A comparative study for the isolation of rabbit phenotypically defined candidate HSPCs based on CD45 or CD34 marker expression

Rabbit hematopoietic stem/progenitor cells (rHSPCs) residing in bone marrow are still poorly characterized. Usually, human and animal HSPCs can be obtained by depletion of mature lineage-committed hematopoietic cells or via positive selection of primitive cells expressing CD34 antigen. Previously, we reported possible enrichment of potential rHSPCs by depletion of rabbit CD45 + cells. This study describes an improved depletion protocol with more satisfying cell purity. Additionally, enrichment of phenotypically defined candidate rHSPCs using new anti-rabbit CD34 monoclonal antibodies and MACS or FACS sorting is presented here to thoroughly compare these three methods according to the quality of isolated rHSPCs. Moreover, human bone marrow CD34 + cells were used to compare their specific features and abilities with enriched rabbit cell populations. We achieved high sorting efficiency and purity after rabbit CD45 depletion ( p < 0.001), as well as for MACS and FACS enrichment of CD34 + cells ( p < 0.0001). Satisfying viability was observed for rabbit CD45 - and FACS CD34 + cells (> 90%, p < 0.05), but not for MACS CD34 + cells (< 50%; p < 0.0001). Rabbit CD45 - ( p < 0.05) and human MACS CD34 + cells ( p < 0.01) expanded well, while rabbit FACS and MACS CD34 + cells did not. All three differently enriched rabbit cell populations exhibited phenotype like human CD34 + cells with slight differences. Almost all enriched rabbit cell populations (except for MACS sorted) showed high clonogenic capacity comparable to human CD34 + cells and produced BFU-E, CFU-E, CFU-GM and CFU-GEMM colonies. In addition, TEM analysis revealed same morphology and ultrastructure for all enriched rabbit cell populations as for human CD34 + cells. Here we isolated phenotypically defined candidate rHSPCs subpopulations with slightly different purity, viability, phenotype, expansion rate and clonogenic capacity. On the other hand, enriched rabbit cell populations showed very similar morphology and ultrastructure to human HSPCs. This study allowed us to isolate, cryopreserve and store rabbit phenotypically defined candidate HSPCs from native Holic Blue and Slovak Grey-Blue Rex rabbits in animal gene bank for future generations. Regarding CD34 enrichment, mAb 58/47/34 seems to be more suitable for the sorting procedures than other tested antibodies. However, further studies are required to improve the expansion method specific for rabbit HSPCs or to analyse transcriptome or secretome of these cells. Moreover, the major limitation of this study is the lack of in vivo repopulation experiments, which might prove the real HSPCs origin of the enriched rabbit cell populations.