br Results br Discussion Despite the widespread

Results
Discussion Despite the widespread clinical use of hMSCs, there are fundamental gaps in our knowledge of basic MSC biology. In contrast to HSCs, the molecular mechanisms that maintain MSCs in their undifferentiated state or determine their differentiation pathways are still poorly understood. Due to the limitations of the methods used to isolate MSCs, it has not been possible to obtain a sufficiently pure MSC population to address these questions. In the current study, we have demonstrated that LNGFR+THY-1+ purinergic receptors in human BM constitute an extremely pure MSC population. The clonogenic potential of single-seeded LNGFR+THY-1+ MSCs, cultured in standard medium (Dulbecco’s modified Eagle’s medium [DMEM] + 20% fetal bovine serum [FBS]) exceeds that of other reported isolation methods (STRO-1+VCAM-1+ [Gronthos et al., 2003], MSCA-1+CD56− [Battula et al., 2009], LNGFR+CD140b+ [Bühring et al., 2007], and LNGFR+CD146+ [Tormin et al., 2011]). Murine HSCs can be isolated based on CD34−, c-Kit+, Sca-1+, and Lineage− expression, which yields one HSC per five cells (Matsuzaki et al., 2004). Our isolation method for hMSCs achieves a level of purity similar to that of mouse HSCs. Moreover, because LNGFR+THY-1+ cells contain the least number of hematopoietic cells, this combination enables the convenient isolation of MSCs based on the surface expression of only two antigens. This simple and easy isolation method should facilitate further studies and improve our basic understanding of the primary characteristics of MSCs. Using highly enriched primary MSCs, one can perform clonal experiments easily and efficiently. Several groups have reported that MSCs are heterogeneous with respect to their morphological appearance (Kucia et al., 2007a, 2007b; Pittenger et al., 1999; Prockop et al., 2001). Our findings confirm that CFU-Fs derived from a single LNGFR+THY-1+ cell can be divided into three functionally distinct subpopulations termed RECs, MECs, and SECs. RECs exhibited robust multilineage differentiation and self-renewal potency. We therefore consider RECs to be the most primitive and undifferentiated population in CFU-Fs. In a previous report, upregulation of p16Ink4a and p19Arf by loss of the transcriptional repressor Bmi-1 led to defective self-renewal of adult HSCs and neural stem cells, but was less critical for generating the differentiated progeny of these cell types (Park et al., 2003; Molofsky et al., 2003). In our results, MECs and SECs had increased expression of the two commonly known biomarkers for cellular senescence, SA-β-gal and P16INK4a. In contrast, P14ARF, which is generated by alternative splicing from the INK4a locus and P21, was not affected. We therefore speculate that the self-renewal capacity of cultured MSCs is mediated by an unknown mechanism rather than by BMI-1 regulation. VCAM-1 encodes a leukocyte adhesion molecule whose expression is restricted to endothelial cells and subpopulations of BM cells (Osborn et al., 1989). VCAM-1 binds to the integrin α4β1 and integrin α4β7 on circulating monocytes, granulocytes, and lymphocytes (Elices et al., 1990; Osborn et al., 1989). Increased expression of VCAM-1 has been reported in various cancer cell types, including breast, gastric, renal carcinoma, and melanomas (Ding et al., 2003; Minn et al., 2005). It has been suggested that the VCAM-1-CD49d interaction promotes cancer cell survival and migration (Klemke et al., 2007). In this study, we demonstrated that RECs have higher expression of VCAM-1 compared with MECs or SECs, and blockade of the VCAM-1-CD49d interaction significantly diminished transwell migration and increased stress-fiber formation. The functional difference between the groups relates to the organization of the F-actin cytoskeleton, since the rigid cytoskeletal structure inhibits their ability to transmigrate across the vascular endothelium (Morales-Ruiz et al., 2000). Therefore, we speculate that RECs interact with each other through VCAM-1-CD49d, thereby maintaining their primitive state, including self-renewal, multilineage potential, and migratory capacity.