Embryonic Stem Research Articles

Zygotic activin a is dispensable for the mouse preimplantation embryo development and for the derivation and pluripotency of embryonic stem cells†.

In this work, we aimed to determine the role of activin A during crucial events of mouse embryogenesis and distinguish the function of the protein of zygotic origin and the one secreted by the maternal reproductive tract. To this end, we recorded the progression of development and phenotype of Inhba knockout embryos and compared them with the heterozygotes and wild-type embryos using time-lapse imaging and detection of lineage-specific markers. We revealed that the zygotic activin A deficiency does not impair the course and rate of development of embryos to the blastocyst stage. Inhba knockout embryos form functional epiblast, as evidenced by their ability to give rise to embryonic stem cells. Our study is the first to show that derivation, maintenance in culture, and pluripotency of embryo-derived embryonic stem cells are exogenous and endogenous activin A-independent. However, the implantation competence of activin A-deficient embryos may be compromised as indicated in the outgrowth assay.

CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length

Mammalian tail length is controlled by several genetic determinants, among which are Hox13 genes, whose function is to terminate the body axis. Accordingly, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing a high number of CTCF sites, potentially isolating Hoxb13 from the rest of the cluster and thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes. These defects were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant Embryonic Stem (ES) cells grown as pseudoembryos in vitro, which allowed us to examine in detail the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB, and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations were already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

Generation of self-renewing neuromesodermal progenitors with neuronal and skeletal muscle bipotential from human embryonic stem cells.

Progress has been made in generating spinal cord and trunk derivatives from neuromesodermal progenitors (NMPs). However, maintaining the self-renewal of NMPs invitro remains a challenge. In this study, we developed a cocktail of small molecules and growth factors that induces human embryonic stem cells to produce self-renewing NMPs (srNMPs) under chemically defined conditions. These srNMPs maintain the state of neuromesodermal progenitors in prolonged culture and have the potential to generate mesodermal cells and neurons, even at the single-cell level. Additionally, suspended srNMP aggregates can spontaneously differentiate into all tissue types of early embryonic trunks. Furthermore, transplanted srNMP-derived muscle satellite cells or progenitors of motor neurons were integrated into skeletal muscle or the spinal cord, respectively, and contributed to regeneration in mouse models. In summary, srNMPs hold great promise for applications in developmental biology and as renewable cell sources for cell therapy for trunk and spinal cord injuries.

A Contemporary Perspective on Embryonic Stem Cell Research

Stem cells serve as an internal repair system by replacing other cells that are worn out or damaged, essentially always keeping the body at maximum capacity. However, the primary method of obtaining human embryonic stem cells (hESC) is by harvesting embryos not used by In Vitro Fertilization clinics, which raises the question if this shows a lack of respect for human life. From one perspective, the leftover embryos from fertility treatment will never develop into a child unless it is implanted in the uterine wall and using them to help treat devastating diseases is better than disposing of them as biological waste. However, others believe the embryos are human beings and that this type of research is tortuous. hESC research has the potential to revolutionize medical treatment and save millions of lives, but we must ensure that we push the boundaries of science while maintaining the utmost respect for human life.

Stem cell transplantation extends the reproductive life span of naturally aging cynomolgus monkeys

The ovary is crucial for female reproduction and health, as it generates oocytes and secretes sex hormones. Transplantation of mesenchymal stem cells (MSCs) has been shown to alleviate pathological ovarian aging. However, it is unclear whether MSCs could benefit the naturally aging ovary. In this study, we first examined the dynamics of ovarian reserve of Chinese women during perimenopause. Using a naturally aging cynomolgus monkey (Macaca fascicularis) model, we found that transplanting human embryonic stem cells-derived MSC-like cells, which we called M cells, into the aging ovaries significantly decreased ovarian fibrosis and DNA damage, enhanced secretion of sex hormones and improved fertility. Encouragingly, a healthy baby monkey was born after M-cell transplantation. Moreover, single-cell RNA sequencing analysis and in vitro functional validation suggested that apoptosis, oxidative damage, inflammation, and fibrosis were mitigated in granulosa cells and stromal cells following M-cell transplantation. Altogether, these findings demonstrate the beneficial effects of M-cell transplantation on aging ovaries and expand our understanding of the molecular mechanisms underlying ovarian aging and stem cell-based alleviation of this process.

Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases.

When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

Copper Oxide Nanoparticles Impair Mouse Preimplantation Embryonic Development through Disruption of Mitophagy-Mediated Metabolism.

