Human monocytes have been grouped into classical (CD14++CD16−), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

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Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O(2) consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.

Preservation of adult stem cells pools is critical for maintaining tissue homeostasis into old age. Exhaustion of adult stem cell pools as a result of deranged metabolic signaling, premature senescence as a response to oncogenic insults to the somatic genome, and other causes contribute to tissue degeneration with age. Both progeria, an extreme example of early-onset aging, and heritable longevity have provided avenues to study regulation of the aging program and its impact on adult stem cell compartments. In this review, we discuss recent findings concerning the effects of aging on stem cells, contributions of stem cells to age-related pathologies, examples of signaling pathways at work in these processes, and lessons about cellular aging gleaned from the development and refinement of cellular reprogramming technologies. We highlight emerging therapeutic approaches to manipulation of key signaling pathways corrupting or exhausting adult stem cells, as well as other approaches targeted at maintaining robust stem cell pools to extend not only lifespan but healthspan.

In vivo real-time imaging of the cavernous nerves is possible with no measurable toxicity, allowing the maintenance of erection. This novel imaging modality may allow urologists to identify cavernous nerves during pelvic surgery.

i Abstract Preservation of adult stem cells pools is critical for maintaining tissue homeostasis into old age. Exhaustion of adult stem cell pools as a result of deranged metabolic signaling and premature senescence as a response to oncogenic insults to the somatic genome both contribute to tissue degeneration with age. Both progeria, an extreme example of early-onset aging, and heritable longevity have provided avenues to study regulation of the aging program and its impact on adult stem cell compartments.Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types.Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multi-lineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor (VEGF) secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was only detected in MSCs preconditioned in normoxic conditions. Osteogenesisassociated genes were up-regulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in 3-dimensional (3D) pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia ii applied during subsequent pellet culture enhanced chondrogenesis. While hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high quality differentiated tissue.Media formulations for MSC culture and differentiation vary between labs; two notable variables addressed inconsistently in the field are ascorbate supplementation and glucose concentration in MSC expansion medium. Glucose and ascorbate effects are coupled to the Hif1α pathway. Therefore, we examined the effects of ascorbate supplementation and low versus high glucose medium in the settings of hypoxia and normoxia on colony formation, colony morphology, proliferation, VEGF secretion, and senescence of MSCs. We found that there is no one medium formulation that maximizes MSC performance for all of these assays.Depending on the desired outcome of the culture, t...

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