ALK5 INHIBITORS FOR EFFICIENT DERIVATION OF MESENCHYMAL STEM CELLS FROM HUMAN EMBRYONIC STEM CELLS.

Objectives Successful tissue regeneration requires a clinically viable source of MSC. We explored ALK5 inhibitors to rapidly derive an MSC-like phenotype with high cartilage forming capacity from a xeno-free human embryonic cell line. Methods ESC cell lines (H9 and HADC100) were treated with ALK-5 inhibitor SB431542; HADC100 cells were additionally treated with ALK-5 inhibitors SB525334 or GW788388. Cells were then seeded upon human fibronectin in the presence of FGF in serum free medium. Flow cytometry was used to assess MSC markers (positive for CD73, CD90, CD105; negative for CD34, CD45). Differentiation status was assessed via qPCR. Cartilage forming capacity was determined in high density pellet cultures, in fibrin gels containing extracellular matrix (fibrin-ECM), and after implantation in ex vivo human osteoarthritic cartilage. Gene expression, histology and immunostaining was used to assess cartilage phenotype, tissue regeneration and integration. Results Exposure to all 3 ALK5 inhibitors lead to expression of mesodermal gene markers and differentiation into MSC-like cells (ES-MSC) based on surface marker expression. ES-MSC in pellet cultures or in fibrin-ECM gels expressed high levels of chondrogenic genes: COL2A1, ACAN, COMP; and low levels of COL1A1 and RUNX2. Cell pellets or fibrin constructs implanted into ex vivo human osteoarthritic cartilage defects produced GAG-rich (Safranin O positive) and collagen type II positive neo-cartilage tissues that integrated well with native diseased tissue. Conclusions We developed a protocol for rapid differentiation of xeno-free ESC into MSC-like cells with high cartilage forming capacity with potential for clinical applications.