Porcine Kidney Organoids Derived from Naïve-like Embryonic Stem Cells.

The scarcity of donor kidneys greatly impacts the survival of patients with end-stage renal failure. Pigs are increasingly becoming potential organ donors but are limited by immunological rejection. Based on the human kidney organoid already established with the CHIR99021 and FGF9 induction strategy, we generated porcine kidney organoids from porcine naïve-like ESCs (nESCs). The derived porcine organoids had a tubule-like constructure and matrix components. The porcine organoids expressed renal markers including AQP1 (proximal tubule), WT1 and PODO (podocyte), and CD31 (vascular endothelial cells). These results imply that the organoids had developed the majority of the renal cell types and structures, including glomeruli and proximal tubules. The porcine organoids were also identified to have a dextran absorptive function. Importantly, porcine organoids have a certain abundance of vascular endothelial cells, which are the basis for investigating immune rejection. The derived porcine organoids might serve as materials for immunosuppressor screening for xenotransplantation.

Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells.

The incomplete silencing of exogenous transcription factors (TFs) and the lack of endogenous counterpart activation hampers the application of porcine induced pluripotent stem cells (piPSCs). We used porcine, bovine and murine TFs to reprogram porcine fetal fibroblasts. Porcine TFs-derived piPSCs (ppiPSCs) showed the highest levels of endogenous pluripotency markers activation, were able to differentiate into three germ layers and primordial germ cell-like cells (PGCLCs) and integrated into neural ectoderm of E7.5 mouse embryos in vitro. The bovine TFs derived piPSCs (bpiPSCs) expressed endogenous pluripotency markers higher than murine TFs derived piPSCs (mpiPSCs), but both had limited differentiation ability in vitro and depended on continuous expression of exogenous TFs for the maintenance. RNA sequencing confirmed ppiPSCs had distinct global transcriptional profiling, upregulated Hippo, PI3K-Akt, MAPK and relevant pluripotency signaling pathways as porcine blastocyst inner cell mass and expressed PGC early related genes. In addition, a positive and a negative correlation between exogenous and endogenous TFs' expression level were observed in ppiPSCs and bpiPSCs lines, respectively. The TFs' protein structures in pig were more similar to cattle than to mouse. In conclusion, the species affinity of the exogenous TFs is a key element, and the own species origin of TFs is optimal for iPSCs generation and application.

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.

The efficacy and safety of osimertinib in treating nonsmall cell lung cancer: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is the primary treatment in treating with EGFR mutant nonsmall cell lung cancer (NSCLC). This systematic review and meta-analysis aimed to evaluate the efficacy and safety of the third-generation EGFR-TKI, osimertinib, and summarize the risk factors associating with outcome after osimertinib treatment. METHOD: The Ovid Medline, Embase, Cochrane Library, and Pubmed were systematically searched due to December 10, 2019. All the studies that mentioned the overall survival (OS), progression-free survival (PFS), treatment response, and adverse events (AEs) of osimertinib were involved in our study. Hazard ratio (HR) with 95% confidence intervals was used for comparing OS and PFS. RESULT: A total of 47 studies were included in the systematic review, of which 14 studies were used to compare the efficacy between osimertinib and other EGFR-TKI or chemotherapy. Patients treating with osimertinib favors a higher OS and PFS in all the patients (HR = 0.56 and 0.38, P < .001, respectively), and in subgroup analysis, compared with other treatments. Median 55% T790 mutant NSCLC patients might experience partial response, and 25% of patients remained as stable disease. The incidence of severe AE ranged from 0% to 5%, and the most common severe AE was pneumonia (3%). Patients with the T858R mutation may have a better OS than Del 19 mutation (HR = 0.55, P = .037), while patients who have a smoking history may have a higher risk of progression than never-smoker patients (HR = 1.47, P = .028). CONCLUSION: Osimertinib has an impressive antitumor activity compared with prior EGFR-TKI and chemotherapy with an acceptable response and tolerable AEs. EGFR mutation type and smoking status were the risk factors for mortality and progression in NSCLC patients.

Vitamin D inhibits the Staphylococcal enterotoxin B-induced expression of tumor necrosis factor in microglial cells.

