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.
Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells.
2020; 11 (1): 1545
Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.
View details for DOI 10.1038/s41467-020-15174-3
View details for PubMedID 32210226
- Platelet-Rich Plasma (PRP) From Older Males With Knee Osteoarthritis Depresses Chondrocyte Metabolism and Upregulates Inflammation JOURNAL OF ORTHOPAEDIC RESEARCH 2019; 37 (8): 1760–70 Hide More
Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.
Cell stem cell
2019; 24 (5): 821
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
View details for PubMedID 31051134
- Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination CELL STEM CELL 2019; 24 (5): 821-+ Hide More
Platelet-Rich Plasma (PRP) from Older Males with Knee Osteoarthritis Depresses Chondrocyte Metabolism and Upregulates Inflammation.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
There is intense clinical interest in the potential effects of platelet-rich plasma (PRP) for the treatment of osteoarthritis (OA). This study tested the hypotheses that (1) 'lower' levels of the inflammatory mediators (IM) interleukin-1-beta (IL-1beta) and tumor-necrosis-factor-alpha (TNF-alpha), and (2) 'higher' levels of the growth factors (GF) insulin-like-growth-factor-1 and transforming-growth-factor-beta-1 within leukocyte-poor PRP correlate with more favorable chondrocyte and macrophage responses in vitro. Samples were collected from ten 'healthy' young male (23-33 years old) human subjects (H-PRP) and nine older (62-85 years old) male patients with severe knee OA (OA-PRP). The samples were separated into groups of 'high' or 'low' levels of IM and GF based on multiplex cytokine and ELISA data. Three-dimensional (3D) alginate bead chondrocyte cultures and monocyte-derived macrophage cultures were treated with 10% PRP from donors in different groups. Gene expression was analyzed by qPCR. Contrary to our hypotheses, the effect of PRP on chondrocytes and macrophages was mainly influenced by the age and disease status of the PRP donor as opposed to the IM or GF groupings. While H-PRP showed similar effects on expression of chondrogenic markers (Col2a1 and Sox9) as the negative control group (p>0.05), OA-PRP decreased chondrocyte expression of Col2a1 and Sox-9 mRNA by 40% and 30%, respectively (Col2a1, p=0.015; Sox9, p=0.037). OA-PRP also upregulated TNF-alpha and MMP-9 (p<0.001) gene expression in macrophages while H-PRP did not. This data suggests that PRP from older individuals with OA contain factors that may suppress chondrocyte matrix synthesis and promote macrophage inflammation in vitro. This article is protected by copyright. All rights reserved.
View details for PubMedID 31042308