RESUMO
STUDY QUESTION: Can endometrial stromal stem/progenitor cell markers, SUSD2 and CD146/CD140b, enrich for human myometrial and fibroid stem/progenitor cells? SUMMARY ANSWER: SUSD2 enriches for myometrial and fibroid cells that have mesenchymal stem cell (MSC) characteristics and can also be induced to decidualise. WHAT IS KNOWN ALREADY: Mesenchymal stem-like cells have been separately characterised in the endometrial stroma and myometrium and may contribute to diseases in their respective tissues. STUDY DESIGN, SIZE, DURATION: Normal myometrium, fibroids and endometrium were collected from hysterectomies with informed consent. Primary cells or tissues were used from at least three patient samples for each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Flow cytometry, immunohistochemistry and immunofluorescence were used to characterise tissues. In vitro colony formation in normoxic and hypoxic conditions, MSC lineage differentiation (osteogenic and adipogenic) and decidualisation were used to assess stem cell activity. Xenotransplantation into immunocompromised mice was used to determine in vivo stem-like activity. Endpoint measures included quantitative PCR, colony formation, trichrome, Oil Red O and alkaline phosphatase activity staining. MAIN RESULTS AND THE ROLE OF CHANCE: CD146+CD140b+ and/or SUSD2+ myometrial and fibroid cells were located in the perivascular region and formed more colonies in vitro compared to control cells and differentiated down adipogenic and osteogenic mesenchymal lineages in vitro. SUSD2+ myometrial cells had greater in vitro decidualisation potential, and SUSD2+ fibroid cells formed larger tumours in vivo compared to control cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Markers used in this study enrich for cells with stem/progenitor cell activity; however, they do not distinguish stem from progenitor cells. SUSD2+ myometrial cells express markers of decidualisation when treated in vitro, but in vivo assays are needed to fully demonstration their ability to decidualise. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest a possible common MSC for the endometrial stroma and myometrium, which could be the tumour-initiating cell for uterine fibroids. STUDY FUNDING/COMPETING INTEREST(S): These studies were supported by NIH grants to JMT (R01OD012206) and to ALP (F32HD081856). The authors certify that we have no conflicts of interest to disclose.
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Leiomioma , Células-Tronco Mesenquimais , Animais , Endométrio , Feminino , Humanos , Camundongos , Miométrio , Células-Tronco , Células EstromaisRESUMO
OBJECTIVE: Participant willingness to share electronic health record (EHR) information is central to success of the National Institutes of Health All of Us Research Program (AoURP). We describe the demographic characteristics of participants who decline access to their EHR data. MATERIALS AND METHODS: We included participants enrolling in AoURP between June 6, 2017 and December 31, 2019 through the Trans-American Consortium for the Health Care Systems Research Network (TACH). TACH is a consortium of health care systems spanning 6 states, and an AoURP research partner. RESULTS: We analyzed data for 25â852 participants (89.3% of those enrolled). Mean age = 52.0 years (SD 16.8), with 66.5% White, 18.7% Black/African American, 7.7% Hispanic, 32.5% female, and 76% with >a high school diploma. Overall, 2.3% of participants declined to share access to their EHR data (range across TACH sites = 1.3% to 3.5%). Younger age, female sex, and education >high school were significantly associated with decline to share EHR data, odds ratio (95% confidence interval) = 1.26 (1.19-1.33), 1.74 (1.42-2.14), and 2.44 (1.86-3.21), respectively. Results were similar when several sensitivity analyses were performed. DISCUSSION: AoURP seeks a dataset reflecting our nation's diversity in all aspects of participation. Those under-represented in biomedical research may be reluctant to share access to their EHR data. CONCLUSION: In our data, race and ethnicity were not independently related to participant decision to decline access to their EHR information. Results suggest that the value of the AoURP dataset is unlikely to be constrained by the size or the racial/ethnic composition of this subgroup.
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Registros Eletrônicos de Saúde , Saúde da População , Etnicidade , Feminino , Hispânico ou Latino , Humanos , Masculino , Pessoa de Meia-Idade , Grupos Raciais , Estados UnidosRESUMO
BACKGROUND: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. RESULTS: An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. RESULTS: The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.
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Citosina , Metilação de DNA , Biblioteca Gênica , Humanos , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Genomic sequencing technology may identify personalized treatment options for patients with pancreatic adenocarcinoma. METHODS: The study was conducted using tissue specimens obtained from 2012 to 2014. Patients with resected pancreatic adenocarcinoma were identified. Next-generation sequencing was performed from paraffin-tumor blocks. Mutational profiles were reviewed to determine available targeted therapies and clinical trial eligibility. RESULTS: Thirty patients were identified. The incidence of mutations was: Kirsten rat sarcoma viral oncogene homolong (KRAS) = 87%, tumor protein 53 (TP53) = 63%, cyclin-dependent kinase inhibitor 2A (CDKN2A) = 20%, Mothers Against Decapentaplegic Homolog 4 (SMAD4) = 20%, epidermal growth factor receptor (EGFR) = 7%. Multiple mutations were found in 73%. All CDKN2A mutations occurred in male patients (P = .06), and there was a trend toward younger patient age in this group (P = .13). Potential for Federal Drug Administration (FDA)-approved targeted therapies was identified in 8 of 30 (27%). In addition, 29 of 30 (97%) had mutations applicable for ongoing phase I or II clinical trials. CONCLUSIONS: Next-generation sequencing of resected pancreatic adenocarcinoma specimens can determine common genetic mutations and identify patients who may be eligible for off-label use of targeted therapies or clinical trial enrollment.