Therapeutic potential of fetal liver cells transplantation in hemophilia A mice

: Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA-mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice for obtaining a suitable "proof of concept" for the development of a new HA treatment in neonates. Hence, we transplanted FLE11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. The engraftment levels and FVIII activity was assessed starting from 6 weeks after transplantation. FLE11-E13+BU-transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FLE13 cells showed engraftment ability even in absence of BU preconditioning, while FLE11 cells did not. BM+BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients

Phenotypic characterization of immune cell populations in homeostatic and FVIII-challenged severe HA mice

Development of FVIII-neutralizing antibodies (inhibitors) is one of the main complications occurring in 30% of severe hemophilia A (HA) patients undergoing replacement therapy. Both lack of central tolerance, due to the congenital absence of FVIII, and the presence of danger signals are factors involved in inhibitors development, which is definitely related to the immune cells activation. Since recent studies have been proposing new extra-coagulative roles for FVIII in the immuno-hematological biology and have shown the presence of increased levels of several inflammatory cytokines in untreated HA patients, we sought to investigate the effect of FVIII absence on the immune cell populations before and after challenge with a specific (FVIII) or general (OVA) immunological challenge. We also sought to investigate the capability of naïve CD4 T cells to differentiate into T regulatory cells. We employed a mouse model of severe HA and we did not observe any significative change in number and percentage of primary immune populations (e.g. T and B cells) and in the quantitative humoral response against FVIII or OVA when compare to control age- and treatment-matched animals. On the other hand, HA mice challenged with both antigens showed higher conversion of naïve to memory T cells. Interestingly, these differences were detected only when the immunological challenge included an adjuvant while it was absent when FVIII was injected intravenously without any supplemental stimulus, as it happens in the normal therapeutic regimen. Overall, the results obtained in this thesis suggest that immunogenic versus tolerogenic response to FVIII in HA patients could depend on the reciprocal interactions of extrinsic and intrinsic factors among which the pro-inflammatory microenvironment determined by the recurrent microbleedings should be taken in consideration

Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells