Neurosphere based differentiation of human iPSC improves astrocyte differentiation

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6(+)/NESTIN(+) cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

[Thyroid hormone T3 triggers the developmental loss of axonal regenerative capacity]

International audienceno abstrac

Bmi1 Loss in the Organ of Corti Results in p16^ink4a Upregulation and Reduced Cell Proliferation of Otic Progenitors <i>In Vitro</i>

<div><p>The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16^ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16^ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16^ink4a <i>in vitro</i>. Viral vector-mediated overexpression of p16^ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres <i>in vitro</i>. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16^ink4a.</p></div

Sphere-forming capacity of the neonatal Bmi1 knockout organ of Corti.

<p><b>(A)</b> Average number of spheres per OC, expressed as a percentage of the WT control (mean ± standard deviation). The data represent the results from a total of 9 WT, 19 Het and 6 KO p0 mice pooled from 4 independent experiments. Cultures from the KO mice generated significantly fewer spheres compared to their WT littermates (one-way ANOVA followed by Tukey‘s post-hoc test, p<0.05). The difference between the WT and Het mice is not significant. (<b>B, D, F and H</b>) Quantification of DAPI, Ki67, EdU and pHH3. Each data point represents one sphere. Mean and standard deviation values are shown in the dot plots. <b>(B)</b> Number of DAPI-labeled cells per sphere. The number of cells per sphere is significantly reduced for the Bmi1 KO spheres compared to the WT spheres (p<0.001, Dwass-Steel test; n = 300 spheres per group). The difference between WT and Het mice is not statistically significant. (<b>C, E and G</b>) Representative images of the WT and KO spheres, labeled for Ki67 (<b>C</b>), EdU (<b>E</b>) and pHH3 (<b>G</b>). (<b>D</b>) Percentage of Ki67-positive cells in the spheres: the KO spheres harbor a significantly lower percentage of Ki67-positive cells compared with the WT spheres (p<0.01, Dwass-Steel; n = 100 spheres per group). There was no significant difference between the WT and Het mice, or between Het and KO mice. (<b>F</b>) Percentage of EdU-incorporating cells in the spheres: the KO spheres contain a significantly lower percentage of EdU-positive cells compared with the WT spheres and Het spheres (p<0.001, Dwass-Steel test; n = 100 spheres per group). The difference between the WT and Het mice is not significant. (<b>H</b>) Percentage of pHH3-positive cells in the spheres: the percentage of pHH3-expressing cells is significantly lower in the KO spheres compared with the WT spheres (p<0.05, Dwass-Steel test; n = 100 spheres per group). There was no statistically significant difference between the WT and Het spheres. WT: wildtype, Het: heterozygous, KO: knockout. *p<0.05, **p<0.01, ***p<0.001.</p

Effect of viral vector-mediated p16^ink4a overexpression on the sphere-forming capacity of the organ of Corti.

<p><b>(A)</b> Quantitative analysis of p16^ink4a mRNA levels in otospheres, derived from wildtype organ of Corti specimens after 5 days <i>in vitro</i>. The organ of Corti-derived cells were incubated with either of two viral vectors: i) Ad-GFP to induce the expression of GFP, or Ad-p16-GFP to induce the expression of both GFP and p16^ink4a. The spheres incubated with Ad-p16-GFP showed a 765-fold increase in p16^ink4a mRNA levels compared to the spheres incubated with Ad-GFP (n = 2 independent samples, measured in triplicate, for both groups). The difference in p16^ink4a mRNA levels between the Ad-GFP and Ad-p16-GFP groups was highly statistically significant (Student’s t-test, p<0.001). <b>(B)</b> Average number of spheres generated after 5 days <i>in vitro</i> per 50000 cells plated (mean ± standard deviation). Cells incubated with Ad-p16-GFP generated significantly fewer spheres, after 5 days <i>in vitro</i>, compared to the cells incubated with Ad-GFP (N = 3 independent experiments, n = 5 replicates per experiment, Student’s t-test, p<0.001). ***p<0.001.</p

Gene expression changes in the Bmi1 knockout organ of Corti and otospheres.

<p><b>(A)</b> Representative high-magnification images of Bmi1 WT and KO spheres after 5DIV, stained for Bmi1 (green) and counterstained with DAPI (blue). Nuclear Bmi1 signal was detected in the WT, but not the KO, spheres. <b>(B)</b> Quantitative analysis of Bmi1 mRNA levels in the WT, Het and KO spheres by qRT-PCR. The Het spheres showed Bmi1 mRNA levels that are 0.59-fold relative to the WT spheres. Limited amounts of mRNA were detected in the KO spheres, representing 0.07-fold compared with the WT control. <b>(C)</b> Quantitative analysis of Bmi1 transcript levels in the neonatal WT, Het and KO OC by qRT-PCR. When normalized to the WT control, the Bmi1 mRNA levels in the Het OC were 0.67-fold of the WT level. In the KO OC, the level of mRNA detected was 0.06-fold of the WT expression level. <b>(D)</b> Quantitative analysis of p16^ink4a transcript levels in the WT, Het and KO spheres by qRT-PCR. In the Het spheres, p16^ink4a is upregulated 1.82-fold relative to the WT spheres. The KO spheres show an 18.38-fold upregulation of p16^ink4a compared to the WT spheres. In the neonatal KO OC, only scarce amounts of p16^ink4a mRNA were detected, representing 0.05-fold compared to the WT spheres. WT: wildtype, Het: heterozygous, KO: knockout. n.s.: not significant. ***p<0.001.</p

Bmi1 expression in the cochlear sensory epithelium.

<p><b>(A, B, and D)</b> Immunohistochemical staining was performed using an anti-Bmi1 antibody on wildtype mice (Bmi1^WT/WT, referred to as WT). <b>(E and G)</b> Alternatively, immunolabeling was performed using an anti-GFP antibody on Bmi1-GFP heterozygous mice (Bmi1^GFP/WT, referred to as Het). OC sections <b>(A-C, E and F)</b> were co-stained for Sox2 (red) and Myosin7a (white) as markers of supporting cells and hair cells, respectively. Hair cells are marked by hollow white arrows, and supporting cells are indicated by solid white arrows. Spiral ganglion sections <b>(D and G)</b> were co-labeled with Sox10 (red) and NeuN (white) to serve as markers of glial and neuronal cells, respectively. Spiral ganglion neurons are labeled with hollow white arrowheads, and glial cells are marked with solid white arrowheads. <b>(A)</b> Bmi1 is expressed in the immature OC at p0. Both hair cells and supporting cells were labeled for Bmi1. <b>(B)</b> Bmi1 expression was detected in hair and supporting cells of the functionally mature OC at p28. <b>(C)</b> Cochleae of homozygous Bmi1-GFP mice (Bmi1^GFP/GFP, referred to as KO) served as a negative control for Bmi1 immunohistochemistry. <b>(D and G)</b> Bmi1 expression co-localized with the neuronal marker NeuN in spiral ganglion neurons. Sox10-positive glial cells did not show Bmi1 expression. <b>(E)</b> Bmi1-GFP signal was observed in hair and supporting cells of the OC at p28. <b>(F)</b> Cochleae of WT mice served as a negative control for the Bmi1-GFP signal. Nuclei were labeled with DAPI in all sections. Solid white arrows indicate supporting cells, while hollow arrows point to inner and outer hair cells. IHC: inner hair cells, OHC: outer hair cells, SC: supporting cells, SG: spiral ganglion, SGN: spiral ganglion neurons. WT: wildtype, Het: heterozygous, KO: knockout. Scale: 20 μm.</p