Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real–time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14⁻CD29⁺CD31⁻CD34⁻CD44⁺CD45⁻CD49f⁺CD73⁺CD90⁺CD105⁺CD133⁻CD146⁻CD166⁺VE-cadherin⁺VEGFR-2⁺SSEA-4⁺MSCA-1⁻vWF⁺eNOS⁺AcLDL⁺ALDH⁺vimentin⁺desmin⁺α-SMA⁺, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-β 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-β 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-β 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-β) and rabbit EPCs (TGF-β 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor 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.