Bio

Administrative Appointments


  • Co-Director PhD Program in Stem Cell Biology and Regenerative Medicine, Stanford School of Medicine (2017 - Present)
  • Director, Transgenic Knockout and Tumor Model Service (TKTC) Stanford Cancer Institute, Stanford School of Medicine (2014 - Present)
  • Director, Human Pluripotent Stem Cells Core Facility Stanford Institute for SCBRM, Stanford School of Medicine (2010 - Present)

Honors & Awards


  • AFAR Junior Investigator Awardee, American Federation for Aging Research (2017)
  • Siebel Stem Cell Institute Scholar, Stanford School of Medicine (2014)
  • Postdoctoral Training Grant, California Institute for Regenerative Medicine, Stanford School of Medicine (2009)
  • Postdoctoral Training Grant, Max Planck for Molecular Biomedicine, Max Plank Institute for Molecular Biomedicine (2004)
  • Honorary fellow member, Examination Board of Zoology and Developmental Biology, University of Pavia, ITALY (2001)
  • Scholarship “Borsa Jacobi-Mazzocchi” for the best B.S. thesis in Applied Biomedicine, University of Pavia, ITALY (2001)

Research & Scholarship

Current Research and Scholarly Interests


Germ cells, preimplantation embryos and pluripotent stem cells at first glance seem to have nothing in common. A more careful look, though, reveals that they are very closely linked to each other. The zygote originates from the fusion of two highly specialized germ cells (the sperm and the oocyte) and in a few days develops into a blastocyst with a pluripotent cell population (the inner cell mass). These cells diverge from the extraembryonic cells of the trophoectoderm and can give rise to embryonic stem cells, in which a perpetual pluripotent and undifferentiated state is maintained.
The correct establishment of pluripotency guarantees the correct onset of development and therefore its acquisition is a fundamental biological process; any mistake associated with it has profound impact on gestation. A detailed understanding of the mechanisms that induce and regulate pluripotency is critical for the basic understanding of fundamental developmental processes that depend from it like the onset of differentiation and cellular plasticity. This is particularly relevant in consideration of the potential clinical application of human pluripotent stem cells (hPSCs).

Teaching

Stanford Advisees


Publications

All Publications


  • Honey bee Royalactin unlocks conserved pluripotency pathway in mammals. Nature communications Wan, D. C., Morgan, S. L., Spencley, A. L., Mariano, N., Chang, E. Y., Shankar, G., Luo, Y., Li, T. H., Huh, D., Huynh, S. K., Garcia, J. M., Dovey, C. M., Lumb, J., Liu, L., Brown, K. V., Bermudez, A., Luong, R., Zeng, H., Mascetti, V. L., Pitteri, S. J., Wang, J., Tu, H., Quarta, M., Sebastiano, V., Nusse, R., Rando, T. A., Carette, J. E., Bazan, J. F., Wang, K. C. 2018; 9 (1): 5078

    Abstract

    Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.

    View details for DOI 10.1038/s41467-018-06256-4

    View details for PubMedID 30510260

  • Efficient scarless genome editing in human pluripotent stem cells. Nature methods Ikeda, K., Uchida, N., Nishimura, T., White, J., Martin, R. M., Nakauchi, H., Sebastiano, V., Weinberg, K. I., Porteus, M. H. 2018; 15 (12): 1045–47

    Abstract

    Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.

    View details for DOI 10.1038/s41592-018-0212-y

    View details for PubMedID 30504872

  • A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells. Nature communications Fang, F., Xia, N., Angulo, B., Carey, J., Cady, Z., Durruthy-Durruthy, J., Bennett, T., Sebastiano, V., Reijo Pera, R. A. 2018; 9 (1): 4384

    Abstract

    Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm.

