animal models for hepatocyte transplantation

7/27/2019 animal models for hepatocyte transplantation

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Animal models for hepatocyte

transplantation

Pradeep B Patil


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Need for HTx

Metabolic diseases-alb,bil,Cu, cholestasis,

tyrosemia

Bridge between OLT

Prolongation survival

Ameliorate specific complicationse.g. ICP

Unique opportunity for Tx variety of diseases Study fundamental mechanism of cell biology


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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


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History of HTx

Liver the 1st organ of isolated cell Tx

30 yrs ago--Gunn rat-CN syndrome


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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 -


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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


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Immortalization

Differentiated nontransformed hepatocyte

SV 40LT

HSV-tk Reversible-gancyclovir


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Fate of hepatocyte

20-40um

Proximal hepatic sinusoid (6-9um)

Periportal regions of hepatic lobules

Leads portal hypertension2-3hoursand 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)


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Encapsulation

Fructose

Trehalose

Vitamin E

University of winconsin

BM cells

Collagen matrix (urathin sodium aginate)

p/c- encapsulate with alginate or collagen coatedbeads

N-acetyl-l-cysteine


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Models for Htx

Syngenic and immunodeficient models--useful


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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


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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?


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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


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Length of expt

Gunn rat12 monthsMatas et al 1976

We did 4 week 6 week studies

Now runing project with SV will be for 3months


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Small animal mdels

Molecular mechanism

Engraftment Factors

Cell to cell interaction Functionality for long term

Rat best easy to operate

Rabbitvery sensitive


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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


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Success rate

Fresh than cryo

Third generation lentivral vectors


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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


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Approaches to induce proliferation of

engrafted hepatocytes

Microenvironment

Only after impairment of native liver for a periodof time

Selective ablation of native heaptocyteCCl4,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


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Tracing

Moloney murine luekemia virus

SV40LT

Radiolabel GFP


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Mortility and mobidity

?


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Samples collected

Bilesensitive to light--

GFP tissuepoly-l-lysinecoated slides


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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 ?


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Recently, it has been shown in several animal

models that more than 90% of host

hepatocytes can be replaced by a small number oftransplanted 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

Principles of Therapeutic Liver Repopulation

Markus Grompe1, Ezio Laconi2, David A. Shafritz31 Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon2Istituto 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


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Liver Repopulation by Transplanted Hepatocytes and Risk of Hepatocellular CarcinomaLaconi, 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 overtwo thirds of the life span of the recipient animal.


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References

Documents

animal models for hepatocyte transplantation