Single-cell RNA sequencing reveals cancer stem-like cells and dynamics in tumor microenvironment during cholangiocarcinoma progression.

Cholangiocarcinoma is a malignancy of the bile ducts that is driven by activities of cancer stem-like cells and characterized by a heterogeneous tumor microenvironment. To better understand the transcriptional profiles of cancer stem-like cells and dynamics in the tumor microenvironment during the progression of cholangiocarcinoma, we performed single-cell RNA analysis on cells collected from three different timepoints of tumorigenesis in a YAP/AKT mouse model. Bulk RNA sequencing data from TCGA (The Cancer Genome Atlas program) and ICGC cohorts were used to verify and support the finding. In vitro and in vivo experiments were performed to assess the stemness of cancer stem-like cells. We identified Tm4sf1high malignant cells as cancer stem-like cells. Across timepoints of cholangiocarcinoma formation in YAP/AKT mice, we found dynamic change in cancer stem-like cell/stromal/immune cell composition. Nevertheless, the dynamic interaction among cancer stem-like cells, immune cells, and stromal cells at different timepoints was elaborated. Collectively, these data serve as a useful resource for better understanding cancer stem-like cell and malignant cell heterogeneity, stromal cell remodeling, and immune cell reprogramming. It also sheds new light on transcriptomic dynamics during cholangiocarcinoma progression at single-cell resolution.

Ionizable Lipid Nanoparticles for Therapeutic Base Editing of Congenital Brain Disease.

Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.

Maternal dendritic cells influence fetal allograft response following murine in-utero hematopoietic stem cell transplantation.

BACKGROUND: Intrauterine hematopoietic stem cell transplantation (IUT), potentially curative in congenital haematological disease, is often inhibited by deleterious immune responses to donor cells resulting in subtherapeutic donor cell chimerism (DCC). Microchimerism of maternal immune cells (MMc) trafficked into transplanted recipients across the placenta may directly influence donor-specific alloresponsiveness, limiting DCC. We hypothesized that dendritic cells (DC) among trafficked MMc influence the development of tolerogenic or immunogenic responses towards donor cells, and investigated if maternal DC-depletion reduced recipient alloresponsiveness and enhanced DCC. METHODS: Using transgenic CD11c.DTR (C57BL/6) female mice enabled transient maternal DC-depletion with a single dose of diphtheria toxin (DT). CD11c.DTR females and BALB/c males were cross-mated, producing hybrid pups. IUT was performed at E14 following maternal DT administration 24 h prior. Bone marrow-derived mononuclear cells were transplanted, obtained from semi-allogenic BALB/c (paternal-derived; pIUT), C57BL/6 (maternal-derived; mIUT), or fully allogenic (aIUT) C3H donor mice. Recipient F1 pups were analyzed for DCC, while maternal and IUT-recipient immune cell profile and reactivity were examined via mixed lymphocyte reactivity functional assays. T- and B-cell receptor repertoire diversity in maternal and recipient cells were examined following donor cell exposure. RESULTS: DCC was highest and MMc was lowest following pIUT. In contrast, aIUT recipients had the lowest DCC and the highest MMc. In groups that were not DC-depleted, maternal cells trafficked post-IUT displayed reduced TCR & BCR clonotype diversity, while clonotype diversity was restored when dams were DC-depleted. Additionally, recipients displayed increased expression of regulatory T-cells and immune-inhibitory proteins, with reduced proinflammatory cytokine and donor-specific antibody production. DC-depletion did not impact initial donor chimerism. Postnatal transplantation without immunosuppression of paternal donor cells did not increase DCC in pIUT recipients; however there were no donor-specific antibody production or immune cell changes. CONCLUSIONS: Though maternal DC depletion did not improve DCC, we show for the first time that MMc influences donor-specific alloresponsiveness, possibly by expanding alloreactive clonotypes, and depleting maternal DC promotes and maintains acquired tolerance to donor cells independent of DCC, presenting a novel approach to enhancing donor cell tolerance following IUT. This may have value when planning repeat HSC transplantations to treat haemoglobinopathies.

Music-based Intervention for Improving Sleep Quality of Adults without Sleep Disorder: A Systematic Review and Meta-analysis.

