Optimized media and workflow for the expansion of human pluripotent stem cells as aggregates in suspension cultures

3D suspension culture enables scale-up of human pluripotent stem cell (hPSC) manufacturing. However, media and methods optimized for 2D adherent cultures can lead to low volumetric productivity and laborious workflow in suspension cultures. To overcome these limitations we developed fed-batch media based on either mTeSRTM1 (BSA-containing) or TeSRTM-E8TM (animal component-free) for hPSC expansion as aggregates in suspension cultures. Fed-batch feeding protocols are more efficient and cost-effective than batch media changes because only exhausted components are replenished. Optimization studies were performed using human embryonic (H7 and H9), and human induced pluripotent (WLS-1C and STiPS-M001) stem cell lines. Suspension cultures were fed daily using either 50% medium exchanges of standard 2D media, or fed-batch optimized media and protocols. hPSC aggregate diameter must be kept below 350 μm to maintain cell viability and phenotype. With observed growth rates, aggregates required passaging every 3 or 4 days into clumps of 5-10 cells with Gentle Cell Dissociation Reagent. Clumps were re-seeded into fresh test medium plus 10 μM Y-27632. Passaging and feeding cycles were repeated for at least 5 passages. Optimization was performed by iteratively modifying the feed solution to maintain consistent nutrient levels and maximal growth rate while maintaining cell quality. Control and optimized fed-batch formulations demonstrated between 1.4 and 1.8-fold expansion per day, \u3e90% viability, Oct4 and TRA-1-60 expression \u3e90%, in vitro trilineage differentiation, and normal karyotype (n=8 independent cultures). Suspension culture optimized mTeSRTM-3D or TeSRTM-E8TM3D fed-batch media enables the cost-effective production of hPSCs as aggregates with efficient workflow and high cell quality

Optimized media and workflow for the expansion of human pluripotent stem cells as aggregates in suspension

3D suspension culture enables the efficient and cost-effective scale-up of human pluripotent stem cell (hPSCs) manufacturing. However, media optimized for 2D adherent cultures can lead to low volumetric productivity and inefficient workflow. To overcome these limitations we developed mTeSRTM3D, a defined medium based on mTeSRTM1, and novel protocols for fed-batch culture of hPSC aggregates. Human embryonic stem cell (hESC) lines (H1 or H9) or human induced pluripotent stem cell (hiPSC) lines (WLS-1C or STiPS-M001) that were previously maintained in 2D mTeSRTM1 culture were seeded into multiple suspension culture vessels containing mTeSRTM3D Seed Medium plus 10 μM Y-27632 ROCK inhibitor. 3D cultures were maintained using either daily 50% mTeSRTM1 medium exchanges (control) or using a fed-batch protocol whereby the culture medium was supplemented daily with mTeSRTM3D Feed Medium. After 3 or 4 days in suspension culture, aggregates were harvested, dissociated into small clumps with Gentle Cell Dissociation Reagent (GCDR) or single cell suspensions enzymatically, and re-seeded in mTeSRTM3D Seed Medium plus 10 μM Y-27632. Passaging and feeding cycles were repeated for at least 5 passages. 3D cultures were assessed for growth, viability, hPSC marker expression, in vitro differentiation potential, and karyotype. In addition, media was analyzed for molar glucose to lactate yield to characterize metabolism. By day 4, aggregates cultured in mTeSRTM3D typically grew to a mean diameter of 350 μm, with a 5-fold increase in cell number. Using mTeSRTM3D up to 109 cells can be produced from a single plate within 2-3 weeks representing a greater than 500-fold expansion. hPSC cultures maintained in mTeSRTM3D differentiated into all 3 germ layers with high efficiency. The average volumetric productivities were 0.7, 3.1 and 6.9 (x105) viable cells / mL in 2D, daily 50% media exchange, and mTeSRTM3D cultures, respectively. Using the GCDR clump passaging protocol, mTeSRTM3D cultured hPSCs retained normal karyotypes. Culture performance was evaluated in shaker bottles, spinner flasks and bioreactors. Performance in each culture system was comparable confirming straightforward scale-up and wide applicability. Typical growth rates were on the order of 1.5-fold expansion per day. Metabolic activity as assessed by the moles lactate produced to glucose consumed was 1.7, consistent with a primarily glycolytic metabolism. Image analysis was performed to estimate aggregate size during growth. Adaptation times for cells moving from 2D to 3D aggregate culture varied with different cell lines; typically one passage in 3D was required before consistent expansion passage over passage was obtained. Additionally, protocols were developed for use on a Hamilton® robotic platform for reproducible, matrix-free, high-throughput hPSC suspension culture at a small scale. mTeSRTM3D enables efficient scale-up and scale-down of hPSC cultures with greatly simplified workflow

Simple Viscous Flows: from Boundary Layers to the Renormalization Group

The seemingly simple problem of determining the drag on a body moving through a very viscous fluid has, for over 150 years, been a source of theoretical confusion, mathematical paradoxes, and experimental artifacts, primarily arising from the complex boundary layer structure of the flow near the body and at infinity. We review the extensive experimental and theoretical literature on this problem, with special emphasis on the logical relationship between different approaches. The survey begins with the developments of matched asymptotic expansions, and concludes with a discussion of perturbative renormalization group techniques, adapted from quantum field theory to differential equations. The renormalization group calculations lead to a new prediction for the drag coefficient, one which can both reproduce and surpass the results of matched asymptotics

