymal stem cells (MSCs) are multipotent, self-renewing cells with the capacity to differentiate into cells of great therapeutic value (Pitteneger et al.) and are also relatively easy to isolate and cultivate in vitro. However, this long term growth may be detrimental to their overall therapeutic value and therefore optimal growth conditions are being sought. Towards this goal Gharibi and Hughes from King's College London, United Kingdom, in a study published in Stem Cells Translational Medicine, have carried out a comprehensive investigation into the effects of cytokines on the cultivation of MSCs in vitro and demonstrate that medium supplemented with fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)- BB, ascorbic acid (AA), and epidermal growth factor (EGF) leads to the enhancement of the in vitro expansion capacity of MSC cultures.

Initial experiments analysed the long term growth of purchased primary bone marrow-derived MSCs in media with differing combinations of AA, EGF, FGF-2, IL (Interleukin)-6, PDGF-BB, transferrin, and Wnt3a. Individually, FGF-2, AA, PDGF-BB, EGF, and Wnt3a mediated an increased proliferation rate and increased cell doublings over the first 4 passages after expansion following thawing and plating. These supplements led to a greater than 1,000-fold increase in total cell numbers compared with controls (a-Minimal Essential Medium, Glutamax and fetal bovine serum). However, FGF-2 combined with AA and/or PDGF-BB further increased the proliferation rate and cell doublings. Analysis of positive (CD105, CD90, CD44, CD71, and CD146) and negative (CD34 and CD45) MSC cell surface markers and under control conditions found that CD90 and CD44 remained constant during the culture periods observed under all conditions, while the negative markers stayed negative. However, MSCs with FGF-2 exhibited significantly reduced levels of CD146 and Alkaline phosphatase (ALP) at every passage, while addition of AA, EGF or PDGF-BB separately also led to a reduction in CD146 expression, but ALP was enhanced in MSCs cultured with AA, EGF or PDGF-BB.

Osteoblastic or adipocytic differentiation was analysed by examination of mRNA markers and the accumulation of calcium deposits and lipid droplets. MSCs at the initial passage were optimal for differentiation, and reduced significantly with increasing passage number. While IL-6, transferrin and PDGF-BB had little effect, the other cytokines elicited a variety of responses. FGF-2 addition led to an increase in the expression of the osteogenic marker Runx-2 but also a decrease in ALP expression, also associated with osteogenesis. No other alteration in osteogenic gene expression was noted during differentiation. AA addition led to increased Runx-2 and ALP but upon differentiation Runx-2 expression was reduced. EGF also reduced ALP but had no effect on osteogenesis; however addition of Wnt3a reduced osteogenic potential as observed by a reduction in Runx-2 and ALP. FGF-2 addition enhanced the expression of PPAR-g, which controls adipogenesis, but not other adipogenic markers (CEBP-a or FAB-4) but during differentiation FGF-2 or PDGF-BB treated cells showed a reduction in adipogenesis. AA addition reduced adipogenic marker expression and inhibited differentiation, while cells grown in AA, EGF, FGF-2, PDGF-BB, and Wnt3a were also inhibited with regards to adipogenesis.

Finally, the effect of growth factors in maintenance of the stem cell phenotype (OCT4, NANOG, KLF4 and TERT), cell cycle (CDK2, CDK4, Cyclin D and E), senescence (P16, P21, RB2), and DNA repair (POLD3, ERCC1, and MRE11A) was studied through mRNA analysis. While MSCs did not express SOX2 or TERT, they did express NANOG, OCT4 and KLF4, although the level decreased with increasing passage number. Addition of FGF-2 and AA enhanced OCT4 and KLF4 expression at first passage while addition of AA was also associated with an increase in CDK2, while CDK4 was highest in FGF-2 and EGF treated cells. All cell cycle genes saw a reduction in expression over time, while P16 expression increased; an effect which was enhanced in the presence of FGF-2, PDGF-BB, and AA at the last passage (passage 10), while AA induced P21 and RB2 also at the last passage. POLD3 was expressed at a higher level in AA, EGF, FGF-2, and PDGF-BB treated cells while AA treatment led to an increase in ERCC2 and MRE11A.

These data represent an interesting attempt to delineate the effect of several combinations of factors on the growth of adult stem cells during long term exposure. The study also shows a definite link between the loss of differentiation capacity of MSCs and long-term growth in vitro as cells move towards replicative senescence (Bonab et al.). Additionally, the lack of change in cell surface markers as differentiation potential is lost suggests that while these may useful for cell sorting, they do not give a readout of the functionality of MSCs. Finally the authors note that a potential limitation of this study was the use of commercially obtained MSCs rather than primary sources, although it seems certain that FGF-2, AA, PDGFBB, and EGF supplementation is highly beneficial for achieving large-scale production of MSCs.

 

References

Pittenger MF et al.
Multilineage potential of adult human mesenchymal stem cells.
Science 1999;284:143–147.

Bonab MM et al.
Aging of mesenchymal stem cell in vitro.
BMC Cell Biol 2006;7:14.

 

 

Study originally appeared in Stem Cells Translational Medicine.

Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.