Embryonic Stem Research Articles

Redundancy of p75NTR neurotrophin receptor function in development, growth and fertility in the rat

The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-Δ alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. p75NTR function is therefore largely dispensable for normal development, growth and basal homeostasis in the rat. However, the availability of constitutive and conditional p75NTREx2-Δ rats provides new opportunities to investigate specific roles of p75NTR upon injury and during tissue repair.

The dynamic expression of YAP is essential for the development of male germ cells derived from human embryonic stem cells

YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.

Stem Cell Therapies for Spinal Cord Injury - A Review

The escalating incidence of Spinal Cord Injury (SCI), with approximately 0.9 million cases globally, underscores its growing public health concern. Traumatic SCI, often prevalent in developing nations due to factors like motor vehicle accidents and falls, leads to secondary damage involving inflammation, neuronal death, and ionic dysregulation. Despite the absence of an effective treatment for SCI, Stem Cell Therapy (SCT) emerges as a promising avenue, harnessing stem cells' unique capabilities for regeneration and replacement. The review explores various stem cell types, such as Neural Stem/Progenitor Cells (NS/PCs), Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs), and Mesenchymal Stem Cells (MSCs), detailing their potential in preclinical and clinical contexts. Specifically, NSCs exhibit therapeutic promise by modulating astrocyte contribution, enhancing differentiation, and promoting growth factors. ESCs, despite their pluripotency, face ethical concerns and potential teratocarcinoma formation. IPSCs, reprogrammed from somatic cells, showcase potential in treating SCI without ethical issues. MSCs, with diverse sources like bone marrow, umbilical cord, and adipose tissue, offer versatility in differentiation and therapeutic benefits. Clinical trials with MSCs, especially BM-MSCs, UC-MSCs, and AD-MSCs, demonstrate improvements in motor and sensory functions, highlighting their regenerative potential. Despite promising results, challenges such as potential tumorigenesis and high costs persist, warranting further exploration and clinical translation of these stem cell therapies for SCI.

Hypoxia exposure induces lactylation of Axin1 protein to promote glycolysis of esophageal carcinoma cells

The hypoxic microenvironment in esophageal carcinoma is an important factor promoting the rapid progression of malignant tumor. This study was to investigate the lactylation of Axin1 on glycolysis in esophageal carcinoma cells under hypoxia exposure. Hypoxia treatment increases pan lysine lactylation (pan-kla) levels of both TE1 and EC109 cells. Meanwhile, ECAR, glucose consumption and lactate production were also upregulated in both TE1 and EC109 cells. The expression of embryonic stem cell transcription factors NANOG and SOX2 were enhanced in the hypoxia-treated cells. Axin1 overexpression partly reverses the induction effects of hypoxia treatment in TE1 and EC109 cells. Moreover, lactylation of Axin1 protein at K147 induced by hypoxia treatment promotes ubiquitination modification of Axin1 protein to promote glycolysis and cell stemness of TE1 and EC109 cells. Mutant Axin1 can inhibit ECAR, glucose uptake, lactate secretion, and cell stemness in TE1 and EC109 cells under normal or hypoxia conditions. Meanwhile, mutant Axin1 further enhanced the effects of 2-DG on inhibiting glycolysis and cell stemness. Overexpression of Axin1 also inhibited tumor growth in vivo, and was related to suppressing glycolysis. In conclusion, hypoxia treatment promoted the glycolysis and cell stemness of esophageal carcinoma cells, and increased the lactylation of Axin1 protein. Overexpression of Axin1 functioned as a glycolysis inhibitor, and suppressed the effects of hypoxia exposure in vitro and inhibited tumor growth in vivo. Mechanically, hypoxia induces the lactylation of Axin1 protein and promotes the ubiquitination of Axin1 to degrade the protein, thereby exercising its anti-glycolytic function.

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

BackgroundBone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll–Plate–Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs).MethodsBy using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans’.ResultsThe NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the “stemness” of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.ConclusionsOverall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Proteome-scale tagging and functional screening in mammalian cells by ORFtag

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

Loss of Tet hydroxymethylase activity causes mouse embryonic stem cell differentiation bias and developmental defects.