    View details for DOI 10.1038/s41467-018-06908-5

    View details for PubMedID 30349051

  • Influenza virus infection causes global RNAPII termination defects NATURE STRUCTURAL & MOLECULAR BIOLOGY Zhao, N., Sebastiano, V., Moshkina, N., Mena, N., Hultquist, J., Jimenez-Morales, D., Ma, Y., Rialdi, A., Albrecht, R., Fenouil, R., Sanchez-Aparicio, M., Ayllon, J., Ravisankar, S., Haddad, B., Ho, J., Low, D., Jin, J., Yurchenko, V., Prinjha, R. K., Tarakhovsky, A., Squatrito, M., Pinto, D., Allette, K., Byun, M., Smith, M., Sebra, R., Guccione, E., Tumpey, T., Krogan, N., Greenbaum, B., van Bakel, H., Garcia-Sastre, A., Marazzi, I. 2018; 25 (9): 885-+

    Abstract

    Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

    View details for DOI 10.1038/s41594-018-0124-7

    View details for Web of Science ID 000443839200019

    View details for PubMedID 30177761

  • Do Induced Pluripotent Stem Cell Characteristics Correlate with Efficient In Vitro Smooth Muscle Cell Differentiation? A Comparison of Three Patient-Derived Induced Pluripotent Stem Cell Lines. Stem cells and development Zhou, Y., Kang, G., Wen, Y., Briggs, M., Sebastiano, V., Pederson, R., Chen, B. 2018

    Abstract

    Human induced pluripotent stem cells (iPSCs) have the potential to repair/regenerate smooth muscle cells (SMCs) in different organs. However, there are many challenges in their translation to clinical therapies. In this study, we describe our observations of in vitro SMC differentiation in three iPSC lines derived from human fibroblasts using retroviral, episomal, and mRNA/miRNA reprogramming methods. We sought to elucidate correlations between differentiation characteristics and efficiencies that can facilitate large-scale production of differentiated cells for clinical applications, and to report differences in pluripotency marker expression in differentiated cells from different iPSC lines. A standardized SMC differentiation protocol was used to induce the CD31+/CD34+ vascular progenitor cell phenotype. These were sorted by magnetic-activated (MACS) and fluorescence-activated cell sorting (FACS), and then treated with PDGF-BB and smooth muscle growth medium for further differentiation into smooth muscle progenitor cells (pSMCs). The expression of SMC and pluripotency markers in early- and late-passage (P1 and P4) pSMCs was analyzed. A total of 36 differentiation runs was performed on the three patient iPSC lines. All pSMC populations expressed SMC markers and Ki67 consistent with the progenitor phenotype. Initial iPSC density correlated positively with the sorted cell FACS efficiency, and this correlation could be fit to a quadratic equation. We also observed that a specific "honeycomb" pattern of the starting cultured iPSCs cultured correlated with higher efficiency in all three iPSC lines. Pluripotency marker expression decreased significantly to nearly undetectable levels in all three lines. There was no significant change in SMC and pluripotent marker expression between passage 1 and 4. In summary, our observations suggest that the method of iPSC reprogramming does not affect iPSC differentiation into pSMCs. Protocol efficiency can be modeled mathematically and coupled with the initial "honeycomb" cell pattern to optimize production of large cell numbers for clinical therapies.

    View details for DOI 10.1089/scd.2018.0031

    View details for PubMedID 30153084

  • NKX3-1 is required for induced pluripotent stem cell reprogramming and can replace OCT4 in mouse and human iPSC induction. Nature cell biology Mai, T., Markov, G. J., Brady, J. J., Palla, A., Zeng, H., Sebastiano, V., Blau, H. M. 2018

    Abstract

    Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is now routinely accomplished by overexpression of the four Yamanaka factors (OCT4, SOX2, KLF4, MYC (or OSKM))1. These iPSCs can be derived from patients' somatic cells and differentiated toward diverse fates, serving as a resource for basic and translational research. However, mechanistic insights into regulators and pathways that initiate the pluripotency network remain to be resolved. In particular, naturally occurring molecules that activate endogenous OCT4 and replace exogenous OCT4 in human iPSC reprogramming have yet to be found. Using a heterokaryon reprogramming system we identified NKX3-1 as an early and transiently expressed homeobox transcription factor. Following knockdown of NKX3-1, iPSC reprogramming is abrogated. NKX3-1 functions downstream of the IL-6-STAT3 regulatory network to activate endogenous OCT4. Importantly, NKX3-1 substitutes for exogenous OCT4 to reprogram both mouse and human fibroblasts at comparable efficiencies and generate fully pluripotent stem cells. Our findings establish an essential role for NKX3-1, a prostate-specific tumour suppressor, in iPSC reprogramming.

