Publications by authors named "Minoru Ko"

119 Publications

Synthetic mRNA-based differentiation method enables early detection of Parkinson's phenotypes in neurons derived from Gaucher disease-induced pluripotent stem cells.

Stem Cells Transl Med 2021 Apr 20;10(4):572-581. Epub 2020 Dec 20.

Neuroscience Drug Discovery Unit, Research, Takeda Pharmaceutical Company Limited, Fujisawa, Japan.

Gaucher disease, the most prevalent metabolic storage disorder, is caused by mutations in the glucocerebrosidase gene GBA1, which lead to the accumulation of glucosylceramide (GlcCer) in affected cells. Gaucher disease type 1 (GD1), although defined as a nonneuronopathic subtype, is accompanied by an increased risk of Parkinson's disease. To gain insights into the association of progressive accumulation of GlcCer and the Parkinson's disease phenotypes, we generated dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) derived from a GD1 patient and a healthy donor control, and measured GlcCer accumulation by liquid chromatography-mass spectrometry. We tested two DA neuron differentiation methods: a well-established method that mimics a step-wise developmental process from iPSCs to neural progenitor cells, and to DA neurons; and a synthetic mRNA-based method that overexpresses a transcription factor in iPSCs. GD1-specific accumulation of GlcCer was detected after 60 days of differentiation by the former method, whereas it was detected after only 10 days by the latter method. With this synthetic mRNA-based rapid differentiation method, we found that the metabolic defect in GD1 patient cells can be rescued by the overexpression of wild-type GBA1 or the treatment with an inhibitor for GlcCer synthesis. Furthermore, we detected the increased phosphorylation of α-synuclein, a biomarker for Parkinson's disease, in DA neurons derived from a GD1 patient, which was significantly decreased by the overexpression of wild-type GBA1. These results suggest that synthetic mRNA-based method accelerates the analyses of the pathological mechanisms of Parkinson's disease in GD1 patients and possibly facilitates drug discovery processes.
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http://dx.doi.org/10.1002/sctm.20-0302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980209PMC
April 2021

Fatty Acid Synthesis Is Indispensable for Survival of Human Pluripotent Stem Cells.

iScience 2020 Sep 6;23(9):101535. Epub 2020 Sep 6.

Department of Cardiology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.

The role of lipid metabolism in human pluripotent stem cells (hPSCs) is poorly understood. We have used large-scale targeted proteomics to demonstrate that undifferentiated hPSCs express different fatty acid (FA) biosynthesis-related enzymes, including ATP citrate lyase and FA synthase (FASN), than those expressed in hPSC-derived cardiomyocytes (hPSC-CMs). Detailed lipid profiling revealed that inhibition of FASN resulted in significant reduction of sphingolipids and phosphatidylcholine (PC); moreover, we found that PC was the key metabolite for cell survival in hPSCs. Inhibition of FASN induced cell death in undifferentiated hPSCs via mitochondria-mediated apoptosis; however, it did not affect cell survival in hPSC-CMs, neurons, or hepatocytes as there was no significant reduction of PC. Furthermore, we did not observe tumor formation following transplantation of FASN inhibitor-treated cells. Our findings demonstrate the importance of FA synthesis in the survival of undifferentiated hPSCs and suggest applications for FASN inhibition in regenerative medicine.
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http://dx.doi.org/10.1016/j.isci.2020.101535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509212PMC
September 2020

Generation and Profiling of 2,135 Human ESC Lines for the Systematic Analyses of Cell States Perturbed by Inducing Single Transcription Factors.

Cell Rep 2020 05;31(7):107655

Department of Systems Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan. Electronic address:

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.
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http://dx.doi.org/10.1016/j.celrep.2020.107655DOI Listing
May 2020

MEIOSIN Directs the Switch from Mitosis to Meiosis in Mammalian Germ Cells.

Dev Cell 2020 02 6;52(4):429-445.e10. Epub 2020 Feb 6.

Department of Pluripotent Stem Cell Biology, IMEG, Kumamoto University, Kumamoto 860-0811, Japan.

