CD133⁺ Pluripotent Stem Cells for the Treatment of Chronic Liver Failure.

The potential role of bone marrow (BM)-derived stem cells (SCs) in patients with end-stage liver disease has been addressed by our group in four studies. Main objectives were: 1) to assess stem/progenitor cell mobilization in 24 patients receiving orthotopic liver transplantation (OLT); 2) to evaluate whether G-CSF can be safely administered to patients with liver cirrhosis in order to expand and mobilize BM-derived SCs; 3) to investigate the effects of transplantation of human G-CSF-mobilized CD34⁺ and CD133⁺ SCs in mice with chronic liver injury and fibrosis; 4) to evaluate the feasibility and the safety of the purification and intrahepatic reinfusion of increasing numbers of autologous BM-derived G-CSF-mobilized CD133⁺ SCs in patients with end-stage liver disease.

1) Flow cytometry analysis, clonogenic assays and RT-PCR have been performed after OLT; 2) 18 patients with advanced liver disease were consecutively treated with increasing doses of G-CSF starting from 2 μg/kg/daily; 3) C57BL/6N mice received CCl₄ by inhalation for thirteen weeks and were treated with Cyclosporin-A. Transplantation was performed by injection (tail vein) of 10⁶ CD34⁺ or CD133⁺ SCs of three cirrhotic patients. After four weeks from transplantation all mice were sacrificed; 4) G-CSF at 7.5μg/Kg/b.i.d. is administered subcutaneosly (sc) from day 1 until the completion of peripheral blood stem cells (PBSC) collection. Collection of PBSC will begin on day + 4 only if the concentration of CD133⁺ cells is 38/mL. PB mononuclear cells obtained from mobilized standard-volume leukapheresis will be incubated with Macs colloidal superparamagnetic CD133 microbeads. CliniMacs device is used for the positive selection of CD133⁺ SCs under GMP conditions. At least 4 weeks after SC mobilization, collection and cryopreservation, highly purified autologous G-CSF-mobilized CD133⁺ cells are re-infused through the hepatic artery by transfemoral or transbranchial arteriography. CD133⁺ cells are administered to patients starting from 5×10⁴/Kg patient's body weight and increased every 3 patients. The maximum infused cell dose will be 1×10⁶/kg. G-CSF at 5μg/Kg/day is administered sc for 3 days after the reinfusion of SCs for their expansion and to induce a selective proliferative advantage of reinfused cells in vivo.

1) We demonstrated that both early subsets of the hematopoietic SC compartment (CD34⁺/CD90⁺ cells) and more mature committed progenitors (CFU-C) were mobilized into PB after OLT. We also demonstrated the release from the BM of liver-committed HSCs co-expressing epithelial markers after OLT; 2) We show that the administration of G-CSF to patients with liver cirrhosis is safe and feasible and allows the mobilization and collection of BM-derived SCs at the dose of 15 mg/kg/day. 3) We demonstrated that mice transplanted with either CD133⁺ or CD34⁺ human cells appear to have less fibrotic septa than mice without SC transplantation, suggesting the potential therapeutic role of human SCs on the recovery of liver fibrosis. 4) Up to date, three patients with end stage liver disease have been successfully mobilized with G-CSF and highly purified autologous CD133+ SCs have been re-infused. The number of collected CD133+ SCs is 0,7, 0,2 and 0.35×10⁶/Kg, respectively. The number of the re-infused highly purified CD133+ SCs is 4.7, 5.0 and 5.4×10⁴/Kg, respectively. No adverse events have been recorded during mobilization or intrahepatic SCs re-infusion. Updated results on current patients and future patients will be presented at the Meeting.

No relevant conflicts of interest to declare.