Abstract
The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.
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Acknowledgments
We would like to thank the generous support from the National Institutes of Health Director’s New Innovator Award 1DP2CA250006-01. Research reported in this publication was also supported by the Flow Cytometry Core grants from the Office of the Director of the National Institutes of Health under Award Number S10OD026959 and NCI Award Number 5P30CA042014-24.
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Lewis, M.R., Deans, T.L. (2024). In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells. In: Ceroni, F., Polizzi, K. (eds) Mammalian Synthetic Systems. Methods in Molecular Biology, vol 2774. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3718-0_19
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DOI: https://doi.org/10.1007/978-1-0716-3718-0_19
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