TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.

Alternative pre-mRNA splicing switch controls hESC pluripotency and differentiation.

Alternative splicing (AS) of pre-mRNAs is a ubiquitous process in mammals that is tightly regulated in a cell type- and cell state-dependent manner. However, the details of how splicing is regulated to impact specific cell fate decisions remains incompletely understood. A study by Yamazaki and colleagues (pp. 1161-1174) in this issue of Genes & Development provides exciting new insight into the role and regulation of splicing in the maintenance of pluripotency of human embryonic stem cells (hESCs). In brief, they show that AS of several genes is robustly regulated upon differentiation of hESCs. One of these genes, T-cell factor 3 (TCF3), is regulated at least in part through the activity of heterogeneous nuclear ribonucleoproteins H1 and F (hnRNP H/F) to control the mutually exclusive expression of the encoded E12 and E47 transcription regulators. The investigators demonstrate that reduced expression of hnRNP H/F favors expression of E47, which in turn decreases E-cadherin expression to promote hESC differentiation. In contrast, high levels of hnRNP H/F induce expression of E12 to maintain pluripotency. Thus, this work provides at least one new link between AS and control of human stem cell fate and suggests a broader role of splicing in pluripotency.

From theory to therapy: a bibliometric and visual study of stem cell advancements in age-related macular degeneration.

BACKGROUND AIMS: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer groundbreaking therapeutic potential for degenerative diseases and cellular repair. Despite their significance, a comprehensive bibliometric analysis in this field, particularly in relation to age-related macular degeneration (AMD), is yet to be conducted. This study aims to map the foundational and emerging areas in stem cell and AMD research through bibliometric analysis. METHODS: This study analyzed articles and reviews on stem cells and AMD from 2000 to 2022, sourced from the Web of Science Core Collection. We used VOSviewer and CiteSpace for analysis and visualization of data pertaining to countries, institutions, authors, journals, references and key words. Statistical analyses were conducted using R language and Microsoft Excel 365. RESULTS: In total, 539 publications were included, indicating an increase in global literature on stem cells and AMD from 2000 to 2022. The USA was the leading contributor, with 239 papers and the highest H-index, also the USA had the highest average citation rate per article (59.82). Notably, 50% of the top 10 institutions were from the USA, with the University of California system being the most productive. Key authors included Masayo Takahashi, Michiko Mandai, Dennis Clegg, Pete J. Coffey, Boris Stanzel, and Budd A. Tucker. Investigative Ophthalmology & Visual Science published the majority of relevant papers (n = 27). Key words like "clinical trial," "stem cell therapy," "retinal organoid," and "retinal progenitor cells" were predominant. CONCLUSIONS: Research on stem cells and AMD has grown significantly, highlighting the need for increased global cooperation. Current research prioritizes the relationship between "ipsc," "induced pluripotent stem cell," "cell culture," and "human embryonic stem cell." As stem cell culture and safety have advanced, focus has shifted to prognosis and complications post-transplantation, signifying the movement of stem cell research from labs to clinical settings.

LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation.

Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.

Functional reconstruction of the basal ganglia neural circuit by human striatal neurons in hypoxic-ischaemic injured brain.

