Biology subjects

Pellegrini, G.

Publications and source records attributed to Pellegrini, G..

5 recordsLinked to original sources

bioRxiv2023

Heterogeneous potential of human airway basal cells: Holoclone-stem cell identification in a clinical-grade system

BackgroundDespite their life-saving potential, tissue engineering approaches for the treatment of extensive tracheal and bronchial defects still face significant limitations. A major challenge is the inability to regenerate a functional airway epithelium containing the appropriate amount of stem cells required for long-term tissue renewal following transplantation of the bioengineered graft. In this scenario, validation assays and quality controls are needed to guide each step of the regeneration process. MethodsStem cell depletion is often due to suboptimal culture conditions, therefore we tested the ability of a clinical-grade culture system to support the safe and efficient in vitro expansion and differentiation of primary human tracheal and bronchial epithelial cells. Single-cell clonal analysis was used to unravel the heterogeneity of airway basal cells and to understand tissue-specific regeneration and differentiation mechanisms. Functional assays were used to investigate the wound healing ability and tightness of the regenerated epithelium under the selected culture conditions. ResultsPrimary tracheobronchial epithelial cells showed an impressive proliferative potential, allowing the regeneration of a mature and functional epithelium without immortalisation events. Analysis at the single cell level allowed the identification of the subpopulation of basal cells endowed with in vitro self-renewal, distinguishing them from transient amplifying cells. This approach has further defined the hierarchy of cellular differentiation and its correlation with regenerative and differentiation potential. ConclusionsOur results show that primary airway epithelial cell cultures can maintain stem cells together with their differentiation lineages in vitro. Airway cells can be safely and effectively used in autologous tissue engineering approaches when cultured under appropriate and well-standardised conditions. In addition to the validation assays proposed for the development of new advanced therapy products, this study outlines possible quality controls to enhance therapeutic success and maximise patient safety in future clinical applications.

cell biology↗

bioRxiv2022

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of -SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed CHIR99021 induced downregulation of EnMT markers (SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the {beta}-catenin and TGF{beta} pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT and brings a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

cell biology↗

bioRxiv2022

Nanoneedles for targeted siRNA silencing of p16 in the Human Corneal Endothelium

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNAi aimed at enhancing its proliferative capacity, necessary for tissue regeneration. Here we develop a strategy for siRNA nanoninjection of the human corneal endothelium. We show that nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is non-toxic ex vivo and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNAi therapy for the treatment of endothelial corneal dysfunction.

bioengineering↗

bioRxiv2020

SF3B1 promotes tumor malignancy through splicing-independent co-activation of HIF1α

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between expression levels of wildtype SF3B1 and tumor malignancy, but underlying mechanisms remain elusive. Here, we report that SF3B1 acts as an activator of HIF signaling through a splicing-independent mechanism. We demonstrate that SF3B1 forms a heterotrimer with HIF1 and HIF1{beta}, facilitating binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. We further validate the relevance of this mechanism for tumor progression. Monoallelic deletion of Sf3b1 impedes formation and progression of hypoxic pancreatic cancer via impaired HIF signaling, but is well tolerated in normoxic chromophobe renal cell carcinoma. Our work uncovers an essential role of SF3B1 in HIF1 signaling, providing a causal link between high SF3B1 expression and aggressiveness of solid tumors.

cell biology↗

bioRxiv2019

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune check-point inhibitors

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 anti-body in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively-spliced EDB domain of fibronectin), retained the activity of the parental cyto-kine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent anti-tumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with check-point blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anti-cancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.\n\nNOVELTY AND IMPACT STATEMENTIn this study, we describe the generation of a novel fusion protein consisting of murine inter-leukin-12 fused to the L19 antibody (specific to the EDB domain of fibronectin). L19-mIL12 revealed favourable tumor to organ ratios 24h after intravenous administration and it was able to cure 60% of CT26 tumor-bearing mice. From a clinical perspective, the rapid clearance from circulation should ease the administration to patients as infusions could be stopped upon onset of side-effects.

cancer biology↗