304 THE DEVELOPMENTAL TOXICITY EVALUATION OF 5-FLUOROURACIL AND INDOMETHACIN IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells have pluripotency and differentiate into and constitute the cells and tissues of our body. In this study, using human embryonic stem cells (hESC), we evaluated novel methods for screening toxicological chemicals during developmental process. We elucidated developmental toxicity of two well-known chemicals, 5-fluorouracil (5-FU) and indomethacin (Indo) in hESC. The undifferentiated hESC were treated with the chemicals (10–4 to 104 µM of 5-FU and Indo) in a dose-dependent manner during 1 to 3 days. Surface markers (SSEA-4, TRA-1-60, and TRA-1-81) expressed only in undifferentiated hESC were monitored by immunocytochemistry to ensure the characterisation of undifferentiated hESC. Moreover, expression of embryonic stem cell-specific genes was assessed with real-time PCR after treatment of 5-FU and Indo (10–2, 100, and102 µM of 5-FU and Indo). The expression of surface markers was not significantly affected by treatment of 5-FU and Indo. The expression of transcription factors (Oct-4, Sox-2, Nanog, and hTERT) was significantly decreased by high concentrations of 5-FU and Indo (102 µM). However, no difference was observed in treatment of low concentration of 5-FU and Indo (10–2 µM). Taken together, these results suggest that 5-FU and Indo have cytotoxic effects, and modulate the expression of transcription factors that have pivotal roles in undifferentiated hESC. Therefore, we suggest that hESC may have potential to test toxicity of chemicals during embryonic developmental stage.

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. Disclosures: No relevant conflicts of interest to declare.

Megakaryocyte Production From Feeder Cell-Free Cultures of Human Embryonic Stem Cells (hESC).

Abstract 2528 Poster Board II-505 Introduction: As the understanding of hESC biology and technology improves, one long-term goal is to utilize expanded numbers of undifferentiated hESC to produce clinically relevant numbers of blood cells in vitro to reduce the need for donors for red blood cell and platelet transfusions. At one end of the production system is the need to create systems for efficient culture of very large numbers of hESC. Methods have been developed using various cytokine combinations to expand end-stage hematopoietic cells from CD34+ cells from bone marrow, peripheral blood, or umbilical cord blood, although little work has been done on megakaryocytopoiesis. Previous systems for bridging the differentiation gap from undifferentiated hESC to mesoderm to hematopoietic progenitor cells have required xenogeneic or in some cases allogeneic supportive cells ± serum at various stages of culture. We have developed a culture system that utilizes feeder cell-free matrix for undifferentiated hESC growth, followed by embryoid body formation, with subsequent shedding of individual hematopoietic progenitor cells that expand and mature to platelet-shedding megakaryocytes. Methods: After extensive evaluations of culture conditions, we arrived at the following optimized conditions. Culture dishes coated with extracellular matrix residual of chemically-digested human foreskin fibroblasts (HFF1 cells from ATCC) support expansion of undifferentiated hESC for more than 30 passages in the presence of DMEM/F12 medium with serum replacement and basic fibroblast growth factor (bFGF). Clusters of cells (∼2 × 105 cells per 2 ml in 6 well plates) from these hESC cultures were transferred to ultra-low attachment culture dishes to form embryoid bodies (EB) in Stemline II medium with (1) basic fibroblast growth factor (bFGF) + vascular endothelial growth factor (VEGF) + bone morphogenic protein (BMP)4 for 2 days; (2) then Stemline II medium with VEGF + BMP4 + thrombopoietin (TPO) + stem cell factor (SCF) + flt3 ligand (FL) for 2 days; (3) then a defined medium with VEGF + BMP4 + TPO + SCF + FL + interleukin (IL)3 + IL6 + IL11 + granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 days; (4) then cells were transferred to single cell suspension in defined medium with GM-CSF + SCF + FL + TPO + IL6 + Heparin for up to 3 additional weeks. Markers of all stages of differentiation from hESC through megakaryocytes were analyzed by immunofluorescence throughout the course of the cultures. Results: Expression of hESC markers Oct4 and Sox2 was decreased by EB day 7 and was undetectable by EB day 10, although SSEA4 expression persisted until EB day 14. The early mesoderm markers brachyury and Mixl1 were barely detectable until EB day 14. Somewhat surprisingly, hemangioblast/hematopoietic progenitor markers, CD34, GATA1, and CD31, were readily detectable in nearly 100% of cells several days earlier than brachyury and Mixl1, by EB day 7. By EB day 14, there was approximately a 10-fold expansion of cell numbers from the beginning of the EB cultures, with large numbers of single almost exclusively mononuclear cells routinely being found in suspension outside of the EBs. Virtually 100% of these cells were strongly CD34+/GATA1+, a majority were brachyury+ and Mixl1+, and the majority of free floating cells, but not cells within the day 14 EBs were mildly positive for the megakaryocytic lineage markers CD41, CD42a, and CD42b, with 5–10% of these cells outside of the EBs being very strongly positive for these megakaryocytic lineage markers. Differentiation toward megakaryocytes progressed from 7 to 14 days after the EB day 14 suspension cells were transferred to new feeder-free, non-adherent culture dishes. Nearly 100% of cells still were CD34+/GATA1+ (decreased intensity compared to EB Day 14), and >80% of cells became CD41+/CD42a+/CD42b+/CD61+/vWF+/PF4+, and many were shedding platelet-like particles by 14 days after transfer from EB cultures. Conclusions: We have developed a multi-stage culture system that, in the presence of appropriate growth factors, supports specific differentiation from hESC to EB to mature megakaryocytes, avoiding any need for xenogeneic or allogeneic feeder cells or serum. Disclosures: No relevant conflicts of interest to declare.