    View details for DOI 10.1038/s41556-018-0136-x

    View details for PubMedID 30013107

  • Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers. Journal of assisted reproduction and genetics Gustin, S. L., Wang, G., Baker, V. M., Latham, G., Sebastiano, V. 2018

    Abstract

    OBJECTIVE: Create a model, using reprogrammed cells, to provide a platform to identify the mechanisms of CGG repeat instability amongst female fragile X mental retardation 1 gene (FMR1) premutation (PM) carriers.METHODS: Female PM carriers (with and without POI) and healthy controls were enrolled from June 2013 to April 2014. Patient-derived fibroblasts (FB) were reprogrammed to induced pluripotent stem cells (iPSC) using viral vectors, encoding KLF4, OCT4, SOX2, and MYC. FMR1 CGG repeat-primed PCR was used to assess the triplet repeat structure of the FMR1 gene. FMR1 promoter methylation (%) was determined using FMR1 methylation PCR (mPCR). Quantification of FMR1 transcripts by RT-qPCR was used to evaluate the effect of reprogramming on gene transcription, as well as to correlate patient phenotype with FMR1 expression. Production of FMR1 protein (FMRP) was determined using a liquid bead array-based immunoassay.RESULTS: Upon induction to pluripotency, all control clones exhibited maintenance of progenitor cell CGG repeat number, whereas 10 of 12 clones derived from PM carriers maintained their input CGG repeat number, one of which expanded and one contracted. As compared to parent FB, iPSC clones exhibited a skewed methylation pattern; however, downstream transcription and translation appeared unaffected. Further, the PM carriers, regardless of phenotype, exhibited similar FMR1 transcription and translation to the controls.CONCLUSIONS: This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.

    View details for DOI 10.1007/s10815-018-1237-y

    View details for PubMedID 29926373

  • Single cell expression analysis of primate-specific retroviruses-derived HPAT lincRNAs in viable human blastocysts identifies embryonic cells co-expressing genetic markers of multiple lineages HELIYON Glinsky, G., Durruthy-Durruthy, J., Wossidlo, M., Grow, E. J., Weirather, J. L., Au, K., Wysocka, J., Sebastiano, V. 2018; 4 (6): e00667

    Abstract

    Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50-80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype.

    View details for DOI 10.1016/j.heliyon.2018.e00667

    View details for Web of Science ID 000437801800006

    View details for PubMedID 30003161

    View details for PubMedCentralID PMC6039856

  • Human Ipsc-Derived Thymic Epithelial Progenitor Cells as Stem Cell-Based Therapyto Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients Gai, H., Sebastiano, V., Weinacht, K. G. ELSEVIER SCIENCE INC. 2018: S364
  • CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep SCIENTIFIC REPORTS Vilarino, M., Rashid, S., Suchy, F., McNabb, B., van der Meulen, T., Fine, E. J., Ahsan, S., Mursaliyev, N., Sebastiano, V., Diab, S., Huising, M. O., Nakauchi, H., Ross, P. J. 2017; 7: 17472

    Abstract

    One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

    View details for DOI 10.1038/s41598-017-17805-0

    View details for Web of Science ID 000417689400096

    View details for PubMedID 29234093

    View details for PubMedCentralID PMC5727233

  • A semi-interpenetrating network of polyacrylamide and recombinant basement membrane allows pluripotent cell culture in a soft, ligand-rich microenvironment. Biomaterials Price, A. J., Huang, E. Y., Sebastiano, V., Dunn, A. R. 2017; 121: 179-192

