Vittorio Sebastiano lab

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 thread of Ariadne that connects germ cells, preimplatation development and pluripotent stem cells is the focus of my research, with a specific interest in human development. My long-term goals are several fold: 1. Understanding the biology of germ cells and and their ability to sustain early phases of preimplantation development; 2. Understanding the mechanisms that regulate very early cell fate decisions in human embryos; 3. Understanding the biology of Pluripotent Stem Cells and the mechanisms that lead to their formation also in the context of iPSCs derivation.

Assistant Professor (Research) of Obstetrics and Gynecology (Reproductive and Stem Cell Biology)
(650) 724-8084

Publications

  • 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