No abstract

The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell–cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics.

Adult stem cells from human bone marrow stroma, referred to as mesenchymal stem cells or marrow stromal cells (hMSCs), are attractive candidates for clinical use. The optimal conditions for hMSC expansion require medium supplemented with fetal calf serum (FCS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FCS proteins. By a sensitive fluorescence-based assay we determined that 7 to 30 mg of FCS proteins are associated with a standard preparation of 100 million hMSCs, a dosage that probably will be needed for clinical therapies. Here we present ex vivo growth conditions for hMSCs that reduce the FCS proteins to less than 100 ng per 100 million hMSCs, approximately a 100,000-fold reduction. The cells maintain their proliferative capacity and sustain their ability for multilineage differentiation. Experiments in rats demonstrate that rat MSCs grown in 20% FCS induce a substantial humoral response after repeated administrations, whereas cells grown under the conditions described in this study reduce the immunogenicity in terms of IgG response over 1000-fold to barely detectable levels. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs and perhaps other cells.

Most recent evidence suggests that the process of tissue repair is driven by stem-like cells that reside in multiple tissues but are replenished by precursor cells from bone marrow. Among the candidates for the reparative cells are the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). We recently found that after MSCs were replated at very low densities to generate single-cell-derived colonies, they did not exit a prolonged lag period until they synthesized and secreted considerable quantities of Dickkopf-1, an inhibitor of the canonical Wnt signaling pathway. We also found that when the cells were cocultured with heat-shocked pulmonary epithelial cells, they differentiated into epithelial cells. Most of the MSCs differentiated without evidence of cell fusion but up to one-quarter underwent cell fusion with the epithelial cells. A few also underwent nuclear fusion. The results are consistent with the interesting possibility that MSCs and similar cells repair tissue injury by three different mechanisms: creation of a milieu that enhances regeneration of endogenous cells, transdifferentiation, and perhaps cell fusion.

Adult human mesenchymal stem cells from bone marrow stroma (hMSCs) differentiate into numerous mesenchymal tissue lineages and are attractive candidates for cell and gene therapy. When early passage hMSCs are plated or replated at low density, the cultures display a lag phase of 3-5 days, a phase of rapid exponential growth, and then enter a stationary phase without the cultures reaching confluence. We found that as the cultures leave the lag phase, they secrete high levels of dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling pathway. The addition of recombinant Dkk-1 toward the end of the lag period increased proliferation and decreased the cellular concentration of ␤-catenin. The addition of antibodies to Dkk-1 in the early log phase decreased proliferation. Also, expression of Dkk-1 in hMSCs decreased during cell cycle arrest induced by serum starvation. The results indicated that high levels of Dkk-1 allow the cells to reenter the cell cycle by inhibiting the canonical Wnt/␤-catenin signaling pathway. Since antibodies to Dkk-1 also increased the lag phase of an osteosarcoma line that expressed the gene, Dkk-1 may have a similar role in some other cell systems.Human bone marrow contains two main populations of stem cells: hematopoietic stem cells usually identified by a CD 34 ϩ phenotype and a population of CD 34 Ϫ cells of mesenchymal origin. The population of human nonhematopoietic mesenchymal stem cells or marrow stromal cells (hMSCs) 1 can differentiate into numerous mesenchymal tissue lineages including osteoblasts, chondrocytes, adipocytes, and neural precursors (1-8). hMSCs are easily obtained from bone marrow aspirates and are readily separated from hematopoietic cells by virtue of their adherence to tissue culture plastic (1). Under the appropriate conditions, hMSCs can be propagated manifold in vitro while retaining their multipotentiality, a feature that makes them attractive candidates for stem cell and gene therapy (2, 5, 9 -12). Although some of the in vitro growth characteristics of hMSCs have been documented, the molecular mechanisms by which hMSCs regulate their own growth in culture are poorly understood. In particular, there is no apparent explanation for the observation that when early passage hMSCs are replated at low density, they display a lag period of 3-5 days, followed by a phase of rapid exponential growth, and then enter a stationary phase without reaching confluence (8,11,13).Preliminary observations (15) suggested to us that conditioned medium from cultures of hMSCs increased the rate of proliferation when added to freshly plated cultures of hMSCs.In the experiments described here, we demonstrate that hMSCs in the early log phase of growth synthesize and secrete dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway (16 -18).The Wnt signaling pathway controls patterning and cell fate determination in the development of a wide range of organisms, from Drosophila to mammals (19). The signaling can occur by at least three different pathways (20). In the canonic...