Reevaluation of Bone Marrow-Derived Cells as a Source for Hepatocyte Regeneration

Abstract

We have investigated the contribution of intrasplenic bone marrow transplants or in vivo mobilized hematopoietic stem cells to the formation of hepatocytes in normal and injured liver. Direct intrasplenic injections of bone marrow mononuclear cells (5 × 10 cells), Sca1+/lin- hematopoietic stem cells (5 × 103) cells, and highly purified “side population” hematopoietic stem cells (5 × 103) derived from enhanced green fluorescent protein (EGFP)-transgenic mice [C57Bl/6-TgN(ActbEGFP)1Osb] were performed in normal C57Bl/6 mice (n = 6) and in C57Bl/6 mice following two thirds hepatectomy (n = 8). To test the effect of mobilized stem cells on transdifferentiation, C57Bl/6 mice (n = 12) were lethally irradiated and reconstituted with EGFP-positive bone marrow mononuclear cells in a second series of experiments. Eight to 12 weeks after bone marrow transplantation a subset of mice (n = 3 in each group) received either rhG-CSF for hematopoietic stem cell mobilization, rhG-CSF combined with an intraperitoneal application of carbon tetrachloride (CCl4) as hepatocyte regeneration stimulus, or CCl4 alone. All mice were completely perfused with PBS to remove circulating nonorgan cells for analyses 4 weeks later. Liver as well as heart, intestine, spleen, and kidney tissue was analyzed for the presence of EGFP-transgenic cells. In 100 sections (2.3 × 107 cells) of any recipient mouse no EGFP-positive hepatocytes were detected either by analysis of native EGFP fluorescence or by immunofluorescence analysis with anti-EGFP and antidipeptidyl peptidase (DPP) IV antibodies. EGFP-transgenic cells resembling heart, kidney, or intestinal cells could also not be proven. The results demonstrate that there is little or no contribution of bone marrow-derived cells to the regeneration of normal and injured liver in the animal models used. Thus, potential therapeutic prospects of hematopoietic stem cell therapy for liver disease have to be critically reassessed.