BACKGROUND: Listening to music is often used as a self-help intervention to improve sleep quality, but its efficacy among individuals without sleep disorder remains unclear. METHODS: A search was performed on five databases to identify for studies that examined the use of music-based intervention to improve sleep quality among individuals without sleep disorder. Random-effects meta-analysis was performed, and the certainty of evidence was evaluated using GRADE (Grading of Recommendations Assessment, Development and Evaluation). RESULTS: Twenty-two articles which recruited 1,514 participants were included for review. Meta-analysis of six studies including 424 participants did not find an improvement in sleep quality among recipients of music-based intervention compared to those with standard care (mean difference: -0.80; 95% CI: -2.15 to 0.54, low-quality evidence). Subgroup analysis showed a clear improvement in sleep quality when interventions were administered for at least 3 weeks (-2.09; -3.84 to -0.34, n = 3). No difference in terms of sleep onset latency (standardized mean difference (SMD) -0.32; 95% CI -0.88 to 0.25, n = 4, very-low quality evidence) and sleep efficiency (SMD: -0.59; 95% CI -3.15 to 1.97, n = 2, very-low quality evidence) were observed. The effect of music-based intervention on anxiety, depression and quality of life were mixed with suggestions of possible benefits. CONCLUSION: Music-based intervention in addition to standard care appears to be a promising strategy to improve sleep quality when delivered for 3 week or longer. However, effects are inconsistent across studies and larger randomized controlled studies reporting long-term outcomes are needed before it can be recommended for routine use. PROSPERO REGISTRATION: CRD42018081193.

The Health Effects of Chocolate and Cocoa: A Systematic Review.

Chocolate has a history of human consumption tracing back to 400 AD and is rich in polyphenols such as catechins, anthocyanidins, and pro anthocyanidins. As chocolate and cocoa product consumption, along with interest in them as functional foods, increases worldwide, there is a need to systematically and critically appraise the available clinical evidence on their health effects. A systematic search was conducted on electronic databases such as MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) using a search strategy and keywords. Among the many health effects assessed on several outcomes (including skin, cardiovascular, anthropometric, cognitive, and quality of life), we found that compared to controls, chocolate or cocoa product consumption significantly improved lipid profiles (triglycerides), while the effects of chocolate on all other outcome parameters were not significantly different. In conclusion, low-to-moderate-quality evidence with short duration of research (majority 4-6 weeks) showed no significant difference between the effects of chocolate and control groups on parameters related to skin, blood pressure, lipid profile, cognitive function, anthropometry, blood glucose, and quality of life regardless of form, dose, and duration among healthy individuals. It was generally well accepted by study subjects, with gastrointestinal disturbances and unpalatability being the most reported concerns.

Use of Composite Outcomes in Neonatal Trials: An Analysis of the Cochrane Reviews.

INTRODUCTION: Composite outcomes are used to increase the power of a study by combining event rates. Many composite outcomes in adult clinical trials have components that differ substantially in patient importance, event rate, and effect size, making interpretation challenging. Little is known about the use of composite outcomes in neonatal randomized controlled trials (RCTs). METHODS: We assessed the use of composite outcomes in neonatal RCTs included in Cochrane Neonatal reviews published till November 2017. Two authors reviewed the components of the composite outcomes to compare their patient importance and computed the ratios of effect sizes and event rates between the components, with an a priori threshold of 1.5, indicating a substantial difference. Descriptive statistics were presented. RESULTS: We extracted 7,766 outcomes in 2,134 RCTs in 312 systematic reviews. Among them, 55 composite outcomes (0.7%) were identified in 46 RCTs. The vast majority (92.7%) of composite outcomes had 2 components, with death being the most common component (included 51 times [92.7%]). The components in nearly three-quarters of the composite outcomes (n = 40 [72.7%]) had different patient importance, while the effect sizes and event rates differed substantially between the components in 27 (49.1%) and 35 (63.6%) outcomes, respectively, with up to 43-fold difference in the event rates observed. CONCLUSIONS: The majority of composite outcomes in neonatal RCTs had different patient importance with contrasting effect sizes and event rates between the components. In patient communication, clinicians should highlight individual components, rather than the composites, with explanation on the relationship between the components, to avoid misleading impression on the effect of the intervention. Future trials should report the estimates of all individual components alongside the composite outcomes presented.

