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Somatic cells can be reprogramed into induced pluripotent stem (iPS) cells by defined factors, which provide a powerful basis for personalized stem-cell based therapies. However, cellular reprograming is an inefficient and metabolically demanding process commonly associated with obstacles that hamper further use of this technology. Spontaneous differentiation of iPS cells cultures represents a significant hurdle that hinder obtaining high quality iPS cells for further downstream experimentation. In this study, we found that a natural compound, vitamin C, augmented pluripotency in iPS cells and reduced unwanted spontaneous differentiation during iPS cells maintenance. Gene expression analysis showed that vitamin C increased the expression of the histone demethylase JARID1A. Furthermore, through gain- and loss-of-function approaches, we show that JARID1A is a key effector in promoting pluripotency and reducing differentiation downstream of vitamin C. Our results therefore highlight a straightforward method for improving the pluripotency and quality of iPS cells; it also shows a possible role for H3K4me2/3 in cell fate determination and establishes a link between vitamin C and epigenetic regulation.

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG- Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

Somatic cells can be reprogramed into induced pluripotent stem (iPS) cells by defined factors, which provide a powerful basis for personalized stem-cell based therapies. However, cellular reprograming is an inefficient and metabolically demanding process commonly associated with obstacles that hamper further use of this technology. Spontaneous differentiation of iPS cells cultures represents a significant hurdle that hinder obtaining high quality iPS cells for further downstream experimentation. In this study, we found that a natural compound, vitamin C, augmented pluripotency in iPS cells and reduced unwanted spontaneous differentiation during iPS cells maintenance. Gene expression analysis showed that vitamin C increased the expression of the histone demethylase JARID1A. Furthermore, through gain- and loss-of-function approaches, we show that JARID1A is a key effector in promoting pluripotency and reducing differentiation downstream of vitamin C. Our results therefore highlight a straightforward method for improving the pluripotency and quality of iPS cells; it also shows a possible role for H3K4me2/3 in cell fate determination and establishes a link between vitamin C and epigenetic regulation.

The chromobox homolog 2 (CBX2) was found to be important for human testis development, but its role in the human ovary remains elusive. We conducted a genome-wide analysis based on DNA adenine methyltransferase identification (DamID) and RNA sequencing strategies to investigate CBX2 in the human granulosa cells. Functional analysis revealed that CBX2 was upstream of genes contributing to ovarian function like folliculogenesis and steroidogenesis (i.e. ESR1, NRG1, AKR1C1, PTGER2, BMP15, BMP2, FSHR and NTRK1/2). We identified CBX2 regulated genes associated with polycystic ovary syndrome (PCOS) such as TGFβ, MAP3K15 and DKK1, as well as genes implicated in premature ovarian failure (POF) (i.e. POF1B, BMP15 and HOXA13) and the pituitary deficiency (i.e. LHX4 and KISS1). Our study provided an excellent opportunity to identify genes surrounding CBX2 in the ovary and might contribute to the understanding of ovarian physiopathology causing infertility in women.

Somatic cells can be reprogramed into induced pluripotent stem (iPS) cells by defined factors, which provide a powerful basis for personalized stem-cell based therapies. However, cellular reprograming is an inefficient and metabolically demanding process commonly associated with obstacles that hamper further use of this technology. Spontaneous differentiation of iPS cells cultures represents a significant hurdle that hinder obtaining high quality iPS cells for further downstream experimentation. In this study, we found that a natural compound, vitamin C, augmented pluripotency in iPS cells and reduced unwanted spontaneous differentiation during iPS cells maintenance. Gene expression analysis showed that vitamin C increased the expression of the histone demethylase JARID1A. Furthermore, through gain- and loss-of-function approaches, we show that JARID1A is a key effector in promoting pluripotency and reducing differentiation downstream of vitamin C. Our results therefore highlight a straightforward method for improving the pluripotency and quality of iPS cells; it also shows a possible role for H3K4me2/3 in cell fate determination and establishes a link between vitamin C and epigenetic regulation.

Sex development, a complex and indispensable process in all vertebrates, has still not been completely elucidated, although new genes involved in sex development are constantly being discovered and characterized. Chromobox Homolog 2 (CBX2) is one of these new additions and has been identified through a 46,XY girl with double heterozygous variants on CBX2.1, causing Differences of Sex Development (DSD). The mutated CBX2.1 failed to adequately regulate downstream targets important for sex development in humans, specifically steroidogenic factor 1 (NR5A1/SF1). To better place CBX2.1 in the human sex developmental cascade, we performed siRNA and CBX2.1 overexpression experiments and created a complete CRISPR/Cas9- CBX2 knockout in Sertoli-like cells. Furthermore, we deployed Next Generation Sequencing techniques, RNA-Sequencing and DamID-Sequencing, to identify new potential CBX2.1 downstream genes. The combination of these two next generation techniques enabled us to identify genes that are both bound and regulated by CBX2.1. This allowed us not only to expand our current knowledge about the influence of CBX2.1 in human sex development, but also to advance our insight in the mechanisms governing one of the most important decisions during embryonal development, the commitment to either female or male gonads.

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG- Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

Background One of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown. Methods The genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild-type and variant CBX2.2 on selected downstream targets. Results Here, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad. Conclusion Our study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.

Somatic cells can be reprogramed into induced pluripotent stem (iPS) cells by defined factors, which provide a powerful basis for personalized stem-cell based therapies. However, cellular reprograming is an inefficient and metabolically demanding process commonly associated with obstacles that hamper further use of this technology. Spontaneous differentiation of iPS cells cultures represents a significant hurdle that hinder obtaining high quality iPS cells for further downstream experimentation. In this study, we found that a natural compound, vitamin C, augmented pluripotency in iPS cells and reduced unwanted spontaneous differentiation during iPS cells maintenance. Gene expression analysis showed that vitamin C increased the expression of the histone demethylase JARID1A. Furthermore, through gain- and loss-of-function approaches, we show that JARID1A is a key effector in promoting pluripotency and reducing differentiation downstream of vitamin C. Our results therefore highlight a straightforward method for improving the pluripotency and quality of iPS cells; it also shows a possible role for H3K4me2/3 in cell fate determination and establishes a link between vitamin C and epigenetic regulation.

CBX2 (Chromobox homolog 2) is a chromatin modifier that plays an important role in sexual development and its disorders (disorders of sex development, DSD), yet the exact rank and function of human CBX2 in this pathway remains unclear. Here, we performed large-scale mapping and analysis of in vivo target loci of the protein CBX2 in Sertoli-like NT-2D1cells, using the DNA adenine methyltransferase (DamID) technique. We identified close to 1600 direct targets for CBX2. Intriguingly, validation of selected candidate genes using qRT-PCR in cells overexpressing CBX2 or in which CBX2 has been knocked down indicated that several CBX2-responsive genes encode proteins that are involved in DSD. We further validated these effects on the candidate genes using a mutated CBX2 causing DSD in human patient. Overall, our findings suggest that CBX2 role in the sex development cascade is to stimulate the male pathway and concurrently inhibit the female pathway. These data provide fundamental insights into potential etiology of DSD.