Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells.

BACKGROUND: Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. METHODS: This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers. RESULTS: Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media. CONCLUSIONS: These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.

The impact of early adversity and education on genetic and brain morphological predictors of cognitive ability.

Cognitive ability is a strong predictor of occupational achievement, quality of life and physical health. While variation in cognition is strongly heritable and has been robustly associated with early environment and brain morphology, little is known about how these factors combine and interact to explain this variation in cognition. To address this, we modelled the relationship between common genetic variation, grey matter volume, early life adversity and education and cognitive ability in a UK Biobank sample of N = 5237 individuals using structural equation modelling. We tested the hypotheses that total grey matter volume would mediate the association between genetic variation and cognitive ability, and that early life adversity and educational attainment would moderate this relationship. Common genetic variation, grey matter volume and early life adversity were each significant predictors in the model, explaining ~15% of variation in cognitive ability. Contrary to our hypothesis, grey matter volume did not mediate the relation between genetic variation and cognition performance. Neither did early life adversity or educational attainment moderate this relation, although educational attainment was observed to moderate the relationship between grey matter volume and cognitive performance. We interpret these findings in terms of the modest explanatory value of currently estimated polygenic scores accounting for variation in cognitive performance (~5%), making potential mediating and moderating variables difficult to confirm.

Serologic reactivity of unidentified specificity in antenatal testing and hemolytic disease of the fetus and newborn: The BEST collaborative study.

BACKGROUND: The clinical significance of serologic reactivity of unidentified specificity (SRUS) in pregnancy is not clear based on available literature. The aim of this study is to determine if SRUS is associated with hemolytic disease of the fetus and newborn (HDFN). STUDY DESIGN AND METHODS: Retrospective data were collected from eight institutions over an 11-year study period (2010-2020), when available (5/8 sites). The outcome of the pregnancies with SRUS-no, mild, moderate, or severe HDFN-was determined. RESULTS: SRUS was demonstrated in 589 pregnancies. After excluding those with incomplete data, a total of 284 pregnancies were included in the primary HDFN outcome analysis. SRUS was detected in 124 (44%) pregnancies in isolation, and none were affected by HDFN. Of 41 pregnancies with SRUS and ABO incompatibility, 37 (90%) were unaffected, and 4 (10%) were associated with mild HDFN. Of 98 pregnancies with SRUS and concurrent identifiable antibody reactivity(s), 80 (81%) were unaffected, and 19 (19%) were associated with mild to severe HDFN. There was 1 case of mild HDFN and 1 case of severe HDFN in the 21 pregnancies with SRUS, ABO incompatibility, and concurrent identifiable antibody reactivity(s), and 19 (90%) were unaffected by HDFN. Among all patients with repeat testing, newly identified alloantibodies or other antibodies were identified in 63 of 212 (30%) patients. Although most were not clinically significant, on occasion SRUS preceded clinically significant antibody(s) associated with HDFN (3%, 5/188). CONCLUSION: The antenatal serologic finding of SRUS in isolation is not associated with HDFN but may precede clinically significant antibodies.

The effect of clinician education on blood transfusion practice in obstetrics: completing the audit cycle.

BACKGROUND: With blood products being a limited and expensive resource within the healthcare system, there is an ever-increasing emphasis on judicial and appropriate use. AIMS: To evaluate whether implementing contemporary society recommendations on restrictive transfusion policies would reduce inappropriate use of red blood cell transfusions, by evaluating the effect of a staff educational campaign. METHODS: An audit of peri-partum red cell concentrate (RCC) transfusion practice within a tertiary obstetric unit was undertaken, covering a 1-year period (2015), examining data related to transfusion prescribing practices. Subsequently, an educational programme was held for clinical and laboratory staff which aimed to bring practice in line with society guidelines. A repeat audit covering another 1-year period (2018) was undertaken. RESULTS: The number of RCC units of transfused reduced by 49% between 2015 and 2018 (426 to 218). The number of patients receiving transfusion dropped from 166 in 2018 (1.8% of births) to 119 in 2015 (1.5% of births). Among stable patients who were transfused, the proportion receiving a single unit increased from 6.9 to 53.9%. (p < 0.001). Haematological reassessment between units rose from 13.8 to 80.4% (p < 0.001). Written consent documentation improved (68% in 2018 vs. 38% in 2015) (p < 0.001). CONCLUSIONS: The implementation of guidelines has resulted in substantial reduction in RCC transfusions between 2015 and 2018. Fewer women received a blood transfusion, and those who did received fewer units. There is a higher proportion of patients being reassessed between units and receiving single unit transfusions. Recording of consent has improved.

Toxicological Assessment of Pure Lolitrem B and Ryegrass Seed Infected with the AR37 Endophyte Using Mice.

Fungal endophytes in perennial ryegrass are essential to New Zealand's pastoral system due to anti-insect effects. However, endophytes also produce compounds which can be detrimental to animals. Furthermore, as these toxins have been detected in the milk and fat of animals grazing common-toxic (containing lolitrem B) or AR37 endophyte-infected herbage they could enter the human food chain. To assess the risk to human health mice were fed for 90 days with three dose rates of lolitrem B and of AR37. Parameters indicative of animal health were measured as well as chemical, hematological and histological analysis of samples collected on day 90. Since endophyte toxin residues have been detected in milk, they could be transferred from mother to offspring via breast milk. To evaluate possible effects on reproduction two complete generations of mice were fed lolitrem B or AR37. At the dose rates given no adverse effects were observed in either study. The 100-fold safety factor to allow the use of animal data in human health assessments was applied and by considering the concentrations of lolitrem B or AR37 metabolites which could be ingested by a consumer it is highly unlikely that they pose any risk to human health.

