Abstract
Endothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.
Highlights
Regenerative medicine offers several promising solutions in the development of cures of some untreatable diseases ranging from diabetes mellitus and heart infarct to Parkinson’s disease and premature ovarian failure
Immunofluorescence analysis for antigens typically expressed by mesenchymal stem cells (MSCs) and cell morphology confirmed that Porcine ovarian putative stem cells (poPSCs), obtained by immunomagnetic isolation from the porcine ovarian cortex represent a true, but nonhomogeneous, MSC population
The statistically significant (**P < 0.05) higher expression of CD29, CD90, and CD105 was found in poPSC cells compared to control ovarian cortex homogenates (Fig. 1)
Summary
Regenerative medicine offers several promising solutions in the development of cures of some untreatable diseases ranging from diabetes mellitus and heart infarct to Parkinson’s disease and premature ovarian failure. In this aspect, stem cell application, including adult stem cells (ASCs), seems to be one of the most promising therapeutic strategies. ASCs are rare tissue-specific cells of the postnatal organism into which they are committed to differentiate (Grompe 2002; Raff 2003). The role of ASCs lies in maintaining, generating, and replacing terminally differentiated cells within their specific tissue lost due to physiological cell turnover or tissue damage caused by injury (Weissman 2000). Based on the expression of some marker genes, such as Oct-4 or Nanog, and the ability of a population of ASCs to differentiate into various cell types, ASC pluripotency is postulated (Jiang et al 2002; D’Ippolito et al 2004)
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