Fibrinogen Mediates Osteogenic Development from Human Induced Pluripotent Stem Cells

Objectives: Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) show robust differentiation into diverse cell populations, including bone. This makes these pluripotent stem cells optimal to study mediators of early human osteogenesis. The purpose of this study was to examine the osteogenic effect of fibrinogen on iPSC lines derived from human umbilical cord blood. Here, a hESC line engineered to express YFP from the RUNX2 promoter was used to identify culture conditions that mediate osteogenic differentiation. Once optimized with these hESCs, these conditions were tested on iPSCs.
Methods: iPSC cells were plated for either six days or 28 days to assess cellular differentiation. iPSCs were grown in serum-free osteogenic media, with fibrinogen applied 24 hours after plating the cells. After six days, mRNA was analyzed for osteogenic-specific gene expression. Cells plated for 28 days were assessed by protein analysis for mature osteoblastic makers.
Results: These studies demonstrated that fibrinogen has a significant osteogenic effect on iPSCs in serum free conditions. On day 6 of fibrinogen treatment, osteogenic genes were found to be up-regulated relative to undifferentiated iPSCs. The up-regulated genes analyzed include RUNX2 (6.12 (±0.21)), DLX5 (4.5 (±0.1)), and OSTERIX (16.2 (±0.16)). In addition, the pluripotency gene OCT3/4 was down regulated (0.08 (±0.02)). All gene expression was relative to the undifferentiated control iPSCs. Positive and negative control groups were also analyzed. On day 28, the fibrinogen treated iPSCs had made early bone proteins osteopontin and osteocalcin, and showed the presence of bone development and calcium ions using Von Kossa staining.

Conclusions: These findings suggest that fibrinogen plays a vital role in osteogenic differentiation of iPSCs in serum-free conditions. More efficient and defined differentiation of hESCs and iPSCs into bone-forming cells will enhance potential therapeutic applications.