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Dive into the research topics where Mark J. Tomishima is active.

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Featured researches published by Mark J. Tomishima.


Nature Biotechnology | 2009

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Stuart M. Chambers; Christopher A. Fasano; Eirini P. Papapetrou; Mark J. Tomishima; Michel Sadelain; Lorenz Studer

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5+ epiblast-like stage followed by PAX6+ neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.


Cell Stem Cell | 2013

Human iPSC-Based Modeling of Late-Onset Disease via Progerin-Induced Aging

Justine Miller; Yosif Ganat; Sarah Kishinevsky; Robert L. Bowman; Becky Liu; Edmund Y. Tu; Pankaj K. Mandal; Elsa Vera; Jae-won Shim; Sonja Kriks; Tony Taldone; Noemi Fusaki; Mark J. Tomishima; Dimitri Krainc; Teresa A. Milner; Derrick J. Rossi; Lorenz Studer

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinsons disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.


Proceedings of the National Academy of Sciences of the United States of America | 2009

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

Eirini P. Papapetrou; Mark J. Tomishima; Stuart M. Chambers; Yvonne Mica; Evan Reed; Jayanthi Menon; Viviane Tabar; Qianxing Mo; Lorenz Studer; Michel Sadelain

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.


Nature Biotechnology | 2010

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Daylon James; Hyung Song Nam; Marco Seandel; Daniel J. Nolan; Tyler Janovitz; Mark J. Tomishima; Lorenz Studer; Gabsang Lee; David Lyden; Robert Benezra; N. Zaninovic; Z. Rosenwaks; Sina Y. Rabbany; Shahin Rafii

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.


Cell Stem Cell | 2010

Efficient Derivation of Functional Floor Plate Tissue from Human Embryonic Stem Cells

Christopher A. Fasano; Stuart M. Chambers; Gabsang Lee; Mark J. Tomishima; Lorenz Studer

The floor plate (FP) is a critical signaling center during neural development located along the ventral midline of the embryo. Little is known about human FP development because of the lack of tissue accessibility. Here we report the efficient derivation of human embryonic stem cell (hESC)-derived FP tissue capable of secreting Netrin-1 and SHH and patterning primary and hESC derived tissues. FP induction in hESCs is dependent on early SHH exposure and occurs at the expense of anterior neurectoderm (AN). Global gene expression and functional studies identify SHH-mediated inhibition of Dkk-1 as key factor in FP versus AN specification. hESC-derived FP tissue is shown to be of anterior SIX6+ character but is responsive to caudalizing factors suppressing SIX6 expression and inducing a shift in usage of region-specific SHH enhancers. These data define the early signals that drive human FP versus AN specification and determine regional identity in hESC-derived FP.


Proceedings of the National Academy of Sciences of the United States of America | 2009

Chromosomal translocations induced at specified loci in human stem cells

Erika Brunet; Deniz Simsek; Mark J. Tomishima; Russell DeKelver; Vivian M. Choi; Philip D. Gregory; Fyodor D. Urnov; David M. Weinstock; Maria Jasin

The precise genetic manipulation of stem and precursor cells offers extraordinary potential for the analysis, prevention, and treatment of human malignancies. Chromosomal translocations are hallmarks of several tumor types where they are thought to have arisen in stem or precursor cells. Although approaches exist to study factors involved in translocation formation in mouse cells, approaches in human cells have been lacking, especially in relevant cell types. The technology of zinc finger nucleases (ZFNs) allows DNA double-strand breaks (DSBs) to be introduced into specified chromosomal loci. We harnessed this technology to induce chromosomal translocations in human cells by generating concurrent DSBs at 2 endogenous loci, the PPP1R12C/p84 gene on chromosome 19 and the IL2Rγ gene on the X chromosome. Translocation breakpoint junctions for t(19;X) were detected with nested quantitative PCR in a high throughput 96-well format using denaturation curves and DNA sequencing in a variety of human cell types, including embryonic stem (hES) cells and hES cell-derived mesenchymal precursor cells. Although readily detected, translocations were less frequent than repair of a single DSB by gene targeting or nonhomologous end-joining, neither of which leads to gross chromosomal rearrangements. While previous studies have relied on laborious genetic modification of cells and extensive growth in culture, the approach described in this report is readily applicable to primary human cells, including mutipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.


Journal of Clinical Investigation | 2012

Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment.

Yosif Ganat; Elizabeth L. Calder; Sonja Kriks; Jenny Nelander; Edmund Y. Tu; Fan Jia; Daniela Battista; Neil L. Harrison; Malin Parmar; Mark J. Tomishima; Urs Rutishauser; Lorenz Studer

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.


Genome Research | 2013

Cancer translocations in human cells induced by zinc finger and TALE nucleases

Marion Piganeau; Hind Ghezraoui; Anne De Cian; Lionel Guittat; Mark J. Tomishima; Loïc Perrouault; Oliver René; George E. Katibah; Lei Zhang; Michael C. Holmes; Yannick Doyon; Jean-Paul Concordet; Carine Giovannangeli; Maria Jasin; Erika Brunet

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.


Cell Reports | 2013

Modeling Neural Crest Induction, Melanocyte Specification, and Disease-Related Pigmentation Defects in hESCs and Patient-Specific iPSCs

Yvonne Mica; Gabsang Lee; Stuart M. Chambers; Mark J. Tomishima; Lorenz Studer

Melanocytes are pigment-producing cells of neural crest (NC) origin that are responsible for protecting the skin against UV irradiation. Pluripotent stem cell (PSC) technology offers a promising approach for studying human melanocyte development and disease. Here, we report that timed exposure to activators of WNT, BMP, and EDN3 signaling triggers the sequential induction of NC and melanocyte precursor fates under dual-SMAD-inhibition conditions. Using a SOX10::GFP human embryonic stem cell (hESC) reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human NC induction. Subsequent maturation of hESC-derived melanocytes yields pure populations that match the molecular and functional properties of adult melanocytes. Melanocytes from Hermansky-Pudlak syndrome and Chediak-Higashi syndrome patient-specific induced PSCs (iPSCs) faithfully reproduce the ultrastructural features of disease-associated pigmentation defects. Our data define a highly specific requirement for WNT signaling during NC induction and enable the generation of pure populations of human iPSC-derived melanocytes for faithful modeling of pigmentation disorders.


Stem Cells | 2009

BAC Transgenesis in Human Embryonic Stem Cells as a Novel Tool to Define the Human Neural Lineage

Dimitris G. Placantonakis; Mark J. Tomishima; Fabien Lafaille; Sabrina Desbordes; Fan Jia; Nicholas D. Socci; Agnes Viale; Hyojin Lee; Neil L. Harrison; Viviane Tabar; Lorenz Studer

Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC‐derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)+ neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP+ cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP+ and HB9::GFP+ populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease. STEM CELLS 2009;27:521–532

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Lorenz Studer

Memorial Sloan Kettering Cancer Center

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Gabsang Lee

Johns Hopkins University School of Medicine

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Viviane Tabar

Memorial Sloan Kettering Cancer Center

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Faranak Fattahi

Memorial Sloan Kettering Cancer Center

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Christopher A. Fasano

Rensselaer Polytechnic Institute

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Eirini P. Papapetrou

Icahn School of Medicine at Mount Sinai

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