Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells

Abstract

We propose a novel method for the efficient production of hematopoietic progenitors from human embryonic stem cells (hESC) via coculture with murine fetal liver-derived stromal cells, in which embryonic hematopoiesis dramatically expands at midgestation. We generated various hematopoietic progenitors in coculture, and this hematopoietic activity was concentrated in cobblestone-like cells derived from differentiated hESC. The cobblestone-like cells mostly expressed CD34 and retained an endothelial cell potential. They also contained hematopoietic colony-forming cells, especially erythroid and multilineage colony-forming cells at high frequency. The multipotential hematopoietic progenitors abundant among the cobblestone-like cells produced almost all types of mature blood cells, including adult-type alpha-globin-expressing erythrocytes and tryptase/chymase double-positive mast cells. These progenitors showed neither the immature properties of ESC nor the potential to differentiate into endoderm and ectoderm at a clonal level. The coculture system developed for hESC can provide a novel source of hematopoietic and blood cells for applications in cellular therapy and drug screening.

Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis

“…1C). Genes for initial development of endothelial/ hematopoietic cells were expressed early in the coculture, as reported in our previous work (8). The expression of ␤-globin could be detected by day 10, consistent with the immunostaining results.…”

“…In a previous study (8), we found that the production of erythroid progenitors from hESCs was greatly enhanced by coculture with mFLSCs. This experimental paradigm provides the opportunity to conduct large-scale investigations of hESC-derived erythropoiesis.…”

“…In our previous report (8), hESC/mFLSC cocultures generated a large number of human hematopoietic progenitors, particularly erythroid and multipotential cells. In the current study, using these clonal erythroid cells, we demonstrated that most hESC-derived E colonies, E bursts, and Mix colonies contained adult-type ␤-globinexpressing erythroid cells, and the percentages of ␤-globin- Progressive maturation of hESC/mFLSC coculture-derived clonal erythroid cells.…”

“…Establishment of Murine FL-Derived Stromal Cells. mFLSCs were prepared by as described (8,10). Briefly, FLs were removed from embryonic day 15 (E15) BCL/ Black 6 mice.…”

“…The in vitro generation of hematopoietic cells from hESCs has been reported by several studies (4)(5)(6)(7). We also reported a method of hESC coculture with mouse FL-derived stromal cells (mFLSCs) that generated a large quantity of hematopoietic progenitors that could give rise to erythroid cells, providing a means to characterize hESC-derived erythropoiesis (8).…”