Perinatal hypoxic-ischaemic encephalopathy is the leading cause of neonatal death and permanent neurological deficits, while the basal ganglia is one of the major nuclei that is selectively and greatly affected in the brains of hypoxic-ischaemic encephalopathy patients, especially in severe cases. Human embryonic stem cell-derived neurons have shown great potential in different types of brain disorders in adults. However, it remains unknown whether and how grafted human embryonic stem cell-derived neurons can repair immature brains with hypoxic-ischaemic encephalopathy. Here, by administrating genetically labelled human embryonic stem cell-derived striatal neural progenitors into the ipsilateral striatum of hypoxic-ischaemic encephalopathy-injured mice, we found that the grafted cells gradually matured into GABA spiny projection neurons morphologically and electrophysiologically, and significantly rescued the area loss of hypoxic-ischaemic encephalopathy-injured brains. Intriguingly, using immunohistochemical staining combined with enhanced ascorbate peroxidase-based immunoelectron microscopy and rabies virus-mediated trans-synaptic tracing, we show that the grafts start to extend axonal projections to the endogenous target areas (globus pallidus externa, globus pallidus internus, substantia nigra), form synapses with host striatal, globus pallidus and nigra neurons, and receive extensive and stable synaptic inputs as early as 2 months post-transplantation. Importantly, we further demonstrated functional neural circuits re-established between the grafted neurons and host cortical, striatal and substantial nigra neurons at 3-6 months post-transplantation in the hypoxic-ischaemic encephalopathy-injured brain by optogenetics combined with electrophysiological recording. Finally, the transplanted striatal spiny projection neurons but not spinal GABA neurons restored the motor defects of hypoxic-ischaemic encephalopathy, which were reversed by clozapine-N-oxide-based inhibition of graft function. These findings demonstrate anatomical and functional reconstruction of the basal ganglia neural circuit including multiple loops by striatal spiny projection neurons in hypoxic-ischaemic encephalopathy-injured immature brains, which raises the possibility of such a cell replacement therapeutic strategy for hypoxic-ischaemic encephalopathy in neonates.

Functional assessment of donated human embryos for the generation of pluripotent embryonic stem cell lines.

RESEARCH QUESTION: Can discarded embryos at blastocyst stage, donated to research because of genetic abnormalities and poor morphological quality, become a reliable source of human embryonic stem cell (HESC) lines? DESIGN: This study was consecutively conducted with 23 discarded embryos that were donated to research between February 2020 and April 2021. All embryos, except one, were morphologically evaluated and underwent trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing (NGS), and then vitrified. After warming, the embryos were placed in appropriate culture conditions for the generation of HESCs, which was functionally assessed with immunofluorescence and flow cytometry for pluripotency capacity and spontaneous in-vitro differentiation. Cytogenetic assessment of the HESC was conducted with multiplex ligation-dependent probe amplification, and micro array comparative genomic hybridization. RESULTS: From the 23 embryos initially included, 17 survived warming, and 16 of them presented viability. Overall, the embryos presented poor morphological quality after warming. Only the previously untested embryo was capable of generating a new HESC line. Further characterization of this line revealed fully functional, euploid HESCs with preserved pluripotency, becoming a useful resource for research into human development and therapeutic investigation. CONCLUSIONS: None of the donated blastocysts with poor morphological quality in association with genetic abnormalities detected by NGS had the capacity for further in-vitro expansion to originate pluripotent HESC lines. This finding seems to provide extra support to genetic counselling on the suitability of this type of embryo for clinical use.

Lnc-ing pluripotency maintenance and early differentiation in human pluripotent stem cells.

Pluripotency maintenance and lineage differentiation are two major characteristics of human embryonic and induced pluripotent stem cells. The determination of self-renewal or differentiation is under the exquisite control of the gene regulatory network, which is composed of transcription factors, signaling pathways, metabolic factors, chromatin or histone modifiers, miRNAs, and lncRNAs. Growing evidence has shown that long noncoding RNAs (lncRNAs) play important roles in epigenetic, transcriptional, and posttranscriptional gene regulation during the cell fate determination of pluripotent stem cells. Here, we summarize recent reports of lncRNA functions in pluripotency maintenance/exit and the early germ layer specification of human pluripotent stem cells. We also illustrate four major lncRNA functional mechanisms according to different types of cofactors: chromatin or histone modifiers, transcription factors, canonical and noncanonical RNA-binding proteins, and miRNAs. Further understanding of lncRNA-based regulation will provide more insights into the drivers manipulating cell fate and promote the therapeutic and research potential of human embryonic and induced pluripotent stem cells.

Glycosphingolipids in human embryonic stem cells and breast cancer stem cells, and potential cancer therapy strategies based on their structures and functions.

