Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells.

Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

Human mulipotential mesenchymal/stromal stem cells are derived from a discrete subpopulation of STRO-1bright/CD34 /CD45(-)/glycophorin-A-bone marrow cells.

Magnetic and flow cytometry-based methods were used to characterize clonogenic stromal cells in human bone marrow. STRO-1(bright) stromal cells were found to lack expression of CD34, CD45 and glycophorin-A markers associated with hematopoietic progenitor cells. These studies support the view that these are two distinct stem cell compartments in adult bone marrow.

Defective osteogenesis of the stromal stem cells predisposes CD18-null mice to osteoporosis.

Osteogenesis by the bone marrow stromal stem cells (BMSSCs) supports continuous bone formation and the homeostasis of the bone marrow microenvironment. The mechanism that controls the proliferation and differentiation of BMSSCs is not fully understood. Here, we report that CD18, a surface protein present primarily on hematopoietic cells, but not on differentiated mesenchymal cells, is expressed by the stromal stem cells and plays a critical role in the osteogenic process. Constitutive expression of CD18 on BMSSCs using a retroviral promoter significantly enhances bone formation in vivo, whereas genetic inactivation of CD18 in mice leads to defective osteogenesis due to decreased expression of the osteogenic master regulator Runx2/Cbfa1. The defective osteogenesis of the CD18-null BMSSCs can be restored by expressing full-length, but not cytoplasmic domain-truncated, CD18. Radiographic analyses with dual-energy x-ray absorptiometry and 3D microcomputed tomography show that mice lacking CD18 have decreased bone mineral density and exhibit certain features of osteoporosis. Altogether, this work demonstrates that CD18 functions critically in the osteogenesis of BMSSCs, and thus lack of CD18 expression in the leukocyte adhesion deficiency patients may predispose them to osteoporosis.

Elevated serum levels of stromal-derived factor-1alpha are associated with increased osteoclast activity and osteolytic bone disease in multiple myeloma patients.

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1alpha (SDF-1alpha) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1alpha protein and exhibit elevated plasma levels of SDF-1alpha when compared with normal, age-matched subjects. The level of SDF-1alpha positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1alpha in osteoclast precursor recruitment and activation. To examine this further, peripheral blood-derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1alpha. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1alpha mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1alpha was associated with an increase in the expression of a number of osteoclast activation-related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1alpha by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1alpha axis may provide an effective means of treatment for MM-induced osteolysis.

Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells.

The maintenance of bone marrow stromal stem cells (BMSSCs) is tightly controlled by the local microenvironment and by autocrine regulatory factors secreted by BMSSCs. To identify such factors, a cDNA subtraction library was generated from purified BMSSCs, based on their high expression of the STRO-1 antigen. Stromal-derived factor-1 (SDF-1) was one differentially expressed gene highly expressed by purified BMSSCs prior to culture. In vitro, immature preosteogenic cells expressed greater levels of SDF-1 when compared with mature cell types representative of osteoblasts and osteocytes/bone lining cells. Furthermore, SDF-1 expression was rapidly down-regulated when BMSSCs were cultured under osteoinductive conditions. BMSSCs were also shown to express functional cell surface SDF-1 receptors (CXCR4). Transduced BMSSC lines, secreting high SDF-1 levels, displayed an enhanced ability to form ectopic bone in vivo, in comparison with control BMSSC lines. Moreover, high SDF-1-expressing BMSSCs displayed an increased capacity for cellular growth and protection against interleukin-4-induced apoptosis. Similarly, fibroblast colony-forming units (CFU-Fs) also displayed increased growth and resistance to alpha-interferon-2a-induced apoptosis, in synergy with platelet-derived growth factor BB (PDGF-BB) and SDF-1 in vitro. These studies indicate that the chemokine, SDF-1, may play a role in the maintenance, survival, and osteogenic capacity of immature BMSSC populations.

Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow.

Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.