Study of Human Amnion Cell Differentiation in Three-Dimensional Culture in Fibrin Matrix

Aims: Recent investigations by us and others have suggested that human amnion cells may possess an ability to acquire different cell fates, i.e. mesodermal (e.g. osteogenic, chondrogenic, adipogenic) and neural lineage, as well as to turn into insulin-producing beta cells when grown in 2D culture in vitro in specific differentiation media. Here we sought to explore a potential utility of human amnion cell transplants in approaches to bone or cartilage healing. For this purpose, we tested whether human amnion cells can differentiate to mesodermal lineage cells in 3D fibrin wound healing matrix as it would be encountered by cells when transplanted in vivo. Materials and Methods: Human amnion epithelial and mesenchymal cell isolates were characterized by flow cytometry and reverse transcriptase (RT)-PCR for expression of stem cell (SC)-markers as various cell surface markers of differentiated cells. The cell isolates were embedded in 3D matrices prepared from the clinically relevant fibrin glue Tissucol, and cultured for 2 weeks in specific differentiation media (osteogenic, chondrogenic, adipogenic). Differentiation was assessed by measurements of gene expression of marker proteins by RT-PCR, IHC, and biochemical stains. Results: Amnion epithelial and mesenchymal cells express surface markers normally present on embryonic SCs such as SCF, c-Kit/CD117 and Oct-3/4). Our studies of amnion cells embedded 3D fibrin gels show that these cells possess a capability of acquiring the osteogenic and chondrogenic cell fate. Conclusion: Both amnion epithelial and mesenchymal cells can adopt osteogenic and chondrogenic cell fate when cultured in 3D fibrin matrix under osteogenic and chondrogenic growth conditions in vivo. Thus, these cells constitute a potentially valuable cell source for cell-based approaches to bone or cartilage regeneration.