Cytokine Stimulation for Large Scale Expansion of MSCs In Vitro.

Methods: Human MSCs were grown in continuous culture in the presence of Ascorbic Acid (AA), EGF, FGF-2, IL-6, PDGF-BB, transferrin or Wnt3a. Cell number was counted at each passage and changes in stem cell surface markers, gene expression and differentiation potential was determined by flow cytometry, qRT-PCR, Alizarin red and Oil red O staining, until cells reached senescence.

Results: Cultures were maintained for up to 8 passages before reaching senescence. Cells grown in the presence of AA, EGF, FGF-2, PDGF-BB and Wnt3a all showed enhanced proliferation and total cell doublings were estimated as up to 21, 16, 24, 22 and 17 respectively in comparison to 11 in culture medium alone. MSCs were positive (90-100%) for CD105, CD90, CD44, CD71 and CD146 and remained negative for CD34 and CD45 throughout. They were able to give rise to cells of osteoblast and adipocyte lineages, although their differentiation potential was decreased at late passages. Cells expanded with FGF-2 showed reduced expression of CD146 and alkaline phosphatase (ALP), while AA and EGF induced ALP. Interestingly FGF-2 and AA induced Runx-2 expression. Long term culture with AA, EGF, FGF-2 and PDGF-BB resulted in reduction of mineralisation. Culture with FGF-2 and AA also reduced the expression of adipogenic genes PPARγ, CEBPα and FAB-4 and lipid accumulation.

Conclusion: Supplementing MSC cultures, particularly with FGF-2, markedly increased proliferative potential whilst preserving undifferentiated phenotype and differentiation potentials. However, late stage cultures showed some loss of stem cell features.