Introduction: Repopulation of decellularised whole organ scaffolds with appropriate cells of autologous origin offers a theoretically attractive solution towards the problem of organ shortages due to end-stage liver disease, potentially allowing reliable and timely organ sourcing without the need for immunosuppression.
In the rat model, following hepatectomy or chemical injury, a significant proportion of Liver sinusoidal endothelial cells (LSECs) proliferating in the regenerating liver are derived from CD133+ bone marrow progenitors, which proliferate in the bone marrow, migrate into the blood stream, and engraft into the liver, where they differentiate into mature LSECS, and express high levels of Hepatocyte Growth Factor, thus promoting hepatocyte proliferation.
We hypothesised that the CD133+ population may be an effective cell population to repopulate the sinusoids of decellularised rat liver scaffolds.
The aim of our study was to isolate CD133+ cells from rat bone marrow 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 sorting with FITC labelled rat specific CD133, and compare efficacy and practicality of these techniques.
Methods: Bone marrow cells were obtained from Lewis’s rat femora and tibiae. Red cell lysis was performed, and remaining cells were washed and labelled with FITC conjugated rat specific anti CD133 antibody. These were then further processed for either (1) direct magnetic bead separation with anti-human CD133 magnetic beads, (2) indirect magnetic bead separation with anti FITC magnetic beads or (3) Fluorescence-activated cell sorting. We compared the efficacy of all three techniques including financial implications.
Results: Direct magnetic bead separation with anti-human CD133 magnetic beads produced poor enrichment of rat CD133+ cells. This is likely due to poor affinity of human magnetic beads towards rat CD133 molecule resulting in loss of cells. In contrast, indirect magnetic bead separation with rat specific FITC labelled CD133 antibody and anti FITC magnetic beads, or Fluorescence-activated cell sorting both resulted in enhanced rat CD133+ cell enrichment.
Conclusions: Although similar in amino acid sequence (75% homology), the differences between human and rat CD133 molecules are 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.