Copper oxide nanoparticles (CuONPs) have been widely applied, posing potential risks to human health. Although the toxicity of CuONPs on the liver and spleen has been reported, their effects on reproductive health remain unexplored. In this study, we investigate the effects of CuONPs on embryonic development and their potential mechanisms. Our results demonstrate that CuONPs exposure impairs mouse preimplantation embryonic development, particularly affecting the morula-to-blastocyst transition. Additionally, CuONPs were found to reduce the pluripotency of the inner cell mass (ICM) and mouse embryonic stem cells (mESCs). Mechanistically, CuONPs block autophagic flux and impair mitophagy, leading to the accumulation of damaged mitochondria. This mitochondrial dysfunction leads to reduced tricarboxylic acid (TCA) cycle activity and decreased α-ketoglutarate (α-KG) production. Insufficient α-KG induces the failure of DNA demethylation, reducing corresponding chromatin accessibility and consequently inhibiting ICM-specific genes expressions. Similar reduced development and inhibitions of pluripotency gene expression were observed in CuONPs-treated human blastocysts. Moreover, in women undergoing assisted reproductive technology (ART), a negative correlation was found between urinary Cu ion concentrations and clinical outcomes. Collectively, our study elucidates the mitophagy-mediated metabolic mechanisms of CuONPs embryotoxicity, improving our understanding of the potential reproductive toxicity associated with it.

Regulation of de novo and maintenance DNA methylation by DNA methyltransferases in post-implantation embryos.

DNA methylation is mainly catalyzed by three DNA methyltransferase (DNMT) proteins in mammals. Usually DNMT1 is considered the primary DNMT for maintenance DNA methylation, whereas DNMT3A and DNMT3B function in de novo DNA methylation. Interestingly, we found DNMT3A and DNMT3B exerted maintenance and de novo DNA methylation in post-implantation mouse embryos. Together with DNMT1, they maintained DNA methylation at some pluripotent genes and lineage marker genes. Germline-derived DNA methylation at the imprinting control regions (ICRs) is stably maintained in embryos. DNMT1 maintained DNA methylation at most ICRs in post-implantation embryos. Surprisingly, DNA methylation was increased at five ICRs after implantation, and two DNMT3 proteins maintained the newly acquired DNA methylation at two of these five ICRs. Intriguingly, DNMT3A and DNMT3B maintained pre-existing DNA methylation at four other ICRs, similar to what we found in embryonic stem (ES) cells before. These results suggest that DNA methylation is more dynamic than originally thought during embryogenesis including the ICRs of the imprinted regions. DNMT3A and DNMT3B exert both de novo and maintenance DNA methylation functions after implantation. They maintain large portions of newly acquired DNA methylation at variable degrees across the genome in mouse embryos, together with DNMT1. Furthermore, they contribute to maintenance of pre-existing DNA methylation at a subset of ICRs as well as in the CpG islands (CGIs) and certain lineage marker gene. These findings may have some implications for the important roles of DNMT proteins in development and human diseases.

Differential modulation of polycomb-associated histone marks by cBAF, pBAF, and gBAF complexes.

Chromatin regulators alter the physical properties of chromatin to make it more or less permissive to transcription by modulating another protein's access to a specific DNA sequence through changes in nucleosome occupancy or histone modifications at a particular locus. Mammalian SWI/SNF complexes are a group of ATPase-dependent chromatin remodelers. In mouse embryonic stem cells, there are three primary forms of mSWI/SNF: canonical BAF (cBAF), polybromo-associated BAF (pBAF), and GLTSCR-associated BAF (gBAF). Nkx2-9 is bivalent, meaning nucleosomes at the locus have active and repressive modifications. In this study, we used unique BAF subunits to recruit each of the three complexes to Nkx2-9 using dCas9-mediated inducible recruitment (FIRE-Cas9). We show that recruitment of cBAF complexes leads to a significant loss of the polycomb repressive-2 H3K27me3 histone mark and polycomb repressive-1 and repressive-2 complex proteins, whereas gBAF and pBAF do not. Moreover, nucleosome occupancy alone cannot explain the loss of these marks. Our results demonstrate that cBAF has a unique role in the direct opposition of polycomb-associated histone modifications that gBAF and pBAF do not share.

Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality.

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown. Therefore, we conducted comprehensive genomic, single-cell transcriptomic, and single-cell ATAC-seq analyses of an isogenic hESC model displaying definitive 'culture-adapted phenotypes'. We found that hESCs lacking TP53, in which loss-of-function mutations were identified in human pluripotent stem cells (hPSCs), presented a surge in somatic mutations. Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present 'culture-adapted phenotypes' or BCL2L1 induction. Single-cell RNA-seq and ATAC-seq analyses revealed active transcriptional regulation at the 20q11.21 locus. Furthermore, the induction of BCL2L1 and TPX2 to trigger 'culture-adapted phenotypes' was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that 20q11.21 copy number gain and additional epigenetic changes are necessary for expressing 'culture-adapted phenotypes' by activating gene transcription at this specific locus.

Cysteamine functionalized gold nanoparticles exhibit high efficiency delivery of genetic materials in embryonic stem cells majorly via clathrin mediated endocytosis

Efficient and safe gene delivery is vital for genetic manipulation of stem cells for regenerative medicine. Gold nanoparticles have been used for various biomedical applications in the past, and are currently being researched as transfection agents. In this study, we report a simple one-pot synthesis of positively charged gold nanoparticles functionalized with cysteamine. The nanoparticles exhibit no cytotoxicity and can bind to both plasmid DNA (pDNA) as well as small interference RNA (siRNA). We observed that a five fold lower concentration of pDNA was sufficient for achieving comparable overexpression as that of a commercial transfection agent. We also observed that about 70 % transient silencing of the target gene was achieved with only 25 nM siRNA delivered by our nano-vehicle. To better understand the fate of the nanoparticle, we attempted to identify its uptake mechanism. The results indicate that while all the mechanisms contribute to the uptake, the clathrin-dependent pathway plays a major role. This is the first study on understanding the mechanism of uptake of CA-AuNPs conjugated to pDNA by embryonic stem cells. This is also the first study, where a successful transfection using gold based nanoparticles has been achieved in ESCs at a concentration as low as 0.5 µg/ml for pDNA and 25ƞM siRNA.

Charge-neutralized polyethylenimine-lipid nanoparticles for gene transfer to human embryonic stem cells

Charge-neutralized polyethylenimine-lipid nanoparticles for gene transfer to human embryonic stem cells

STAG2 promotes naive-primed transition via activating Lin28a transcription in mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) exist in two distinct pluripotent states: the naive and the primed. Mainly by inducing differentiation of mESCs in vitro, conducting RNA sequencing analyses, and specifying expression of the regulatory genes, we explored the regulatory mechanisms underlying the transition between the naive and primed states. We found that, under the defined differentiation-inducing conditions, the naive state of mESCs shifted to the primed state within two days of differentiation induction, during which the cell cycle- and differentiation-related proteins changes significantly. Specifically, we uncovered that the expression of STAG2, a subunit of the Cohesin complex, was upregulated. We further revealed that knockout of STAG2 resulted in upregulation of the naive gene sets and downregulation of the primed gene sets, indicating importance of STAG2 in regulating the naive-primed transition. More importantly, STAG2 knockout led to a reduction in number of the bivalent genes, a decrease in Lin28a transcription, and a reduced cytoplasmic localization of Lin28a. Overexpressing Lin28a or a Lin28a variant lacking the nucleolar localization signal (Lin28aΔNoLS) in STAG2 knockout cells rescued the downregulation of the primed marker genes Dnmt3a/3b. Collectively, we conclude that STAG2 facilitates the naive-primed transition of mESCs by activating Lin28a transcription and that this work may offer a new insight into the regulation of pluripotency in mESCs.

PIAS4 regulates pluripotency exit and cell fate commitment in porcine embryonic stem cells

Post-translational modifications (PTMs) are dynamic processes that regulate cell states by enhancing proteome diversity. However, the overall impact of PTMs on pluripotency exit in porcine embryonic stem cells (pESCs) remains largely unknown. Here, we present a systematic assay to identify E3 ubiquitin ligases for pluripotency exit by using CRISPR/Cas9 pooled screening and identified PIAS4 as a major regulator of pluripotency exit, as the cell differentiation was significantly impaired upon PIAS4 depletion in pESCs. PIAS4 shows a high degree of genomic occupation in promoter regions, particularly in key pluripotency maintenance genes. Moreover, we found that PIAS4 was recruited to the gene promoter marked by H3K4me3 and interacted with lysine demethylase KDM5B via SUMOylation, thereby affecting the stability of KDM5B and further facilitating the regulation of H3K4me3-mediated lineage-specific genes. Together, our findings reveal a regulatory mechanism by which PIAS4 modulates H3K4me3 modification on development-related genes, subsequently influencing pluripotency exit and cell fate commitment by interacting with KDM5B in pESCs.

A Single Cell Transcriptomic Fingerprint of Stressed Premature, Imbalanced Differentiation of Embryonic Stem Cells.

Miscarriages cause a greater loss-of-life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra-embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri-implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation. Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF - , ND). Sorbitol and RA increase subpopulations of 2-cell embryo-like (2CEL) and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE-head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects.

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Progress in Transdifferentiation of Autologous Alternative Cell Sources into Corneal Epithelial Cells.

Corneal limbal epithelial stem cells (LESCs) play a crucial role in corneal epithelium regeneration. Severe damage to these cells can result in limbal stem cell deficiency (LSCD), characterized by repeated corneal conjunctivalization, leading to corneal turbidity and scar formation. Restoring functional LESCs and their ecological location are essential for treating LSCD. The goal of this review is to provide researchers and clinicians with key insights into LESCs biology and to conclude the current cell-based therapies advancement in LSCD treatments. Therapeutic cell resources mainly include mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), skin keratinocyte stem cells (SKCs), and oral mucosal epithelial cells (OMECs).

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programmes and repeats in pluripotent cells.

H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A 'binary switch' governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMT depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening and exit from pluripotency within 12 h. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3 heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

Human Pluripotent Stem Cell Colony Migration Is Related to Culture Environment and Morphological Phenotype

Human pluripotent stem cells (hPSCs) are an important tool in the field of regenerative medicine due to their ability to differentiate towards all tissues of the adult organism. An important task in the study of hPSCs is to understand the factors that influence the maintenance of pluripotent and clonal characteristics of colonies represented by their morphological phenotype. Such factors include the ability of colonies to migrate during growth. In this work, we measured and analyzed the migration trajectories of hPSC colonies obtained from bright-field images of three cell lines, including induced hPSC lines AD3 and HPCASRi002-A (CaSR) and human embryonic stem cell line H9. To represent the pluripotent status, the colonies were visually phenotyped into two classes having a “good” or “bad” morphological phenotype. As for the migration characteristics, we calculated the colony speed and distance traveled (mobility measures), meandering index (motion persistence measures), outreach ratio (trajectory tortuosity characteristic), as well as the velocity autocorrelation function. The analysis revealed that the discrimination of phenotypes by the migration characteristics depended on both the cell line and growth environment. In particular, when the mTESR1/Matrigel culture environment was used, “good” AD3 colonies demonstrated a higher average migration speed than the “bad” ones. The reverse relationship between average speeds of “good” and “bad” colonies was found for the H9 line. The CaSR cell line did not show significant differences in the migration speed between the “good” and “bad” phenotypes. We investigated the type of motion exhibited by the colonies by applying two diffusion models to the mean squared displacement dynamics, one model corresponding to normal and the other to anomalous diffusion. The type of diffusion and diffusion parameter values resulting from the model fitting to data demonstrated a similar cell line, environment, and phenotype dependency. Colonies mainly showed a superdiffusive behavior for the mTESR1/Matrigel culture conditions, characterized by longer migration steps compared to the normal random walk. The specific properties of migration features and the patterns of their variation demonstrated in our work can be useful for the development and/or improvement of automated solutions for quality control of hPSCs.

Integrating Microarray Data and Single-Cell RNA-Seq Reveals Key Gene Involved in Spermatogonia Stem Cell Aging.

The in vitro generation of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) offers a viable approach for addressing male infertility. A multitude of molecules participate in this intricate process, which requires additional elucidation. Despite the decline in SSCs in aged testes, SSCs are deemed immortal since they can multiply for three years with repeated transplantation. Nonetheless, the examination of aging is challenging due to the limited quantity and absence of precise indicators. Using a microarray, we assessed genome-wide transcripts (about 55,000 transcripts) of fibroblasts and SSCs. The WGCNA approach was then used to look for SSC-specific transcription factors (TFs) and hub SSC-specific genes based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from the GEO databases as well as gene expression data (RNA-seq and microarray data). The microarray analysis of three human cases with different SSCs revealed that 6 genes were upregulated, and the expression of 23 genes was downregulated compared to the normal case in relation to aging genes. To reach these results, online assessments of Enrich Shiny GO, STRING, and Cytoscape were used to forecast the molecular and functional connections of proteins before identifying the master routes. The biological process and molecular function keywords of cell-matrix adhesion, telomerase activity, and telomere cap complex were shown to be significantly altered in upregulated differentially expressed genes (DEGs) by the functional enrichment analysis. According to our preliminary research, cell-specific TFs and TF-mediated GRNs are involved in the creation of SSCs. In order to maximize the induction efficiency of ESC differentiation into SSCs in vitro, hub SSC-specific genes and important SSC-specific TFs were identified, and sophisticated network regulation was proposed. According to our research, these genes and the hub proteins that they interact with may be able to shine a light on the pathophysiologies of infertility and aberrant germ cells.