The mechanisms regulating meiotic initiation in mammals are enigmatic. It is known that retinoic acid (RA) signaling plays a pivotal role during meiotic initiation. STRA8, which is expressed in response to RA, is thought to be a key factor promoting meiotic initiation. However, the specific role of STRA8 in meiotic initiation has remained elusive. Here, we identified MEIOSIN as a germ-cell-specific factor that associates with STRA8. MEIOSIN, like STRA8, is expressed in response to RA and plays an essential role in meiotic initiation in both males and females. Functional analyses revealed that MEIOSIN acts as a transcription factor together with STRA8, and that both factors are critical for driving meiotic gene activation. Furthermore, temporally restricted expression of MEIOSIN leads to meiotic entry decision during spermatogenesis. The present study demonstrates that MEIOSIN, in collaboration with STRA8, plays a central role in regulating the mitosis to meiosis germ cell fate decision in mammals.
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http://dx.doi.org/10.1016/j.devcel.2020.01.010DOI Listing
February 2020

Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression.

Stem Cell Reports 2019 02 31;12(2):305-318. Epub 2019 Jan 31.

Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Electronic address:

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.
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http://dx.doi.org/10.1016/j.stemcr.2018.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373546PMC
February 2019

Induction of human pluripotent stem cells into kidney tissues by synthetic mRNAs encoding transcription factors.

Sci Rep 2019 01 29;9(1):913. Epub 2019 Jan 29.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan.

The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2SALL1 nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8LHX1 pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.
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http://dx.doi.org/10.1038/s41598-018-37485-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351687PMC
January 2019

Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors.

Stem Cell Res Ther 2018 10 25;9(1):277. Epub 2018 Oct 25.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan.

Background: Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)-PDX1 and NKX6.1-has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages.

Methods: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin.

Results: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13.

Conclusions: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.
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http://dx.doi.org/10.1186/s13287-018-1038-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203190PMC
October 2018

Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing.

Sci Rep 2018 01 19;8(1):1189. Epub 2018 Jan 19.

Department of Systems Medicine, Keio University School of Medicine, Tokyo, 160, Japan.

Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.
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http://dx.doi.org/10.1038/s41598-017-19114-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775307PMC
January 2018

Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

NPJ Aging Mech Dis 2017 24;3. Epub 2017 Jan 24.

Department of Systems Medicine, Keio University School of Medicine, Shinjuku, Tokyo 160-8582 Japan.

Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining , , and caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as , and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.
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http://dx.doi.org/10.1038/s41514-016-0001-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445629PMC
January 2017

Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Biochem Biophys Res Commun 2017 08 10;490(2):296-301. Epub 2017 Jun 10.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Electronic address:

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.
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http://dx.doi.org/10.1016/j.bbrc.2017.06.039DOI Listing
August 2017

Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells.

Stem Cells Int 2017 9;2017:7215010. Epub 2017 Apr 9.

Department of Systems Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan.

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.
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http://dx.doi.org/10.1155/2017/7215010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401757PMC
April 2017

Salt suppresses IFNγ inducible chemokines through the IFNγ-JAK1-STAT1 signaling pathway in proximal tubular cells.

Sci Rep 2017 04 20;7:46580. Epub 2017 Apr 20.

Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.

The mechanisms of immunoactivation by salt are now becoming clearer. However, those of immunosuppression remain unknown. Since clinical evidence indicates that salt protects proximal tubules from injury, we investigated mechanisms responsible for salt causing immunosuppression in proximal tubules. We focused on cytokine-related gene expression profiles in kidneys of mice fed a high salt diet using microarray analysis and found that both an interferon gamma (IFNγ) inducible chemokine, chemokine (C-X-C motif) ligand 9 (CXCL9), and receptor, CXCR3, were suppressed. We further revealed that a high salt concentration suppressed IFNγ inducible chemokines in HK2 proximal tubular cells. Finally, we demonstrated that a high salt concentration decreased IFNGR1 expression in the basolateral membrane of HK2 cells, leading to decreased phosphorylation of activation sites of Janus kinase 1 (JAK1) and Signal Transducers and Activator of Transcription 1 (STAT1), activators of chemokines. JAK inhibitor canceled the effect of a high salt concentration on STAT1 and chemokines, indicating that the JAK1-STAT1 signaling pathway is essential for this mechanism. In conclusion, a high salt concentration suppresses IFNγ-JAK1-STAT1 signaling pathways and chemokine expressions in proximal tubules. This finding may explain how salt ameliorates proximal tubular injury and offer a new insight into the linkage between salt and immunity.
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http://dx.doi.org/10.1038/srep46580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397865PMC
April 2017

SCODE: an efficient regulatory network inference algorithm from single-cell RNA-Seq during differentiation.

