Basic fibroblast growth factor support of human embryonic stem cell self-renewal

Area of Science:

• Stem Cell Biology
• Developmental Biology

Background:

• Human embryonic stem cells (hESCs) are typically cultured with basic fibroblast growth factor (FGF2) on feeder cells or conditioned medium.
• Recent findings suggest high FGF2 concentrations may support hESC culture independently of feeder cells.

Purpose of the Study:

• To evaluate the efficacy of varying FGF2 concentrations in unconditioned medium (UM) for sustaining low-density hESC cultures.
• To determine the optimal FGF2 concentration for long-term hESC maintenance without feeder support.

Main Methods:

• Culturing two hESC lines (H1 and H9) in UM supplemented with FGF2 at concentrations of 4, 24, 40, 80, 100, and 250 ng/ml.
• Assessing culture viability through multiple passages and long-term maintenance (up to 164 population doublings).
• Evaluating pluripotency markers and teratoma formation post-transplantation.

Main Results:

• FGF2 concentrations of 4, 24, and 40 ng/ml were insufficient for sustained hESC culture.
• 100 ng/ml FGF2 in UM supported hESC cultures as effectively as conditioned medium (CM).
• FGF2 degrades faster in UM than CM, necessitating higher concentrations for effective signaling.

Conclusions:

• 100 ng/ml FGF2 in unconditioned medium is sufficient for extensive, feeder-free culture of human embryonic stem cells.
• Fibroblasts may support hESCs by stabilizing FGF signaling, maintaining it above a critical threshold.
• These findings simplify large-scale, routine hESC culture and advance stem cell research.