When we fall asleep, consciousness fades yet the brain remains active. Why is this so? To investigate whether changes in cortical information transmission play a role, we used transcranial magnetic stimulation together with high-density electroencephalography and asked how the activation of one cortical area (the premotor area) is transmitted to the rest of the brain. During quiet wakefulness, an initial response (approximately 15 milliseconds) at the stimulation site was followed by a sequence of waves that moved to connected cortical areas several centimeters away. During non-rapid eye movement sleep, the initial response was stronger but was rapidly extinguished and did not propagate beyond the stimulation site. Thus, the fading of consciousness during certain stages of sleep may be related to a breakdown in cortical effective connectivity.

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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.

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...