Bioinformatics 2017 Aug;33(15):2314-2321

Bioinformatics Research Unit, Advanced Center for Computing and Communication, RIKEN, Wako, Saitama 351-0198, Japan.

Motivation: The analysis of RNA-Seq data from individual differentiating cells enables us to reconstruct the differentiation process and the degree of differentiation (in pseudo-time) of each cell. Such analyses can reveal detailed expression dynamics and functional relationships for differentiation. To further elucidate differentiation processes, more insight into gene regulatory networks is required. The pseudo-time can be regarded as time information and, therefore, single-cell RNA-Seq data are time-course data with high time resolution. Although time-course data are useful for inferring networks, conventional inference algorithms for such data suffer from high time complexity when the number of samples and genes is large. Therefore, a novel algorithm is necessary to infer networks from single-cell RNA-Seq during differentiation.

Results: In this study, we developed the novel and efficient algorithm SCODE to infer regulatory networks, based on ordinary differential equations. We applied SCODE to three single-cell RNA-Seq datasets and confirmed that SCODE can reconstruct observed expression dynamics. We evaluated SCODE by comparing its inferred networks with use of a DNaseI-footprint based network. The performance of SCODE was best for two of the datasets and nearly best for the remaining dataset. We also compared the runtimes and showed that the runtimes for SCODE are significantly shorter than for alternatives. Thus, our algorithm provides a promising approach for further single-cell differentiation analyses.

Availability And Implementation: The R source code of SCODE is available at https://github.com/hmatsu1226/SCODE.

Contact: hirotaka.matsumoto@riken.jp.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860123PMC
August 2017

Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors.

Sci Rep 2017 02 13;7:42367. Epub 2017 Feb 13.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan.

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.
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http://dx.doi.org/10.1038/srep42367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304326PMC
February 2017

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells.

Development 2016 10;143(20):3674-3685

Department of Systems Medicine, Keio University School of Medicine, Tokyo 160, Japan

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings.
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http://dx.doi.org/10.1242/dev.139360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087640PMC
October 2016

Induction of specific neuron types by overexpression of single transcription factors.

In Vitro Cell Dev Biol Anim 2016 Oct 1;52(9):961-973. Epub 2016 Jun 1.

Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.

Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.
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http://dx.doi.org/10.1007/s11626-016-0056-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518781PMC
October 2016

Zscan4 is expressed specifically during late meiotic prophase in both spermatogenesis and oogenesis.

In Vitro Cell Dev Biol Anim 2017 Feb 3;53(2):167-178. Epub 2016 Oct 3.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan.

Mouse zinc finger and SCAN domain containing 4 (Zscan4) proteins, which are encoded by multiple copies of Zscan4 genes, are expressed specifically in preimplantation embryos in vivo and embryonic stem (ES) cells in vitro. However, the expression patterns of mouse Zscan4 in vivo have been largely elusive. Here, we show that Zscan4 proteins are expressed in adult ovaries and testes. In ovaries, Zscan4 proteins were detected in germinal vesicle (GV) stage oocytes in antral follicles, indicating that Zscan4 genes are activated during the diplotene/dictyate stage in meiotic prophase I. Remarkably, Zscan4 showed different spatial localization patterns between two distinct GV oocytes, which can be distinguished by global chromatin organization-surrounded nucleolus (SN) and non-surrounded nucleolus (NSN). These spatiotemporal differences in Zscan4 localizations correlated with the transition of RNA polymerase II-mediated transcriptional status during GV oocyte maturation. In testes, Zscan4 proteins were detected in spermatocytes at late pachytene/diplotene stages and in Sertoli cells. These results suggest that Zscan4 may play critical roles during late meiotic prophase in both males and females.
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http://dx.doi.org/10.1007/s11626-016-0096-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311088PMC
February 2017

Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos.

