SRS298 - Isolation of CD133 positive cells from rat bone marrow by immune-magnetic beads and Fluorescence-activated cell sorting

Abstract In the rat model, following liver injury, a significant proportion of liver sinusoidal endothelial cells (LSECs) proliferating in regenerating liver are derived from CD133+ bone marrow (BM) progenitors. These cells proliferate in the BM, migrate into the bloodstream, engraft into the liver, differentiate into mature LSECs, and express high levels of Hepatocyte Growth Factor, thus promoting hepatocyte proliferation. The study aimed to isolate CD133+ cells from rat BM using (1) direct magnetic bead separation with human CD133 antibody conjugated magnetic beads, (2) indirect magnetic bead separation using FITC-labelled rat specific CD133 antibody and anti-FITC conjugated magnetic beads and (3) Fluorescence-activated cell (FACS) sorting with FITC labelled rat specific CD133, and compare efficacy and practicality of these techniques. Rat BM cells were labelled with FITC conjugated rat specific anti CD133 antibody and subjected to each method. Direct magnetic bead separation resulted in poor enrichment of CD133+ cells, likely due to poor affinity of human magnetic beads towards rat CD133 molecule. In contrast, both indirect magnetic bead separation and FACS sorting yielded significantly better enrichment of CD133+ cells. Although similar in amino acid sequence, the differences between human and rat CD133 molecules are may be sufficient to result in poor binding of the human CD133 antibody to the rat homolog molecule. Consequently, whilst effective in enriching human CD133+ cells, magnetic beads conjugated to human CD133 do not produce effective enrichment of rat CD1133+ cells. In contrast, rat specific CD133 antibody-based methods result in effective CD133+ cell enrichment from rat BM.