Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Boehringer Ingelheim, Vienna, Austria.
Methods Mol Biol. 2022;2377:1-27. doi: 10.1007/978-1-0716-1720-5_1.
Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.
人多能干细胞（hPSCs）具有自我更新和分化为大多数细胞类型的能力，与广泛使用的细胞系相比，其染色体核型正常且未转化。因此，hPSCs 被认为是疾病建模的金标准系统，特别是在再生医学领域。尽管 hPSCs 和诱导多能干细胞（iPSCs）得到了广泛的研究应用，但对多能性和谱系分化机制的系统理解仍不完整。在通过遗传筛选解决谱系分化的复杂性之前，至关重要的是要对多能状态下细胞适应性和增殖的一般遗传要求进行编目，并评估它们在常用培养条件下的可塑性。我们描述了一种通过在诱导型 S. pyogenes Cas9 H1 hPSC 系中进行全基因组规模的功能丧失 CRISPR 筛选，来绘制 hPSC 适应性和多能性的关键遗传决定因素图谱的方法，这里将适应性和多能性定义为细胞繁殖。为了解决与上下文相关的基因必需性问题，我们包括了在饲养细胞和层粘连蛋白上培养 hPSCs 的筛选方案，这两种是常用的生长基质。该方法为 hPSCs 中的全基因组筛选建立了参数，使人类干细胞能够进行功能基因组学方法，从而促进对 hPSC 生物学的研究。
