Isolation of Mesenchymal Stem Cells from Human Bone Marrow Using Aldehyde Dehydrogenase as a Marker.

Mesenchymal stem cells (MSCs) represent multipotent stem cells that are capable of self-renewal while maintaining their multipotence and can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells. However, the origin and properties of human MSCs remain to be elucidated. Aldehyde dehydrogenase (ALDH) is known to be useful for isolation of hematopoietic stem cells as well as neural stem cells and endothelial progenitors. In this study, we attempted to isolate MSCs from human bone marrow (BM) using ALDH as a marker for MSCs. Mononuclear cells (MNCs) in BM were first sorted to ALDH^negative, ALDH^low, and ALDH^high cell fractions. ALDH^negative and ALDH^low cells had no colony forming units-fibroblast (CFU-F). ALDH^high cells represented 1.8±0.8% of MNCs in human BM (n=4) and contained CD45 negative cells (9.9±5.3%). When ALDH^high CD45⁺ cells and ALDH^highCD45⁻ cells were cultured with MF start® (TOYOBO, Osaka, Japan), CFU-F was found in the latter cells but not in the former cells. The frequency of CFU-F in ALDH^highCD45⁻ cells and MNCs in BM was 1 in 330 and 13,600, respectively. We found that CFU-F differentiated to adipocytes and osteocytes as examined using the human mesenchymal stem cell functional kit (R&D, Minneapolis) and immunological staining with FABP-4- and osteocalsin-specific antibodies. We also tried to isolate MSCs in human cord blood (CB) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (PB). Proportion of ALDH^high cells was 2.1±0.8% in CB (n=3) and 0.6±0.2% in G-CSF-mobilized PB (n=4). CB ALDH^high cells contained CD45⁻ cells (14.9±2.8%), but G-CSF-mobilized PB ALDH^high cells contained only few CD45⁻ cells (1.3±0.8%). CFU-F was seen in CB, while no CFU-F was identified in G-CSF-mobilized PB. The current data demonstrate that ALDH^highCD45⁻ cells can differentiate into adipocytes and osteocytes and that ALDH is a useful marker for isolation of human MSCs.