Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45⁺ Bone Marrow Cells

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45(+)) cells. The cells were depleted with overall purity about 60-70% and then cultured in a special medium designed for the expansion of CD34(+) cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p < 0.05) and CD45 decreased (p < 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45(+) cells, as identified by flow cytometry. After gating on CD45(-) cells the MHCI(+)MHCII(-)CD38(+)CD49f(+)CD90(-)CD117(-) phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required.