Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells.

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.

Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas.

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.

Pluripotent stem cell models of Blau syndrome reveal an IFN-γ-dependent inflammatory response in macrophages.

BACKGROUND: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. OBJECTIVES: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. METHODS: Blau syndrome-specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. RESULTS: IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ-dependent inflammatory response. CONCLUSIONS: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation.

Inhibitors of Mek1/2 and Gsk3ß, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

An EWS-FLI1-Induced Osteosarcoma Model Unveiled a Crucial Role of Impaired Osteogenic Differentiation on Osteosarcoma Development.

EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation.

Induced pluripotent stem cell technology for dissecting the cancer epigenome.

Cancer arises through the accumulation of both genetic and epigenetic alterations. Although the causal role of genetic mutations on cancer development has been established in vivo, similar evidence for epigenetic alterations is limited. Moreover, mutual interactions between genetic mutations and epigenetic alterations remain unclear. Cellular reprogramming technology can be used to actively modify the epigenome without affecting the underlying genomic sequences. Here we introduce recent studies that have utilized this property for cancer research. We propose that just as it has potential for regenerative medicine and disease modeling, cell reprogramming could also be a powerful tool for dissecting the role of the cancer epigenome in the development and maintenance of cancer cells.

[Application of reprogramming technology for cancer research].

Cancer is generally developed through accumulation of multiple genetic mutations. Epigenetic abnormalities of DNA methylation and histone modification patterns were also found in most cancer cells. Although induced pluripotent stem cells (iPSCs) can be generated through epigenetic reorganization without affecting the underlying genomic sequencing, they have some shared characteristics with cancer cells, which include unlimited growth potential. Taking advantages of such properties of iPSC derivation, the reprogramming technology is applicable not only for regenerative medicine but also for cancer research. Here, we introduce the potential application of iPSC technology for better understandings of cancer biology. Especially, we would like to propose the role of cellular identity in cancer development.

Reducing DNA methylation suppresses colon carcinogenesis by inducing tumor cell differentiation.

The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.

Application of iPS cell technology to cancer epigenome study: uncovering the mechanism of cell status conversion for drug resistance in tumor.

Recent studies imply that cancer cells possess the ability to reversibly change their properties between a drug sensitive state and a drug resistant state accompanied by epigenetic changes. This evidence indicates that better understanding of cancer epigenetics is important for efficient cancer therapies. Nevertheless, it had been difficult to deeply examine the epigenetic mechanisms because of lack of the tools to actively modify coordinated epigenetic events. In this stagnant situation, the reprogramming technology established by Yamanaka and coworkers have shed a new light. The novel reprogramming technology has made it possible for researchers to artificially introduce epigenetic remodeling into somatic cells. Accordingly, we might be able to use this technology as a tool to introduce the coordinated epigenetic reorganization. In this review, we introduce the idea of cell state interconversion in cancer cells that is attributable to altered epigenetic regulations. We then depict the epigenetic modifications observed during the process of somatic cell reprogramming and give some examples of the difficulty in cancer cell reprogramming. Finally, we discuss how we can translate this reprogramming refractoriness of cancer cells into uncovering unique epigenetic regulations in cancer cells, which might be applicable eventually to the development of novel cancer therapeutics against drug resistant cancer cells.

Epigenetic regulation leading to induced pluripotency drives cancer development in vivo.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotency in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.

Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation.

Cancer is believed to arise primarily through accumulation of genetic mutations. Although induced pluripotent stem cell (iPSC) generation does not require changes in genomic sequence, iPSCs acquire unlimited growth potential, a characteristic shared with cancer cells. Here, we describe a murine system in which reprogramming factor expression in vivo can be controlled temporally with doxycycline (Dox). Notably, transient expression of reprogramming factors in vivo results in tumor development in various tissues consisting of undifferentiated dysplastic cells exhibiting global changes in DNA methylation patterns. The Dox-withdrawn tumors arising in the kidney share a number of characteristics with Wilms tumor, a common pediatric kidney cancer. We also demonstrate that iPSCs derived from Dox-withdrawn kidney tumor cells give rise to nonneoplastic kidney cells in mice, proving that they have not undergone irreversible genetic transformation. These findings suggest that epigenetic regulation associated with iPSC derivation may drive development of particular types of cancer.

EWS/ATF1 expression induces sarcomas from neural crest-derived cells in mice.

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest-derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.

Cellular reprogramming and cancer development.

Cancer develops through the accumulation of genetic and epigenetic abnormalities. The role of genetic alterations in cancer development has been demonstrated by reverse genetic approaches. However, evidence indicating the functional significance of epigenetic abnormalities remains limited due to the lack of means to actively modify coordinated epigenetic regulations in the genome. Application of the reprogramming technology may help researchers to overcome this limitation and shed new light on cancer research. Reprogramming is accompanied by dynamic changes of epigenetic modifications and is therefore considered to be a useful tool to induce global epigenetic changes in cancer genomes. We herein discuss the similarities between reprogramming processes and carcinogenesis and propose the potential use of reprogramming technology to help understanding of the significance of epigenetic regulations in cancer cells. We, also discuss the application of induced pluripotent stem cell technology to cancer modeling based on the similar characteristics between pluripotent stem cells and cancer cells.

Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells.

Because of their ability to self-renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long-term, self-renewing, neuroepithelial-like stem cells from human iPS cells (hiPS-lt-NES cells), which can provide a homogeneous and well-defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS-lt-NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS-lt-NES cell-derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS-lt-NES transplantation represents a promising avenue for effective cell-based treatment of SCI.