Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Abstract

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells

“…Nevertheless, unlike previous studies based on compound inhibitors or overexpression of genes that interfere with cell cycle regulation in 3T3-L1 cells, we believe that direct inhibition of cell proliferation by transiently downregulating players of the core cell cycle control machinery, the Cdk/Cyclin complexes, offers a more straightforward interpretation of the relationship between cell proliferation and adipogenesis. FGF2 (bFGF) belongs to a large family of FGFs and has long been used in human pluripotent stem cell growth media to maintain cell self-renewal and inhibit differentiation (reviewed in the introduction of Quang et al [49]). Bone marrow-derived MSCs isolated from bFGF -/knockout mice demonstrated increased potency in adipogenic differentiation and reduced potency in osteogenic differentiation compared to wild-type MSCs, indicating that bFGF inhibits adipogenesis [50].…”

Systematic optimization of human pluripotent stem cells media using Design of Experiments

“…For maintaining hPSC in feeder-free system, many groups have claimed best concentration of bFGF at 100 ng/mL 34 35 for over decades and of NRG1β1 at 10 ng/mL 15 . Regular methods to define best concentrations in hPSC field usually starts with random dictates of the concentration developed around the previously known values, followed by the test of each concentration 34 36 . The one with the best readouts, e.g.…”

Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells

“…Detailed procedure can be found in our previously published study [ 89 ]. Briefly, hMSCs at P4 passage were collected and centrifuged at 1000 rpm for five minutes.…”