To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heatshocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

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...

Attempts have been made to develop cell and gene therapies using the adult stem cells from bone marrow referred to as mesenchymal stem cells or marrow stromal cells (MSCs). However, the results have been variable in part because there are no standardized protocols for preparing and characterizing MSCs. In the experiments presented here, we developed a standardized assay by light scattering to measure the content of rapidly self-renewing cells (RS cells) in preparations of MSCs. The assay quickly identifies preparations of MSCs that replicate rapidly in subsequent culture. In addition, the standardized assay enabled us to isolate RS cells that were up to 90% clonogenic and that generated single cell-derived colonies that differentiated into either mineralizing cells or adipocytes with appropriate additions to the medium.

Summary Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produce Protein S (Pros1) that signals through the TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells leads to increased expression of co-stimulatory molecules and cytokines in DCs, enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 is expressed in activated human T cells and its ability to regulate DC activation is conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.