Bone marrow transplantation (BM-Tx) of CD117+ cells in Abcb4 knockout mice

Introduction: We demonstrated positive effects of bone marrow transplantation (BM-Tx) on the long term which were attributed to enhanced fibrolysis in consequence of graft-versus-host induced Th2→Th1 shift (1). In the follow up study we intended to avoid graft versus host effects and focused on reconstitution of Abcb4-transporter function in recipient's hepatocytes by fusion with Abcb4+ transplanted hematopoietic stem cells (2). With the present study we analyzed regenerative potential of transplantation of desialylated and sorted CD117+ hematopoietic stem cells in Abcb4-/- mice, a model of cholangitis.

Methods: Abcb4-/- recipient mice at the age of six weeks were lethally irradiated and transplanted with desialylated and MACS-sorted CD117+ bone marrow stem cells from green fluorescent protein (GFP) transgenic-donor mice via tail vein injection. Livers and serum of transplanted mice and untreated littermates were harvested at the age of 8 and 20 weeks, i.e. 2 and 20 weeks after BM-Tx. We performed quantitative hydroxylproline assay and sirius-red staining to analyze collagen accumulation and measured serum transaminases to assess liver integrity. Immunostaining was implemented to identify GFP positive donor derived cells in recipient's liver and to identify other cells by specific markers. RT-PCR and Western blotting was carried out to measure transcriptional and protein expression. Pathological grading was performed with H&E-stained liver slices.

Results: Serum aminotransferases, hepatic collagen accumulation, and portal infiltration of inflammatory cells were significantly enhanced two weeks after BM-Tx but not significantly altered on the long term. GFP-specific-immunohistochemistry demonstrated very rare events of stem cell/hepatocyte fusion but abundant infiltration of GFP+ bone marrow derived cells around periportal fields. Co-immunostaining of GFP with CD3 and CD11c indicated the substantial appearance of T-cells and dendritic cells from donor derived bone marrow while these cells barely infiltrated control livers. Significant upregulation of proinflammatory (Th1) and profibrogenic (Th2) cytokines revealed enhanced stimuli for disease even on the long term.

Conclusion: In contrast to previous studies where Abcb4-/–mice were transplanted with unsorted BM, in the present study reduced temporary graft versus host disease and unaltered liver integrity and fibrosis on the long term appeared. Fusion of transplanted cells to host hepatocytes was a rare event while lots of GFP+ cells including GFP+-dendritic cells and T-cells infiltrated around portal fields. Taken together these results suggest that BM-Tx is not suitable for the reconstitution of hepatocyte's function in the case of Abcb4-/-. Nevertheless, further studies will provide functional insight in the pathological role of donor BM-derived dendritic- and T-cells.

1. Roderfeld M et al. Gut 2012 Jun;61(6):907–916.

2. Wang X et al. Nature 2003 Apr 24;422(6934):897–901.