Adult BM is composed of hematopoietic stem cells (HSCs) and tissue stem cells, which are often referred to as fibroblast CFUs (CFU-Fs), marrow stromal cells/mesenchymal stem cells (MSCs), or mesenchymal progenitor cells (MPCs; Friedenstein et al., 1974
; Prockop, 1997
; Conget and Minguell, 1999
; Pittenger et al., 1999
). As information is gathered about MSCs, parallels are often drawn between them and the extensively characterized HSCs. HSCs were initially identified by Till and McCulloch (1961)
, who called them spleen CFUs (CFU-Ss), and MSCs were first described by Friedenstein et al. (1974)
, who called them CFU-Fs. There has since been a major divergence in the way the two stem cell types are studied.
HSCs can be identified prospectively by surface markers, isolated by flow cytometry, and transplanted in vivo without being cultured in vitro (Smith et al., 1991
; Spangrude et al., 1995
; Osawa et al., 1996
; Matsuzaki et al., 2004
). In contrast, MSCs, which can give rise to multiple mesenchymal cell lineages, including adipocytes, chondrocytes, and osteocytes (Prockop, 1997
; Pittenger et al., 1999
), are currently isolated by culturing tissues from humans and other species (da Silva Meirelles et al., 2006
; Beltrami et al., 2007
). Therefore, most information about MSCs comes from in vitro studies (Pittenger et al., 1999
) of heterogeneous populations of adherent cells that contain unidentified, putative stem cells. This is a critical difference because it is the ability to isolate HSCs prospectively that has facilitated the rapid progress in understanding their biology. In contrast, because our knowledge of MSCs is based solely on the characterization of cultured cells, it has been virtually impossible to study many of their properties, particularly their function, in vivo.
Recent studies consistently show that MSCs not only differentiate into mesenchymal lineage cells but also into neurons (Kohyama et al., 2001
; Kondo et al., 2005
), skeletal muscle (Dezawa et al., 2005
), and myocardium (Makino et al., 1999
; Miyahara et al., 2006
). Therefore, MSCs are now considered a potentially effective source for stem cell therapy (Jin et al., 2002
; Hoffmann et al., 2006
). However, safety issues still need to be clarified before their clinical use, particularly because so many biological aspects of MSCs, such as their exact identity and in vivo function, are still unknown.
One disadvantage of the conventional in vitro method for isolating MSCs is the unavoidable contamination by hematopoietic cells and the cellular heterogeneity of the cultures, including various fibroblastic cells. In fact, depending on the study, cultured MSCs express a different subset of various cell lineage–specific antigens, adhesion molecules, integrins, and growth factor receptors (Jiang et al., 2002
; da Silva Meirelles et al., 2006
). Another problem with the current technique is that the cultured cells may acquire different characteristics from their in vivo state, which could include changes in the cell surface markers they express. One example of adherent culture-induced change is seen when MSCs, which are readily expanded in culture without loss of multipotency, show poor tissue tropism when transplanted, including a failure to migrate to the BM (Rombouts and Ploemacher, 2003
; Wang et al., 2005
; Muguruma et al., 2006
; Sackstein et al., 2008
), which limits their therapeutic usefulness. In contrast, some studies (including the current one) show that primary BM-derived MSCs (assayed as CFU-Fs) show a low but efficient seeding of the BM upon injection into lethally irradiated hosts (Rombouts and Ploemacher, 2003
; Koide et al., 2007
). Because these changes affect fundamental properties of the cells, it is difficult to know whether they have retained or lost their original characteristics, including their apparent multipotency, in vitro.
Most studies on MSCs have been done using human cells, not murine cells. Murine MSCs (mMSCs) are far more difficult to isolate from the BM and to expand in culture than human MSCs (Phinney et al., 1999
; Sun et al., 2003
; Peister et al., 2004
). Thus, there is much less information on mMSCs than on human MSCs. This reliance on human material means that MSCs cannot be studied in genetic mouse models, which greatly hinders the study of their basic biology, engraftment, and therapeutic potential. In fact, without some means for the prospective isolation and purification of MSCs, it becomes extremely difficult to ascribe any attribute to them with certainty. Hence, there is a clear need for specific markers and methods of detection, enumeration, and isolation of MSCs from the BM and other tissues where they reside.
In a previous study, we identified several mesenchymal lineage specific markers in murine BM cells (Koide et al., 2007
), and after initial screening, we determined two possible MSC markers (Morikawa et al., 2009
), platelet-derived growth factor receptor α (PDGFRα; an early mesodermal marker; Takakura et al., 1997
) and stem cell antigen-1 (Sca-1; a known stem cell marker; Ortega et al., 1986
Here, we report a method for identifying and isolating MSCs from the adult murine BM, using flow cytometry in combination with in vitro function assays. We also report our findings on their physiological role, obtained through existing in vivo precursor cell transplantation assays. We describe cell type–specific markers for MSCs, which are useful for their prospective isolation as a highly enriched population that gives rise to mesenchymal cells at the clonal level with high frequency, and demonstrate that these cells are capable of in vivo grafting when transplanted systemically.