In Vitro Cell Dev Biol Anim 2017 Feb 3;53(2):179-190. Epub 2016 Oct 3.

Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan.

Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.
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http://dx.doi.org/10.1007/s11626-016-0097-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311086PMC
February 2017

Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines.

Sci Rep 2016 05 6;6:25667. Epub 2016 May 6.

Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs.
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http://dx.doi.org/10.1038/srep25667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858678PMC
May 2016

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.

In Vitro Cell Dev Biol Anim 2016 May 29;52(5):616-24. Epub 2016 Apr 29.

National Institute on Aging (NIA), National Institutes of Health, Baltimore, MD, 21224, USA.

Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.
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http://dx.doi.org/10.1007/s11626-016-0013-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884469PMC
May 2016

Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells.

Biol Open 2016 Feb 12;5(3):311-22. Epub 2016 Feb 12.

Department of Tissue and Organ Development, Regeneration, and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0076, Japan.

Neural crest cells (NC cells) are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs) into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+) cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.
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http://dx.doi.org/10.1242/bio.015735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810742PMC
February 2016

Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells.

DNA Res 2015 Oct 31;22(5):307-18. Epub 2015 Aug 31.

Department of Systems Medicine, Keio University School of Medicine, Tokyo 160, Japan Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD 21224, USA

Mouse embryonic stem cells (mESCs) have a remarkable capacity to maintain normal genome stability and karyotype in culture. We previously showed that infrequent bursts of Zscan4 expression (Z4 events) are important for the maintenance of telomere length and genome stability in mESCs. However, the molecular details of Z4 events remain unclear. Here we show that Z4 events involve unexpected transcriptional derepression in heterochromatin regions that usually remain silent. During a Z4 event, we see rapid derepression and rerepression of heterochromatin leading to a burst of transcription that coincides with transient histone hyperacetylation and DNA demethylation, clustering of pericentromeric heterochromatin around the nucleolus, and accumulation of activating and repressive chromatin remodelling complexes. This heterochromatin-based transcriptional activity suggests that mESCs may maintain their extraordinary genome stability at least in part by transiently resetting their heterochromatin.
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http://dx.doi.org/10.1093/dnares/dsv013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596397PMC
October 2015

Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures.

DNA Res 2015 Oct 31;22(5):331-42. Epub 2015 Aug 31.

Elixirgen, LLC, Science + Technology Park at Johns Hopkins, 855 N Wolfe Street, Suite 621, Baltimore MD 21205-1511, USA Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160, Japan

Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.
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http://dx.doi.org/10.1093/dnares/dsv016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596399PMC
October 2015

ExAtlas: An interactive online tool for meta-analysis of gene expression data.

J Bioinform Comput Biol 2015 Dec 9;13(6):1550019. Epub 2015 Jun 9.

† Department of Systems Medicine, The Sakaguchi Laboratory, Keio University School of Medicine, Tokyo 160-8582, Japan.

We have developed ExAtlas, an on-line software tool for meta-analysis and visualization of gene expression data. In contrast to existing software tools, ExAtlas compares multi-component data sets and generates results for all combinations (e.g. all gene expression profiles versus all Gene Ontology annotations). ExAtlas handles both users' own data and data extracted semi-automatically from the public repository (GEO/NCBI database). ExAtlas provides a variety of tools for meta-analyses: (1) standard meta-analysis (fixed effects, random effects, z-score, and Fisher's methods); (2) analyses of global correlations between gene expression data sets; (3) gene set enrichment; (4) gene set overlap; (5) gene association by expression profile; (6) gene specificity; and (7) statistical analysis (ANOVA, pairwise comparison, and PCA). ExAtlas produces graphical outputs, including heatmaps, scatter-plots, bar-charts, and three-dimensional images. Some of the most widely used public data sets (e.g. GNF/BioGPS, Gene Ontology, KEGG, GAD phenotypes, BrainScan, ENCODE ChIP-seq, and protein-protein interaction) are pre-loaded and can be used for functional annotations.
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http://dx.doi.org/10.1142/S0219720015500195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518779PMC
December 2015

SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1).