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tetm/m). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tetm/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

P-794 Unraveling the function of GATA2 and GATA3 in mouse blastocyst formation via CRISPR/Cas9 gene editing

Abstract Study question What is the effect of the CRISPR/Cas9 mediated knock-out (KO) of Gata2 and Gata3 on mouse preimplantation development/blastocyst formation? Summary answer Efficient generation of Gata3/Gata2 KO mouse embryos was achieved. Gata3 and Gata2 KO embryos could form blastocysts, suggesting a possible dispensability for blastocyst formation. What is known already GATA2 and GATA3 are important trophectoderm markers in mouse and human embryos. Prior studies, involving RNAi knockdown or conditional KO of Gata3, demonstrated high rates of morula arrest of Gata3-/- embryos, indicating its essential role in blastocyst formation. However, conflicting results showed embryonic lethality in Gata3-/- embryos only at 11-12 dpf. Besides, conditional KO of Gata2 suggested its dispensability in blastocyst formation, as Gata2-/- embryos could form blastocysts. It is of interest to validate these findings and potential functional redundancy between GATA2 and GATA3 in mouse embryos, by generating CRISPR/Cas9-mediated single and double KOs of Gata2 and Gata3. Study design, size, duration Upon assessment of the on-/ off-target effects of CRISPR/Cas9 components in mouse embryonic stem cells (mESC), CRISPR/Cas9 ribonucleoprotein (RNP) complexes were electroporated in mouse zygotes. Suitable control groups (non-targeted and scrambled targeted (inactive crRNA)) were incorporated. Embryos were genotyped by next generation sequencing (NGS), to assess the effect of gene KO on embryo morphology. Additionally, the expression pattern of Gata2 and Gata3 was examined in wild type and CRISPR/Cas9 targeted embryos, with immunofluorescent staining (IFS). Participants/materials, setting, methods Following CRISPR/Cas9 mediated targeting of Gata2 and Gata3, mouse embryos were cultured and morphologically monitored until day 4.5, alongside relevant control groups (scrambled, media control). Subsequently, DNA was extracted, and embryos were genotyped by PCR amplification of the region surrounding the target site, followed by NGS. IFS of mouse embryos at different developmental stages (E2.5, E3.0, E3.5 and E4.5) was performed to assess expression patterns and localization of both Gata2 and Gata3 throughout development. Main results and the role of chance Following the design of two gRNAs targeting either Gata2 or Gata3, on-target editing efficiency was tested in mESCs. The selected Gata2-targeting design resulted in a frameshift efficiency of 75%, whereas the Gata3-targeting design resulted in a frameshift efficiency of 78%. No off-target effects were observed in Gata2 and Gata3 targeted mESCs when examining in silico predicted off-target sites through PCR and NGS. Furthermore, 51% of Gata3 targeted and 41% of Gata2 targeted mouse embryos contained frameshift mutations in all cells. Notably, the majority of Gata3-/- (93%, n = 22) and Gata2-/- (94%, n = 16) embryos developed to blastocysts, similar to media control and scrambled groups. No statistically significant difference between Gata3-/- (n = 24, p = 0.326) and Gata2-/- (n = 17, p = 0.978) embryo morphology and scrambled (n = 96) embryo morphology was detected. IFS of control embryos (n = 16) showed that nuclear expression of GATA2 precedes nuclear expression of GATA3, indicating that GATA2 might control the expression of Gata3. Following IFS of Gata3 targeted embryos (n = 4), no nuclear signal for both GATA2 and GATA3 was observed, indicating that GATA3 could regulate the expression of Gata2. Furthermore, we created 100% double KO mouse embryos for Gata2 and Gata3 in two ways, enabling comprehensive exploration of functional redundancy in future studies. Limitations, reasons for caution Mosaicism (>1 genotype present in one embryo), off-target effects and chromosomal arrangements pose challenges regarding CRISPR/Cas9 germline genome editing. Zygote-stage delivery of RNP complexes is used to address mosaicism, and off-target effects are screened in mESCs via NGS. Future efforts aim to increase the sample sizes for stronger support. Wider implications of the findings Our goal is to identify the molecular pathways underlying unsuccessful implantation in mouse and human embryos. Utilizing CRISPR/Cas9 to generate KO embryos enhances our understanding of key molecular mechanisms, potentially shedding light on pregnancy failure due to failure of trophectoderm specification, formation or maintenance. Trial registration number not applicable

