Generation of Megakaryocytes from Human Embryonic Stem Cells.

The ability to generate multiple cell types from human embryonic stem cells (hESC) in culture offers an unprecedented opportunity to produce an unlimited supply of cells for research and clinical purposes. Megakaryocytes comprise only 0.02–0.05% of the nucleated cell population in the bone marrow, thus making them a difficult cell to isolate and study. The ES cell technology provides a resource for generating megakaryocyte progenitors for in vitro and in vivo analyses. For this approach to be successful, reproducible differentiation schemes that generate sufficient numbers of cells are necessary. With an embryoid body (EB)-based protocol, in serum-free media we are able to generate hematopoietic populations with megakaryocyte potential from two different hESC lines; H1 and HES2. Using CD41 as an early marker of definitive hematopoiesis, we found that 4 to 12% of the day 11–12 EB population expressed high levels of the marker (CD41Hi) whereas 10 to 20% expressed low levels of the marker (CD41Lo). Cells from the CD41Hi but not the CD41Lo population expressed the megakaryocyte marker GPIb. To further analyze these populations, both were isolated from day 12 EBs by cell sorting and cultured in media containing TPO and SCF. Following several days in culture, the CD41Hi population expressed increasing levels of GPIb, as determined by flow cytometry and real time PCR. PF4 was detected in the sorted CD41Hi population and levels increased 10-fold following culture. These cells also adhered to fibrinogen and collagen and stained positive for von Willebrand factor (vWF) and P-selectin. Following activation by PAR1, the cells bound soluble fibrinogen and expressed P-selectin on their surface. Few to no progenitors were detected in the CD41Hi fraction by colony assays suggesting that this population contained cells that were already committed to the megakaryocyte lineage. Incubation of the CD41Lo population of cells in the presence of TPO resulted in 70–80% of the cells expressing increased levels of CD41 and GPIb. Immunostaining revealed vWF and P-selectin expression following adhesion of the cells to fibrinogen and collagen. Both fibrinogen binding and P-selectin surface expression were detected following activation with PAR1. Quantitative PCR analysis demonstrated low levels of PF4 message in the sorted population with an increase of 3-fold following incubation in the presence of TPO. Progenitor cells were detected in colony assays suggesting that this population of cells contained megakaryocyte-like progenitors. Taken together, these finding demonstrate the efficient and reproducible generation of megakaryocytes from hESC using a serum-free EB protocol.