    Abstract

    The physical properties of the extracellular matrix play an essential role in guiding stem cell differentiation and tissue morphogenesis both in vivo and in vitro. Existing work to investigate the role of matrix mechanics in directing stem cell proliferation, self-renewal, and differentiation has been limited by the poor attachment and survival of human pluripotent cells cultured on soft matrices (Young's modulus E ≲ 1000 Pa). To address this limitation we developed a protocol for generating semi-interpenetrating networks of polyacrylamide and recombinant basement membrane. Using these materials, we found that human embryonic stem cells (hESCs) remained proliferative and pluripotent even when grown in small colonies and on surfaces ranging in stiffness from 150 to 12000 Pa, spanning the range of tissue stiffnesses likely to be encountered in the embryo. Considerable recent attention has focused on the role of the transcriptional coactivator and Hippo effector YAP in regulating differentiation and cell proliferation both in the early embryo and in vitro. We found that while YAP localized to the nucleus on substrates of E ≳ 1000 Pa, its localization was heterogeneous on substrates of moduli ≲ 450 Pa, with predominantly nuclear localization at the colony periphery and mixed cytoplasmic and nuclear localization for cells in the colony interior, a pattern reminiscent of YAP subcellular localization in the inner cell mass (ICM) of the early embryo. In addition, hESC colony dynamics were highly responsive to substrate stiffness, with cells assembling into monolayers, multilayer structures, and transient, hollow rosettes in response to decreasing substrate stiffnesses in the range of 12000 to 150 Pa. We suggest that soft, ligand-rich substrates such as are described here provide a promising means of recapitulating aspects of early mammalian development that are otherwise inaccessible, and more broadly may be useful in the derivation of complex tissues from pluripotent cells in an in vitro setting.

    View details for DOI 10.1016/j.biomaterials.2016.12.005

    View details for PubMedID 28088685

  • A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circulation research Karakikes, I., Termglinchan, V., Cepeda, D. A., Lee, J., Diecke, S., Hendel, A., Itzhaki, I., Ameen, M., Shrestha, R., Wu, H., Ma, N., Shao, N., Seeger, T., Woo, N. A., Wilson, K. D., Matsa, E., Porteus, M. H., Sebastiano, V., Wu, J. C. 2017

    Abstract

    Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

    View details for DOI 10.1161/CIRCRESAHA.116.309948

    View details for PubMedID 28246128

    View details for PubMedCentralID PMC5429194

  • Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency DEVELOPMENTAL CELL Durruthy-Durruthy, J., Wossidlo, M., Pai, S., Takahashi, Y., Kang, G., Omberg, L., Chen, B., Nakauchi, H., Pera, R. R., Sebastiano, V. 2016; 38 (1): 100-115

    Abstract

    Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology.

    View details for DOI 10.1016/j.devcel.2016.06.014

    View details for Web of Science ID 000380073400013

    View details for PubMedID 27404362

  • YAP Induces Human Naive Pluripotency. Cell reports Qin, H., Hejna, M., Liu, Y., Percharde, M., Wossidlo, M., Blouin, L., Durruthy-Durruthy, J., Wong, P., Qi, Z., Yu, J., Qi, L. S., Sebastiano, V., Song, J. S., Ramalho-Santos, M. 2016; 14 (10): 2301-2312

    Abstract

    The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

    View details for DOI 10.1016/j.celrep.2016.02.036

    View details for PubMedID 26947063

    View details for PubMedCentralID PMC4807727

  • The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming. Nature genetics Durruthy-Durruthy, J., Sebastiano, V., Wossidlo, M., Cepeda, D., Cui, J., Grow, E. J., Davila, J., Mall, M., Wong, W. H., Wysocka, J., Au, K. F., Reijo Pera, R. A. 2016; 48 (1): 44-52

