Abstract
BackgroundDifferentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.Methodology/Principal FindingsESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34+ cells. ESC-derived CD34+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34− cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Conclusions/SignificanceShort-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.
Highlights
Embryonic stem cells (ESC) are derived from the inner cell mass of the pre-implantation blastocyst [1]
Two studies showed that human induced pluripotent stem cells (iPSC) could be differentiated toward hematopoiesis [15,16], as for earlier studies with embryonic stem cells (ESC), experiments were performed in bovine serum-supplemented cocultures, in the presence of murine stroma
We show that human ESC and iPSC maintained over mitotically-inactivated human foreskin fibroblasts (HFF) [14] can differentiate toward the hematopoietic lineage in fully defined medium devoid of animal proteins when sequentially exposed to recombinant bone morphogenic protein-4 (BMP-4), a factor known to enhance mesodermal differentiation of human ESC in vitro [17,18,19], and to a cocktail of early acting hematopoietic factors comprising FMS-like tyrosin kinase-3 ligand (FLT3-L), stem cell factor (SCF), thrombopoietin (TPO) and vascular endothelial growth factor (VEGF)
Summary
Embryonic stem cells (ESC) are derived from the inner cell mass of the pre-implantation blastocyst [1]. Several studies have already identified defined culture conditions for directing ESC differentiation toward the mesodermal hematopoietic lineage [2,3,4,5,6], and terminally differentiated erythrocytes and leukocytes have been generated through these strategies [2,3,7,8,9]. These studies used mouse embryonic fibroblasts as feeder support and poorly defined xenogenic compounds including serum prior to lineage-specific differentiation. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells
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