Animal models for hepatocyte transplantation

Pradeep B Patil

Need for HTx

Metabolic diseases-alb,bil,Cu, cholestasis, tyrosemia Bridge between OLT Prolongation survival Ameliorate specific complications e.g. ICP Unique opportunity for Tx variety of diseases Study fundamental mechanism of cell biology

Impediments
Lung embolism Portal vein hypertension Need of Repeated tx Early loss of cells Insufficient number of engraftment Lack of differentiation after engraftment Rejection in alloTx Immunosupressive drugs has deleterious effect (SRL) Precoagulatant activity Freezing protocol

History of HTx
Liver the 1st organ of isolated cell Tx 30 yrs ago--Gunn rat-CN syndrome

Cell Dose
Rat 2x107 cells ( 3%) ---7,5 x 107 (12,5%) NHP 410 x 106 (4%) Pig-1 x 109 Mouse -2x 106 Human -

Routes for Htx

ALFarchitecture goodintraportal CLF architecture lost prolonged hypertension and embolization of lung pertoneal or s/c i/s the best Neovascularised s/c

Immortalization
Differentiated nontransformed hepatocyte SV 40LT HSV-tk Reversible-gancyclovir

Fate of hepatocyte
20-40um Proximal hepatic sinusoid (6-9um) Periportal regions of hepatic lobules Leads portal hypertension 2-3hours and IRI ---12 hours 70% trapped=phagocytes Disruption of sinusoidal endothelium Remodelling-3 to 7days i/s---26% spleen+72% in liver+2% lung (pulmonary capp cleaned in 24hr)

Encapsulation
Fructose Trehalose Vitamin E University of winconsin BM cells Collagen matrix (urathin sodium aginate) p/c- encapsulate with alginate or collagen coated beads

N-acetyl-l-cysteine

Models for Htx

Syngenic and immunodeficient models--useful

Models for liver Disease

Acute liver failure
90% hepatectomy Lethal dose of acetaminophen D-gal

Chronic liver failure

Dog--portal hypertension-sephadex microsperes Ligation of portal vein

PH
In normal mouse 70% --2 weeks restored 20% --NHP--not sufficient 70%--high risk of mortality 30% -- by us ---no mortility or mobidity with enough engraftment?

Disease model
Gunn rat inherited-- no heaptocyte damage
(absence of UDP glucoronyltransferase-high level of toxic bile in circulation)

Abcd4 k/oinheritedwith damage Immunodeficient micehuman hepatocyte

Length of expt
Gunn rat 12 monthsMatas et al 1976 We did 4 week 6 week studies Now runing project with SV will be for 3 months

Small animal mdels

Molecular mechanism Engraftment Factors Cell to cell interaction Functionality for long term Rat best easy to operate Rabbit very sensitive

Large animal models

Pigsuitable for assessment of HTx and BAL and reliable model for liver failure NHP-closely related with liver anatomy and vasculature-macaca

Success rate
Fresh than cryo Third generation lentivral vectors

Approaches to augment hepatocyte engraftment

Microenvironment Portal vein partial occlusion/ligation(R/R/P/NHP) rat portal br for ant lobe (70%) Liver conditioning by irradiation Unlike to animal model in human partial hepatectomy and toxins cant be used

Approaches to induce proliferation of engrafted hepatocytes

Microenvironment Only after impairment of native liver for a period of time Selective ablation of native heaptocyte CCl4, genotoxic hepatic damage Retrorsine /T3/PH induces hepatic polyploidy Liver conditioning by irradiation Unlike to animal model in human partial hepatectomy and toxins cant be used

Tracing
Moloney murine luekemia virus SV40LT Radiolabel GFP

Mortility and mobidity

Samples collected
Bilesensitive to light- GFP tissue poly-l-lysine coated slides

Why further study?

Source for Htx
Unsuitable OLT Immortalised hepatocytes Stem cell derived heaptocytes xeno

HTx cost effective, easy and safe ptocedure QOL Less chance of GvHD In situ proliferation partially understood Long term ?

Principles of Therapeutic Liver Repopulation

Markus Grompe1, Ezio Laconi2, David A. Shafritz3 1 Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 2 Istituto di Pathologia Sperimentale, Universita di Cagliari and Ospedale Oncologico A. Businco, Cagliari, Italy 3 Departments of Medicine, Cell Biology, and Pathology and The Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York

Recently, it has been shown in several animal models that more than 90% of host hepatocytes can be replaced by a small number of
transplanted donor cells in a process we term therapeutic liver repopulation. This phenomenon is analogous to repopulation of the hematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. Here we review the current knowledge of this process and discuss the hopeful implications for treatment of liver diseases.

https://www.thieme-connect.com/DOI/DOI?10.1055/s-2007-1007093

Liver Repopulation by Transplanted Hepatocytes and

Risk of Hepatocellular Carcinoma

Laconi, Sergio; Montisci, Stefania; Doratiotto, Silvia; Greco, Marianna; Pasciu, Daniela; Pillai, Sara; Pani, Paolo; Laconi, Ezio
Abstract Background. Transplantation of isolated hepatocytes in rats treated with retrorsine (RS) results in massive repopulation of the host liver. In this study, the long-term fate of hepatocytes transplanted into RS-treated recipients was followed for up to two years.

Methods. Dipeptidyl-peptidase type IV-deficient (DPPIV-) Fischer 344 rats were given two injections of RS (30 mg/kg), followed by transplantation of 2 million hepatocytes, isolated from a syngenic, DPPIV+ donor. Results. Extensive (917%) liver replacement by transplanted hepatocytes was observed in animals sacrificed 18 months posttransplantation. Similar levels of repopulation persisted at two years (875%). No evidence of preneoplastic and/or neoplastic evolution of the transplanted cell population was present in the RS-treated and repopulated livers at any time point considered. Furthermore, serum parameters related to hepatocyte function and integrity were in the normal range. In control groups given cell transplantation in the absence of prior treatment with RS, only small clusters of donor-derived, DPPIV+ hepatocytes were discerned.

Conclusions. These

results indicate that liver repopulation in this model is largely stable, persisting for up to two years and allowing for a normal liver function. In addition, no increased risk of neoplastic transformation appears to be associated with the process of liver repopulation for as long as over two thirds of the life span of the recipient animal.

References