Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources

Abstract

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.

Rabbit Endothelial Progenitor Cells Derived From Peripheral Blood and Bone Marrow: An Ultrastructural Comparative Study

“…First, mononuclear cells (MCs) as starting cell populations were collected from peripheral blood (PB-MCs) and bone marrow (BM-MCs) of humanely sacrificed rabbits. The isolation of MCs was performed using Biocoll (Biochrom, Berlin, Germany) density-gradient centrifugation as it was described previously (Vašíček et al, 2018(Vašíček et al, , 2020(Vašíček et al, , 2021. Then, the MCs from both biological sources were resuspended in endothelial growth medium (EGM-2) containing endothelial basal medium (EBM-2), recombinant growth factors: bFGF, EGF, R3-IGF-1, and vitamin C (Lonza, Walkersville, MD, USA), 20% fetal calf serum (Biowest, Riverside, MO, USA), and penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA).…”

Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study

“…These features make them both interesting for stem cell therapy, tissue engineering, and regenerative medicine. For such studies, rabbit stem and progenitor cells became also a favorable choice [2,4,5,12].…”

“…Peripheral blood and subcutaneous fat were harvested from humanely sacrificed rabbits as described in our previous studies [5,12]. Briefly, mononuclear cells from peripheral blood were isolated using density-gradient centrifugation and resuspended in endothelial basal (EBM-2) medium supplemented with recombinant growth factors (Lonza, Walkersville, MD, USA); fetal calf serum (Biowest, Riverside, MO, USA), and penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Briefly, mononuclear cells from peripheral blood were isolated using density-gradient centrifugation and resuspended in endothelial basal (EBM-2) medium supplemented with recombinant growth factors (Lonza, Walkersville, MD, USA); fetal calf serum (Biowest, Riverside, MO, USA), and penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). Then, the cells were plated into culture flasks and cultured until the 3rd passage, as described previously [12], in order to obtain pure cell culture of rabbit endothelial progenitor cells (rEPCs).…”

“…Human cell lines were obtained from ATCC (Primary Umbilical Vein Endothelial Cells; Normal, Human (HUVECs) (ATCC ® PCS-100-010 TM ) and Adipose-Derived Mesenchymal Stem Cells; Normal, Human (hAT-MSCs) (ATCC ® PCS-500-011 TM ; ATCC, Manassas, VA, USA). Both frozen cell lines were thawed in a water bath and cultured in a proper culture medium with supplements (EBM-2 for HUVECs and α-MEM for hAT-MSCs) until the 3rd passage, as described previously [5,12].…”