Microglial cells play a crucial role in inflammatory responses in neural tissue. Vitamin D (VitD3) deficiency is associated with the pathogenesis of inflammation. Staphylococcal enterotoxin B (SEB) is a superantigen and can initiate inflammation. This study tests a hypothesis that VitD3 deficiency upregulates the expression of tumor necrosis factor (TNF) in microglial cells. Microglial cells were isolated from the mouse brain. The microglial cells were cultured in the presence or absence of VitD3 or/and SEB. The expression of TNF in the microglial cells was assessed by RT-qPCR and Western blotting. We observed that SEB increased the expression of TNF in microglial cells. The presence of calcitriol (an active form of VitD3) inhibited the SEB-induced TNF production by microglial cells in a dose-dependent manner. Exposure to SEB increased the binding of vitamin D receptor (VDR) to the TNF promoter, which was inhibited by the presence of calcitriol with the mechanism being the formation of a complex of VDR and LITAF (a transcription factor of TNF) in microglial cells; the complex prevented the binding of LITAF to the promoter of the TNF gene. Exposure to calcitriol also increased the expression of IL-10 in microglial cells; the inhibition of SEB-induced TNF expression was partially due to the IL-10 induced by calcitriol (Heine G, Niesner U, Chang HD, teinmeyer A, Zügel U, Zuberbier T, et al. 1,25-dihydroxyvitamin D(3) promotes IL-10 production in human B cells. Eur J Immunol. 2008;38(8):2210-8). Hormonal vitamin D [calcitriol] can inhibit the SEB-induced TNF expression in microglial cells.

Micro RNA-550a interferes with vitamin D metabolism in peripheral B cells of patients with diabetes.

The pathogenesis of diabetes is to be further investigated. Vitamin D3 (VitD3) can improve diabetes. Micro RNAs (miR) are involved in regulating cell activities. This study tests a hypothesis that miR-550a interferes with the metabolism of VitD3 in peripheral B cells. In this study, blood samples were collected from patients with diabetes and healthy persons. The B cells were isolated from the blood samples to be treated with tumor necrosis factor (TNF)-α. The B cells were then collected and analyzed for the expression of miR-550a and cyp27b1. The results showed that B cells from healthy subjects were capable of converting VitD metabolite calcidiol to calcitriol, which was impaired in B cells collected from diabetic patients. The diabetic patients showed lower bone mineral density than that in healthy subject. The miR-550a was negatively correlated with bone mineral density and the Levels of cyp27b1 in peripheral B cells of patients with diabetes. In vitro study showed that TNF-α increased miR-550a expression and inhibited the expression of cyp27b1 in B cells. miR-550a mediated the effects of TNF-α on inducing chromatin remodeling at the cyp27b1 gene locus. In conclusion, miR-550a mediates the TNF-α-induced suppression of cyp27b1 expression in peripheral B cells of patients with diabetes, which can be blocked by inhibition of miR-550a.

The association of metabolic syndrome with left ventricular mass and geometry in community-based hypertensive patients among Han Chinese.

BACKGROUND: The association of metabolic syndrome (MS) with left ventricular (LV) hypertrophy is controversial. The objective of our study was to investigate the influence of MS on LV mass and geometry in community-based hypertensive patients among Han Chinese. MATERIALS AND METHODS: This study included 1733 metabolic syndrome patients according to the International Diabetes Federation (IDF) definition and 2373 non-MS hypertension patients. LV hypertrophy was diagnosed by the criteria of LV mass ≥49.2 g/m(2.7) for men and 46.7 g/m(2.7) for women. LV geometric patterns (normal, concentric remodeling, concentric or eccentric hypertrophy) were calculated according to LV hypertrophy and relative wall thickness. Logistic regression analysis was used to determine odds ratio (OR) and 95% confidence interval (CI) of MS for LV hypertrophy and LV geometry abnormality. RESULTS: The LV mass and LV mass index were higher in the MS group than in the non-MS group. In multiple adjusted models. LV mass index, LV mass, interventricular septum, and post wall were raised with the increased number of MS disorders. MS was associated with increased LV hypertrophy risk (unadjusted OR 1.38; 95% CI 1.21-1.57); age, sex, and blood pressure (BP; adjusted OR 1.39; 95% CI 1.22-1.59). MS was also associated with increased risk of eccentric hypertrophy in male and female patients. MS was only associated with increased risk of concentric hypertrophy in female patients; and MS was not associated with concentric remodeling. CONCLUSION: LV mass and LV mass index were associated with the increased number of MS disorders in the Chinese community-based hypertensive population. MS was not only associated with increased LV hypertrophy risk, but also associated with concentric and eccentric LV geometry abnormality, especially in females.