Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo.

Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state.

Learning common and specific patterns from data of multiple interrelated biological scenarios with matrix factorization.

High-throughput biological technologies (e.g. ChIP-seq, RNA-seq and single-cell RNA-seq) rapidly accelerate the accumulation of genome-wide omics data in diverse interrelated biological scenarios (e.g. cells, tissues and conditions). Integration and differential analysis are two common paradigms for exploring and analyzing such data. However, current integrative methods usually ignore the differential part, and typical differential analysis methods either fail to identify combinatorial patterns of difference or require matched dimensions of the data. Here, we propose a flexible framework CSMF to combine them into one paradigm to simultaneously reveal Common and Specific patterns via Matrix Factorization from data generated under interrelated biological scenarios. We demonstrate the effectiveness of CSMF with four representative applications including pairwise ChIP-seq data describing the chromatin modification map between K562 and Huvec cell lines; pairwise RNA-seq data representing the expression profiles of two different cancers; RNA-seq data of three breast cancer subtypes; and single-cell RNA-seq data of human embryonic stem cell differentiation at six time points. Extensive analysis yields novel insights into hidden combinatorial patterns in these multi-modal data. Results demonstrate that CSMF is a powerful tool to uncover common and specific patterns with significant biological implications from data of interrelated biological scenarios.

Considerations on chemistry, manufacturing, and control of stem cell products for Investigational New Drug application in China.

Tremendous progress has been made in recent years to produce functional cells for cell therapy products. Hundreds of clinical trials of stem cell products (SCPs) have shown promising therapeutic potential worldwide, including the products derived from human pluripotent stem cells (hPSCs), adult stem cells and mesenchymal stem cells (MSC). Before starting a clinical trial, comprehensive chemistry, manufacturing and control (CMC) study is required to assure the safety and quality consistency of SCPs. The heterogeneity of stem cell products arises from the variability in the donor tissues, isolation of cells and differentiation processes, and appropriate testing approaches are needed to characterize and release SCPs. Here we summarize the regulatory considerations of CMC study in Investigational New Drug (IND) application of SCPs in China based on the current knowledge, and they will be updated in the future with the advance of stem cell biology and regulatory science.

Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.

The significance of the pluripotency and cancer stem cell-related marker NANOG in diagnosis and treatment of ovarian carcinoma.

Ovarian cancer is among the most common gynecologic cancers and unfortunately the most common cause of death from gynecologic malignancies. Due to few early symptoms and insufficient screening programs, an early diagnosis of ovarian cancer is very difficult and new biomarkers related to early ovarian carcinogenesis are needed. In the last years a growing scientific knowledge about cancer stem cells and their markers opened a new perspective on screening and early diagnosis of ovarian cancer. The transcription factor NANOG is not only a pluripotency and cancer stem cell-related marker, but also promotes cancer stem cell-like characteristics of tumor, tumor growth, dissemination, immune evasion, and resistance to conventional therapy. The recent data showed that small stem cells resembling very small embryonic-like stem cells are present in the ovarian surface epithelium of adult human ovaries. These cells expressed several genes related to primordial germ cells, germinal lineage, and pluripotency, including NANOG, therefore their involvement in the manifestation of ovarian cancer are not excluded. As majority of cancer cells within a tumor are non tumorigenic, the therapies targeting these cells cause tumor regression, but the survived cancer stem cells regenerate the tumor, so tumor relapse or reoccur. The eradication of cancer actually requires the elimination of cancer stem cells, therefore new strategies in treatment that specifically target cancer stem cells are urgently needed. Although the therapeutic efficacy of targeting NANOG as a cancer treatment method is still in experimental phase, the gene therapy with small interfering RNA or short hairpin RNA have already shown some promising therapeutic potential. The authors can conclude that NANOG represents a promising diagnostic marker and agent for target therapy of ovarian cancer.

Histone variant macroH2A confers resistance to nuclear reprogramming.

How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveals differentially expressed microRNAs and novel IsomiRs with distinct targets.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells.

Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.

[Differentiation of pluripotent stem cells into pancreatic lineages]. / Différenciation des cellules souches pluripotentes en cellules pancréatiques.

Diabetes mellitus is the leading metabolic disease and represents a major public health concern worldwide. Whereas the transplantation of pancreas donor-derived islets significantly improves the quality of life of diabetic patients who become insulin independent for few years, it can unfortunately be provided only to few patients in an advanced stage of the disease. This situation is related to the severe shortage in pancreas donors and has prompted the hunt for alternative sources of islet cells. Beside many other strategies aiming at producing new beta cells in vitro or in vivo, a particular focus has been on the plupiropent stem cells because of their abundant availability and their extreme plasticity. Progress in understanding small vertebrates embryonic development has tremendously contributed to the design of differentiation strategies applied to pluripotent stem cells. Nowadays, definitive endoderm and pancreatic progenitors can be efficiently induced from human embryonic stem cells and from human induced pluripotent stem cells. Although we are still lacking the knowledge required for deriving functional beta cells in vitro, transplantation experiments have demonstrated that stem cell-derived pancreas progenitors further generate this phenotype in vivo. All these findings gathered during the last decade witness the closer clinical application of pluripotent stem cell progenies in diabetes cell therapy.

The characterisation of pluripotent and multipotent stem cells using Fourier transform infrared microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods.

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture.

BACKGROUND: Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS: We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS: Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.

Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Microplastics exposure affects neural development of human pluripotent stem cell-derived cortical spheroids.

Plastics have been part of our ecosystem for about a century and their degradation by different environmental factors produce secondary microplastics (MPs). To date, the impact of MPs on human health has not been well investigated. To understand the possible effects of polystyrene-MPs (PS-MPs) on the human brain, a 3D model of human forebrain cortical spheroids has been derived, which mimics early development of human cerebral cortex. The spheroids were exposed to 100, 50, and 5 µg/mL of 1 µm and 10 µm PS-MPs during day 4-10 and day 4-30. The short-term MP exposure showed the promoted proliferation and high gene expression of Nestin, PAX6, ATF4, HOXB4 and SOD2. For long-term exposure, reduced cell viability was observed. Moreover, changes in size and concentration of PS-MPs altered the gene expression of DNA damage and neural tissue patterning. In particular, ß-tubulin III, Nestin, and TBR1/TBR2 gene expression decreased in PS-MP treated conditions compare to the untreated control. The results of this study suggest that the size- and concentration-dependent exposure to PS-MPs can adversely affect embryonic brain-like tissue development in forebrain cerebral spheroids. This study has significance in assessing environmental factors in neurotoxicity and degeneration in human.

OCT4 expression in outgrowth colonies derived from porcine inner cell masses and epiblasts.

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM- and epiblast-derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)-like morphology. A total of 104 zona pellucida-enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC-like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC-like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC-like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT-PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC-like morphology. In conclusion, we have established a robust system for derivation of ESC-like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC-like morphology although this relationship is lost during early passages.

5-Hydroxymethylcytosine, the sixth base of the genome.

5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection--most notably LC-MS and glucosyltransferase assays--helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker.

Comparative cytotoxicity studies of halophenolic disinfection byproducts using human extended pluripotent stem cells.

2,4,6-trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are a new class of halophenolic disinfection byproducts (DBPs) which have been widely detected in drinking water. In recent years, their developmental toxicity has got increasing public attention due to their potential toxic effects on embryo development towards lower organisms. Nonetheless, the application of human embryos for embryonic toxicologic studies is rendered by ethical and moral considerations, as well as the technical barrier to sustaining normal development beyond a few days. Human extended pluripotent stem (EPS) cells (novel totipotent-like stem cells) represent a much more appropriate cellular model for studying human embryo development. In this study, we utilized human EPS cells to study the developmental toxicity of TCP, TBP and TIP, respectively. All three halophenolic DBPs showed cytotoxicity against human EPS cells in an obvious dose-dependent manner, among which TIP was the most cytotoxic one. Notably, the expression of pluripotent genes in human EPS cells significantly declined after 2,4,6-trihalophenol exposure. Meanwhile, 2,4,6-trihalophenol exposure promoted ectodermal differentiation of human EPS cells in an embryoid bodies (EBs) differentiation assay, while both endodermal and mesodermal differentiation were impaired. These results implied that phenolic halogenated DBPs have specific effects on human embryo development even in the early stage of pregnancy. In summary, we applied human EPS cells as a novel research model for human embryo developmental toxicity study of environmental pollutants, and demonstrated the toxicity of phenolic halogenated DBPs on early embryo development of human beings.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.