In vivo formation of unstable heterokaryons following liver damage and HSC/Progenitor transplantation

¹ Hematopoietic Stem Cell Laborator, Cencer Research UK, London Research Institute, London, United Kingdom

^* To whom correspondence should be addressed. E-mail: dominique.bonnet{at}cancer.org.uk.

	Abstract

Following reports of lineage plasticity in human hematopoietic stem cells (HSCs) we investigated the potential of human cord blood HSC-enriched cells to create hepatocytes in hosts after inducing liver damage. Carbon tetrachloride (CCl4) induces severe liver damage and subsequent repair via mitosis of resident hepatocytes. It additionally leads to a three-fold increase in homing of human mononuclear cells to bone marrow and liver, and subsequently to a substantial enhancement of bone marrow engraftment. Eight-weeks after liver damage and infusion of an eGFP lentivirus-transduced human HSC-enriched cell population, we observed eGFP positive cells with clear hepatocyte morphology in the livers of animals. These eGFP positive cells co-expressed human albumin and RT-PCR analysis demonstrated the presence of human albumin and alpha-anti-trypsin mRNA. However, two antibodies against human mitochondria and human nuclei failed to mark eGFP positive hepatocyte-like cells but did give clear staining of donor-derived hematopoietic cells. Subsequent FISH analysis revealed the presence of mouse Y chromosome in eGFP positive hepatocyte-like cells. To resolve this discrepancy we performed single cell PCR analysis of micro-dissected eGFP positive hepatocyte-like cells and found that these cells contained mostly mouse and little human genomic material. FISH analysis highlighting the centromeres of all human chromosomes revealed indeed only few human chromosomes in these cells. From these results we conclude that similarly to the murine system, human hematopoietic cells have the potential to fuse with resident host hepatocytes. As no selective pressure is applied to retain the human genomic material, it is gradually lost over time leading to a variable phenotype of the chimeric cells making their detection difficult.

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