Liver Repopulation and Gene Therapy Using Fetal Liver Stem/Progenitor Cells and Genetically Re-engineered Adult Hepatocytes

Dr. Shafritz’ research has focused on regulation of liver gene expression, cell growth control, liver regeneration and liver reconstitution through cell transplantation.  A number of years ago, he discovered that bipotent stem cells from the rat fetal liver expand several thousand fold after their transplantation into a normal adult liver, differentiate into both hepatocytes and bile ducts, replace ~25% of hepatic mass, become structurally and functionally integrated into the liver tissue and form completely new liver lobules.  Liver replacement occurs by cell competition, during which the faster growing fetal liver cells repopulate the liver by inducing apoptosis in much slower growing host hepatocytes, a mechanism originally described in Drosophila during wing development. 

Dr. Shafritz and his research staff recently reproduced the rat liver repopulation model using normal adult hepatocytes that have been transduced with a lentivirus containing a human Yap gene linked to the estrogen receptor (lenti-Yap ERT2).  Expression of Yap, the effector gene of the Hippo kinase signaling pathway, induces proliferation of cells and controls organ size.  This leads to liver hyperplasia and hepatic tumors in Yap transgenic mice, but in the Yap ERT2 transduced hepatocyte transplantation system, biological activity of Yap was controlled by linking it to the estrogen receptor which keeps expressed Yap protein in the cytoplasm in a non-functional state.  When tamoxifen is administered, Yap ERT2 protein transfers to the nucleus, where it binds to chromatin, serves as a transcriptional coactivator of TEADs and induces expression of genes involved in cell cycle progression and cell proliferation.  Thus, Yap function is controlled by a novel protein trafficking mechanism that is regulated by tamoxifen administration.

Repopulation of the normal rat liver by lenti Yap ERT2 transduced hepatocyte is ~10% at 6 mo. after cell transplantation and more than 20% after one year.  This has significant medical implications, because this level of repopulation is more than twice that necessary to treat a number of genetic-based metabolic disorders of the liver that do not have ongoing or underlying liver injury, but nonetheless lead to serious clinical diseases.  Among these diseases are Crigler-Najjar Syndrome, Type 1 which causes hyperbilirubinemia, brain damage and mental retardation, ornithine transcarbamylase deficiency which causes ammonia toxicity, coma and death, LDL-receptor deficiency which causes heart attacks and strokes in children and adolescents, phenylketonuria which causes brain damage and mental retardation, and Factor IX deficiency which causes severe hemophilia.  These diseases cannot be cured by transplanting unmodified adult hepatocytes because the transplanted cells have no proliferative advantage over host hepatocytes and will not expand and effectively repopulate the liver after their transplantation.  Based on the above studies, Dr. Shafritz and colleagues have obtained new NIH funding to pursue this research. 

Ongoing projects include:

1)      Determining the mechanism by which Yap transduced hepatocytes repopulate the liver.  Whether they behave as stem cells, whether they are serially transplantable and whether they can be maintained in culture and subsequently used for repopulation, which cannot be achieved with non-transduced adult hepatocytes. 

2)      Developing an in vitro cell competition assay to identify the factor(s) produced by fetal liver stem/progenitor cells that induce apoptosis in normal adult hepatocytes (an important component in cell competition) and the genes expressed in fetal liver cells or in Yap transduced hepatocytes that render these cells resistant to apoptosis (further enhancing their repopulation potential).

3)      Conducting laser capture microdissection studies in conjunction with RNA seq to identify specific genes that control the proliferative potential of fetal liver stem cells and lenti-Yap ERT2 transduced hepatocytes vs. host hepatocytes in the cell transplanted liver and regulate cell competition during liver repopulation.

4)      Determining whether Yap transduced hepatocytes that have repopulated the liver can be serially passaged, whether these cells revert to a less differentiated or oncogenic phenotype and assess the level of tumor risk incurred by introducing lenti Yap ERT2 into adult hepatocytes and transplanting these cells into a normal or diseased liver. 

5)      Determining whether effective repopulation of the liver by lenti Yap transduced hepatocytes can be achieved in animal models of hepatic fibrosis caused by chronic liver injury.  Recent results reported in collaboration with former colleague, Dr. Michael Oertel, showing very modest repopulation of the fibrotic rat liver by transplanting normal hepatocytes suggest that in this chronic injury/fibrotic model repopulation could be markedly increased using lenti-Yap ERT2 transduced hepatocytes. It may also be possible to couple Yap ERT2 hepatocyte transplantation with anti-fibrotic therapy to cure advanced hepatic fibrosis and restore liver function.  Success in these experiments would have major implications in the potential use of hepatic cell therapy in liver fibrosis/cirrhosis for which the only currently available alternative is whole liver transplantation.

6)      Determining whether human hepatocytes transduced with Yap can repopulate the liver a mouse model system that accepts human xenographs (the Nod/SCID/gc null mouse).

Kollet, O., Shivtiel, S., Chen, Y.-Q., Suriawinata, J., Thung, S.N., Dabeva, M.D., Kahn, J., Spiegel, A. , Dar, A., Samira, S., Goichberg, P., Kalinkovich,A., Arenzana-Seisdedos, F., Nagler, A., Hardan, I., Revel, M., Shafritz, D.A. and Lapidot, T. HGF, SDF-1 and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest. 2003 Jul;112(2):160-9.

Oertel, M., Menthena, A., Dabeva, M.D., and Shafritz, D.A.  Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology. 2006 Feb;130(2):507-20;

Gouon-Evans, V., Boussemart, L., Gadue, P., Nierhoff, D., Koehler, C.I., Kubo, A., Shafritz, D.A., and Keller. G. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol. 2006 Nov;24(11):1402-11.

Shafritz, D.A. A human hepatocyte factory.  Nat Biotechnol. 2007 Aug;25(8):871-2.

Oertel, M., Menthena, A., Chen, Y.Q., Teisner, B., Harken-Jensen, C., and Shafritz, D.A. Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver. Gastroenterology. 2008 Mar;134(3):823-32.

Shafritz, D.A. and Oertel, M.  Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells.  Int J Biochem Cell Biol. 2011 Feb;43(2):198-213.

Menthena, A., Koehler, C.I., Sandhu, J.S., Yovchev, M.I., Hurston, E., Shafritz, D.A. and Oertel, M.  Activin A, p15INK4b signaling, and cell competition promote stem/progenitor cell repopulation of livers in aging rats. Gastroenterology. 2011 Mar;140(3):1009-20.

Yovchev, M.I., Xue, Y., Shafritz, D.A., Locker, J. and Oertel, M. Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes. Hepatology. 2014 Jan;59(1):284-95.

Yovchev M, Jaber FL, Lu Z, Patel S, Locker J, Rogler LE, Murray JW, Sudol M, Dabeva MD, Zhu L, Shafritz DA.  Experimental model for successful liver cell therapy by lenti TTR-YapERT2 transduced hepatocytes with tamoxifen control of Yap subcellular location. Sci Rep. 2016 Jan 14;6:19275.