    Abstract

    Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

    View details for DOI 10.1038/ng.3449

    View details for PubMedID 26595768

  • Derivation of GMP-Compliant Integration-Free hiPSCs Using Modified mRNAs. Methods in molecular biology (Clifton, N.J.) Durruthy, J. D., Sebastiano, V. 2015; 1283: 31-42

    Abstract

    The clinical use of human induced pluripotent stem cells (hiPSCs) and the development of patients-specific gene and cell therapies rely on the development of fast, reliable, and integration-free methods of derivation of pluripotent stem cells from somatic tissues. Here we describe an integration-free protocol for the rapid derivation of hiPSCs from dermal fibroblasts using modified mRNAs. This method is inexpensive, highly efficient, and makes use of reagents that are xeno-free and chemically defined and can therefore be adopted by any Good Manufacturing Practice (GMP) facility.

    View details for DOI 10.1007/7651_2014_124

    View details for PubMedID 25304205

  • Patenting parthenotes in the US and Europe. Nature biotechnology Senatore, V., Scott, C. T., Sebastiano, V. 2015; 33 (12): 1232–34

    View details for DOI 10.1038/nbt.3421

    View details for PubMedID 26650007

  • Lift NIH restrictions on chimera research. Science (New York, N.Y.) Sharma, A., Sebastiano, V., Scott, C. T., Magnus, D., Koyano-Nakagawa, N., Garry, D. J., Witte, O. N., Nakauchi, H., Wu, J. C., Weissman, I. L., Wu, S. M. 2015; 350 (6261): 640

    View details for DOI 10.1126/science.350.6261.640-a

    View details for PubMedID 26542560

  • Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa SCIENCE TRANSLATIONAL MEDICINE Sebastiano, V., Zhen, H. H., Derafshi, B. H., Bashkirova, E., Melo, S. P., Wang, P., Leung, T. L., Siprashvili, Z., Tichy, A., Li, J., Ameen, M., Hawkins, J., Lee, S., Li, L., Schwertschkow, A., Bauer, G., Lisowski, L., Kay, M. A., Kim, S. K., Lane, A. T., Wernig, M., Oro, A. E. 2014; 6 (264)

    Abstract

    Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

    View details for DOI 10.1126/scitranslmed.3009540

    View details for Web of Science ID 000345595200003

    View details for PubMedCentralID PMC4428910

  • The transcriptome of human pluripotent stem cells. Current opinion in genetics & development Au, K. F., Sebastiano, V. 2014; 28: 71-77

    Abstract

    Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify the transcriptome of hESCs. Lately, we developed a new tool (IDP) to integrate the hybrid sequencing data to characterize a more reliable and comprehensive hESC transcriptome with discoveries of many novel transcripts.

    View details for DOI 10.1016/j.gde.2014.09.012

    View details for PubMedID 25461453

  • Germ Cell Nuclear Factor Regulates Gametogenesis in Developing Gonads PLOS ONE Sabour, D., Xu, X., Chung, A. C., Le Menuet, D., Ko, K., Tapia, N., Arauzo-Bravo, M. J., Gentile, L., Greber, B., Huebner, K., Sebastiano, V., Wu, G., Schoeler, H. R., Cooney, A. J. 2014; 9 (8)
  • Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing. Cell reports Hendel, A., Kildebeck, E. J., Fine, E. J., Clark, J. T., Punjya, N., Sebastiano, V., Bao, G., Porteus, M. H. 2014; 7 (1): 293-305

    Abstract

    Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.

