Antibodies to CD49f and PODXL, a sialomucin in the CD34 family, were the most robust for FACScan assays. RNA interference caused aggregation of the cells. Furthermore, PODXLhi/CD49fhi MSCs were less prone to produce lethal pulmonary emboli, and larger numbers were recovered in heart and kidney after intravenous infusion Temanogrel into mice with myocardial infarcts. Introduction Among the cells being used for cell therapies for nonhematopoietic tissues are the stem/progenitor cells from bone marrow that were referred to initially as fibroblast colony-forming units,1 subsequently as marrow stromal cells, then as mesenchymal stem cells2 and, most recently, as multipotent mesenchymal stromal cells or MSCs. 3 Clinical trials with MSCs are now in progress, 4C9 but several questions are still unresolved as to how the cells should be isolated, expanded in culture, and characterized. One view is that confluent cultures of MSCs (Figure 1A) are useful and perhaps the optimal preparations for therapy. An opposing view is that confluent cultures of MSCs are partially committed to differentiation or even senescence. Therefore, they lack some of the therapeutic potentials of low-density cultures that contain a subpopulation of rapidly self-replicating cells10C13 that display a different pattern of expressed genes,14 that have a greater capacity to generate single-cellCderived clones,10,11 and that more efficiently engraft in vivo.15 Open in a separate window Number 1 Microarrays as a preliminary display for useful surface epitopes. (A) Schematic of 2 protocols used to prepare human being MSCs. (B) Phase-contrast photomicrographs of viable MSCs from passage 1/donor 1 plated at 100 cells/cm2 and incubated for 5 or 9 days to generate passage 2 MSCs. (C) Assay by ahead and part scatter of light of MSCs from panel B. Vertical and horizontal lines were generated with microbeads to standardize the assay.10 (D) Microarray assays of mRNAs from viable MSCs from passage 1/donor 6 plated at 100 cells/cm2 and incubated for 5 days to approximately 50% confluency, 10 days to 100% confluency, and 15 days to overconfluency. The ideals were normalized to mRNA signals on day time 15 (remaining) or on day time 5 (right). MSCs were originally explained by Friedenstein et al1 Temanogrel and Owen and Friedenstein,16 who isolated the cells by their ready adherence to MTF1 cells culture surfaces, an isolation technique that consequently was followed by most investigators.17 The cells were characterized primarily by their ability to generate colonies in culture and to differentiate into adipocytes, osteoblasts, and chondrocytes. Several attempts were made to develop more specific methods for isolation and characterization of the cells by preparing antibodies to surface epitopes on MSC. The 1st antibody was the monoclonal immunoglobulin M antibody STRO-1, which was raised against confluent cultures of human being MSCs that were used as feeder layers for hematopoietic stem cells.18 STRO-1 alone or in combination with other antibodies subsequently was used extensively to identify and isolate MSCs.19C26 Also, a series of additional monoclonal antibodies were prepared to MSCs.27C30 In addition, antibodies initially prepared to other cell types were used to characterize MSCs.31C35 Even though published antibodies to MSCs are useful, none distinguishes 2 major subpopulations that are present in early-passage human MSCs plated at low density: (1) spindle-shaped and rapidly self-renewing cells referred to as type I cells13 or as RS-MSCs,11 and (2) larger, slowly replicating type II Temanogrel cells or SR-MSCs that arise from type I or RS-MSCs as the cultures increase to confluency. Recently, we searched for antibodies to surface proteins that determine early progenitors in cultures of MSCs. We found 6 helpful antibodies to proteins that were previously linked to cell trafficking and tumor progression. Methods Isolation and tradition of human being MSCs MSCs from bone marrow aspirates were from the National Institutes of Health (NIH)/National Center for Study Resources (NCRR)Cfunded Tulane Center for the Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribute.shtml). In brief, the MSCs were prepared from 2- to 4-mL bone marrow aspirates of the iliac crest of normal adult volunteers as explained previously (Table S1.
