Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal ...signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct‐4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions. STEM CELLS 2011;29:1176–1185
Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system ...in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their ...contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.
Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human ...teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.
Achieving health equity (where every person has the opportunity to attain their full health potential) requires the removal of obstacles to health, including barriers to high-quality medical care. ...Innovations in service delivery can inadvertently maintain, worsen, or introduce inequities. As such, implementation of innovations must be accompanied by a dual commitment to evaluate impact on marginalized groups and to restructure systems that obstruct people from health and healthcare. Understanding the impact innovations have on access to high-quality care is central to this effort. In this Perspective, we join conceptual models of healthcare access and quality with health equity frameworks to conceptualize healthcare receipt as a series of interactions between people and systems unfolding over time. This synthesized model is applied to illustrate the effects of telemedicine on patient, population, and system outcomes. Telemedicine may improve or worsen health equity by altering access to care and by altering quality of care once it is accessed. Teasing out these varied effects is complex and requires considering multilevel influences on the outcome of a care-seeking episode. This synthesized model can be used to inform research, practice, and policy surrounding the equity implications of care delivery innovations more broadly.
To establish the correlation between clinical grading of papilloedema and diffusion abnormalities of optic nerve head (ONH) on diffusion-weighted imaging (DWI).
Brain magnetic resonance imaging ...(MRI), including readout segmented echo planar imaging-based DWI, was performed in 32 patients with papilloedema and the same number of age- and sex-matched controls. Clinical grading of papilloedema was done according to the modified Frisén scale. Two neuroradiologists independently evaluated the MRI for ONH hyperintensity and apparent diffusion coefficient (ADC) value of ONH. The comparison between papilloedema clinical grade and qualitative grade of ONH hyperintensity and its presence between cases and control groups were done using the Chi-square test and Fisher's exact test, respectively. The comparison between mean ADC value of ONH among different grades and between cases and controls were done using analysis of variance (ANOVA)-F-test and Student's t-test, respectively. Receiver operating characteristic (ROC) analysis was done to calculate a cut-off ADC value between the case and control groups.
Significant correlation between ONH hyperintensity and mean ADC value of ONH with clinical grades of papilloedema and between cases and control groups were found. ONH hyperintensity was found to be a highly sensitive (87.5% for both) and specific (specificity 97.1% and 98.6% for two observers) sign of papilloedema. A mean cut-off ONH ADC value was found to have high sensitivity (96.83%) and specificity (95.31%) to distinguish between the cases and controls.
Diffusion parameters of ONH have significant correlation with clinical grading of papilloedema and can serve as a surrogate marker for intracranial pressure.
•Optic nerve head (ONH) hyperintensity is a definite imaging sign of papilloedema.•Diffusion parameters of ONH correlate well with clinical grade of papilloedema.•ONH diffusion parameters can be a surrogate marker of raised intracranial pressure.
Permanent supportive housing (PSH) integrates long-term housing and supports for families and individuals experiencing homelessness. Although PSH is frequently provided to families with children, ...little is known about the impacts of PSH among children. We examined changes in health care visits among children receiving PSH compared with similar children who did not receive PSH.
We analyzed Pennsylvania Medicaid administrative data for children entering PSH between 2011 and 2016, matching to a comparison cohort with similar demographic and clinical characteristics who received non-PSH housing services. We conducted propensity score-weighted difference-in-differences (DID) analyses to compare changes in health care visits 3 years before and after children entered PSH versus changes in the comparison cohort.
We matched 705 children receiving PSH to 3141 in the comparison cohort. Over 3 years following PSH entry, dental visits among children entering PSH increased differentially relative to the comparison cohort (DID: 12.70 visits per 1000 person-months, 95% confidence interval: 3.72 to 21.67). We did not find differential changes in preventive medicine visits, hospitalizations, or emergency department (ED) visits overall. When stratified by age, children ≤5 years old at PSH entry experienced a greater decrease in ED visits relative to the comparison cohort (DID: -13.16 visits per 1000 person-months, 95% confidence interval: -26.23 to -0.10). However, emergency visit trends before PSH entry differed between the cohorts.
Children in PSH had relatively greater increases in dental visits, and younger children entering PSH may have experienced relative reductions in ED visits. Policymakers should consider benefits to children when evaluating the overall value of PSH.
A physician workforce that reflects the patient population is associated with improved patient outcomes and promotes health equity. Notwithstanding, racial and ethnic disparities persist within US ...medical schools, making some individuals underrepresented in medicine (URM). We sought to increase the percentage of URM residents who matched into our pediatric residency programs from a baseline of 5% to 35% to achieve demographic parity with our patients. We developed a multifaceted approach using multiple iterative tests of change, with the primary strategy being increased visibility of URM trainees and faculty to residency applicants. Strategies included applicant interviews with URM faculty, interview dinners with URM residents, visibility at academic conferences for URM trainees, development of targeted marketing materials, and a visiting student program supported by networking with URM residents. The primary outcome measure was the percentage of matched residents in the categorical pediatrics, child neurology, and medical genetics training programs who identified as URM. The percentage of URM residents increased to 16% (6 of 37) in 2018, 26% (11 of 43) in 2019, 19% (8 of 43) in 2020, and 21% (9 of 43) in 2021 (a four-year average of 22% URM residents; P = .0002). This progress toward a more representative residency program was met by challenges, such as pipeline concerns, the minority tax, and recruitment during a pandemic. We were able to implement small, low-resource strategies that had a large cumulative impact and could be implemented in other residency programs. Specific tactics and challenges encountered are discussed in this special article.
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