    View details for DOI 10.1016/j.celrep.2014.02.040

    View details for PubMedID 24685129

  • Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell stem cell Loh, K. M., Ang, L. T., Zhang, J., Kumar, V., Ang, J., Auyeong, J. Q., Lee, K. L., Choo, S. H., Lim, C. Y., Nichane, M., Tan, J., Noghabi, M. S., Azzola, L., Ng, E. S., Durruthy-Durruthy, J., Sebastiano, V., Poellinger, L., Elefanty, A. G., Stanley, E. G., Chen, Q., Prabhakar, S., Weissman, I. L., Lim, B. 2014; 14 (2): 237-252

    Abstract

    Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

    View details for DOI 10.1016/j.stem.2013.12.007

    View details for PubMedID 24412311

  • Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

    View details for DOI 10.1371/journal.pone.0094231

    View details for PubMedID 24718618

  • Germ cell nuclear factor regulates gametogenesis in developing gonads. PloS one Sabour, D., Xu, X., Chung, A. C., Le Menuet, D., Ko, K., Tapia, N., Araúzo-Bravo, M. J., Gentile, L., Greber, B., Hübner, K., Sebastiano, V., Wu, G., Schöler, H. R., Cooney, A. J. 2014; 9 (8)

    Abstract

    Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

    View details for DOI 10.1371/journal.pone.0103985

    View details for PubMedID 25140725

  • Characterization of the human ESC transcriptome by hybrid sequencing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Au, K. F., Sebastiano, V., Afshar, P. T., Durruthy, J. D., Lee, L., Williams, B. A., van Bakel, H., Schadt, E. E., Reijo-Pera, R. A., Underwood, J. G., Wong, W. H. 2013; 110 (50): E4821-E4830

    Abstract

    Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.

    View details for DOI 10.1073/pnas.1320101110

    View details for Web of Science ID 000328061700004

    View details for PubMedID 24282307

    View details for PubMedCentralID PMC3864310

  • SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients. Nature Shcheglovitov, A., Shcheglovitova, O., Yazawa, M., Portmann, T., Shu, R., Sebastiano, V., Krawisz, A., Froehlich, W., Bernstein, J. A., Hallmayer, J. F., Dolmetsch, R. E. 2013; 503 (7475): 267-271

    Abstract

    Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.

    View details for DOI 10.1038/nature12618

    View details for PubMedID 24132240

  • SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients NATURE Shcheglovitov, A., Shcheglovitova, O., Yazawa, M., Portmann, T., Shu, R., Sebastiano, V., Krawisz, A., Froehlich, W., Bernstein, J. A., Hallmayer, J. F., Dolmetsch, R. E. 2013; 503 (7475): 267-?

    View details for DOI 10.1038/nature12618

    View details for Web of Science ID 000326894200052

    View details for PubMedID 24132240

  • Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status STEM CELL RESEARCH & THERAPY Awe, J. P., Lee, P. C., Ramathal, C., Vega-Crespo, A., Durruthy-Durruthy, J., Cooper, A., Karumbayaram, S., Lowry, W. E., Clark, A. T., Zack, J. A., Sebastiano, V., Kohn, D. B., Pyle, A. D., Martin, M. G., Lipshutz, G. S., Phelps, P. E., Pera, R. A., Byrne, J. A. 2013; 4

    Abstract

    The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

    View details for DOI 10.1186/scrt246

    View details for Web of Science ID 000323720300001

    View details for PubMedCentralID PMC3854769

  • Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells. Stem cells and development Ge, X., Wang, I. E., Toma, I., Sebastiano, V., Liu, J., Butte, M. J., Reijo Pera, R. A., Yang, P. C. 2012; 21 (15): 2798-2808

    Abstract

    Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.

    View details for DOI 10.1089/scd.2011.0435

    View details for PubMedID 22530853

    View details for PubMedCentralID PMC3464077

  • Human Amniotic Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells May Generate a Universal Source of Cardiac Cells STEM CELLS AND DEVELOPMENT Ge, X., Wang, I. E., Toma, I., Sebastiano, V., Liu, J., Butte, M. J., Pera, R. A., Yang, P. C. 2012; 21 (15): 2798-2808
  • Ultrastructural Characterization of Mouse Embryonic Stem Cell-Derived Oocytes and Granulosa Cells STEM CELLS AND DEVELOPMENT Psathaki, O. E., Huebner, K., Sabour, D., Sebastiano, V., Wu, G., Sugawa, F., Wieacker, P., Pennekamp, P., Schoeler, H. R. 2011; 20 (12): 2205-2215

    Abstract

    Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.