Maternal microchimerism and cell-mediated immune-modulation enhance engraftment following semi-allogenic intrauterine transplantation.

Successful intrauterine hematopoietic cell transplantation (IUT) for congenital hemoglobinopathies is hampered by maternal alloresponsiveness. We investigate these interactions in semi-allogenic murine IUT. E14 fetuses (B6 females × BALB/c males) were each treated with 5E+6 maternal (B6) or paternal (BALB/c) bone marrow cells and serially monitored for chimerism (>1% engraftment), trafficked maternal immune cells, and immune responsiveness to donor cells. A total of 41.0% of maternal IUT recipients (mIUT) were chimeras (mean donor chimerism 3.0 ± 1.3%) versus 75.0% of paternal IUT recipients (pIUT, 3.6 ± 1.1%). Chimeras showed higher maternal microchimerism of CD4, CD8, and CD19 than non-chimeras. These maternal cells showed minimal responsiveness to B6 or BALB/c stimulation. To interrogate tolerance, mIUT were injected postnatally with 5E+6 B6 cells/pup; pIUT received BALB/c cells. IUT-treated pups showed no changes in trafficked maternal or fetal immune cell levels compared to controls. Donor-specific IgM and IgG were expressed by 1%-3% of recipients. mIUT splenocytes showed greater proliferation of regulatory T cells (Treg) upon BALB/c stimulation, while B6 stimulation upregulated the pro-inflammatory cytokines more than BALB/c. pIUT splenocytes produced identical Treg and cytokine responses to BALB/c and B6 cells, with higher Treg activity and lower pro-inflammatory cytokine expression upon exposure to BALB/c. In contrast, naïve fetal splenocytes demonstrated greater alloresponsiveness to BALB/c compared to B6 cells. Thus pIUT, associated with increased maternal cell trafficking, modulates fetal Treg, and cytokine responsiveness to donor cells more efficiently than mIUT, resulting in improved engraftment. Paternal donor cells may be considered alternatively to maternal donor cells for intrauterine and postnatal transplantation to induce tolerance and maintain engraftment.

Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta.

The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony-forming cells (ECFCs) from human term placenta and to compare them to the well-established donor-matched umbilical cord blood (UCB)-derived ECFCs. A sorting strategy was devised to enrich for CD45-CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL-ECFCs) upon culture. UCB-ECFCs were derived using a well-described assay. PL-ECFCs were fetal in origin and expressed the same cell surface markers as UCB-ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL-ECFCs and UCB-ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL-ECFCs and UCB-ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL-ECFCs and UCB-ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL-ECFCs have significant bio-banking and clinical translatability potential.

CD166(pos) subpopulation from differentiated human ES and iPS cells support repair of acute lung injury.

Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient's own iPS cells to prevent immune rejection which arise from allogenic transplantation.

The male seahorse synthesizes and secretes a novel C-type lectin into the brood pouch during early pregnancy.

The male seahorse incubates its young in a manner resembling that of a mammalian pregnancy. After the female deposits her eggs into the male's brood pouch they are fertilized and the embryos develop and grow for several weeks until they are able to withstand the external environmental conditions independently, at which point they are irreversibly released. Although the precise function of the brood pouch is not clear, it is probably related to providing a suitable protective and osmotic environment for the young. The aim of this project was to construct and characterize a cDNA library made from the tissue lining the pouch, in order to help understand the molecular mechanisms regulating its development and function. The library profile indicates expression of genes encoding proteins involved in metabolism and transport, as well as structural proteins, gene regulatory proteins, and other proteins whose function is unknown. However, a large portion of the library contained genes encoding C-type lectins (CTLs), of which three full-length proteins were identified and found to contain a signal peptide and a single C-lectin domain, possessing all the conserved structural elements. We have produced recombinant protein for one of these and raised antisera; we have shown, using Western analysis and 2D electrophoresis, that this protein is secreted in significant quantities into the pouch fluid specifically during early pregnancy. Preliminary functional studies indicate that this CTL causes erythrocyte agglutination and may help to repress bacterial growth.