Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

Genome-wide screening reveals essential roles for HOX genes and imprinted genes during caudal neurogenesis of human embryonic stem cells.

Mapping the essential pathways for neuronal differentiation can uncover new therapeutics and models for neurodevelopmental disorders. We thus utilized a genome-wide loss-of-function library in haploid human embryonic stem cells, differentiated into caudal neuronal cells. We show that essential genes for caudal neurogenesis are enriched for secreted and membrane proteins and that a large group of neurological conditions, including neurodegenerative disorders, manifest early neuronal phenotypes. Furthermore, essential transcription factors are enriched with homeobox (HOX) genes demonstrating synergistic regulation and surprising non-redundant functions between HOXA6 and HOXB6 paralogs. Moreover, we establish the essentialome of imprinted genes during neurogenesis, demonstrating that maternally expressed genes are non-essential in pluripotent cells and their differentiated germ layers, yet several are essential for neuronal development. These include Beckwith-Wiedemann syndrome- and Angelman syndrome-related genes, for which we suggest a novel regulatory pathway. Overall, our work identifies essential pathways for caudal neuronal differentiation and stage-specific phenotypes of neurological disorders.

EpiTyping: analysis of epigenetic aberrations in parental imprinting and X-chromosome inactivation using RNA-seq.

Human pluripotent stem cells (hPSCs) hold a central role in studying human development, in disease modeling and in regenerative medicine. These cells not only acquire genetic modifications when kept in culture, but they may also harbor epigenetic aberrations, mainly involving parental imprinting and X-chromosome inactivation. Here we present a detailed bioinformatic protocol for detecting such aberrations using RNA sequencing data. We provide a pipeline designed to process and analyze RNA sequencing data for the identification of abnormal biallelic expression of imprinted genes, and thus detect loss of imprinting. Furthermore, we show how to differentiate among X-chromosome inactivation, full activation and aberrant erosion of X chromosome in female hPSCs. In addition to providing bioinformatic tools, we discuss the impact of such epigenetic variations in hPSCs on their utility for various purposes. This pipeline can be used by any user with basic understanding of the Linux command line. It is available on GitHub as a software container ( https://github.com/Gal-Keshet/EpiTyping ) and produces reliable results in 1-4 d.

Differentiation of uniparental human embryonic stem cells into granulosa cells reveals a paternal contribution to gonadal development.

Genomic imprinting underlies the mammalian requirement for sexual reproduction. Nonetheless, the relative contribution of the two parental genomes during human development is not fully understood. Specifically, a fascinating question is whether the formation of the gonad, which holds the ability to reproduce, depends on equal contribution from both parental genomes. Here, we differentiated androgenetic and parthenogenetic human pluripotent stem cells (hPSCs) into ovarian granulosa-like cells (GLCs). We show that in contrast to biparental and androgenetic cells, parthenogenetic hPSCs present a reduced capacity to differentiate into GLCs. We further identify the paternally expressed gene IGF2 as the most upregulated imprinted gene upon differentiation. Remarkably, while IGF2 knockout androgenetic cells fail to differentiate into GLCs, the differentiation of parthenogenetic cells supplemented with IGF2 is partly rescued. Thus, our findings unravel a surprising essentiality of genes that are only expressed from the paternal genome to the development of the female reproductive system.

Large-scale analysis of imprinting in naive human pluripotent stem cells reveals recurrent aberrations and a potential link to FGF signaling.

Genomic imprinting is a parent-of-origin dependent monoallelic expression of genes. Previous studies showed that conversion of primed human pluripotent stem cells (hPSCs) into naive pluripotency is accompanied by genome-wide loss of methylation that includes imprinted loci. However, the extent of aberrant biallelic expression of imprinted genes is still unknown. Here, we analyze loss of imprinting (LOI) in a large cohort of both bulk and single-cell RNA sequencing samples of naive and primed hPSCs. We show that naive hPSCs exhibit high levels of non-random LOI, with bias toward paternally methylated imprinting control regions. Importantly, we show that different protocols used for the primed to naive conversion led to different extents of LOI, tightly correlated to FGF signaling. This analysis sheds light on the process of LOI occurring during the conversion to naive pluripotency and highlights the importance of these events when modeling disease and development or when utilizing the cells for therapy.

Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells.

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.