title: Identification and propagation of haploid human pluripotent stem cells.
abstract: 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.
authors.affiliation: The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.
authors.forename: Ido
authors.initials: I
authors.lastname: Sagi
authors.affiliation: Department of Pediatrics, Columbia University, New York, New York, USA.|The New York Stem Cell Foundation Research Institute, New York, New York, USA.
authors.forename: Dieter
authors.initials: D
authors.lastname: Egli
authors.affiliation: The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.
authors.forename: Nissim
authors.initials: N
authors.lastname: Benvenisty
mesh_terms: D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D006238:Haploidy; D006801:Humans; D008677:Metaphase; D010312:Parthenogenesis; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*