    View details for DOI 10.1089/scd.2010.0575

    View details for Web of Science ID 000297740200017

    View details for PubMedID 21244227

  • In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases STEM CELLS Sebastiano, V., Maeder, M. L., Angstman, J. F., Haddad, B., Khayter, C., Yeo, D. T., Goodwin, M. J., Hawkins, J. S., Ramirez, C. L., Batista, L. F., Artandi, S. E., Wernig, M., Joung, J. K. 2011; 29 (11): 1717-1726

    Abstract

    The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.

    View details for DOI 10.1002/stem.718

    View details for Web of Science ID 000296565500009

    View details for PubMedID 21898685

    View details for PubMedCentralID PMC3285772

  • Induction of human neuronal cells by defined transcription factors NATURE Pang, Z. P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D. R., Yang, T. Q., Citri, A., Sebastiano, V., Marro, S., Suedhof, T. C., Wernig, M. 2011; 476 (7359): 220-U122

    Abstract

    Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

    View details for DOI 10.1038/nature10202

    View details for Web of Science ID 000293731900039

    View details for PubMedID 21617644

    View details for PubMedCentralID PMC3159048

  • Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells NATURE Batista, L. F., Pech, M., Zhong, F. L., Nguyen, H. N., Xie, K. T., Zaug, A. J., Crary, S. M., Choi, J., Sebastiano, V., Cherry, A., Giri, N., Wernig, M., Alter, B. P., Cech, T. R., Savage, S. A., Pera, R. A., Artandi, S. E. 2011; 474 (7351): 399-?

    Abstract

    The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

    View details for DOI 10.1038/nature10084

    View details for Web of Science ID 000291647100050

    View details for PubMedID 21602826

    View details for PubMedCentralID PMC3155806

  • Embryonic Stem Cells, Derived Either after In Vitro Fertilization or Nuclear Transfer, Prolong Survival of Semiallogeneic Heart Transplants JOURNAL OF IMMUNOLOGY Imberti, B., Casiraghi, F., Cugini, D., Azzollini, N., Cassis, P., Todeschini, M., Solini, S., Sebastiano, V., Zuccotti, M., Garagna, S., Redi, C. A., Noris, M., Morigi, M., Remuzzi, G. 2011; 186 (7): 4164-4174

    Abstract

    Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

    View details for DOI 10.4049/jimmunol.1000654

    View details for Web of Science ID 000288751200041

    View details for PubMedID 21389254

  • Oct1 regulates trophoblast development during early mouse embryogenesis DEVELOPMENT Sebastiano, V., Dalvai, M., Gentile, L., Schubart, K., Sutter, J., Wu, G., Tapia, N., Esch, D., Ju, J., Huebner, K., Arauzo-Bravo, M. J., Schoeler, H. R., Cavaleri, F., Matthias, P. 2010; 137 (21): 3551-3560

    Abstract

    Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

    View details for DOI 10.1242/dev.047027

    View details for Web of Science ID 000283669300003

    View details for PubMedID 20876643

  • Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes EMBO JOURNAL Wossidlo, M., Arand, J., Sebastiano, V., Lepikhov, K., Boiani, M., Reinhardt, R., Schoeler, H., Walter, J. 2010; 29 (11): 1877-1888

    Abstract

    In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as gammaH2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.

    View details for DOI 10.1038/emboj.2010.80

    View details for Web of Science ID 000278235100010

    View details for PubMedID 20442707

  • Induction of Pluripotency in Adult Unipotent Germline Stem Cells CELL STEM CELL Ko, K., Tapia, N., Wu, G., Kim, J. B., Arauzo-Bravo, M. J., Sasse, P., Glaser, T., Ruau, D., Han, D. W., Greber, B., Hausdoerfer, K., Sebastiano, V., Stehling, M., Fleischmann, B. K., Bruestle, O., Zenke, M., Schoeler, H. R. 2009; 5 (1): 87-96

