HSCs isolated by the set of markers which

HSCs isolated by the set of markers which are Lin−/Sca-1+/C-kit+ (LSK) have been also reported to be heterogeneous (Osawa et al., 1996). Moreover, Sca-1 expression differs between mouse strains and therefore is not very effective to enrich HSCs (Spangrude and Brooks, 1993). Likewise, the SLAM family member CD150 is not useful for emerging and developing HSCs in embryos (McKinney-Freeman et al., 2009). Other markers such as CD11b/Mac1 is often expressed in the early HSPCs (Matsubara et al., 2005; Mikkola and Orkin, 2006). The inconsistencies of surface markers on BMSCs and HSPCs led us to identify alternative markers with greater specificity to sort the BMSC and HSPC populations from bone marrow. Maintenance of stemness of adult stem cells in their niches is governed by unique combinations of epigenetic regulators (Oh and Humphries, 2012). Epigenetic mechanisms, including Histone acetylation/deacetylation, play a crucial role in transcriptional regulation via remodelling of chromatin architecture (Huang et al., 2015; Oh and Humphries, 2012). Histone deacetylases (HDACs), classified into four classes, (Gregoretti et al., 2004; Telles and Seto, 2012; Yang and Seto, 2003) catalyzes a wide spectrum of physiological process including proliferation, differentiation, apoptosis and INNO-406 regulation (Choudhary et al., 2009). Mammalian cell cycle progression through four distinct phases, G1/S/G2/M are mediated via cyclins and cyclin dependent kinases (CDKs) (Nurse, 1994). These CDKs are successively regulated by Cdk inhibitors (CKIs) (Sherr and Roberts, 1999). HDACs deacetylate and regulate the activity of key cell cycle proteins (Glozak et al., 2005). In the past, studies have been performed to assess the role of HDACs in modulating the differentiation ability of stem cells, including Embryonic Stem Cells (ESCs) and adult stem cells (Qiao et al., 2015). However, studies are lacking in identification of HDACs regulation of adult stem cell proliferation. Often in the event of an injury, adult stem cells migrate from their niche to regenerate the damaged tissues, which is directly dependent on its proliferative capacity. Thus, understanding the molecular mechanisms involved during HDAC-mediated cell cycle gene regulation may provide a better insight into the proliferative potential of these adult stem/progenitor cells. In the present study, we have isolated and characterized both BMSCs and HSPCs from mouse bone marrow based on the presence and absence of CD11b, Ter119 and CD133 markers. The two resultant sorted cell populations, CD11b−/Ter119− and CD11b+/CD133+, were successfully differentiated into skeletal and hematopoietic lineages, respectively. CDK and CKI gene expression correlated well with the cell cycle analysis depicting a relatively higher percentage of BMSCs in G2/M phase, and HSPCs in the G0 phase of the cell cycle. The expression profile of HDACs in these populations suggests the plausible mechanism of differential proliferation. Finally, HDAC inhibition led to an increase in acetylated Histones and Cyclins B1/D2 levels in these cell populations. This study suggests the translational role of epigenetic modifiers in regulating the proliferative potential of these adult stem/progenitor cells.
Materials and methods
Results
Discussion BMSCs and HSPCs are the most intensely studied adult stem cell types because of their great potential in the field of regenerative medicine. Isolation of murine bone marrow derived BMSCs has been performed by immuno-depletion of CD11b cells (Kopen et al., 1999). But, use of a single marker for isolation of BMSCs often leads to contamination with other cells of hematopoietic origin which lack CD11b. Moreover, reports of isolation of murine BMSCs using combinatorial markers have revealed these to be Ter119 negative (Morikawa et al., 2009). Therefore, in our experimental approach we combined double negative selection of Ter119, a marker of erythroid cells ranging from early pro-erythroblasts to mature erythrocytes, along with CD11b for enrichment of BMSCs. The CD11b−/Ter119− cell population thus obtained appeared morphologically similar to BMSCs in previously published literature (Kopen et al., 1999). CD11b+ cell fractions were earlier reported to contain short term HSPCs. We have therefore, further enriched HSPCs using CD133 (prominin-1), a stem cell marker known to be expressed in primitive hematopoietic progenitor cells (Hess et al., 2006). Studies by Hess et al., demonstrated that CD133+/ALDHhigh/lin− HSC population significantly engrafted in NOD/SCID mice as compared to the CD133− population. Our sorted CD11b+/CD133+ HSPC populations depicted morphological similarity with the aforementioned cell populations (Hess et al., 2006). The expression profile of mRNA and protein, examined in our sorted cell populations to gauge their biological activity, matched with expression profile of markers observed by Terskikh et al., in purified HSPCs and committed progenitors (Terskikh et al., 2003). The pluripotent marker expression on BMSCs has been studied in detail only in human BMSCs. Riekstina et al., have shown a differential expression pattern of embryonic stem cell markers including Oct-4, Nanog and SOX-2 in stromal cells derived from various sources such as bone marrow, adipose tissue, dermis and heart (Riekstina et al., 2009). Similarly, Palma et al. have also shown that enforced expression of Oct-4 in human BMSCs increases the expression of other pluripotent genes including KLF-4, SOX-2, FOXD3, NANOG and c-MYC (Palma et al., 2013). Our studies revealed a similar differential profile of pluripotency gene expression in murine bone marrow-derived BMSCs and HSPCs; although the levels are generally much lower than in ES cells.