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

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.

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.

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.

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.

No relevant conflicts of interest to declare.