FGF-2 Supplementation Enhances Hematopoietic Differentiation of Rhesus ESC’s.

Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC’s (hESC’s), efforts to induce conclusive hematopoietic differentiation from rhesus ESC’s (rESC’s) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC’s and hESC’s. We recently reported that although rESC’s have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC’s. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC’s in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC’s and rESC’s has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC’s could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC’s were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC’s (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC’s. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.