Expression profiles of glycosphingolipids (GSLs) in human embryonic stem cell (hESC) lines and their differentiated embryoid body (EB) outgrowth cells, consisting of three germ layers, were surveyed systematically. Several globo- and lacto-series GSLs were identified in undifferentiated hESCs and during differentiation of hESCs to EB outgrowth cells, and core structure switching of these GSLs to gangliosides was observed. Such switching was attributable to altered expression of key glycosyltransferases (GTs) in GSL biosynthetic pathways, reflecting the unique stage-specific transitions and mechanisms characteristic of the differentiation process. Lineage-specific differentiation of hESCs was associated with further GSL alterations. During differentiation of undifferentiated hESCs to neural progenitor cells, core structure switching from globo- and lacto-series to primarily gangliosides (particularly GD3) was again observed. During differentiation to endodermal cells, alterations of GSL profiles were distinct from those in differentiation to EB outgrowth or neural progenitor cells, with high expression of Gb4Cer and low expression of stage-specific embryonic antigen (SSEA)-3, -4, or GD3 in endodermal cells. Again, such profile changes resulted from alterations of key GTs in GSL biosynthetic pathways. Novel glycan structures identified on hESCs and their differentiated counterparts presumably play functional roles in hESCs and related cancer or cancer stem cells, and will be useful as surface biomarkers. We also examined GSL expression profiles in breast cancer stem cells (CSCs), using a model of epithelial-mesenchymal transition (EMT)-induced human breast CSCs. We found that GD2 and GD3, together with their common upstream GTs, GD3 synthase (GD3S) and GD2/GM2 synthase, maintained stem cell phenotype in breast CSCs. Subsequent studies showed that GD3 was associated with epidermal growth factor receptor (EGFR), and activated EGFR signaling in breast CSCs and breast cancer cell lines. GD3S knockdown enhanced cytotoxicity of gefitinib (an EGFR kinase inhibitor) in resistant MDA-MB468 cells, both in vitro and in vivo. Our findings indicate that GD3S contributes to gefitinib resistance in EGFR-positive breast cancer cells, and is a potentially useful therapeutic target in drug-resistant breast cancers.

The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene.

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

Evaluation of tools for identifying large copy number variations from ultra-low-coverage whole-genome sequencing data.

BACKGROUND: Detection of copy number variations (CNVs) from high-throughput next-generation whole-genome sequencing (WGS) data has become a widely used research method during the recent years. However, only a little is known about the applicability of the developed algorithms to ultra-low-coverage (0.0005-0.8×) data that is used in various research and clinical applications, such as digital karyotyping and single-cell CNV detection. RESULT: Here, the performance of six popular read-depth based CNV detection algorithms (BIC-seq2, Canvas, CNVnator, FREEC, HMMcopy, and QDNAseq) was studied using ultra-low-coverage WGS data. Real-world array- and karyotyping kit-based validation were used as a benchmark in the evaluation. Additionally, ultra-low-coverage WGS data was simulated to investigate the ability of the algorithms to identify CNVs in the sex chromosomes and the theoretical minimum coverage at which these tools can accurately function. Our results suggest that while all the methods were able to detect large CNVs, many methods were susceptible to producing false positives when smaller CNVs (< 2 Mbp) were detected. There was also significant variability in their ability to identify CNVs in the sex chromosomes. Overall, BIC-seq2 was found to be the best method in terms of statistical performance. However, its significant drawback was by far the slowest runtime among the methods (> 3 h) compared with FREEC (~ 3 min), which we considered the second-best method. CONCLUSIONS: Our comparative analysis demonstrates that CNV detection from ultra-low-coverage WGS data can be a highly accurate method for the detection of large copy number variations when their length is in millions of base pairs. These findings facilitate applications that utilize ultra-low-coverage CNV detection.

Long Noncoding RNA SPRY4-IT1 Modulates Ketamine-Induced Neurotoxicity in Human Embryonic Stem Cell-Derived Neurons through EZH2.

