Phenotypical Characterization and Neurogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchymal Stem Cells

Abstract

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.

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).…”

“…Then, the cells were resuspended in α-MEM culture medium (Gibco TM , Thermo Fisher Scientific, Waltham, MA, USA) supplemented with fetal bovine serum (Sigma Aldrich, Gillingham, UK) and penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). Cell suspensions were plated into culture flasks and cultured until the 3rd passage, as described previously [5] in order to obtain a pure cell culture of rabbit adipose tissue-derived mesenchymal stem cells (rAT-MSCs).…”

“…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].…”

“…This specific cell type can be presently obtained from various human as well as rabbit biological sources, most often from bone marrow, amniotic fluid, or others [1][2][3][4]. However, recently, adipose tissue became preferred for the isolation of MSCs as a more feasible biological source from human or rabbit [1,5]. Adipose tissue-derived MSCs (AT-MSCs) are very interesting for the cellular therapy, as they show an immuno-regulatory ability via cytokine secretion or the expression of cytokine receptors [6].…”