Future of stem cell therapy in liver disease Domenico ALVARO, MD Sapienza, University of Rome, Italy
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1 Future of stem cell therapy in liver disease Domenico ALVARO, MD Sapienza, University of Rome, Italy AISF, 17 th Pre Meeting Future Scenarios in Hepatology,
2 CELL THERAPY and LIVER DISEASES The cell therapy for treatment of liver diseases is the object of extensive investigations Targets: 1. Liver cirrhosis 2. Acute Liver Failure, Acute on Chronic 3. Congenital/metabolic diseases
3 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Homing and liver engraftment of administered cells - Immunogenicity - Tracking in vivo the infused cells - Cryopreservation - Teratogenesis and cancerogenesis
4 CELL THERAPY and LIVER DISEASES Cell Sources: Adult cells: Hepatocytes Stem-cells: 1. Embrionic stem cells 2. Fetal stem cells 3. Adult hepatic stem cells 4. Mesenchymal stem cells 5. Amniotic fluid-derived stem cells 6. Induced pluripotent stem cells
5 Required liver replacement by transpl. Hep. > 5%! Discouraging results! In general, only short-term benefits! Replacement level < 1% in clinical hepatocyte There is need for another transplantation since : Current indications source of congenital/metabolic cells! diseases (urea cycle defects, Crigler-Najjar..)! A. most donor hepatocytes cleared.! B. Total proliferation 34 pts treated of surviving donor cells not observed! (22OLT, functional transient improvement)
6 The main features of stem cells (Potten et al. ) 1. Self-renewal; 2. Indefinite proliferative capacity; 3. Production of large number of differentiate progeny; 4. Ability of regenerating injured tissues; 5. Low immunogenicity, scarce rejection.
7 CELL THERAPY and LIVER DISEASES Cell Sources: Adult Hepatocytes Stem/progenitor-cells: 1. Embrionic stem cells 2. Fetal stem cells 3. Adult hepatic stem cells 4. Mesenchymal stem cells 5. Amniotic fluid-derived stem cells 6. Induced pluripotent stem cells (ips)
8 Menstrual blood derived Fetal/adult Hepatic progenitors Fetal/adult stem cells Ethical. Teratoma risk!
9 CELL THERAPY and LIVER DISEASES Cell Sources: Adult cells: Hepatocytes Stem-cells: 1. Embrionic stem cells 2. Fetal stem cells 3. Adult hepatic stem cells 4. Mesenchymal stem cells 5. Amniotic fluid-derived stem cells 6. Induced pluripotent stem cells
10 BM-derived stem cells and liver injury!
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14 Currently, the paracrine effects of MSCs in liver regeneration are widely recognized and research is focusing on the soluble factors reprogramming (change of potential) involved! rather than differentiation(expression of potential)!
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16 Fetal/adult Hepatic progenitors Fetal/adult stem cells Ethical. Teratoma risk!
17 Cardinale V, Carpino G., Alvaro D., Gaudio E., Reid L. Hepatology 2011, J. Anatomy 2012, Nature Rev
18 Ductal plate Canals transitions of Hering to bile ducts and The stem cell Canal niche of Hering the adult liver! Fetal liver Fetal liver Adult liver Ductal Plate Hepatoblasts Canal of Hering HPC 18-week fetus, CK-19 focal duplication ductal plate. Crawford week fetus, tubular bile duct with a CK-19 positive ductular tether to the CK-19 positive ductal plate.
19 2014
20 Science 2014
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22 Ductal plate Canals transitions of Hering to bile ducts and The stem cell Canal niche of Hering the adult liver! Fetal liver Fetal liver Adult liver Ductal Plate Hepatoblasts Canal of Hering HPC 18-week fetus, CK-19 focal duplication ductal plate. Crawford week fetus, tubular bile duct with a CK-19 positive ductular tether to the CK-19 positive ductal plate.
23 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Homing and liver engraftment of administered cells - Immunogenicity - Teratogenesis and cancerogenesis - Cryopreservation - Tracking in vivo the infused cells
24 CELL THERAPY and LIVER DISEASES Route of cell administration - Hepatic artery, Portal vein, Splenic Artery, Intrasplenic, Peripheral vein, Others??? * Portal vein cell spreading outside the liver via collateral veins; cells into hepatic sinudoids * Hepatic artery.cell in portal spaces *Complications (emboli portal vein > hepatic artery) Administration through the hepatic artery safer than the portal vein route (reviewed in Lanzoni G. et al. Stem Cells. 2013; 31(10): ).