Development 2014 Nov;141(22):4254-66

Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo 160-8582, Japan

Upon removal of culture conditions that maintain an undifferentiated state, mouse embryonic stem cells (ESCs) differentiate into various cell types. Differentiation can be facilitated by forced expression of certain transcription factors (TFs), each of which can generally specify a particular developmental lineage. We previously established 137 mouse ESC lines, each of which carried a doxycycline-controllable TF. Among them, Sox9 has unique capacity: its forced expression accelerates differentiation of mouse ESCs into cells of all three germ layers. With the additional use of specific culture conditions, overexpression of Sox9 facilitated the generation of endothelial cells, hepatocytes and neurons from ESCs. Furthermore, Sox9 action increases formation of p21 (WAF1/CIP1), which then binds to the SRR2 enhancer of pluripotency marker Sox2 and inhibits its expression. Knockdown of p21 abolishes inhibition of Sox2 and Sox9-accelerated differentiation, and reduction of Sox2 2 days after the beginning of ESC differentiation can comparably accelerate mouse ESC formation of cells of three germ layers. These data implicate the involvement of the p21-Sox2 pathway in the mechanism of accelerated ESC differentiation by Sox9 overexpression. The molecular cascade could be among the first steps to program ESC differentiation.
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http://dx.doi.org/10.1242/dev.115436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302912PMC
November 2014

Chromatin properties of regulatory DNA probed by manipulation of transcription factors.

J Comput Biol 2014 Aug 11;21(8):569-77. Epub 2014 Jun 11.

1 National Institute on Aging, National Institutes of Health , Baltimore, Maryland.

Transcription factors (TFs) bind to DNA and regulate the transcription of nearby genes. However, only a small fraction of TF binding sites have such regulatory effects. Here we search for the predictors of functional binding sites by carrying out a systematic computational screening of a variety of contextual factors (histone modifications, nuclear lamin-bindings, and cofactor bindings). We used regression analysis to test if contextual factors are associated with upregulation or downregulation of neighboring genes following the induction or knockdown of the 9 TFs in mouse embryonic stem (ES) cells. Functional TF binding sites appeared to be either active (i.e., bound by P300, CHD7, mediator, cohesin, and SWI/SNF) or repressed (i.e., with H3K27me3 histone marks and bound by Polycomb factors). Active binding sites mediated the downregulation of nearby genes upon knocking down the activating TFs or inducing repressors. Repressed TF binding sites mediated the upregulation of nearby genes (e.g., poised developmental regulators) upon inducing TFs. In addition, repressed binding sites mediated repressive effects of TFs, identified by the downregulation of target genes after the induction of TFs or by the upregulation of target genes after the knockdown of TFs. The contextual factors associated with functions of DNA-bound TFs were used to improve the identification of candidate target genes regulated by TFs.
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http://dx.doi.org/10.1089/cmb.2013.0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115781PMC
August 2014

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

Stem Cells Transl Med 2014 Jul 5;3(7):787-91. Epub 2014 Jun 5.

Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA; Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Shinjuku, Tokyo, Japan

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes.
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http://dx.doi.org/10.5966/sctm.2013-0036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073818PMC
July 2014

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

J Pathol 2014 Jul;233(3):228-37

Laboratory of Molecular Biology and Immunology, National Institute on Aging, Baltimore, MD, USA.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.
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http://dx.doi.org/10.1002/path.4353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149901PMC
July 2014

Identification of transcription factors for lineage-specific ESC differentiation.

Stem Cell Reports 2013 27;1(6):545-59. Epub 2013 Nov 27.

Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA ; Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo 160-8582, Japan ; Japan Science and Technology Agency, CREST, Tokyo160-8582, Japan.

A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1-can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.
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http://dx.doi.org/10.1016/j.stemcr.2013.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871400PMC
May 2015