    Abstract

    Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

    View details for DOI 10.1016/j.stem.2009.05.025

    View details for Web of Science ID 000267879200013

    View details for PubMedID 19570517

  • Oct4-Induced Pluripotency in Adult Neural Stem Cells CELL Kim, J. B., Sebastiano, V., Wu, G., Arauzo-Bravo, M. J., Sasse, P., Gentile, L., Ko, K., Ruau, D., Ehrich, M., Van Den Boom, D., Meyer, J., Huebner, K., Bernemann, C., Ortmeier, C., Zenke, M., Fleischmann, B. K., Zaehres, H., Schoeler, H. R. 2009; 136 (3): 411-419

    Abstract

    The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

    View details for DOI 10.1016/j.cell.2009.01.023

    View details for Web of Science ID 000263120600012

    View details for PubMedID 19203577

  • Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors NATURE Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo, M. J., Ruau, D., Han, D. W., Zenke, M., Schoeler, H. R. 2008; 454 (7204): 646-U54

    Abstract

    Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.

    View details for DOI 10.1038/nature07061

    View details for Web of Science ID 000258026500049

    View details for PubMedID 18594515

  • Experimental demonstration that mammalian oocytes are not selective towards X- or Y-bearing sperm MOLECULAR REPRODUCTION AND DEVELOPMENT Zuccotti, M., Sebastiano, V., Garagna, S., Redi, C. A. 2005; 71 (2): 245-246

    Abstract

    Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.

    View details for DOI 10.1002/mrd.20252

    View details for Web of Science ID 000228966300014

    View details for PubMedID 15791593

  • Cloned pre-implantation mouse embryos show correct timing but altered levels of gene expression MOLECULAR REPRODUCTION AND DEVELOPMENT Sebastiano, V., Gentile, L., Garagna, S., Redi, C. A., Zuccotti, M. 2005; 70 (2): 146-154

    Abstract

    Mammalian embryos obtained by somatic nuclear transfer (NT) struggle to survive throughout development, encountering a number of hurdles leading to wrong functional reprogramming of the donor genome. However, despite these obstacles, some of these embryos continue their development, as if the required transcriptional functions are somehow satisfied. The amount of information gathered on the kinetics and quantitative profile of gene expression in NT pre-implantation embryos is still scarce and limited to a handful of genes described in two species, bovine and mouse. Using a single-cell sensitive semi-quantitative RT-PCR, we have compared the onset and profile of abundance of Hprt, Tsx, Bex1, Bax, Cpt2, and Oct4 genes, in in vitro fertilised and NT-derived mouse 1-cell, 2-cell, 4-cell embryos, morulae, and blastocysts. The genes analysed were activated in NT embryos at approximately the correct time compared to control embryos, indicating that the reprogramming phenomenon is developmentally regulated and that the somatic genome is quickly rearranged towards an embryonic-type of expression during the early stages of segmentation. Despite the right timing of genes onset, the high degree of variability in the number of transcripts found in NT embryos at the latest stages of pre-implantation development, suggests that genome reprogramming is incomplete and inaccurate.

    View details for DOI 10.1002/mrd.20144

    View details for Web of Science ID 000226154600004

    View details for PubMedID 15570622

  • Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis JOURNAL OF MOLECULAR HISTOLOGY Garagna, S., Merico, V., Sebastiano, V., Monti, M., Orlandini, G., Gatti, R., Scandroglio, R., Redi, C. A., Zuccotti, M. 2004; 35 (6): 631-638

    Abstract

    Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.

    View details for Web of Science ID 000225903600011

    View details for PubMedID 15614617

  • Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos CYTOGENETIC AND GENOME RESEARCH Gentile, L., Monti, M., Sebastiano, V., Merico, V., Nicolai, R., Calvani, M., Garagna, S., Redi, C. A., Zuccotti, M. 2004; 105 (2-4): 215-221

    Abstract

    Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.

    View details for DOI 10.1159/000078191

    View details for Web of Science ID 000224158000007

    View details for PubMedID 15237209