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.
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
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
Human Ipsc-Derived Thymic Epithelial Progenitor Cells as Stem Cell-Based Therapyto Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients
ELSEVIER SCIENCE INC. 2018: S364
View details for Web of Science ID 000425476000529
CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep
2017; 7: 17472
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.
2017; 121: 179-192
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.
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