Identification of New Rat Bone Marrow‐Derived Population of Very Small Stem Cell with Oct‐4A and Nanog Expression by Flow Cytometric Platforms

Abstract

Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells

“…VSELs represent a unique and rare population of adult stem cells sharing several structural, genetic, biochemical, and functional properties with ESCs [ 2 ]. They have also been identified in several adult murine and human tissues, including ovaries and testicles [ 4 ]. VSELs offer several major advantages over currently available seed cells.…”

“…Finally, since VSELs can be isolated from adult tissues, the use of autologous VSELs not only avoids rejection and other potential immune risks but also avoids ethical issues [ 10 ]. Although VSELs have been isolated from mouse and human tissue for more than a decade, VSELs were not isolated from rat bone marrow until 2016 [ 4 ]. Compared with mouse and humans, isolating VSELs from rats is easier and less ethically controversial.…”

“…Compared with mouse and humans, isolating VSELs from rats is easier and less ethically controversial. In our work, VSELs with high expression of Sox2, Oct-4, and Nanog were successfully isolated from rat BM according to the experimental method of Labedz-Maslowska et al [ 4 ]. VSELs accounted for 0.01-0.02% of the total number of nucleated cells, which was consistent with previous reports [ 1 , 4 , 18 ].…”

“…To obtain a high-purity rat BM-derived population of VSELs, this study adopted the sorting scheme for VSELs proposed by Labedz-Maslowska et al [ 4 ]. All procedures involving the care and use of animals conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the laboratory animal management and experimental animal ethics committee of Yangzhou University.…”

“…By covalently binding to DNA methyltransferase, 5-AzaC reduces DNA methyltransferase activity, thereby reducing the methylation level and inducing cell proliferation and differentiation. Several studies have shown that 5-AzaC could induce differentiation of different types of stem cells into cardiomyocytes [ 4 , 8 , 9 ]. Furthermore, these differentiated cells exhibit spontaneous pulsation and measurable action potentials, consistent with cardiomyocytes [ 4 , 8 , 9 ].…”