TBP facilitates RNA Polymerase I transcription following mitosis

ABSTRACT The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

O-094 Novel stem cell-based twin embryo model reveals inner cell mass division and enhanced endometrial adhesion in monochorionic twin development

Abstract Study question How do monochorionic twin embryos arise and develop during the pre- and peri-implantation stage? Summary answer Twinning involves inner cell mass division during blastocyst expansion and heightened endometrial adhesion, with twin blastoids showing greater implantation potential. What is known already The occurrence of monozygotic twins is between 0.4 and 0.5% of livebirths globally from natural conception, of which the majority are monochorionic. Monochorionic twins are associated with increased risk of complications and higher mortality rates during pregnancy due to twin-to-twin transfusion syndrome, twin anemia-polycythemia sequence, developmental malformations and increased incidence of pregnancy-related diseases in the mother. The rarity of monochorionic twinning, combined with ethical constraints in human embryo research, renders the study of twinning practically infeasible, which presents in a significant knowledge gap regarding the origins and implications of early developmental monochorionic twin embryos development. Study design, size, duration In a stem cell-based model of the blastocyst-stage embryo (i.e., blastoid) conditions were identified that favor the generation of monochorionic twin blastoids. To obtain sufficient statistical power, screening experiments were performed using 3 culture wells per condition, each well containing at least 90 blastoids. Experiments were repeated 3 times and results were pooled. Blastoids were cultured for at least 3 days followed by 2 days on endometrium monolayers on-chip. Participants/materials, setting, methods Human naïve induced pluripotent stem cells and embryonic stem cells were used to form twin blastoids within custom microwell array platforms. Patient-derived endometrium epithelial cells were expanded as organoids and seeded as monolayers in microfluidic chips. Blastoids were infused into the chip, allowed to adhere for 2 days, followed by a controlled stepwise increase of flow rate after which the blastoid adhesion rate to the endometrium layer for both singleton and twin blastoids was quantified. Main results and the role of chance Twin blastoids contained a cystic GATA3+ single layered trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses revealed that twinning occurs during the blastocyst cavitation phase via division of the OCT4+ pluripotent cell mass. After three days of culture, twin blastoids measured 228±32 µm, which is in the upper range of the cyst diameter of a blastocyst. In addition, both sibling ICM-like clusters preserved similar epiblast and hypoblast cell proportions and were consistently positioned nearly 150° (146.6±26°) apart along the blastocoel lining (measured as 2D-projections and in 3D reconstructions), which suggests a minimal angular distance for establishing two separate condensed ICM clusters. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, indicative of implantation readiness. Under defined flow regimes, twin blastoids show increased adhesion capacity to endometrium monolayers compared to singleton blastoids (60% vs 38% at highest flow rate), suggestive of increased implantation potential possibly due to a larger adhesive surface area. Twin blastoids cultured on endometrium monolayers could develop into structures with two distinctive OCT4+ pluripotent cell clumps both delineated by SOX17+ hypoblast-like cells and as a whole encompassed by GATA3+ trophoblast-like cells, suggestive of post-implantation organization. Limitations, reasons for caution While twin blastoids adhered to the endometrium monolayer and maintained two separate OCT4 compartments after 48 hours in post-implantation culture, both singleton and twin blastoids did not establish organized 3D structures associated with later post-implantation development, including amniotic and yolk sac-like cavities. Wider implications of the findings Twin blastoids open new avenues for studying the various aspects of twinning, ranging from their formation processes to the effects of multiple ICMs on trophectoderm maturation and implantation potential. In the future, with more sophisticated endometrium platforms, this model may inform on how clinical complications arise in twin pregnancies. Trial registration number not applicable

P-804 Smchd1 is a key regulator of epigenetic cellular memory induced by morphine in mouse embryonic stem cells (mESC)

