Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency, providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid, their enrichment at an early passage is key for successful derivation. In this protocol, we describe two methodologies, namely metaphase spread analysis and cell sorting, for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages, as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4–6 weeks.
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We thank T. Golan-Lev and O. Yanuka for providing technical details for the protocol, and M. Peretz, A. Yilmaz and S. Bar for critical reading of the manuscript. I.S. is supported by the Adams Fellowships Program of the Israel Academy of Sciences and Humanities, D.E. is a NYSCF – Robertson Investigator, and N.B. is the Herbert Cohn Chair in Cancer Research. This work was partially supported by The Azrieli Foundation (N.B.), by the Russell Berrie Foundation Program in Cellular Therapies of Diabetes and by the New York Stem Cell Foundation (D.E.).
The authors declare no competing financial interests.
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Sagi, I., Egli, D. & Benvenisty, N. Identification and propagation of haploid human pluripotent stem cells. Nat Protoc 11, 2274–2286 (2016). https://doi.org/10.1038/nprot.2016.145
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