The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)-derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271(+) population. The recognized CD271(+) populations were fractionated by fluorescence-activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU-F. The results showed that only the CD271(bright) but not the CD271(dim) population contained CFU-F. Two-color flow cytometry analysis revealed that only the CD271(bright) population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271(bright) population but no other BM cells. The new MSC-specific molecules included the platelet-derived growth factor receptor-beta (CD140b), HER-2/erbB2 (CD340), frizzled-9 (CD349), the recently described W8B2 antigen, as well as cell-surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM-MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

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Very recently, we identified two distinct mesenchymal stem cell (MSC) subsets in primary bone marrow (BM) that differ in their expression pattern (CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-)) and morphology as well as in their clonogenic and differentiation capacity. Here we analyzed the cell surface antigen expression in these subsets in more detail and compared the profiles with the expression pattern on cultured MSCs. Most of the tested antigens, including CD13, CD15, CD73, CD140b, CD144, CD146, and CD164, are expressed at similar levels in both primary BM populations. However, a number of markers were differentially expressed. Of these, CD166 (ALCAM), CD200, and CD106 (VCAM-1) showed an almost selective expression on either CD271(bright)MSCA-1(dim)CD56(+) (increased CD166 and CD200 expression) or CD271(bright)MSCA-1(bright)CD56(-) (increased CD106 expression) MSCs, respectively. Additional markers with elevated expression on CD56(+) MSCs include F9-3C2F1, HEK-3D3, HEK5-1B3, and W1C3 antigens, whereas CD10, CD26, CD106, 7C5G1, 9A3G2, 56A1C2, 66E2D11, HEK-3D6, HEK4-1A1, HEK4-2D6, W1D6, W4A5, W7C6, and W8B2 (MSCA-1) antigens showed increased expression in the CD56(-) population. The majority of the analyzed markers found on primary MSCs were also expressed on cultured MSCs. However, in contrast to primary MSCs, HEK7-1C4, W1C3, W1D6, and W4A5 antigens were absent on the cultured counterparts. 7G5G1 and 9A3G2 antigens showed reduced, and HEK-3D6, F9-3C2, and HEK-3D3 showed increased expression on cultured cells. The extended knowledge about the phenotype of the two subsets and the identification of novel MSC markers may result in the isolation of attractive starting populations for applications in regenerative medicine.