Intestinal crypts reproducibly expand in culture

In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to: first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro

Intestinal stem cells remain viable after prolonged tissue storage

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such they have significant therapeutic potential. However, it is unknown whether ISCs can survive tissue storage. We hypothesized that, although the majority of epithelial cells may die, ISCs would remain viable for at least 24 h at 4°C. To explore this hypothesis, jejuni of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate buffered saline (PBS) at 4°C. Delayed isolations of epithelia were performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact through 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retain high integrity through 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Culture of isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80%. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated with human tissue, storage at 4°C may offer a valuable temporal window for harvest of crypts or ISCs for therapeutic application

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

PTP-S2, a nuclear tyrosine phosphatase, is phosphorylated and excluded from condensed chromosomes during mitosis

PTP-S2 is a tyrosine specific protein phosphatase that binds to DNA and is localized to the nucleus in association with chromatin. It plays a role in the regulation of cell proliferation. Here we show that the subcellular distribution of this protein changes during cell division. While PTP-S2 was localized exclusively to the nucleus in interphase cells, during metaphase and anaphase it was distributed throughout the cytoplasm and excluded from condensed chromosomes. At telophase PTP-S2 began to associate with chromosomes and at cytokinesis it was associated with chromatin in the newly formed nucleus. It was hyperphosphorylated and showed retarded mobility in cells arrested in metaphase.In vitro experiments showed that it was phosphorylated by CK2 resulting in mobility shift. Using a deletion mutant we found that CK2 phosphorylated PTP-S2 in the C-terminal non-catalytic domain. A heparin sensitive kinase from mitotic cell extracts phosphorylated PTP-S2 resulting in mobility shift. These results are consistent with the suggestion that during metaphase PTP-S2 is phosphorylated (possibly by CK2 or a CK2-like enzyme), resulting in its dissociation from chromatin

The role of maternal CREB in early embryogenesis of Xenopus laevis

AbstractIn Xenopus embryos, body patterning and cell specification are initiated by transcription factors, which are themselves transcribed during oogenesis, and their mRNAs are stored for use after fertilization. We have previously shown that the T-box transcription factor VegT is both necessary and sufficient to initiate transcription of all endoderm, and most mesoderm genes. In the absence of maternal VegT, no mesodermal organs (including the heart) or endodermal organs form. A second maternal transcription factor XTcf3 acts as a global repressor of transcription of dorsal genes, whose repression is inactivated on the dorsal side by a maternally encoded Wnt signaling pathway. In the absence of β-catenin, no mesodermal or endodermal organs form. We show here that the maternally encoded transcription factor CREB is also essential for development. It is required for the initiation of expression of several mesodermal genes, including Xbra, Xcad2, and -3 and also regulates the cardiogenic gene Nkx 2-5. We show that maternal CREB-depleted embryos develop gastrulation defects that are rescued by the reintroduction of activated CREB mRNA. We conclude that maternal CREB must be added to the list of essential maternal transcription factors regulating cell specification in the early embryo

Enhanced expression of a chromatin associated protein tyrosine phosphatase during G<SUB>0</SUB> to S transition

The non-transmembrane protein tyrosine phosphatase, PTP-S, is located predominantly in the cell nucleus in association with chromatin. Here we have analysed the expression of PTP-S upon mitogenic stimulation and during cell division cycle. During liver regeneration after partial hepatectomy, PTP-S mRNA levels increased 16-fold after 6 h (G<SUB>1</SUB> phase) and declined thereafter. Upon stimulation of serum starved cells in culture with serum, PTP-S mRNA levels increased reaching a maximum during late G<SUB>1</SUB> phase and declined thereafter. No significant change in PTP-S RNA levels was observed in growing cells during cell cycle. PTP-S protein levels were also found to increase upon mitogenic stimulation. Upon serum starvation for 72 h, PTP-S protein disappears from the nucleus and is seen in the cytoplasm; after 96 h of serum starvation the PTP-S protein disappears from the nucleus as well as cytoplasm. Refeeding of starved cells for 6 h results in reappearance of this protein in the nucleus. Our results suggest a role of this phosphatase during cell proliferation

In vivo human PSC-derived intestinal organoids to study stem cell maintenance

International audienceHuman intestinal organoids (HIOs), derived from pluripotent stem cells, are a new tool to gain insights in gastrointestinal development, physiology and associated diseases. Herein, we present a method for orthotopic renal transplantation of HIOs in immunocompromised mice and subsequent analysis to study intestinal epithelial proliferation. In addition, we describe how to generate enteroids from transplanted HIOs. The method highlights the specific steps to successful engraftment and provides insight into the study of human intestinal stem cells

In Vivo Human PSC-Derived Intestinal Organoids to Study Stem Cell Maintenance

International audienceHuman intestinal organoids (HIOs), derived from pluripotent stem cells, are a new tool to gain insights in gastrointestinal development, physiology, and associated diseases. Herein, we present a method for renal transplantation of HIOs in immunocompromised mice and subsequent analysis to study intestinal epithelial cell proliferation. In addition, we describe how to generate enteroids from transplanted HIOs. The method highlights the specific steps to successful engraftment and provides insight into the study of human intestinal stem cells

Evaluation of transplantation sites for human intestinal organoids.

Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS