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

• Main Authors: Jaromír Vašíček, Andrej Baláži, Miroslav Bauer, Andrea Svoradová, Mária Tirpáková, Ľubomír Ondruška, Vladimír Parkányi, Alexander V. Makarevich, Peter Chrenek
• Format: Article
• Language: English
• Published: MDPI AG 2021-01-01
• Series: Magnetochemistry
• Subjects: rabbit; hematopoietic stem cells; CD45; MACS; CD34 expansion; flow cytometry
• Online Access: https://www.mdpi.com/2312-7481/7/1/11

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 (<i>p <</i> 0.05) and CD45 decreased (<i>p <</i> 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.