Background Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our recent experience isolating and immunophenotypically characterizing ASCs from >60 human subjects of mean age 43.6 and mean body mass index of 27. Methods We examined the ASC yield per unit volume of lipoaspirate tissue, their surface antigen profile based on flow cytometry, their histochemical differentiation potential along the adipogenic and osteogenic pathways, and their expression of adipogenic mRNAs by transcriptomic microarray and RT-PCR. Results The population (n = 64) of predominantly Caucasian (84.3%) female (90.6%) donors had a mean age of 43.6 ± 11.1 years and a mean body mass index of 27.0 ± 3.8. A yield of 375 ± 142 × 103 ASC was obtained per ml of lipoaspirate within a 4.1 ± 0.7 day culture period (n = 62). The ASC population was uniformly CD29+ CD34+CD44loCD45loCD73+CD90+CD105+ and capable of undergoing both adipogenesis and osteogenesis in vitro based on Oil Red O and Alizarin Red staining, respectively. Adipogenic differentiation was associated with the significant induction of multiple mRNAs associated with lipid storage and synthesis based microarray analysis of n = 3 donors. During an adipogenic differentiation time course, representative mRNAs (adiponectin, C/EBPα, leptin, LPL) displayed increases of several orders of magnitude. Discussion These findings demonstrate the reproducibility of subcutaneous lipoaspirates as a consistent and abundant source of functional ASCs from donors across a spectrum of ages and BMIs. These results have relevance to regenerative medical applications exploiting autologous or allogeneic ASCs for soft and hard tissue engineering.

Summary: Erythropoietin, a hemotopoietic growth factor, has brain protective actions. This study investigated the mechanisms of Recombinant Human EPO (rhEPO)-induced brain protection in neonates. An established rat hypoxia-ischemia model was used by ligation of the right common carotid artery of 7-day-old pups, followed by 90 minute of hypoxia (8% 0 2 and 92% N 2 ) at 37°C. Animals were divided into three groups: control, hypoxia-ischemia, and hypoxia-ischemia plus rhEPO treatment. In rhEPO treated pups, 300 units rhEPO was administered intraperitoneally 24 hours before hypoxia. rhEPO treatment (300 units) was administered daily for an additional 2 days. ELISA and immunohistochemistry examined the expression of EPO and EPOR. Brain weight, morphology, TUNEL assay, and DNA laddering evaluated brain protection. rhEPO abolished mortality (from 19% to 0%) during hypoxia insult, increased brain weight from 52% to 88%, reduced DNA fragmentation, and decreased TUNEL-positive cells. Real-time RT-PCR, Western blot, and immunohistochemistry revealed an enhanced expression of heat shock protein 27 (HSP27) in ischemic brain hemisphere. Double labeling of TUNEL with HSP27 showed most HSP27 positive cells were negative to TUNEL staining. rhEPO reduces brain injury, especially apoptotic cell death after neonatal hypoxia-ischemia, partially mediated by the activation of HSP27. Key Words: apoptosisbrain injury-heat shock protein-RT-PCR.

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

No abstract

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.