Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we ...propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.
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•Naive human ESCs share a unique transposon signature with cleavage-stage embryos•Global DNA demethylation in naive human ESCs is reversible except at imprinted loci•The X chromosome status of naive human ESCs resembles the preimplantation embryo•Naive human ESCs incorporate into the mouse morula or blastocyst very inefficiently
Theunissen et al. use molecular criteria based on transposon expression, DNA methylation, and X chromosome status to compare naive human pluripotent cells to human preimplantation embryos. Current approaches yield cells that most closely resemble the morula/early blastocyst stage.
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between ...gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
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•YY1 generally occupies active enhancers and promoters across cell types•YY1 can form dimers and promote DNA interactions•Perturbation of YY1 binding disrupts enhancer-promoter looping and gene expression•YY1’s structural role accounts for diverse functions reported previously
YY1 is a structural regulator of enhancer-promoter interactions and facilitates gene expression.
Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell ...identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.
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•Allele-specific reporters revealed dynamic DNA methylation of Sox2 and miR290 SEs•DNMTs and transcription factor binding regulate methylation dynamics•SE DNA methylation directly regulates transcription in cis•Dynamic DNA methylation is co-regulated with MED1 recruitment and H3K27ac level
Song et al. used an allelic reporter approach to show super-enhancers allelic DNA methylation dynamics underlies locus-specific heterogeneity, which functionally impacts transcription and cellular states of mouse embryonic stem cells.
Intravital imaging of mouse embryos Huang, Qiang; Cohen, Malkiel A; Alsina, Fernando C ...
Science (American Association for the Advancement of Science),
04/2020, Letnik:
368, Številka:
6487
Journal Article
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Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 ...to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.
Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC ...chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.
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•A new platform to study human neuroblastoma using mouse-human neural crest chimeras•Chimeras are created by injecting hNCCs with relevant oncogenes into E8.5 mouse embryos•The chimeras developed tumors that resemble primary neuroblastomas seen in patients•The chimeric mice mount a strong immune response that is blunted by the tumor
Cohen et al. present a novel system to model human cancer in immunocompetent mice. This is accomplished by injecting human pluripotent stem cell-derived neural crest cells (hNCC) into early mouse embryos, generating mouse-human neural crest chimeras. Oncogenes are then induced, allowing transformation of the hNCCs to form neuroblastoma.
Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of ...lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.
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•Defective autophagy in NPC1 disease is caused by failure of SNARE machinery•Loss of NPC1 protein impairs amphisome formation and autophagosome maturation•Cholesterol depletion with HP-β-cyclodextrin blocks autophagic flux•Induced autophagy rescues defects and is a rational therapeutic strategy for NPC1
Autophagy dysfunction is implicated in several human diseases. Now, Jaenisch and colleagues show that autophagic flux is impaired in Niemann-Pick type C1 disease, possibly contributing to disease pathology. Stimulating autophagy rescues the block in basal autophagy without the formation of amphisomes. Cholesterol-depletion treatment with HP-β-cyclodextrin also impedes autophagy, whereas using a lower dose that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy by removing both cholesterol and autophagic cargo.
The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is ...involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (Wsh/Wsh
), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse–human chimeric platform allows for a new approach to study NC development and diseases.
Chimeric mice have been generated by injecting pluripotent stem cells into morula-to-blastocyst stage mouse embryo or by introducing more mature cells into later stage embryos that correspond to the ...differentiation stage of the donor cells. It has not been rigorously tested, however, whether successful chimera formation requires the developmental stage of host embryo and donor cell to be matched. Here, we compared the success of chimera formation following injection of primary neural crest cells (NCCs) into blastocysts or of embryonic stem cells (ESCs) into E8.5 embryos (heterochronic injection) with that of injecting ESCs cells into the blastocyst or NCCs into the E8.5 embryos (isochronic injection). Chimera formation was efficient when donor and host were matched, but no functional chimeric contribution was found in heterochronic injections. This suggests that matching the developmental stage of donor cells with the host embryo is crucial for functional engraftment of donor cells into the developing embryo.
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•Developmental matching of donor cells and host is crucial for chimera formation•Heterochronic injection of ESCs to E8.5 mouse embryos failed to yield chimeras•NCCs injected to blastocyst failed to form chimeras, even when apoptosis is impaired
Cohen at al. compares the efficiency of chimera formation in heterochronic and isochronic injections of ESCs and NCCs. Using two distinct and well-characterized pre- and post-implantation chimeric platforms, they show that matching of developmental age of donor cells and the host is essential for chimera formation.
The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to ...characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord‐like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3–4 × 2 × 1 mm3. A total of 40–60% of these cells was positive for Oct4, Sox2, and Nanog and 60–80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer‐derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis—cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural‐crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal‐ and mesodermal‐derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue. STEM CELLS 2010;28:984–995
Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these ...core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.
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•Core transcription factors (TFs) are predicted for >200 cell types/tissues•Predicted TFs for retinal pigment epithelial (RPE) cells can reprogram fibroblasts•These reprogrammed RPE-like cells are functionally similar to primary RPE•The sets of predicted factors may facilitate studies of control of cell identity
Small sets of core transcription factors establish gene expression programs that determine cellular function. Young, Lee, and colleagues have generated an atlas of candidate core transcription factors for over 200 human cell types. Candidate core transcription factors proved capable of reprogramming cells, suggesting this atlas may facilitate studies of transcriptional regulation and reprogramming for clinically important cell types.
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