Due to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem (iPS) cells. One technique that has not yet been explored is the use of "minicircle" DNA, a novel compact vector that is free of bacterial DNA and capable of persistent high level expression in cells. Here, we report the use of a single minicircle vector to generate transgene-free iPS cells from adult human cells. Keywords minicircle DNA; reprogramming; iPS cells; viral-free; human adipose stem cellsNon-viral methods for generating iPS cells using adenovirus 1 , plasmids 2 , or excision of reprogramming factors using Cre/LoxP 3,4 or piggyBAC transposition 5 have been reported, but in general are restricted to mouse, suffer from low reprogramming efficiencies (<0.003%), and may leave behind residual vector sequences. Recently, successful reprogramming of human neonatal foreskin fibroblasts was reported using episomal vectors derived from the EpsteinBarr virus6. However, this technique required three individual plasmids carrying a total of seven factors, including the oncogene SV40, and has not been shown to successfully reprogram cells from adult donors, a more clinically-relevant target population. Further, expression of the EBNA1 protein, as was required for this technique, may increase immune cell recognition of transfected cells7, thus potentially limiting clinical application if the transgene is not completely removed. Protein-based iPS cell generation in mouse8 and human9 fetal and neonatal cells has also been published, but required either chemical treatment (valproic acid) 8 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript methods require only minimal molecular biology background, and so remain a more attractive option for a wider population of researchers interested in cellular reprogramming.Compared to plasmids, minicircle DNA benefits from higher transfection efficiencies and longer ectopic expression due to its lower activation of exogenous silencing mechanisms10 , 11 , and thus may represent an ideal mechanism for generating iPS cells. We constructed a plasmid (P2PhiC31-LGNSO) that contained a single cassette of four reprogramming factors (Oct4, Sox2, Lin28, Nanog) plus a green fluorescent protein (GFP) reporter gene, each separated by selfcleavage peptide 2A sequences 12, 13 ( Supplementary Fig. 1a,b). We next took advantage of the PhiC31-based intramolecular recombination system that allows the plasmid backbone to be excluded and degraded in bacteria, and the minicircle to be purified and isolated as described 10,11 ( Supplementary Fig. 1c). Expression of individual protein factors was validated in 293FT cells ( Supplementary Fig. 2). To determine the reprogramming ability of the minicircle vector, we chose to induce pluripotency in human adipose stem cells (hASCs). hASCs have a number of advantages over other somatic cell types such as fibroblasts since they can be isolated in large quantities (100 ml of human adipo...

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.differentiation ͉ pluripotency ͉ reprogramming

Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

Students' motivations in choosing a career in the health professions are of great interest for educators and admission committees, particularly in the ield of dentistry. This study conducted in four private dental institutions in India was designed to investigate dental students' motivations in their choice of dentistry as a career and their perceptions regarding dentistry in India. A total of 400 questionnaires were distributed, and 369 students responded in a combination of selected responses to the questions, for a response rate of 92.3 percent. In the results, 53.7 percent of the students reported pursuing dentistry because it offers stable work (p<0.002); 38.7 percent because the profession is highly paid; and 7.6 percent due to the ease in inding a regular job in dental schools or hospitals. The survey also found that 44.4 percent of the students pursued dentistry because they can determine their own hours of work and 36.6 percent said they liked to be their own boss. Among these students, 64.5 percent said they were content to be joining dentistry as a professional course, but 35.5 percent were discontented (p<0.001). Regarding the specialties, 79.1 percent said they want to become specialists in the ield of dentistry (p<0.001); oral surgery was the leading choice followed by orthodontics. Only 11.7 percent reported wanting to pursue dentistry for research purposes. Overall, this study found that inancial and professional factors were the chief criteria for students' pursuing dentistry in India; however, the strongest inluence in the choice of dentistry was the students' parents or family.

In the face of mounting clinical demand, and armed with reconstructive techniques that are technically challenging and frequently result in suboptimal patient outcomes, increasing focus is being placed on tissue engineering and regenerative medicine as a potential source of novel skeletal reconstructive approaches. Specifically, evidence is accumulating that highlights the promise of osteoprogenitor cell-based reconstructive strategies to meet the needs of an expanding patient population. Historically, the study of cell and molecular biology guiding physiologic and pathologic skeletal development, as well as endogenous bone regeneration following injury, has provided a wealth of information that lends insight toward potential parallel processes that may regulate the osteogenic differentiation of progenitor cells. Multiple progenitor cell populations are now known to possess a capacity to undergo robust osteogenic differentiation in the presence of appropriate environmental cues (hESC, BMSC, ASC, etc.) Recent investigations have put forth multiple advantages of ASC relative to BMSC. Of note, ASC exist in relative abundance, lack the need for in vitro expansion prior to utilization, and can be harvested with relative ease and reduced donor morbidity. Collectively, these factors, paired with promising in vitro and in vivo observations that speak toward the substantial osteogenic potential of ASC, have spurred enthusiasm to pursue the application of ASC in the maturation of skeletal tissue engineering applications. Yet, elucidating what structural and functional properties of scaffolds designed for ASC-mediated skeletal tissue engineering applications (porosity, pore size, composition, mechanical stability, degradation kinetics, etc.), as well as evolving our understanding and capacity to deliver spatiotemporally specific pro-osteogenic targeted molecular manipulation to progenitor cells, remain important hurdles to clear. The scope of this review encompasses the current state of ongoing investigations along these fronts, as well as what future direction will be critical to the transition of cell-based skeletal tissue engineering strategies to the bedside.

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We identified 18 cases of acute puerperal uterine inversion after vaginal delivery out of 65,581 deliveries in two university-affiliated hospitals (an incidence of 1:3643). In all cases manual repositioning of the uterus without tocolysis was attempted. In four cases this was successful. Of the 14 patients requiring tocolysis, a single dose of terbutaline 0.25 mg i.v. was tried in eight and was successful in five cases (success rate of 63%), while in three patients general anaesthesia was required. In six other patients, general anaesthesia was chosen as the initial management. There was no associated maternal mortality or morbidity with the exception of one case of acute postpartum endometritis. We suggest that when tocolysis is required, terbutaline should be used first because of its rapid onset of action, short duration, simplicity, availability in the labour suite and familiarity to the obstetrician, and general anaesthesia, with its inherent dangers, if possible, should be avoided.

ABSTRACT:Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-␤, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration.