Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

Metabolic and cell cycle shift induced by the deletion of Dnm1l attenuates the dissolution of pluripotency in mouse embryonic stem cells.

Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.

Long Noncoding RNA Lx8-SINE B2 Interacts with Eno1 to Regulate Self-Renewal and Metabolism of Embryonic Stem Cells.

Long noncoding RNAs (lncRNAs) emerge as important orchestrators of biological processes in embryonic stem cells (ESCs). LncRNA Lx8-SINE B2 was recently identified as an ESC-specific lncRNA that marks pluripotency. Here, we studied the function of lncRNA Lx8-SINE B2 in ESCs. Depletion of Lx8-SINE B2 disrupted ESC proliferation, repressed the expression of pluripotency genes, activated differentiation genes, and inhibited reprogramming to induced pluripotent stem cells. The reduction of the colony formation ability of ESCs upon Lx8-SINE B2 knockdown was accompanied by the elongation of the G1 phase and the shortening of the S phase. Transcriptome analysis revealed that Lx8-SINE B2 deficiency affected multiple metabolic pathways, particularly glycolysis. Mechanistically, Lx8-SINE B2 functions as a cytoplasmic lncRNA and interacts with the glycolytic enzyme Eno1 as shown by RNA pull-down and RNA localization analysis. Lx8-SINE B2 and Eno1 interact with and regulate each other's expression, hence promoting the expression of metabolic genes and influencing glycolysis. In conclusion, we have identified lncRNA Lx8-SINE B2 as a novel regulator of ESC proliferation, cell cycle, and metabolism through working with Eno1.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.

SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina.

The stem cell factor receptor (SCFR) has been demonstrated to be expressed in the neural retina of mice, rat and human for decades. Previous reports indicated that the SCFR correlates with glia differentiation of late retinal progenitor cells (RPCs), retinal vasculogenesis and homeostasis of the blood-retinal barrier. However, the role of SCF/SCFR signaling in the growth and development of the neural retina (NR), especially in the early embryonic stage, remains poorly understood. Here, we show that SCF/SCFR signaling orchestrates invagination of the human embryonic stem cell (hESC)-derived NR via regulation of cell cycle progression, cytoskeleton dynamic and apical constriction of RPCs in the ciliary marginal zone (CMZ). Furthermore, activation of SCF/SCFR signaling promotes neurogenesis in the central-most NR via acceleration of the migration of immature ganglion cells and repressing apoptosis. Our study reveals an unreported role for SCF/SCFR signaling in controlling ciliary marginal cellular behaviors during early morphogenesis and neurogenesis of the human embryonic NR, providing a new potential therapeutic target for human congenital eye diseases such as anophthalmia, microphthalmia and congenital high myopia.

Pluripotent stem cell-based gene therapy approach: human de novo synthesized chromosomes.

A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.

Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modeling.

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson's disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD.

Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy.

The gene therapy approach aiming at curing various human diseases began to develop as a technology from early eighties of the last century. To date the delivery of therapeutic genes are mainly mediated by virus-based, predominantly, non-integrated virus vectors. These gene delivery approaches have several fundamental limitations on the way of efficient deployment in clinical gene therapy. A totally different approach was suggested about 20 years ago when episomal non-integrative artificial chromosome-based vectors featuring large size inserts (even native gene loci) advanced to the stage. Since then numerous human artificial chromosome (HAC) vectors were developed by both de novo synthesis and top-down engineering technology. This approach so far is limited to ex vivo gene transfer and correction within highly proliferative or reversibly immortalized precursor stem cells or pluripotent stem cells. Recent breakthrough in generation of induced pluripotent stem cells and embryonic stem cell manipulation give the additional pivotal stimuli to integrate it with the HAC technology and to develop thereby novel approaches to replacement therapies of human genetic diseases. The HAC technology is complex and time consuming while nowadays it has significantly advanced and become notably closer to medical applications. In this review we discuss current advancements in the HAC technology, in particular, in terms of improvement of chromosome transfer method and achievements in developing mouse-based gene therapy tissue replacement models for several monogenic human diseases. The main progress has been done in elaboration of top-down type HAC technology in modeling and preclinical studies of gene therapy treatment for Duchenne muscular dystrophy (DMD) disease.

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway.

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.

HLA-G Expression in Human Mesenchymal Stem Cells (MSCs) Is Related to Unique Methylation Pattern in the Proximal Promoter as well as Gene Body DNA.

Multipotent human mesenchymal stem cells (MSCs) harbor clinically relevant immunomodulation, and HLA-G, a non-classical MHC class I molecule with highly restricted tissue expression, is one important molecule involved in these processes. Understanding of the natural regulatory mechanisms involved in expression of this elusive molecule has been difficult, with near exclusive reliance on cancer cell lines. We therefore studied the transcriptional control of HLA-G in primary isolated human bone marrow- (BM), human embryonic stem cell-derived (hE-), as well as placenta-derived MSCs (P-MSCs), and found that all 3 types of MSCs express 3 of the 7 HLA-G isoforms at the gene level; however, fibroblasts did not express HLA-G. Protein validation using BM- and P-MSCs demonstrated expression of 2 isoforms including a larger HLA-G-like protein. Interferon-γ (IFN-γ) stimulation upregulated both gene and protein expression in MSCs but not the constitutively expressing JEG-3 cell line. Most interestingly in human MSCs and placental tissue, hypomethylation of CpG islands not only occurs on the HLA-G proximal promoter but also on the gene body as well, a pattern not seen in either of the 2 commonly used choriocarcinoma cell lines which may contribute to the unique HLA-G expression patterns and IFN-γ-responsiveness in MSCs. Our study implicates the importance of using normal cells and tissues for physiologic understanding of tissue-specific transcriptional regulation, and highlight the utility of human MSCs in unraveling the transcriptional regulation of HLA-G for better therapeutic application.

Epigenetic differences between naïve and primed pluripotent stem cells.

It has been 8 years since the concept of naïve and primed pluripotent stem cell states was first proposed. Both are states of pluripotency, but exhibit slightly different properties. The naïve state represents the cellular state of the preimplantation mouse blastocyst inner cell mass, while the primed state is representative of the post-implantation epiblast cells. These two cell types exhibit clearly distinct developmental potential, as evidenced by the fact that naïve cells are able to contribute to blastocyst chimeras, while primed cells cannot. However, the epigenetic differences that underlie the distinct developmental potential of these cell types remain unclear, which is rather surprising given the large amount of active investigation over the years. Elucidating such epigenetic differences should lead to a better understanding of the fundamental properties of these states of pluripotency and the means by which the naïve-to-primed transition occurs, which may provide insights into the essence of stem cell commitment.

Pluripotent Stem Cell Heterogeneity.

Pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, show heterogeneity with respect to their pluripotency, self-renewal ability, and other traits. PSC heterogeneity may exist among cell lines, among cells within a line, and among temporal states of individual cells. Both genetic and epigenetic factors can cause heterogeneity among cell lines. Heterogeneity among cells within a cell line may arise during long-term culturing even when a PSC cell line is derived from a single cell. Moreover, the expression levels of genes and proteins in PSCs fluctuate continuously at a frequency ranging from a few hours to a few days. Such heterogeneity decreases the reproducibility of research. Thus, methods related to the detection, reduction, and control of heterogeneity in experiments involving human PSCs need to be developed. Further, the presupposition that PSCs are highly heterogeneous should be taken into account by all researchers not only when they plan their own studies but also when they review the studies of other researchers in this field.

Establishment of a human embryonic stem cell-based liver differentiation model for hepatotoxicity evaluations.

The liver is one of the major targets of hormones, including thyroid hormones (THs), and many industrial chemicals, such as endocrine-disrupting chemicals. Those compounds may permeate the placenta barrier and pose a risk for embryonic development. Therefore, it is necessary to assess the toxic effects of those kind of industrial chemicals during liver development. In this study, to mimic liver specification in vitro, we differentiated human embryonic stem cells (ESCs) into functional hepatocyte-like cells. We performed this differentiation process in presence of two THs, triiodothyronine (T3) and thyroxine (T4), with the purpose of identifying biomarkers for toxicity screening. TH exposure (3, 30 and 300 nM) yielded to hepatocytes with impaired glycogen storage ability and abnormal lipid droplets' accumulation. Global gene expression analysis by RNA-seq identified a number of genes responsible for hepatic differentiation and function which were affected by 30 nM T3 and T4. Those differentially expressed genes were used to assess the potential developmental liver toxicity of two famous environmental pollutants, 2, 2, 4, 4-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209), at 10 nM to 1 µM treatments. Our findings demonstrate that BDE-47 and BDE-209, dysregulated pathways such as "chemical carcinogenesis", "steroid hormone biosynthesis" and "drug metabolism-cytochrome P450". Moreover, we were able to identify a set of 17 biomarkers, very useful to predict the potential developmental hepatotoxicity of industrial chemicals.

Stem Cells: The Game Changers of Human Cardiac Disease Modelling and Regenerative Medicine.

A comprehensive understanding of the molecular basis and mechanisms underlying cardiac diseases is mandatory for the development of new and effective therapeutic strategies. The lack of appropriate in vitro cell models that faithfully mirror the human disease phenotypes has hampered the understanding of molecular insights responsible of heart injury and disease development. Over the past decade, important scientific advances have revolutionized the field of stem cell biology through the remarkable discovery of reprogramming somatic cells into induced pluripotent stem cells (iPSCs). These advances allowed to achieve the long-standing ambition of modelling human disease in a dish and, more interestingly, paved the way for unprecedented opportunities to translate bench discoveries into new therapies and to come closer to a real and effective stem cell-based medicine. The possibility to generate patient-specific iPSCs, together with the new advances in stem cell differentiation procedures and the availability of novel gene editing approaches and tissue engineering, has proven to be a powerful combination for the generation of phenotypically complex, pluripotent stem cell-based cellular disease models with potential use for early diagnosis, drug screening, and personalized therapy. This review will focus on recent progress and future outcome of iPSCs technology toward a customized medicine and new therapeutic options.

Genomic integrity of ground-state pluripotency.

Pluripotent cells appear to be in a transient state during early development. These cells have the capability to transition into embryonic stem cells (ESCs). It has been reported that mouse pluripotent cells cultivated in chemically defined media sustain the ground state of pluripotency. Because the epigenetic pattern of pluripotent cells reflects their environment, culture under different conditions causes epigenetic changes, which could lead to genomic instability. This study focused on the DNA methylation pattern of repetitive elements (REs) and their activation levels under two ground-state conditions and assessed the genomic integrity of ESCs. We measured the methylation and expression level of REs in different media. The results indicated that although the ground-state conditions show higher REs activity, they did not lead to DNA damage; therefore, the level of genomic instability is lower under the ground-state compared with the conventional condition. Our results indicated that when choosing an optimum condition, different features of the condition must be considered to have epigenetically and genomically stable stem cells.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

Roles of ZIC2 in Regulation of Pluripotent Stem Cells.

Pioneered by the classical mouse embryonic stem cells (ESCs), various stem cell lines representing the peri- and postimplantation stages of embryogenesis have been established. To gain insight into the gene regulatory network operating in these cells, we first investigated epiblast stem cells (EpiSCs), performing ChIP-seq analysis for five major transcription factors (TFs) involved in epiblast regulation. The analysis indicated that SOX2-POU5F1 TF pairs highlighted in mouse ESCs are not the major players in other stem cells. The major acting transcription factors shift from SOX2/POU5F1 in mouse ESCs to ZIC2/OTX2 in EpiSCs, and this shift is primed in ESCs by binding of ZIC2 at relevant genomic positions that later function as enhancers.

Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.

Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. MSCs derived from placental chorionic villi of the first trimester are likely to resemble, biologically, embryonic stem cells (ESC), due to the earlier development stage of placenta. In the present study long-term cultures of MSC-like cells were assessed in order to evaluate MSCs multipotent characteristics and molecular features during the period of culture. CV-cells obtained from 10 samples of chorionic villus displayed typical fibroblastoid morphology, undergone 20 passages during a period of 120 days, maintaining a stable karyotype throughout long term expansion. The cells were positive, for CD90, CD73, CD105, CD29, CD44, HLA ABC antigens and negative for CD14, CD34, AC133, and HLA DR antigens as resulted from the flow cytometry analysis. CV-cells were differentiated in adipocytes, osteoblasts, chondrocytes and neuronal cells under specific culture conditions. The expression of the ESC-gene markers POU5F1 (Oct-4) and NANOG was observed at earliest stages (4-12 passages) and not at the late stages (14-20 passages) by RT-PCR analysis. ZFP42 and SOX2 expression were not detected. Moreover, CV-cells were found to express GATA4 but not NES (Nestin). Chorionic villi-derived cells possess multipotent properties, display high proliferation rate and self-renew capacity, share common surface antigens with adult MSCs and express certain embryonics stem cells gene markers. These characteristics highlight chorionic villi as an attractive source of MSCs for the needs of regenerative medicine.

Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.