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We report here the isolation of a population of nontransformed pluripotent human cells from bone marrow after a unique expansion/selection procedure. This procedure was designed to provide conditions resembling the in vivo microenvironment that is home for the mostprimitive stem cells. Marrow-adherent and -nonadherent cells were co-cultured on fibronectin, at low oxygen tension, for 14 days. Colonies of small adherent cells were isolated and further expanded on fibronectin at low density, low oxygen tension with 2% fetal bovine serum. They expressed high levels of CD29, CD63, CD81, CD122, CD164, hepatocyte growth factor receptor (cMet), bone morphogenetic protein receptor 1B (BMPR1B), and neurotrophic tyrosine kinase receptor 3 (NTRK3) and were negative for CD34, CD36, CD45, CD117 (cKit) and HLA-DR. The embryonic stem cell markers Oct-4 and Rex-1, and telomerase were expressed in all cultures examined. Cell-doubling time was 36 to 72 hours, and cells have been expanded in culture for more than 50 population doublings. This population of cells was consistently isolated from men

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Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing humanderived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment. Cancer Res; 76(2); 491-504. Ó2016 AACR.

We have developed an in vitro system, using embryonic chicken tibiae grown in a serum-free medium, which exhibits simultaneous bone formation and resorption. Tibiae from 8-day embryos increased in mean (±SD) length (4.0 ± 0.4 to 11.0 ± 0.3 mm) and dry weight (0.30 ± 0.04 to 0.84 ± 0.04 mg) during 12 days in vitro. There was increased incorporation of [3H]proline into hydroxyproline (120 ± 20 to 340 ± 20 cpm/mg of bone per 24 hr) as a measure of collagen synthesis, as well as a 62 ± 5% increase in total calcium and 45Ca taken up as an indication of active mineralization. A physiologic concentration (1 pM) of parathyroid hormone was found to stimulate bone resorption over control levels in this system. Parathyroid hormone stimulated the release of [3H]hydroxyproline from the bone shafts but not from the cartilage ends, indicating the specificity of theresponse. With 1 pM parathyroid hormone we observed an acute inhibition of bone formation, followed (after 12-16 hr) by a chronic stimulation of bone formation during the 12-day incubation. Both mineral uptake and matrix formation were enhanced at approximately the same rate during the 12-day incubation. The chronic enhancement of formation required parathyroid hormone only for the initial 8-10 hr of incubation. These results could be explained by the production or release ofa factor from bone to stimulate formation in response to the acute increase in resorption-a "coupling factor." Indeed, dialyzed culture medium conditioned by actively resorbing bones stimulated bone formation over controls when added to organ cultures at a 1:20 dilution. The factor is larger than 12,000 daltons as determined by dialysis. The factor is specific for the bone shaft and did not affect the cartilage ends. Work directed toward a better understanding of the regulation of bone metabolism has shown that bone formation and bone resorption are coupled in vivo (1). This coupling occurs even in some disease states (e.g., hyper-and hypoparathyroidism, Paget disease, and acromegaly) (2). Detailed knowledge of the regulatory mechanism of coupling could lead to better understanding of the problems of metabolic bone disease. Studies toward this end from our laboratory have in the past relied predominantly on in vivo experiments (3,4). Although a large number of investigators have examined bone metabolism in vitro (5-9), none of these studies involved a chemically defined system in which simultaneously enhanced bone formation and resorption could be demonstrated. We have developed such an in vitro system and describe the details herein.We have utilized this coupled system to study the effects of parathyroid hormone (PTH) on bone metabolism in vitro in an attempt to clarify what appears to be a disagreement in the literature concerning these effects. The acute effect in vivo is reported to be the stimulation of bone resorption and inhibition of bone formation (10,11). Chronic in vivo treatment of young growing rats with PTH results in an increase in formation and resorption (4) and a net ga...

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