Astrocytes modulate neuronal activity by releasing chemical transmitters via a process termed gliotransmission. The role of this process in the control of behavior is unknown. Since one outcome of SNARE-dependent gliotransmission is the regulation of extracellular adenosine and because adenosine promotes sleep, we genetically inhibited the release of gliotransmitters and asked if astrocytes play an unsuspected role in sleep regulation. Inhibiting gliotransmission attenuated the accumulation of sleep pressure, assessed by measuring the slow wave activity of the EEG during NREM sleep and prevented cognitive deficits associated with sleep loss. Since the sleep-suppressing effects of the A1 receptor antagonist CPT were prevented following inhibition of gliotransmission and because intracerebroventricular delivery of CPT to wildtype mice mimicked the transgenic phenotype we conclude that astrocytes modulate the accumulation of sleep pressure and its cognitive consequences through a pathway involving A1 receptors.

Stem/progenitor cells from bone marrow and other sources have been shown to repair injured tissues by differentiating into tissue-specific phenotypes, by secreting chemokines, and, in part, by cell fusion. Here we prepared the stem/progenitor cells from human bone marrow (MSCs) and implanted athem into the dentate gyrus of the hippocampus of immunodeficient mice. The implanted human MSCs markedly increased the proliferation of endogenous neural stem cells that expressed the stem cell marker Sox2. Labeling of the mice with BrdUrd demonstrated that, 7 days after implantation of the human MSCs, BrdUrd-labeled endogenous cells migrated throughout the dorsal hippocampus (positive for doublecortin) and expressed markers for astrocytes and for neural or oligodendrocyte progenitors. Subpopulations of BrdUrd-labeled cells exhibited short cytoplasmic processes immunoreactive for nerve growth factor and VEGF. By 30 days after implantation, the newly generated cells expressed markers for more mature neurons and astrocytes. Also, subpopulations of BrdUrd-labeled cells exhibited elaborate processes immunoreactive for ciliary neurotrophic factor, neurotrophin-4/5, nerve growth factor, or VEGF. Therefore, implantation of human MSCs stimulated proliferation, migration, and differentiation of the endogenous neural stem cells that survived as differentiated neural cells. The results provide a paradigm to explain recent observations in which MSCs or related stem/progenitor cells were found to produce improvements in disease models even though a limited number of the cells engrafted.

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

Human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo, and under appropriate conditions they differentiate into osteoblasts ex vivo. The cells provide a convenient cell culture model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose hMSCs to cell cycle entry and prevent osteogenesis. Our investigation of the role of Wnt signaling in osteogenesis by hMSCs ex vivo has demonstrated that osteogenesis proceeds in response to bone morphogenic protein 2 stimulation and is sustained by Wnt signaling. In the presence of Dkk-1, an inhibitor of Wnt signaling, the cascade is disrupted, resulting in inhibition of osteogenesis. Peptide mapping studies have provided peptide Dkk-1 agonists and the opportunity for the production of blocking antibodies. Anti-Dkk-1 strategies are clinically relevant since high serum levels of Dkk-1 are thought to contribute to osteolytic lesion formation in multiple myeloma and possibly some forms of osteosarcoma. Specific inhibitors of glycogen synthetase kinase 3beta (GSK3beta), which mimic Wnt signaling, may also have a therapeutic benefit by enhancing in vitro osteogenesis despite the presence of Dkk-1. Antibodies that block Dkk-1 and GSK3beta inhibitors may provide novel opportunities for the enhancement of bone repair in a variety of human diseases such as multiple myeloma and osteosarcoma.

Controversies have arisen as to whether adult stem cells or progenitor cells from bone marrow can engraft into nonhematopoietic tissues in vivo. To resolve some of the controversies, we developed a highly sensitive polymerase chain reactionbased single nucleotide polymorphism (PCR-SNP) assay for competitive engraftment of mixtures of stem/progenitor cells. We used the assay to follow engraftment in immunodeficient mice of subpopulations of the stem/progenitor cells from human bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). The engraftment into adult mice without induced tissue injury was low and variable, but there was preferential engraftment of a subpopulation of rapidly self-renewing MSCs (RS-MSCs) compared with a subpopulation of slowly renewing MSCs (SR-MSCs). After intravenous infusion, there was a tendency for the cells to engraft into the hippocampal region that was previously designated a "vascular niche." Migration assays suggested that preferential engraftment of RS-MSCs was in part explained by their expression of CXCR4 and CX3R1, the receptors for SDF-1 and fractalkine.