研究者詳細 - 洪　実

論文 - 洪　実

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SCODE

Matsumoto Hirotaka, Kiryu Hisanori, Furusawa Chikara, Ko Minoru S.H., Ko Shigeru B.H., Gouda Norio, Hayashi Tetsutaro, Nikaido Itoshi

Bioinformatics 33 （ 15 ） 2314 - 2321 2017年08月

ISSN 1367-4803

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 RNASeq 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 RNASeq 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 singlecell differentiation analyses.

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

Matsushita Misako, Nakatake Yuhki, Arai Itaru, Ibata Keiji, Kohda Kazuhisa, Goparaju Sravan K., Murakami Miyako, Sakota Miki, Chikazawa-Nohtomi Nana, Ko Shigeru B.H., Kanai Takanori, Yuzaki Michisuke, Ko Minoru S.H.

Biochemical and Biophysical Research Communications 2017年05月

ISSN 0006-291X

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

Goparaju Sravan Kumar, Kohda Kazuhisa, Ibata Keiji, Soma Atsumi, Nakatake Yukhi, Akiyama Tomohiko, Wakabayashi Shunichi, Matsushita Misako, Sakota Miki, Kimura Hiromi, Yuzaki Michisuke, Ko Shigeru B H, Ko Minoru S H

Scientific Reports 7 2017年02月

ISSN 2045-2322

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

Akiyama Tomohiko, Wakabayashi Shunichi, Soma Atsumi, Sato Saeko, Nakatake Yuhki, Oda Mayumi, Murakami Miyako, Sakota Miki, Chikazawa-Nohtomi Nana, Ko Shigeru B.H., Ko Minoru S.H.

Stem Cells International 2017 2017年

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

Akiyama Tomohiko, Wakabayashi Shunichi, Soma Atsumi, Sato Saeko, Nakatake Yuhki, Oda Mayumi, Murakami Miyako, Sakota Miki, Chikazawa-Nohtomi Nana, Ko Shigeru B H, Ko Minoru S H

Development (Cambridge) 143 （ 20 ） 3674 - 3685 2016年10月

ISSN 0950-1991

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

Ishiguro Kei ichiro, Monti Manuela, Akiyama Tomohiko, Kimura Hiromi, Chikazawa-Nohtomi Nana, Sakota Miki, Sato Saeko, Redi Carlo Alberto, Ko Shigeru B H, Ko Minoru S H

In Vitro Cellular and Developmental Biology - Animal 1 - 12 2016年10月

ISSN 1071-2690

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

Ishiguro Kei ichiro, Nakatake Yuhki, Chikazawa-Nohtomi Nana, Kimura Hiromi, Akiyama Tomohiko, Oda Mayumi, Ko Shigeru B H, Ko Minoru S H

In Vitro Cellular and Developmental Biology - Animal 1 - 12 2016年10月

ISSN 1071-2690

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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Induction of specific neuron types by overexpression of single transcription factors

Teratani-Ota Yusuke, Yamamizu Kohei, Piao Yulan, Sharova Lioudmila, Amano Misa, Yu Hong, Schlessinger David, Ko Minoru S H, Sharov Alexei A.

In Vitro Cellular and Developmental Biology - Animal 1 - 13 2016年06月

ISSN 1071-2690

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

Yamamizu Kohei, Sharov Alexei A., Piao Yulan, Amano Misa, Yu Hong, Nishiyama Akira, Dudekula Dawood B., Schlessinger David, Ko Minoru S H

Scientific Reports 6 2016年05月

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

Sharova Lioudmila V., Sharov Alexei A., Piao Yulan, Stagg Carole A., Amano Tomokazu, Qian Yong, Dudekula Dawood, Schlessinger David, Ko Minoru S H

In Vitro Cellular and Developmental Biology - Animal 1 - 9 2016年04月

ISSN 1071-2690

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

Motohashi Tsutomu, Watanabe Natsuki, Nishioka Masahiro, Nakatake Yuhki, Yulan Piao, Mochizuki Hiromi, Kawamura Yoshifumi, Ko Minoru S H, Goshima Naoki, Kunisada Takahiro

Biology Open 5 （ 3 ） 311 - 322 2016年03月

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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Zygotic Genome Activation Revisited

Ko M. S H

Current Topics in Developmental Biology 2016年

ISSN 0070-2153

Zygotic genome activation (ZGA, a.k.a. zygotic gene activation) is a critical event in development, when the paternally derived genome and maternally derived genome begin to be activated and transcribed after fertilization. Major ZGA occurs at the two-cell stage in mice and the four- to eight-cell stage in human preimplantation embryos. It has been thought that ZGA exists to provide RNAs and proteins supporting embryonic development after supplies stored in oocytes are used up; however, this paradigm does not seem to explain recent findings. For example, many ZGA genes-once activated-are quickly turned off, and thus ZGA forms a transient wave of transcriptional activation. In addition, ZGA genes are not evolutionarily conserved. In this review, we address these issues by focusing on Zscan4 (zinc finger and SCAN domain-containing 4), which was identified for its specific expression in preimplantation embryos during ZGA. Detailed molecular analyses of Zscan4 expression and function have revealed common features of Zscan4-associated events (Z4 events) in mouse embryonic stem cells and ZGA in preimplantation embryos. One feature is a rapid derepression and rerepression of constitutive heterochromatin, which includes pericentromeric major satellites and telomeres, and facultative heterochromatin, which includes retrotransposons and Z4 event-associated genes. We propose that the Z4 event superimposed on ZGA plays a critical role in the maintenance of genome and chromosome integrity in preimplantation embryos by promoting correction of DNA damage and chromosome abnormalities.

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ExAtlas

Sharov Alexei A., Schlessinger David, Ko Minoru S H

Journal of Bioinformatics and Computational Biology 13 （ 6 ） 2015年12月

ISSN 0219-7200

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

洪実

DNA Res. 22 （ 5 ） 331 - 342 2015年10月

研究論文（学術雑誌）, 査読有り

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

洪実

DNA Res. 22 （ 5 ） 307 - 318 2015年10月

研究論文（学術雑誌）

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

洪実

J Bioinform Comput Biol. 1550019 2015年06月

研究論文（学術雑誌）, 共著, 査読有り

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

Yamamizu Kohei, Schlessinger David, Ko Minoru S H

Development (Cambridge) 141 （ 22 ） 4254 - 4266 2014年11月

ISSN 0950-1991

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 mouseESClines, 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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Chromatin properties of regulatory DNA probed by manipulation of transcription factors

Sharov Alexei A., Nishiyama Akira, Qian Yong, Dudekula Dawood B., Longo Dan L., Schlessinger David, Ko Minoru S H

Journal of Computational Biology 21 （ 8 ） 569 - 577 2014年08月

ISSN 1066-5277

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. © Copyright 2014, Mary Ann Liebert, Inc. 2014.

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

Piao Yulan, Hung Sandy Shen Chi, Lim Shiang Y., Wong Raymond Ching Bong, Ko Minoru S H

Stem Cells Translational Medicine 3 （ 7 ） 787 - 791 2014年

ISSN 2157-6564

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. © AlphaMed Press 2014.

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

Sherman-Baust Cheryl A., Kuhn Elisabetta, Valle Blanca L., Shih Ie Ming, Kurman Robert J., Wang Tian Li, Amano Tomokazu, Ko Minoru Sh, Miyoshi Ichiro, Araki Yoshihiko, Lehrmann Elin, Zhang Yongqing, Becker Kevin G., Morin Patrice J.

Journal of Pathology 233 （ 3 ） 228 - 237 2014年

ISSN 0022-3417

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. Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA. © Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

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