Generation of Robust Numbers of Functional Human Hepatocytes by Human Hematopoietic Stem Cells in the Non-Injury Fetal Sheep Model.

We have previously shown that human hematopoietic stem cell preparations (HSCs) are able to engraft and transdifferentiate into human hepatocyte in the human/sheep xenograft model (Blood. 2004 Jul 1 Epub). In the present study 1x10⁶ human CD34⁺ cells were transduced with GFP expressing RD114 pseudotyped spleen fociforming retroviral vector in vitro and injected into pre-immune fetal sheep. Two months post-transplantation, immunohistochemistry confirmed production of human hepatocytes that were double positive for GFP and Hepar1. These data indicate that transduced human CD34⁺ cells are able to transdifferentiate into human hepatocyte and still be able to express GFP. In order to retrieve human hepatocytes from the liver of chimeric sheep, we used c-met antibody to sort out human hepatocytes. Flowcytometric analysis demonstrated 8% c-met+ cells in the liver of chimeric sheep. Preliminary data using PCR analysis from c-met+ cells for human beta-2 microglobulin demonstrated presence of human cells in c-met+ population. Livers from chimeric sheep were treated with collagenase to obtain single cell suspension and 3x10⁵ cells/ml were placed in T75 flask covered with 0.2% gelatin and cultured in optimized media designed to support the proliferation and maintenance of human and sheep hepatocytes. In this system, using a weekly passage protocol, we were able to maintain/expand human (and sheep) hepatocytes for up to 3 months. The cells are able to produce human albumin in culture and are CK19 positive, a-fetoprotein negative, and cytochrome P450 negative. These results demonstrate that in this non-injury large animal model human HSCs generate robust numbers of functional human hepatocytes apparently via a “transdifferentiation” mechanism.