25 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Immunogenicity - Homing and liver engraftment of administered cells - Teratogenesis and cancerogenesis - Cryopreservation - Tracking in vivo the infused cells
26 Low or null Immunogenicity of BTSCs (Riccio M. et al. J. Hepatol. 2014)
27 T-Cells apoptosis in hbtscs/t-cells co-cultures (Riccio M. et al. J. Hepatol. 2014). hbtscs are able to modulate the immune response playing an active role in the interactions with microenvironment that promotes their expansion, proliferation and differentiation. Live confocal imaging of hbtscs- T-Cells double labelled with anti-cd4/ AnnexinV or anti-cd8/annexinv. White arrowheads indicate CD4+/annexinV+ T cells; yellow arrowheads indicate CD8+/annexinV+ T cells.
28 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Immunogenicity - Homing and liver engraftment of administered cells - Teratogenesis and cancerogenesis - Cryopreservation - Tracking in vivo the infused cells
29 H-GSCs transplanted (intra-splenic injection, 1 month) in CCL4- cirrhotic mice (Carpino G. et al. J. Hepatol 2014). CCL4+Veh (N=5) CCL4+hHep (N=5) CCL4+hGSC (N=5) Fibrosis(% liver volume) 4.83 ± ± ± 0.46 Necrosis (% liver volume) 3.28 ± ± ± 1.16* Human Hepatocyte Mass (% liver volume) N/D 2.39 ± ± 2.08* Human ALB (ng/dl) N/D N/D ± 0,3* Cholinesterase(U/L) 3787 ± ± ± 209* AST(U/L) 263 ± ± ± 89* ALT(U/L) 60 ± ± ± 9.58*
30 Ongoing studies: IMPROVING hbtscs ENGRAFMENT INTO THE LIVER Hepatology 2013
31 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Immunogenicity - Homing and liver engraftment of administered cells - Tracking in vivo the infused cells - Teratogenesis and cancerogenesis - Cryopreservation
32 Ongoing studies: TRACKING TRANSPLANTED CELLS
33 CELL THERAPY and LIVER DISEASES Issues and problems to be addressed - Cell sources - Route of cell administration - Immunogenicity - Homing and liver engraftment of administered cells - Tracking in vivo the infused cells - Cryopreservation - Teratogenesis and cancerogenesis
34 hbtscs cryopreservation: ongoing studies Kubota Medium, 10% DMSO, 15% Albumin -1 C per minute until -80 C and then long preservation (<3 months) in liquid nitric oxide EASL2014 (-160 C). Engrafment in the liver correlates Viability after thawing= 50 ± 5% M±SD (n= 8) hbtscs one day after plating hbtscs after three days of culture with the platting efficiency of the cells and attachment/long Hyaluronans (HA) term % survival on culture plates! Viability after thawing= 82±5% M±SD (N= 8) Improved expression of adhesion molecules (CD44, hyaluronan receptor, E-cadherin, β4 integrin) BTSCs after seven days of culture hbtscs after ten days of culture
35 CELL THERAPY and LIVER DISEASES Cell Sources: Adult cells: Hepatocytes Stem-cells: 1. Embrionic stem cells 2. Fetal stem cells 3. Adult hepatic (HPC) stem cells 4. Mesenchymal stem cells 5. Amniotic fluid-derived stem cells 6. Induced pluripotent stem cells
36 *HPC + Mesenchymal-SC + Hematopoietic-SC from foetal liver (18-22 week gestational age) *Splenic Artery, transplantation (5 x 10 8 cells twice ) in a patient with Cirrhosis-HCV. Basal MELD = 15 After 18 month F.U. MELD = 10!
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38 HPC (EpCAM+) from foetal liver (18-22 week gestation) Transplanted (80 million cells) through hepatic artery
39 Phase I/II study on advanced cirrhosis transplanted with fetal hbtscs. 1 Paz. BILIRUBINA TOTALE BILIRUBINA DIRETTA Pz. A.G., 72 yrs, Cirrhosis-HCV, Child-Pugh 12, Meld millions freshly isolated fetal BTScs, EpCAM+(52% Lgr5+) injected via hepatic artery Time: 0 12 months Child-Pugh score Meld score Bilirubin mg/dl Albumin g/dl INR Creatinine mg/dl
40 Phase I/II study on advanced cirrhosis transplanted with fetal hbtscs. 2 Paz. BILIRUBINA TOTALE BILIRUBINA DIRETTA Pz. A.P., 71 yrs, Cirrhosis-HCV, Child-Pugh 12, Meld millions freshly isolated fetal BTScs, EpCAM+ (50% Lgr5+) injected via hepatic artery Time: 0 24 months Child-Pugh score Meld score Bilirubin mg/dl Albumin g/dl INR Creatinine
41 Acknowledgments Prof. P. Berloco Prof. M. Nuti/Dr. Napolitano Prof. A.F. Attili/Prof. A. DeSantis Prof. E. Gaudio Prof. N. Caporaso Dr R. Semeraro Thanks for the attention Dr G. Carpino Prof L. Reid at UNC Dr V. Cardinale
Colangiocarcinoma on the rise!
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