The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2

Autoantibodies against type I IFNs in patients with critical influenza pneumonia

In an international cohort of 279 patients with hypoxemic influenza pneumonia, we identified 13 patients (4.6%) with autoantibodies neutralizing IFN-alpha and/or -omega, which were previously reported to underlie 15% cases of life-threatening COVID-19 pneumonia and one third of severe adverse reactions to live-attenuated yellow fever vaccine. Autoantibodies neutralizing type I interferons (IFNs) can underlie critical COVID-19 pneumonia and yellow fever vaccine disease. We report here on 13 patients harboring autoantibodies neutralizing IFN-alpha 2 alone (five patients) or with IFN-omega (eight patients) from a cohort of 279 patients (4.7%) aged 6-73 yr with critical influenza pneumonia. Nine and four patients had antibodies neutralizing high and low concentrations, respectively, of IFN-alpha 2, and six and two patients had antibodies neutralizing high and low concentrations, respectively, of IFN-omega. The patients' autoantibodies increased influenza A virus replication in both A549 cells and reconstituted human airway epithelia. The prevalence of these antibodies was significantly higher than that in the general population for patients 70 yr of age (3.1 vs. 4.4%, P = 0.68). The risk of critical influenza was highest in patients with antibodies neutralizing high concentrations of both IFN-alpha 2 and IFN-omega (OR = 11.7, P = 1.3 x 10(-5)), especially those <70 yr old (OR = 139.9, P = 3.1 x 10(-10)). We also identified 10 patients in additional influenza patient cohorts. Autoantibodies neutralizing type I IFNs account for similar to 5% of cases of life-threatening influenza pneumonia in patients <70 yr old

General scheme of the ‘mESC Plasticity Test’.

<p>(A) mESCs maintained in pluripotency (+LIF) or cultivated without LIF for the indicated period of time in days (d1 to d5) are induced with LIF at different time points. (B) The effect of specific genes was tested by a siRNA strategy in which addition of siRNA was performed the first day of LIF withdrawal. (C) PI3K inhibitor LY was added for 24 hours to the committed or differentiated cells as indicated.</p

The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2

<i>Klf5</i> but not <i>JunB</i> disturbs the LIF-dependent plasticity in early committed cells.

<p>Cells were depleted from LIF for 24 hours and either transfected with siControl (siCtrl) (A) siKlf5, or (B) siJunB and then induced with LIF for three days (d3). Graphs represent the average level of expression and SEM from 3 to 6 independent experiments. Paired sample t-test was performed to determine the significance of the difference in the expression levels observed with the non specific (siCtrl) versus specific siRNA: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant.</p

Hypoxia maintains cell plasticity with a novel equilibrium of <i>Pluri</i> and <i>early Diff</i> genes.

<p>Cells grown under normoxia (N) or hypoxia (H) were depleted from LIF or induced with LIF after a period of LIF depletion as indicated. After 4 days, expression of the selected genes, (A) <i>Master</i>, (B) <i>Pluri</i>, (C) <i>Pluri Adhesion</i> and (D) <i>Diff</i> was analysed by RT-qPCR. Graphs represent the average level of expression and SEM as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146281#pone.0146281.g002" target="_blank">Fig 2</a> (n = 4). One sample t-test was performed for the +LIF condition and paired sample t-test was performed for the other conditions to evaluate the significance of the difference in the expression levels observed in normoxia versus hypoxia: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant.</p

PI3K does not regulate the LIF-dependent plasticity window but stimulates differentiation towards early mesoderm lineage while inhibiting ectoderm and endoderm differentiation processes.

<p>Cells were depleted from LIF or induced with LIF in the presence of LY, for the first 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146281#pone.0146281.g001" target="_blank">Fig 1C</a>. After 5 days, expression of the selected genes (A) <i>Master</i> genes, (B) <i>Pluri</i> genes, (C) early <i>Diff</i> genes and (D) specific germ layer genes, was analyzed by RT-qPCR. Graphs represent the average level of expression and SEM from 4 independent experiments. Paired sample t-test was performed to evaluate the significance of the difference in the expression levels observed with or without LY: *p-value<0.05; **p-value<0.01, ***p-value<0.001; a: pvalue = 0.051; if not stated: not significant.</p

LIF has differential effects on mESC-derived cells, after d1, d2 or d3 of LIF starvation.

