Abstract
BackgroundThe production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a ‘spin embryoid body’ method in serum-free differentiation medium.Methodology and Principal FindingsImmunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a+ cells sorted from these differentiation cultures produced 100–800 Mk progenitors at day 13 and 25–160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a+ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34+ and CD45+ cells.ConclusionsWe describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.
Highlights
Pancytopenia and thrombocytopenia remain significant clinical problems for patients with a range of medical conditions, especially those undergoing repeated cycles of chemotherapy as treatment for cancer
We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells
Our laboratory has established protocols whereby homogenous ‘spin embryoid bodies (EBs)’ generated from human embryonic stem cells (hESCs) [9] were supplemented with bone morphogenetic protein (BMP4), stem cell factor (SCF), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) for 10 days (d) to produce hematopoietic progenitors [10]
Summary
Pancytopenia and thrombocytopenia remain significant clinical problems for patients with a range of medical conditions, especially those undergoing repeated cycles of chemotherapy as treatment for cancer. The ability to differentiate hematopoietic lineages from human embryonic stem cells (hESCs) or induced pluripotent stem cells represents a potential alternate supply of megakaryocytes (Mks) and platelets for research and eventually for the treatment of thrombocytopenia. In our current studies we differentiated hESCs in serum-free medium supplemented with growth factor combinations designed to support the generation of Mks from hematopoietic mesoderm. We showed that the Mk progenitors were found in CD41+ and CD34+ cell fractions when assayed at d13 and d20 of differentiation. The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a ‘spin embryoid body’ method in serum-free differentiation medium
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