Abstract Study question Can morphine induce stable epigenetic changes that persist mitotically in mESC? Summary answer SMCHD1 is a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by mESC and upon embryo development. What is known already Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. X chromosome inactivation (XCI) is an important epigenetic process, which takes places during embryo development and is maintained in the adulthood. Since XCI is part of normal development, changes induced by environmental factors might affect embryo development and lead to health problems. As morphine can easily pass through the placental barrier and reach the embryo, our aim is to elucidate whether morphine causes epigenetic regulation on XCI process. Study design, size, duration Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. To evaluate if morphine can induce cellular epigenetic memory, we analyzed the dynamic epigenetic changes over the time in the absence of morphine. For that purpose, OCT4-reported mESCs were chronically treated with morphine during 24h (10-5 mM) Participants/materials, setting, methods • Transcriptomic analysis by RNA-Sequencing and gene expression validation by qRT-PCR. • Epigenetic analysis by H3K27me3 ChIP-Sequencing and WGBS-Sequencing. • Smchd1 gene silencing by Crisper-Cas9. Main results and the role of chance Morphine induced cellular epigenetic memory upon embryo development. mESCs can memorize morphine exposure and induces transcriptional changes that can persist over the time, even in the absence of morphine. RNA-seq has shown that morphine chronic treatment produces a transcriptional deregulation of 932 genes in P1 just after the treatment and this amount increased after treatment withdrawal (1196 genes, P2 and 2138 in P3). Integrative analyses between P1, P2 and P3 identifies Smchd1 gene as a potential regulator of epigenetic cellular memory induced by morphine. Crisper-Cas9 silencing analyses confirmed that Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1), an architectural factor critical for X-chromosome inactivation (XCI), is a morphine-sensitive target gene. Morphine reduced the gene expression of Smchd1 in mESC at different time points, which is consistent with an increase of H3K27me3 and DNA methylation at promoter. Morphine alters normal X chromosome wide silencing. Our results identified Smchd1 a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by the cell, providing an epigenetic mechanism that is maintained into cell-to-cell memory and cell differentiation process, which might cause diseases or health problems during the adulthood. Limitations, reasons for caution To perform the in-vitro analysis in mouse Wider implications of the findings Our results provide insights into the epigenetic mechanisms induced by morphine and establish the bases to understand how environmental factors can cause epigenetic changes that cause health problems or diseases, since a skewed XCI pattern is behind the higher incidence on different neurological diseases such as Alzheimer or Autism Trial registration number not applicable

A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit

During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing this transition remain elusive. Here, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

O-215 Copper oxide nanoparticles (CuONPs) impairs mitophagy and promotes DNA methylation to suppress mouse embryonic pluripotency

