The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro . Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

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BackgroundConsideration and better understanding of patients’ needs on the part of the healthcare system might help increase the number of people seeking necessary medical care. Many studies have been conducted on patients’ preferences in choosing their health care provider, but the majority of them were conducted in modern western societies, establishing a need to explore other populations. The present study was performed in the Israeli Druze community which is composed of a uniquely traditional and religious population.We assessed the sex preference of Israeli Druze women regarding obstetricians/gynecologists, and identify other features that affect their choice.MethodWe conducted a cross-sectional study that included 196 Israeli Druze women who anonymously completed a 36-item questionnaire between January-July, 2011.ResultsMost (63.8%) of the responders preferred female obstetricians/gynecologists, while 74.5% had no sex preference for their family physicians. 68.6% of the religious women preferred female obstetricians/gynecologists as compared to 51.76% of those women who self-identified as secular. Most of the women (65%) preferred female obstetricians/gynecologists for intimate procedures, such as pelvic examination and pregnancy follow-up. The main reasons given were: feeling more comfortable with a female practitioner (69.7%), the belief that females are more gentle (56.6%), and being more embarrassed with male obstetricians/gynecologists (45.4%). Three factors were associated with the responders’ preferences for female obstetricians/gynecologists: their age and religious status, and the sex of their regular obstetricians/gynecologists. Women who preferred a female obstetrician/gynecologist assigned a lesser weight to the physician’s knowledge when choosing them. Older and religious women as well as those who attributed less weight to the physician’s professional knowledge were more likely to prefer a female obstetrician/gynecologist.ConclusionsThe majority of responders to our survey (Israeli Druze women), like those in other communities where religiousness and modesty are deeply rooted, prefer female obstetricians/gynecologists, with the overwhelming reasons given being feeling more comfortable and less embarrassed with females, and the notion that female obstetricians/gynecologists are more gentle during intimate procedures.