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.

Keywords: HUVECs; bone marrow; cryopreservation; ddPCR; endothelial progenitor cells; flow cytometry; neuro-transdifferentiation; peripheral blood; qPCR; rabbit.

Figure A1

Illustrative flow-cytometric histogram plots comparing expression of surface markers by rabbit and human cells (passage 3). Red peak: Fluorescence minus one (FMO) unstained control, Blue peak: antibody staining of specific marker.

Figure A2

Illustrative flow-cytometric histograms comparing expression of intracellular markers by rabbit and human cells (passage 3). Red peak: Fluorescence minus one (FMO) unstained control, Blue peak: antibody staining of specific marker.

Figure 1

Cell morphology and population doubling time of the cultured endothelial progenitor cells. Upper part: Rabbit endothelial progenitor cells derived from peripheral blood (endothelial progenitor cells (EPCs)) as well as those derived from bone marrow (BEPCs) showed a spindle-shaped morphology (passage 3), whereas human umbilical vein endothelial cells (HUVECs, passage 4) had a cobblestone appearance as observed under a Zeiss Axio Observer.Z1/7 microscope (magnification at 100×; scale bar = 100 μm). Lower part: Both rabbit (EPCs and BEPCs) as well as HUVEC cell cultures were able to double their concentration after 20–30 h of culture independently of the passage number. The data are expressed as the mean ± standard deviation (SD); *—difference is statistically significant at p < 0.05.

Figure 2

Relative expression of selected markers compared among the different passages of rabbit EPCs. P0—initial culture, P1—first passage, P2—second passage, P3—third passage. The data are expressed as the mean ± SD; *—difference is statistically significant at p < 0.05; **—difference is statistically significant at p < 0.01.

Figure 3

Relative expression of selected markers compared among the different passages of rabbit BEPCs. P0—initial culture, P1—first passage, P2—second passage, P3—third passage. The data are expressed as the mean ± SD; **—difference is statistically significant at p < 0.01; ***—difference is statistically significant at p < 0.001.

Figure 4

Comparison of the rabbit EPCs phenotyping using two different biological methods: droplet digital PCR and flow cytometry. ddPCR—droplet digital PCR, FC—flow cytometry. The data are expressed as the mean ± SD.

Figure 5

Comparison of the rabbit BEPCs phenotyping using two different biological methods: droplet digital PCR and flow cytometry. ddPCR—droplet digital PCR, FC—flow cytometry. The data are expressed as the mean ± SD.

Figure 6

Neurogenic induction of rabbit and human endothelial progenitor cells. After induction, the neuron-like cells can be observed in the culture of all cell lines with the typical formation of axon- and dendrite-like cellular structures (Line 2; Zeiss Axio Observer.Z1/7 microscope; magnification at 100× for morphology and at 200× for confocal microscopy; scale bar = 100 μm). Moreover, all induced cell lines expressed the neuronal markers (ENO2 and MAP2) as confirmed by laser scanning confocal microscope Zeiss LSM 700 (Line 3 and 4; magnification at 200×; scale bar = 100 μm) or qPCR (Line 1). Blue—cell nuclei stained with DAPI, green—antibody staining; Con—control (uninduced) cells; Neuro—cells after neurogenic induction. The data are expressed as the mean ± SD; *—difference is statistically significant at p < 0.05; ***—difference is statistically significant at p < 0.001.

Figure 7

Viability and phenotype of the cryopreserved and re-cultured rabbit EPCs and BEPCs. P3—third passage of cells before freezing, P3 thawed—cryopreserved and thawed cells, P4—subsequent passage of the thawed and re-cultured cells. The data are expressed as the mean ± SD; *—difference is statistically significant at p < 0.05.