OBJECTIVE: This study aimed to investigate whether long noncoding RNA sprouty receptor tyrosine kinase signaling antagonist 4-intronic transcript 1 (SPRY4-IT1) is involved in the regulation of ketamine-induced neurotoxicity. METHODS: Human embryonic stem cells (hESCs) were induced into neurons in vitro and treated with ketamine. Apoptosis and neurite degeneration assays were used to determine ketamine-induced neurotoxicity and qRT-PCR to determine SPRY4-IT1 expression. SPRY4-IT1 was downregulated in hESC-induced neurons to examine its regulation on ketamine-induced neurotoxicity. The correlation between enhancer of zeste homolog 2 (EZH2) and SPRY4-IT1 was also examined. EZH2 was upregulated in SPRY4-IT1-downregualted hESC-induced neurons to further examine its participation in SPRY4-IT1-mediated ketamine neurotoxicity. RESULTS: Ketamine-induced dose-dependent apoptosis, neurite degeneration, and SPRY4-IT1 upregulation in hESC-induced neurons. Lentivirus-mediated SPRY4-IT1 downregulation protected ketamine neurotoxicity. EZH2 expression was positively correlated with SPRY4-IT1 in hESC-induced neurons. EZH2 overexpression markedly reversed the protective effects of SPRY4-IT1 knockdown on ketamine neurotoxicity. CONCLUSIONS: SPRY4-IT1 is involved in anesthesia-induced neurotoxicity, possibly through the regulation on EZH2 gene.

Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell-Derived (hESC) Islet-Like Clusters Posttransplantation.

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Hyperglycemia Altered DNA Methylation Status and Impaired Pancreatic Differentiation from Embryonic Stem Cells.

The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1, NKX6-1 and NKX6-2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6-2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6-1. Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro.

AIM: Immunological checkpoint therapy is considered a powerful method for cancer therapy and acts by re-activating autologous T cells to kill the cancer cell. Myocarditis cases have been reported in cancer patients after immunological therapy; for example, nivolumab treatment is a monoclonal antibody that blocks programmed cell death-1/programmed cell death ligand-1 ligand interaction. This project provided insight into the inflammatory response as a benchmark to investigate the potential cardiotoxic effect of T cell response to the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis in regulating cardiomyocyte injury in vitro. METHODS AND RESULTS: We investigated cardiomyopathy resulted from the PD-1/PD-L1 axis blockade using the anti-PD-1 antibody in Rockefeller University embryonic stem cells-derived cardiomyocytes (RUES2-CMs) and a melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory-related proteins, including PD-L1 expression, and did not induce apoptosis, which was contrary to doxorubicin, a cardiotoxic chemotherapy drug. However, nivolumab was able to exacerbate the immune response by increasing cytokine and inflammatory gene expression in RUES2-CMs when co-cultured with CD4+ T lymphocytes and induced apoptosis. This effect was not observed when RUES2-CMs were co-cultured with CD8+ T lymphocytes. The in vivo model showed that the heart function of tumor-bearing mice was decreased after treatment with anti-PD-1 antibody and demonstrated a dilated left ventricle histological examination. The dilated left ventricle was associated with an infiltration of CD4+ and CD8+ T lymphocytes into the myocardium. PD-L1 and inflammatory-associated gene expression were significantly increased in anti-PD-1-treated tumor-bearing mice. Cleaved caspase-3 and mouse plasma cardiac troponin I expressions were increased significantly. CONCLUSION: PD-L1 expression on cardiomyocytes suppressed T-cell function. Blockade of PD-1 by nivolumab enhanced cardiomyocyte inflammation and apoptosis through the enhancement of T-cell response towards cardiomyocytes.

Targeted Delivery of Iron Oxide Nanoparticle-Loaded Human Embryonic Stem Cell-Derived Spherical Neural Masses for Treating Intracerebral Hemorrhage.

This study evaluated the potential of iron oxide nanoparticle-loaded human embryonic stem cell (ESC)-derived spherical neural masses (SNMs) to improve the transportation of stem cells to the brain, ameliorate brain damage from intracerebral hemorrhage (ICH), and recover the functional status after ICH under an external magnetic field of a magnet attached to a helmet. At 24 h after induction of ICH, rats were randomly separated into three experimental groups: ICH with injection of phosphate-buffered saline (PBS group), ICH with intravenous injection of magnetosome-like ferrimagnetic iron oxide nanocubes (FION)-labeled SNMs (SNMs* group), and ICH with intravenous injection of FION-labeled SNMs followed by three days of external magnetic field exposure for targeted delivery by a magnet-embedded helmet (SNMs*+Helmet group). On day 3 after ICH induction, an increased Prussian blue-stained area and decreased swelling volume were observed in the SNMs*+Helmet group compared with that of the other groups. A significantly decreased recruitment of macrophages and neutrophils and a downregulation of pro-inflammatory cytokines followed by improved neurological function three days after ICH were observed in the SNMs*+Helmet group. Hemispheric atrophy at six weeks after ICH was significantly decreased in the SNMs*+Helmet group compared with that of the PBS group. In conclusion, we have developed a targeted delivery system using FION tagged to stem cells and a magnet-embedded helmet. The targeted delivery of SNMs might have the potential for developing novel therapeutic strategies for ICH.