Abstract Study question Does exposure to copper oxide nanoparticle (CuONPs) have adverse effects on early embryonic development? What is the potential mechanism? Summary answer CuONPs exposure can damage mitophagy and lead to α-KG reduction, resulting DNA hypermethylation, thereby reducing the pluripotency of pre-implantation embryos in mice. What is known already Environmental nanomaterials contamination is a great concern for organisms including human. Exposure to nanomaterials have been associated with fecundity and fertility in couples conceiving via assisted reproduction technology. Among these bio-effects, nanomaterials induced epigenetic changes are of particular interest. Copper oxide nanoparticles (CuONPs) are widely used in a huge range of applications which might pose potential risk to organisms. Recent studies reported that CuONPs exposure resulted in transgenerational toxicity in Caenorhabditis elegans associated with possible epigenetic regulation. However, whether CuONPs exposure affect mammalian embryonic development, and if so the underlying mechanisms, are unclear. Study design, size, duration For the human experiments, we measured the copper ion content in women’s urine, and assessed the expression of pluripotent genes in 3PN embryos treated with CuONPs. For the embryo experiments, we collected zygotes from ICR mice and cultured them in KSOM medium with CuONPs (10 μg/mL) until blastocyst stage. For the cell experiments, we treated mouse embryonic stem cells (mESCs) with CuONPs (2 μg/mL) for four days to detect cell proliferation and pluripotency. Participants/materials, setting, methods The ICP-MS method was used to detect copper ion content in women’s urine. Time–lapse incubator was used to document the developmental potential of embryos. To quantify the metabolites of embryo, we employed metabolomics. Single-cell multiomics sequencing that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression were performed to detect genes expression and epigenetic modification. We used real-time PCR, immunofluorescence, and western blot to measure the expression of pluripotency related genes. Main results and the role of chance In human, we discovered a significant negative relationship between the urine’s copper ion content and clinical pregnancy rate. In embryo experiments, CuONPs exposure has been shown to have adverse effects on embryo developmental potential, resulting morula and blastocyst transformation arrest, as well as reduction in blastocyst rate and live birth rate. Furthermore, CuONPs treatment led to a reduction in blastocyst quality, indicating a decrease in blastocyst cell count, particularly in ICM cells. CuONPs also inhibited the pluripotency of blastocysts and mESCs, showing down-regulated expression of pluripotent genes. Mechanistically, CuONPs aggregated in lysosomes and decreased the mitophagic flux of embryos, which caused damaged mitochondria to accumulate and ultimately caused mitochondrial dysfunction. Notably, in embryos treated with CuONPs, there was a decrease in α-ketoglutarate (α-KG) due to the disruption of the tricarboxylic acid (TCA) cycle. α-KG is an essential cofactor for α-KG dependent dioxygenases such as the ten-eleven translocation (TET) family of DNA demethylase. The lack of α-KG in CuONPs-treated embryos led to DNA hypermethylation, which reduced chromatin accessibility, and suppressed the expression of pluripotent genes like sox2, klf2, and klf4. The poor pluripotency ultimately leads to abnormal embryonic development in CuONPs-treated embryos. Limitations, reasons for caution The molecular mechanism by which CuONPs arrest the development of pre-implantation embryos remains to be determined. In addition, future work should be extended to human embryos to determine whether this mechanism is conserved between mice and humans. Wider implications of the findings The findings of this work indicated the relationship between CuONPs and epigenetic modification. The results will support a more thorough understanding into the biosafety of CuONPs, and offer fresh perspectives on the potential uses of nanomaterials in the field of reproductive medicine in the future. Trial registration number Not Applicable

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)—The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells

Background aimsEx vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. MethodsWe generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. ResultsMorphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5–7 days. ConclusionsOur results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Pig blastocyst-like structure models from embryonic stem cells

Pluripotent stem cells have the potential to generate embryo models that can recapitulate developmental processes in vitro. Large animals such as pigs may also benefit from stem-cell-based embryo models for improving breeding. Here, we report the generation of blastoids from porcine embryonic stem cells (pESCs). We first develop a culture medium 4FIXY to derive pESCs. We develop a 3D two-step differentiation strategy to generate porcine blastoids from the pESCs. The resulting blastoids exhibit similar morphology, size, cell lineage composition, and single-cell transcriptome characteristics to blastocysts. These porcine blastoids survive and expand for more than two weeks in vitro under two different culture conditions. Large animal blastoids such as those derived from pESCs may enable in vitro modeling of early embryogenesis and improve livestock species’ breeding practices.

CBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers.

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment

The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explore the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibit growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single-cell transcriptome levels. This mutation induces premature stem-like cell exhaustion, accelerates the maturation of GABAergic neurons, and disrupts neurodevelopmental and synaptic pathways. Additionally, it specifically hinders CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrors the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.

Regenerative mechanisms of stem cells and their clinical applications for degenerative eye diseases

There are different types of treatment for eye diseases. Although the majority of eye diseases are curable with primary treatments and surgery, some of degenerative eye damages need regeneration that is not gained by conventional procedures. Stem cells, such as mesenchymal stem cells, human embryonic stem cell-derived retinal pigmented epithelium, and inducible pluripotent stem cells, are now considered one of the most important and safe methods for regeneration of various damaged tissues or organs. However, how will stem cell therapy contribute to regeneration and overcome degenerative eye diseases? This review discusses the regenerative mechanisms, clinical applications, and advantages of different types of stem cells for restoring degenerative eye diseases.