<p>Cells were depleted from LIF or induced with LIF after a period of LIF depletion as indicated. After 4 days, expression of the selected genes (A) <i>Master</i> genes, (B) <i>Pluri</i> genes and (C) <i>Diff</i> genes was analyzed by RT-qPCR with <i>Hprt</i> used for normalization. Graphs represent the average level of expression and standard error of the mean (SEM) bars, calculated from 4 independent experiments. +LIF condition was arbitrarily set as 1. One sample t-test was performed for each condition versus the +LIF sample: *p-value<0.05; **p-value<0.01, ***p-value<0.001; a: p-value = 0.051; if not stated: not significant. (D) A representative experiment of flow cytometry, performed in the indicated conditions with the Ceacam-PE antibody, is presented: "off set graph" of the MFI of the total P1 gated cell population of each cell growth condition, with the Flo Jo software.</p

mESCs grown under 3% O2 have an impaired LIF signaling, express less ‘pluripotency-involved’ proteins but maintain pluripotency.

<p>Cells were grown with (+LIF) or without LIF for 1 to 3 or 4 days under normoxia or hypoxia as indicated. (A) Protein cell lysates (60 μg) from cells grown under the indicated conditions were analyzed by Western blot. ERK2 was used as a loading control. A representative experiment is shown. Quantification of signals obtained with the different antibodies in the + LIF condition (n = 4) is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146281#pone.0146281.s003" target="_blank">S3 Fig</a> (B) Gene expression of the indicated genes was analyzed by RT-qPCR. Graphs represent the average level of expression and SEM as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146281#pone.0146281.g002" target="_blank">Fig 2</a> (n = 4). Paired sample t-test was performed to evaluate the significance of the difference in the expression levels observed in normoxia versus hypoxia: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant. (C) early lineage markers, (D) specific lineage markers in mESCs induced for <i>in vitro</i> differentiation under normoxia (20% O2, N) or hypoxia (3% O2, H). Gene expression was analyzed by RT-qPCR in cells grown for two days under N or H conditions (+LIFd2), in EBs at day 8 of their formation (EBs d8) and seven days after EBs plating. Y axis is in log scale.</p

Precipitating factors of heart failure decompensation, short-term morbidity and mortality in patients attended in primary care

Objective: To evaluate the precipitating factors for heart failure decompensation in primary care and associations with short-term prognosis. Design Prospective cohort study with a 30-d follow-up from an index consultation. Regression models to determine independent factors associated with hospitalisation or death. Setting: Primary care in ten European countries. Patients Patients with diagnosis of heart failure attended in primary care for a heart failure decompensation (increase of dyspnoea, unexplained weight gain or peripheral oedema). Main outcome measures: Potential precipitating factors for decompensation of heart failure and their association with the event of hospitalisation or mortality 30 d after a decompensation. Results: Of 692 patients 54% were women, mean age 81 (standard deviation [SD] 8.9) years; mean left ventricular ejection fraction (LVEF) 55% (SD 12%). Most frequently identified heart failure precipitation factors were respiratory infections in 194 patients (28%), non-compliance of dietary recommendations in 184 (27%) and non-compliance with pharmacological treatment in 157 (23%). The two strongest precipitating factors to predict 30 d hospitalisation or death were respiratory infections (odds ratio [OR] 2.8, 95% confidence interval [CI] (2.4-3.4)) and atrial fibrillation (AF) > 110 beats/min (OR 2.2, CI 1.5-3.2). Multivariate analysis confirmed the association between the following variables and hospitalisation/death: In relation to precipitating factors: respiratory infection (OR 1.19, 95% CI 1.14-1.25) and AF with heart rate > 110 beats/min (OR 1.22, 95% CI 1.10-1.35); and regarding patient characteristics: New York Heart Association (NYHA) III or IV (OR 1.22, 95% CI 1.15-1.29); previous hospitalisation (OR 1.15, 95% CI 1.11-1.19); and LVEF < 40% (OR 1.14, 95% CI 1.09-1.19). Conclusions: In primary care, respiratory infections and rapid AF are the most important precipitating factors for hospitalisation and death within 30 d following an episode of heart failure decompensation. Key points Hospitalisation due to heart failure decompensation represents the highest share of healthcare costs for this disease. So far, no primary care studies have analysed the relationship between precipitating factors and short term prognosis of heart failure decompensation episodes. We found that in 692 patients with heart failure decompensation in primary care, the respiratory infection and rapid atrial fibrillation (AF) increased the risk of short-term hospital admission or death. Patients with a hospital admission the previous year and a decompensation episode caused by respiratory infection were even more likely to be hospitalized or die within 30 d