[High-level androgen enhances the differentiation of hESCs into male primordial germ cells].

OBJECTIVE: To observe the effects of different concentrations of testosterone on the differentiation of human embryonic stem cells (hESCs) into early male germ cells and investigate the potential impact of high-level androgen exposure in early pregnancy in women with polycystic ovary syndrome (PCOS) on the fertility and primordial germ cell reserve of the male offspring in adulthood. METHODS: We used 2 µmol/L retinoic acid to induce the differentiation of hESCs (46, XY) into male germ cells in vitro and meanwhile treated them with testosterone (T) at 0 mol/L, 3×10－7 mol/L, 5×10－7 mol/L, 15×10－7 mol/L, 45×10－7 mol/L, and 135×10－7 mol/L, respectively. We collected the cell samples at 0, 4, 7 and 14 days to determine the expressions of the specific genes and compare the differentiation process and efficiency of the male germ cells in different stages. RESULTS: There was no difference in the morphology of the hESCs treated with different concentrations of testosterone in the same differentiation stage. The expression of the marker gene DAZL in the primordial germ cells peaked on the 4th day of differentiation, significantly higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.05), and that of the specific gene SCP3 in the early-meiosis germ cells began to increase on the same day, more significantly in the 45×10－7mol/L than in the 3×10－7 mol/L and 5×10－7 mol/L groups (P < 0.01), and peaked on the 7th day, dramatically higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.01). Immunofluorescence staining and flow cytometry showed a T concentration-dependent increase in the expression of DAZL at 4 days and those of SCP3 and VASA at 7 days. Moreover, the expression of the androgen receptor (AR) in the hESCs began to rise on the 4th day and kept going up till the 14th day, higher in the high-concentration than in the low-concentration T groups in the same stage of differentiation, though with no statistically significant difference (P > 0.05). CONCLUSIONS: Exposure to high-level androgen during the differentiation of hESCs into early male germ cells can induce earlier expression of AR and earlier differentiation of hESCs into early male germ cells, which may result in insufficient reserve of male primary germ cells in the male offspring of PCOS women and affect their fertility after adulthood. hESCs can be used as an in vitro model to study the effects of intrauterine hyperandrogen on the reproductive development of male offspring in PCOS patients, which is also contributive to researches on the etiology of male infertility.

Inhibition of Human Y Chromosome Gene, SRY, Promotes Naïve State of Human Pluripotent Stem Cells.

Although males and females have a variety of sexually dimorphic features related to hormonal effects, the genetic basis of dimorphism relies on early embryo development. Two pluripotent states, naïve and primed, emerge during early mammalian development. Identification of signaling pathways that induce differences between these two states can help to modulate conversion of primed cells to naïve cells. Naïve cells have a shorter doubling time and longer survival than their primed counterparts when passaged as single cells. In this study, we sought to explore the role of Y chromosome genes on human pluripotent stem cells (hPSCs) by investigating differential expressions of the male-specific region of the Y chromosome (MSY) genes in primed and naïve cells. Interestingly, we found that several MSY genes, including SRY, showed higher expression levels in primed compared to naïve human embryonic stem cells (hESCs). We hypothesize that SRY prevents WNT/ß-catenin signaling by its interaction and inhibition of ß-catenin activation in the nucleus. Results of the loss-of-function approach conducted by depletion of SRY indicated increased expressions of pluripotency marker genes and alkaline phosphatase (ALP) activity in the primed cells. SRY reduction was associated with overexpression of WNT signaling target genes AXIN2, Brachury, TCF1, TBX2, and TBX3. We suggest that inhibition of SRY may result in activation of ß-catenin and up-regulation of the WNT signaling pathway, both of which are important to naïve conversion.

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer.

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.

Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality.

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.