Thrombopoietin Enhances Generation of CD34+ Cells from Human Embryonic Stem Cells

• Published in: Stem cells (Dayton, Ohio) Ohio), 2007-06, Vol.25 (6), p.1456-1461
• Main Authors: Srivastava, Anand S., Nedelcu, Elena, Esmaeli‐Azad, Babak, Mishra, Rangnath, Carrier, Ewa
• Format: Article
• Language: English
• Subjects: Antigens, CD34 - metabolism; CD31+ progenitors; CD34+ progenitors; Cell Differentiation - drug effects; Cells, Cultured; Embryonic Stem Cells - cytology; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Gene Expression Regulation - drug effects; Hematopoietic progenitors; Hematopoietic Stem Cells - cytology; Human embryonic stem cells; Humans; Primates; Receptors, Thrombopoietin - genetics; Receptors, Thrombopoietin - metabolism; Receptors, Vascular Endothelial Growth Factor - genetics; Receptors, Vascular Endothelial Growth Factor - metabolism; Thrombopoietin - pharmacology; Thrombopoietin - physiology
• ISSN: 1066-5099; 1549-4918
• DOI: 10.1634/stemcells.2006-0701

The role of thrombopoietin (TPO) in adult hematopoiesis is well‐established. A recent report suggests that TPO and vascular endothelial growth factor (VEGF) play a role in promoting formation of early erythropoietic progenitors in a nonhuman primate embryonic stem cell (ES) model. No such report exists for human ES cells as yet. Because 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 an EB‐derived culture system. The emphasis of this work was to demonstrate the molecular mechanisms involved in this process, specifically the role of c‐mpl and its ligand TPO. Human ES cells were cultured to the EB state, and EB‐derived secondary cultures supporting hematopoietic differentiation were established: condition 1, control (stem cell factor [SCF] and Flt3 ligand [Flt3L]); condition 2, SCF, Flt3L, and TPO; and condition 3, SCF, Flt3L, TPO, and VEGF. Cells were harvested daily, starting at day 2 and continuing until day 8, for reverse transcription‐polymerase chain reaction and Western blot. There was no evidence of expression of c‐mpl and VEGF receptor on the gene or protein level until day 8, when the formation of well‐established hematopoietic colonies began. This correlated with the formation of CD34+/CD31− negative progenitors, mostly found in blast‐forming units‐erythroid‐like colonies. We concluded that TPO and VEGF play an important synergistic role in the formation of early ES‐derived hematopoietic progenitors that occurs through the c‐mpl and VEGF receptors. Disclosure of potential conflicts of interest is found at the end of this article.