Predictive value of subclinical autistic traits at age 14–15 months for behavioural and cognitive problems at age 3–5 years

It is unclear whether subclinical autistic traits at very young age are transient or stable, and have clinical relevance. This study investigated the relationship between early subclinical autistic traits and the occurrence of later developmental and behavioural problems as well as problems in cognitive and language functioning. Parents of infants aged 14–15 months from the general population completed the Early Screening of Autistic Traits Questionnaire (ESAT). Three groups of children with high, moderate, and low ESAT-scores (total n = 103) were selected. Follow-up assessments included the CBCL 1½–5 at age 3 years, and the SCQ, the ADI-R, the ADOS-G, a non-verbal intelligence test, and language tests for comprehension and production at age 4–5 years. None of the children met criteria for autism spectrum disorder at follow-up. Children with high ESAT-scores at 14–15 months showed significantly more internalizing and externalizing problems at age 3 years and scored significantly lower on language tests at age 4–5 years than children with moderate or low ESAT-scores. Further, significantly more children with high ESAT-scores (14/26, 53.8%) than with moderate and low ESAT-scores (5/36, 13.9% and 1/41, 2.4%, respectively) were in the high-risk/clinical range on one or more outcome domains (autistic symptoms, behavioural problems, cognitive and language abilities). Subclinical autistic traits at 14–15 months predict later behavioural problems and delays in cognitive and language functioning rather than later ASD-diagnoses. The theoretical implications of the findings lie in the pivotal role of early social and communication skills for the development of self-regulation of emotions and impulses. The practical implications bear on the early recognition of children at risk for behavioural problems and for language and cognitive problems

Stable Genetic Influence on Anxiety-Related Behaviours Across Middle Childhood

We examined the aetiology of anxiety symptoms in an unselected population at ages 7 and 9, a period during which anxiety disorders first begin to develop (mean age at onset is 11 years). Specifically, the aim of the study was to investigate genetic and environmental continuity and change in components of anxiety in middle childhood. Parents of over 3,500 twin pairs completed the Anxiety-Related Behaviours Questionnaire (ARBQ) when twins were 7 and 9 years old. Multivariate-longitudinal analyses were conducted to examine genetic and environmental influences on stability and change in four anxiety scales: Negative Cognition, Negative Affect, Fear and Social Anxiety. We found moderate temporal stability in all four scales from 7 to 9 years (correlations ranging from 0.45 to 0.54) and moderate heritability (average 54%). Both shared and non-shared environmental influences were modest (average 18%–28% respectively). Genetic factors (68%) explained most of the homotypic continuity in anxiety. We show that homotypic continuity of Anxiety-Related Behaviours (i.e. the continuation of one specific type of anxiety over time) was largely driven by genetic factors. In contrast, though more varied, heterotypic continuity between some traits (i.e. the change from one type of anxiety-related behaviour into another over time) was mainly due to shared-environmental factors

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Genome-wide associations for birth weight and correlations with adult disease

Birth weight (BW) has been shown to be influenced by both fetal and maternal factors and in observational studies is reproducibly associated with future risk of adult metabolic diseases including type 2 diabetes (T2D) and cardiovascular disease. These life-course associations have often been attributed to the impact of an adverse early life environment. Here, we performed a multi-ancestry genome-wide association study (GWAS) meta-analysis of BW in 153,781 individuals, identifying 60 loci where fetal genotype was associated with BW ($\textit{P}$  < 5 × 10$^{-8}$). Overall, approximately 15% of variance in BW was captured by assays of fetal genetic variation. Using genetic association alone, we found strong inverse genetic correlations between BW and systolic blood pressure ($\textit{R}$ $_{g}$ = -0.22, $\textit{P}$  = 5.5 × 10$^{-13}$), T2D ($\textit{R}$ $_{g}$ = -0.27, $\textit{P}$  = 1.1 × 10$^{-6}$) and coronary artery disease ($\textit{R}$ $_{g}$ = -0.30, $\textit{P}$  = 6.5 × 10$^{-9}$). In addition, using large -cohort datasets, we demonstrated that genetic factors were the major contributor to the negative covariance between BW and future cardiometabolic risk. Pathway analyses indicated that the protein products of genes within BW-associated regions were enriched for diverse processes including insulin signalling, glucose homeostasis, glycogen biosynthesis and chromatin remodelling. There was also enrichment of associations with BW in known imprinted regions ($\textit{P}$ = 1.9 × 10$^{-4}$). We demonstrate that life-course associations between early growth phenotypes and adult cardiometabolic disease are in part the result of shared genetic effects and identify some of the pathways through which these causal genetic effects are mediated.For a full list of the funders pelase visit the publisher's website and look at the supplemetary material provided. Some of the funders are: British Heart Foundation, Cancer Research UK, Medical Research Council, National Institutes of Health, Royal Society and Wellcome Trust

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead