Modeling X-chromosome inactivation and reactivation during human development.

Stem-cell-based embryo models generate much excitement as they offer a window into an early phase of human development that has remained largely inaccessible to scientific investigation. An important epigenetic phenomenon during early embryogenesis is the epigenetic silencing of one of the two X chromosomes in female embryos, which ensures an equal output of X-linked gene expression between the sexes. X-chromosome inactivation (XCI) is thought to be established within the first three weeks of human development, although the inactive X-chromosome is reactivated in primordial germ cells (PGCs) that migrate to the embryonic gonads. Here, we summarize our current understanding of X-chromosome dynamics during human development and comment on the potential of recently established stem-cell-based models to reveal the underlying mechanisms.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

Induction of Human Naïve Pluripotency Using 5i/L/A Medium.

Prior to implantation, the cells in the mammalian epiblast constitute a naïve pluripotent state, which is distinguished by absence of lineage priming, freedom from epigenetic restriction, and expression of a unique set of transcription factors. However, human embryonic stem cells (hESCs) derived under conventional conditions have exited this naïve state and acquired a more advanced "primed" pluripotent state that corresponds to the post-implantation epiblast. We have developed a cocktail comprising five kinase inhibitors and two growth factors (5i/L/A) that enables induction of defining features of naïve pluripotency in primed hESCs. These conditions can also be applied to induce naïve pluripotency in patient-specific induced pluripotent stem cells (iPSCs). Here, we provide a detailed protocol for inducing naïve pluripotency in primed hESCs and iPSCs and methods for the routine validation of naïve identity. We also outline the use of two fluorescent reporter systems to track acquisition of naïve identity in live cells: (a) a GFP reporter linked to an endogenous OCT4 allele in which the primed-specific proximal enhancer has been deleted (OCT4-ΔPE-GFP); and (b) a dual-color reporter system targeted to both alleles of an X-linked gene that reports on the status of the X chromosome in female cells (MECP2-GFP/tdTomato). The conditions described herein have given insight into various aspects of naïve human pluripotent stem cells (hPSCs), including their unique transposon transcription profile, X chromosome status, and extraembryonic potential.

OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human.

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening.

Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.

Derivation of trophoblast stem cells from naïve human pluripotent stem cells.

Naïve human pluripotent stem cells (hPSCs) provide a unique experimental platform of cell fate decisions during pre-implantation development, but their lineage potential remains incompletely characterized. As naïve hPSCs share transcriptional and epigenomic signatures with trophoblast cells, it has been proposed that the naïve state may have enhanced predisposition for differentiation along this extraembryonic lineage. Here we examined the trophoblast potential of isogenic naïve and primed hPSCs. We found that naïve hPSCs can directly give rise to human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous and syncytiotrophoblast. In contrast, primed hPSCs do not support hTSC derivation, but give rise to non-self-renewing cytotrophoblasts in response to BMP4. Global transcriptome and chromatin accessibility analyses indicate that hTSCs derived from naïve hPSCs are similar to blastocyst-derived hTSCs and acquire features of post-implantation trophectoderm. The derivation of hTSCs from naïve hPSCs will enable elucidation of early mechanisms that govern normal human trophoblast development and associated pathologies.

The placenta is one of the most important human organs, but it is perhaps the least understood. The first decision the earliest human cells have to make, shortly after the egg is fertilized by a sperm, is whether to become part of the embryo or part of the placenta. This choice happens before a pregnancy even implants into the uterus. The cells that commit to becoming the embryo transform into 'naïve pluripotent' cells, capable of becoming any cell in the body. Those that commit to becoming the placenta transform into 'trophectoderm' cells, capable of becoming the two types of cell in the placenta. Placental cells either invade into the uterus to anchor the placenta or produce hormones to support the pregnancy. Once a pregnancy implants into the uterus, the naïve pluripotent cells in the embryo become 'primed'. This prevents them from becoming cells of the placenta, and it poses a problem for placental research. In 2018, scientists in Japan reported conditions for growing trophectoderm cells in the laboratory, where they are known as "trophoblast stem cells". These cells were capable of transforming into specialized placental cells, but needed first to be isolated from the human embryo or placenta itself. Dong et al. now show how to reprogram other pluripotent cells grown in the laboratory to produce trophoblast stem cells. The first step was to reset primed pluripotent cells to put them back into a naïve state. Then, Dong et al. exposed the cells to the same concoction of nutrients and chemicals used in the 2018 study. This fluid triggered a transformation in the naïve pluripotent cells; they started to look like trophoblast stem cells, and they switched on genes normally active in trophectoderm cells. To test whether these cells had the same properties as trophoblast stem cells, Dong et al. gave them chemical signals to see if they could mature into placental cells. The stem cells were able to transform into both types of placental cell, either invading through a three-dimensional gel that mimics the wall of the uterus or making pregnancy hormones. There is a real need for a renewable supply of placental cells in pregnancy research. Animal placentas are not the same as human ones, so it is not possible to learn everything about human pregnancy from animal models. A renewable supply of trophoblast stem cells could aid in studying how the placenta forms and why this process sometimes goes wrong. This could help researchers to better understand miscarriage, pre-eclampsia and other conditions that affect the growth of an unborn baby. In the future, it may even be possible to make custom trophoblast stem cells to study the specific fertility issues of an individual.

MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage.

Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells.

Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

Techniques to access histone modifications and variants in cancer.

Recent years have witnessed an explosion of epigenetic research on the role of histone variants and modifications in cancer. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications, is required. The nucleosome is the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. The differential alterations in diverse histone variants and their accompanying modifications patterns will provide a deeper insight into their biological role in structural and functional properties of chromatin. Here we provide a step-by-step protocol to investigate these aspects, the histone modifications and variants, their localization and dynamics within specific regions of chromatin under distinct condition and the recruitment/retention of epigenetic regulators at their target sites in chromatin to influence cell growth and differentiation.

Cell-type specificity of ß-actin expression and its clinicopathological correlation in gastric adenocarcinoma.

AIM: To investigate cell type specific distribution of ß-actin expression in gastric adenocarcinoma and its correlation with clinicopathological parameters. METHODS: ß-actin is a housekeeping gene, frequently used as loading control, but, differentially expresses in cancer. In gastric cancer, an overall increased expression of ß-actin has been reported using tissue disruptive techniques. At present, no histological data is available to indicate its cell type-specific expression and distribution pattern. In the present study, we analyzed ß-actin expression and distribution in paired normal and tumor tissue samples of gastric adenocarcinoma patients using immunohistochemistry (IHC), a tissue non-disruptive technique as well as tissue disruptive techniques like reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Correlation of ß-actin level with clinicopathological parameters was done using univariate analysis. RESULTS: The results of this study showed significant overexpression, at both mRNA and protein level in tumor tissues as confirmed by RT-PCR (1.47 ± 0.13 vs 2.36 ± 0.16; P < 0.001) and western blotting (1.92 ± 0.26 vs 2.88 ± 0.32; P < 0.01). IHC revealed that ß-actin expression is majorly distributed between epithelial and inflammatory cells of the tissues. Inflammatory cells showed a significantly higher expression compared to epithelial cells in normal (2.46 ± 0.13 vs 5.92 ± 0.23, P < 0.001), as well as, in tumor tissues (2.79 ± 0.24 vs 6.71 ± 0.14, P < 0.001). Further, comparison of immunostaining between normal and tumor tissues revealed that both epithelial and inflammatory cells overexpress ß-actin in tumor tissues, however, significant difference was observed only in inflammatory cells (5.92 ± 0.23 vs 6.71 ± 0.14, P < 0.01). Moreover, combined expression in epithelial and inflammatory cells also showed significant increase (4.19 ± 0.15 vs 4.75 ± 0.14, P < 0.05) in tumor tissues. In addition, univariate analysis showed a positive correlation of ß-actin level of inflammatory cells with tumor grade (P < 0.05) while epithelial cells exhibited negative correlation (P > 0.05). CONCLUSION: In gastric cancer, ß-actin showed an overall higher expression predominantly contributed by inflammatory or tumor infiltrating immune cells of the tissue microenvironment and correlates with tumor grade.

Expression of histone variant, H2A.1 is associated with the undifferentiated state of hepatocyte.

Recent studies suggest the incorporation of histone variants into the chromatin regulate cellular proliferation, differentiation, and de-differentiation. We have earlier reported the increase of H2A.1 variant during sequential de-differentiation of hepatocyte to hepato-cellular carcinoma. Here, we decipher the alterations in expression of H2A.1 and H2A.2 variants during rat liver embryogenesis and regeneration. The expression of H2A.1 and H2A.2, at protein and mRNA level, does not alter in normal cellular proliferation associated with regeneration of liver post PH. In contrast, gradual decrease of H2A.1 with increase of H2A.2 is observed during differentiation of embryonic to adult liver. Furthermore, the accumulation of H2A.1 is higher in embryonic stem cells compared to normal adult liver. Collectively, these data support a strong correlation of H2A.1 expression with undifferentiated cells and overall epigenetic reprogramming in dedifferentiation and maturation of undifferentiated cells, rather than with normal cellular proliferation.