Thrombopoietin Enhances Generation of CD 34+ Cells from Human Embryonic Stem Cells

¹ Moores UCSD Cancer Center, Department of Medicine, University of California San Diego, San Diego, California
² Department of Pathology, University of California Los Angeles, Los Angeles, California
³ Division of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio

^* To whom correspondence should be addressed. E-mail: ecarrier{at}ucsd.edu.

	Abstract

The role of thrombopoietin (TPO) in adult hematopoiesis is well established. Recent report suggest that TPO and VEGF play a role in promoting formation of early erythropoietic progenitors in a non-human, primate embryonic stem cells (ES) model. No such report exists for human ES cells (hES) as yet. As TPO may become an important factor promoting human ES-cell derived hematopoiesis, we sought to investigate whether TPO in combination with VEGF can enhance human ES-derived hematopoiesis in EB-derived culture system. The emphasis of this work was to demonstrate molecular mechanisms involved in this process, specifically the role of c-mpl and its ligand TPO. Human ES cells were cultured to EB state and EB-derived secondary cultures supporting hematopoietic differentiation were established: 1) control (SCF and Flt3L), 2) SCF, Flt3L and TPO, and 3) SCF, FLT3L, TPO and VEGF. Cells were harvested daily, starting at day 2 until day 8 for RT-PCR and Western Blot. There was no evidence of expression of c-mpl and VEGFR on the gene or protein level until day 8, where the formation of well established hematopoietic colonies begun. This correlated with the formation of CD34+/CD31- negative progenitors, mostly found in BFU-E-like colonies. We concluded that TPO and VEGF play an important synergistic role in the formation of early ES-derived hematopoietic progenitors which occurs through the c-mpl and VEGF receptors.