The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss of function provides a growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.
Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.
Highlights d CRISPR screens reveal genes essential for germ layerdifferentiation of human ESCs d Several classes of developmental diseases exhibit early differentiation phenotypes d Germ layer-specific essential transcription factors manifest a functional hierarchy d Exit from pluripotency is orchestrated by proteins within the ER-Golgi network
Recent advances in the generation of haploid embryonic stem cells (ESCs), capable of self-renewal and differentiation, have laid the groundwork for numerous biomedical applications in developmental biology and reproductive medicine. When combined with the power of genetic screening, haploid human ESCs could advance cancer research, regenerative medicine, and disease modeling.
SummaryHuman pluripotent stem cells (hPSCs) acquire genetic changes during their propagation in culture that can affect their use in research and future therapies. To identify the key genes involved in selective advantage during culture adaptation and tumorigenicity of hPSCs, we generated a genome-wide screening system for genes and pathways that provide a growth advantage either in vitro or in vivo. We found that hyperactivation of the RAS pathway confers resistance to selection with the hPSC-specific drug PluriSIn-1. We also identified that inactivation of the RHO-ROCK pathway gives growth advantage during culture adaptation. Last, we demonstrated the importance of the PI3K-AKT and HIPPO pathways for the teratoma formation process. Our screen revealed key genes and pathways relevant to the tumorigenicity and survival of hPSCs and should thus assist in understanding and confronting their tumorigenic potential.
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