Impiego di un vettore lentivirale basato sul virus dell'immunodeficienza umana di tipo 1 per il trasferimento genico di siRNA.

Questo lavoro di tesi si è focalizzato sull’impiego di un vettore lentivirale basato sul Virus dell’Immunodeficienza Umana di tipo 1 (HIV-1) per introdurre un siRNA specifico per il silenziamento del Vascular Endothelial Growth Factor (VEGF) in cellule di epitelio pigmentato retinico umano (ARPE-19).ope

G9a co-suppresses LINE1 elements in spermatogonia

BACKGROUND: Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements. RESULTS: Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules. CONCLUSIONS: Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis

A transit amplifying population underpins the efficient regenerative capacity of the testis

The spermatogonial stem cell (SSC) that supports spermatogenesis throughout adult life resides within the GFRα1-expressing A type undifferentiated spermatogonia. The decision to commit to spermatogenic differentiation coincides with the loss of GFRα1 and reciprocal gain of Ngn3 (Neurog3) expression. Through the analysis of the piRNA factor Miwi2 (Piwil4), we identify a novel population of Ngn3-expressing spermatogonia that are essential for efficient testicular regeneration after injury. Depletion of Miwi2-expressing cells results in a transient impact on testicular homeostasis, with this population behaving strictly as transit amplifying cells under homeostatic conditions. However, upon injury, Miwi2-expressing cells are essential for the efficient regenerative capacity of the testis, and also display facultative stem activity in transplantation assays. In summary, the mouse testis has adopted a regenerative strategy to expand stem cell activity by incorporating a transit-amplifying population to the effective stem cell pool, thus ensuring rapid and efficient tissue repair

Oligoasthenoteratozoospermia and Infertility in Mice Deficient for miR-34b/c and miR-449 Loci

Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility

In vitro effect of retinoic acid and epigenetic modifying drugs on mesenchymal stem cells

Effetto in vitro dell'acido retinoico e dei farmaci epigenetici sulle cellule staminali mesenchimal

Impiego di un vettore lentivirale basato sul virus dell'immunodeficienza umana di tipo 1 per il trasferimento genico di siRNA.

Questo lavoro di tesi si è focalizzato sull’impiego di un vettore lentivirale basato sul Virus dell’Immunodeficienza Umana di tipo 1 (HIV-1) per introdurre un siRNA specifico per il silenziamento del Vascular Endothelial Growth Factor (VEGF) in cellule di epitelio pigmentato retinico umano (ARPE-19)

Vitamin C Limits Multipotent Progenitor Self-Renewal and Clonal Expansion

A fundamental question is whether physiological variations in diet-derived metabolite levels in vivo influence stem and progenitor cell self-renewal. Ascorbate (vitamin C) is enriched in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) compared to all other hematopoietic cells, and ascorbate depletion increases HSC function by reducing Tet2 function (Agathocleous et al., 2017). However, whether ascorbate regulates the self-renewal of other hematopoietic progenitors downstream of HSCs is still unknown. To answer this question, we conditionally deleted Slc23a2, the gene that encodes the main hematopoietic ascorbate transporter, in murine hematopoietic cells using Mx1-Cre. Deletion of Slc23a2 substantially increased the reconstituting potential of bone marrow cells upon competitive transplantation into irradiated mice. Slc23a2 deletion only mildly increased donor chimerism in HSCs (Lin- Kit+ Sca1+ CD150+ CD48-) in the reconstituted mice, but significantly increased donor chimerism in MPPs (Lin- Kit+ Sca1+ CD150- CD48-) and downstream progenitors, suggesting that ascorbate depletion enhanced MPP self-renewal. Deletion of the ascorbate transporter also increased the long-term reconstituting potential of purified MPPs in irradiated mice, but not the long-term reconstituting potential of purified HPC1 (Lin- Kit+ Sca1+ CD150- CD48+) or HPC2 (Lin- Kit+ Sca1+ CD150+ CD48+) progenitor cells. These data showed that ascorbate depletion conferred long-term reconstituting potential upon MPPs. To understand how ascorbate depletion enhanced MPP self-renewal, we measured the composition and proliferation rate of MPPs. Slc23a2 deletion significantly reduced MPP proliferation, and it increased the frequency of the more quiescent MPP1 (CD229- CD244-) subpopulation while decreasing the frequency of less quiescent MPP2 (CD229+ CD244-) and MPP3 (CD229+ CD244+) subpopulations (Oguro et al., 2013). Single-cell RNA-Seq (scRNA-Seq) analysis revealed that Slc23a2 deletion increased the frequency of MPPs with high expression of self-renewal gene signatures (Rodriguez-Fraticelli et al., 2020). Taken together, these data suggested that ascorbate depletion promoted MPP self-renewal through the expansion of a subpopulation of quiescent MPPs. To test this, we evaluated the division history of wild-type and Slc23a2-deficientMPPs using H2B-GFP reporter mice (Foudi et al., 2009). Slc23a2 deletion significantly increased the percentage of H2B-GFP High quiescent MPPs, while reducing the percentage of H2B-GFP Neg proliferating MPPs as compared to controls. Competitive transplantation of purified H2B-GFP Highor H2B-GFP Neg MPPs revealed that Slc23a2 deletion increased the reconstituting potential of H2B-GFP High quiescent MPPs as compared to controls, but not the reconstituting potential of H2B-GFP Neg proliferating MPPs. Our data thus showed that ascorbate depletion enhanced MPP long-term self-renewal by expanding a subpopulation of quiescent MPPs. Overall, we showed that ascorbate cell-autonomously limited MPP self-renewal potential by negatively regulating MPP quiescence. Our study thus points to a central role for diet-derived nutrients in the regulation of HSC and hematopoietic progenitor self-renewal abilities. Additionally, our data suggest that nutrition might directly impact the expansion of mutated cells found in clonal hematopoiesis