Differentiation of Flk-1 positive multipotent adult germline stem cells into endothelial cells in vitro and in vivo

Abstract

Multipotent adult germline stem cells (maGSCs) offer a novel route to cell-based therapy without immune rejection and ethical limitations. The present study showed the generation and expansion of fetal liver kinase-1-positive (Flk-1+) cardiovascular progenitor cells from maGSCs as well as the differentiation of these cells into functional endothelial cells (ECs) in vitro and in vivo. First, we established a culture system that allows maGSC-derived Flk-1+ cells differentiating into functional ECs. These differentiated maGSC-ECs showed a typical endothelial morphology and gene expression patterns. In addition, they continued to proliferate and exhibit angiogenic potential both in vitro and in vivo. Second, we established a culture condition, under which cardiovascular progenitor cells proliferated rapidly and were maintained for at least 15 passages with freeze-thaw cycles. Under this culture condition, approximately 70% of cells retained Flk-1 expression and did not spontaneously differentiate into cardiovascular lineages. They were clonogenic and exhibited angiogenic potential in vitro. Furthermore, they were able to differentiate into all three cardiovascular lineages when cultured on OP9 cells. This is the first study showing that cardiovascular progenitor cells can be expanded and maintained in vitro for long periods. However, the impurity, i.e. the continued existence of pluripotent stem cells, within cultures of both maGSC-ECs and long-term cultured Flk-1+ cardiovascular progenitor cells still limits their usefulness in clinical applications. Thus, future work in this area will have to focus on the purification of both cell types.