טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBoyango Ilanit
SubjectThe Effect of Mouse Liver in Regeneration on Transplanted
Human Embryonic Stem Cells
DepartmentDepartment of Medicine
Supervisor Clinical Professor Yaacov Baruch
Full Thesis textFull thesis text - English Version


Abstract

Background: The need for mature functioning hepatocytes for clinical purposes is essential. Orthotopic liver transplantation is the standard treatment for chronic or inherited metabolic liver diseases. However, this therapy is limited by the availability of donor tissue. Hepatocyte transplantation has been shown to provide temporary liver function until donor organs become available. Utility of human hepatocytes is limited by their availability and isolated hepatocytes have limited proliferation capacity and shorter spectrum of cell functions. The development of hepatocytes from progenitor cells from hepatic and nonhepatic sources has a great potential. One of these sources could be human embryonic stem cells (hESCs).

Aim: To study the effect of regenerating liver microenvironment on hESCs   engraftment, survival and differentiation following transplantation.

Methods: SCID-beige mice were subjected to a single intraperitoneal injection of CCl4. hESCs or human embryoid bodies (hEBs) transplantation was done at 24hrs post CCl4 injury or in another set of experiments hEBs were first transplanted to non treated animals and exposure to CCl4 was done 72hrs later, to allow for maximal stimulation by the regenerating milieu.  HESCs development and engraftment were evaluated by immunohistochemistry and reverse transcriptase-PCR analysis (RT-PCR) for fetal and adult human liver-specific markers. Teratomas formed in non treated animals, in the hind limb were studied as a control for site and regeneration milieu.

Results: Three weeks post transplantation hEBs developed into early teratomas in both spleen and liver. The spleen showed a large single teratoma while more than one, smaller teratomas developed in the liver. In addition, AFP and HEP-PAR1 immunostained cells were seen only within the spleen teratoma, and appeared as clusters of cells.  The fetal and adult liver specific markers by RT-PCR were predominantly expressed in the spleen than in the liver of hEBs day one or four. Adult hepatocytes specific markers were expressed only in the hind limb teratoma. When hEBs day 8 were transplanted before CCl4 exposure, we did not receive teratomas but abundance of uniform cells not resembling hepatocytes. These cells were seen only in regenerating animals. Identification of these cells suggests human ectodermal origin.

Conclusions: The maturation level into hepatocytes within a teratoma correlated best with time spent in-vivo than with growth stimulation or hepatic tissue microenvironment. We have confirmed that the differentiation of early hEBs leads to teratoma formation that differ in size and molecular markers, while late hEBs do not form teratomas under CCl4 exposure.