JAK/STAT Signal Transduction Pathway Activation During Hematopoietic Differentiation From Human Embryonic Stem Cells (hESC)

Abstract 2346

The ability to produce hematopoietic cells from human embryonic stem cells (hESC) has been demonstrated, using different multistage culture systems with multiple growth factor combinations. However, very little is understood about the molecular mechanisms that regulate the differentiation from hESC to hematopoietic stem and progenitor cells and further to specific lineages of differentiated hematopoietic cells. Among many signaling pathways involved in stem and progenitor cell differentiation, the JAK/STAT pathways are known to play critical roles in hematopoietic stem cell maintenance and hematopoietic differentiation. STAT3 activation is known to be essential for maintenance of murine ESC, but not human ESC, but it appears not to play a major role in myeloid cell differentiation. Although different levels of JAK2 and STAT5 signaling are important for erythroid and megakaryocytic differentiation, JAK/STAT signaling is not thought to play a role in hESC maintenance or differentiation and is not known to be essential for early stages of differentiation to hematopoietic stem and progenitor cells (HSC/HPC). We have established a serum-free, feeder cell-free system for maintaining hESC (H1 and H9 cells) and for differentiating the hESC to embryoid bodies (EB), from which end-stage hematopoietic cells, notably megakaryocytes and platelets, are produced. We used a multi-stage culture system to produce megakaryocytes and platelets from EBs, including 2 days with vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP4), 2 more days with VEGF, BMP4, stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO), 10 days with VEGF, BMP4, TPO, SCF, FL, IL3, IL6, and IL11, and 2–6 weeks with TPO, SCF, FL, IL3, IL6, and IL11. We used serial western blots, immunofluorescence with confocal microscopy and systematically observed changes of JAK/STAT signal transduction molecule activation. We found a consistent, dynamic change of STAT5 protein phosphorylation during the hematopoietic differentiation process. Interestingly, although JAK2, STAT3 and STAT5 protein were present, and JAK2 and STAT3 were phosphorylated in hESC, there was no evidence of STAT5 phosphorylation/activation in the undifferentiated hESC. During the early phases of differentiation of hESC-derived EBs toward hematopoietic progenitors in the presence of hematopoiesis-related cytokines, STAT5 was phosphorylated and activated in CD34+ HSCs and in CD61+/CD235a (glycophorin A)+ or CD41+/CD235a+ early megakaryocytic/erythroid progenitor cells (MEP). Although there was no detectable change in total STAT5 protein expression levels through hematopoietic differentiation, there was a slowly progressive decrease in phosphorylated/activated STAT5 with further maturation to megakaryocytes that express CD42b+, platelet factor 4, and von Willebrand factor and form proplatelets and platelets. Thus, in spite of hESC containing abundant phosphorylated JAK2, which is a known activator of STAT5, there was no phosphorylation/activation of STAT5 in undifferentiated hESC or early EBs. However, STAT5 became phosphorylated/activated early in hematopoiesis and declined over the course of progressive differentiation along the megakaryocytic lineage. These findings imply that activated JAK2 does not drive the activation of STAT5 that is an early event in differentiation from EBs and mesoderm to HSC and HPC in vitro.