GMP-Compliant Production of Autologous Adipose-Derived Stromal Cells in the NANT 001 Closed Automated Bioreactor.

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

The rate of decline in fetal hemoglobin following intrauterine blood transfusion in the management of red cell alloimmunization.

OBJECTIVE: Hemolytic disease of the fetus and newborn is characterized by fetal anemia, secondary to maternal alloantibody-mediated fetal erythrocyte destruction. Despite our reliance on intrauterine blood transfusion (IUT) to maintain severely affected pregnancies, it remains difficult to predict the fetal response to an infusion of donor blood. Our objective was to determine the daily rate of decline in fetal hemoglobin following one, two, and three transfusions. We also evaluated the relationship between the fetal hemoglobin level and the corresponding doppler measurement of the fetal middle cerebral artery peak systolic velocity (MCA-PSV). STUDY DESIGN: A prospective observational study of all singleton pregnancies treated with intrauterine transfusion for fetal anemia secondary to maternal alloimmunization at the National Maternity Hospital, a tertiary referral centre, was conducted over a 10-year period (2011-2020). Demographic and clinical data was obtained from the electronic patient records. Ethical approval was granted by the Ethics and Research Committee of the National Maternity Hospital. RESULTS: A total of 90 intrauterine blood transfusions were performed in 41 fetuses affected by maternal alloimmunization, of which 70% (n = 29), 34% (n = 14) and 15% (n = 6) required a 2nd, 3rd, and 4th transfusion, respectively. The mean rate of decline in fetal hemoglobin following the first transfusion was 0.4 g/dl/day (range, 0.12-0.64 g/dl/day). The mean rate of decline was lower after repeat transfusions at 0.27 g/dl/day (range, 0.16-0.45 g/dl/day). The sensitivity of MCA-PSV threshold of 1.5 Multiples of the Median (MoM) to detect moderate-severe anaemia declined with rank of IUT, from 82% after one previous transfusion, to 75% after two or more previous transfusions. No fetal mortality was seen in our series. CONCLUSION: Knowledge of the expected rate of decline in fetal hemoglobin following an IUT aids in the determination of appropriate timing of subsequent transfusions in a fetus affected by red cell alloimmunization. We observed a reducing rate of daily decline in hemoglobin in fetuses requiring successive transfusions. Our findings suggest a reduced accuracy of the MCA-PSV threshold of 1.5 MoM in determining the optimal timing of 2nd, 3rd, and 4th transfusions.

Thirteen Independent Genetic Loci Associated with Preserved Processing Speed in a Study of Cognitive Resilience in 330,097 Individuals in the UK Biobank.

Cognitive resilience is the ability to withstand the negative effects of stress on cognitive functioning and is important for maintaining quality of life while aging. The UK Biobank does not have measurements of the same cognitive phenotype at distal time points. Therefore, we used education years (EY) as a proxy phenotype for past cognitive performance and current cognitive performance was based on processing speed. This represented an average time span of 40 years between past and current cognitive performance in 330,097 individuals. A confounding factor was that EY is highly polygenic and masked the genetics of resilience. To overcome this, we employed Genomics Structural Equation Modelling (GenomicSEM) to perform a genome-wide association study (GWAS)-by-subtraction using two GWAS, one GWAS of EY and resilience and a second GWAS of EY but not resilience, to generate a GWAS of Resilience. Using independent discovery and replication samples, we found 13 independent genetic loci for Resilience. Functional analyses showed enrichment in several brain regions and specific cell types. Gene-set analyses implicated the biological process "neuron differentiation", the cellular component "synaptic part" and the "WNT signalosome". Mendelian randomisation analysis showed a causative effect of white matter volume on cognitive resilience. These results may contribute to the neurobiological understanding of resilience.

Cell-based strategies for IVD repair: clinical progress and translational obstacles.

Intervertebral disc (IVD) degeneration is a major cause of low back pain, a prevalent and chronic condition that has a striking effect on quality of life. Currently, no approved pharmacological interventions or therapies are available that prevent the progressive destruction of the IVD; however, regenerative strategies are emerging that aim to modify the disease. Progress has been made in defining promising new treatments for disc disease, but considerable challenges remain along the entire translational spectrum, from understanding disease mechanism to useful interpretation of clinical trials, which make it difficult to achieve a unified understanding. These challenges include: an incomplete appreciation of the mechanisms of disc degeneration; a lack of standardized approaches in preclinical testing; in the context of cell therapy, a distinct lack of cohesion regarding the cell types being tested, the tissue source, expansion conditions and dose; the absence of guidelines regarding disease classification and patient stratification for clinical trial inclusion; and an incomplete understanding of the mechanisms underpinning therapeutic responses to cell delivery. This Review discusses current approaches to disc regeneration, with a particular focus on cell-based therapeutic strategies, including ongoing challenges, and attempts to provide a framework to interpret current data and guide future investigational studies.

Cognitive Genomics: Recent Advances and Current Challenges.

PURPOSE OF REVIEW: We review recent progress in uncovering the complex genetic architecture of cognition, arising primarily from genome-wide association studies (GWAS). We explore the genetic correlations between cognitive performance and neuropsychiatric disorders, the genetic and environmental factors associated with age-related cognitive decline, and speculate about the future role of genomics in the understanding of cognitive processes. RECENT FINDINGS: Improvements in genomic methods, and the increasing availability of large datasets via consortia cooperation, have led to a greater understanding of the role played by common and rare variants in the genomics of cognition, the highly polygenic basis of cognitive function and dysfunction, and the multiple biological processes involved. Recent research has aided in our understanding of the complex biological nature of genomics of cognition. Further development of data banks and techniques to analyze this data hold significant promise for understanding cognitive ability, and for treating cognitively related disability.

Marine Collagen Substrates for 2D and 3D Ovarian Cancer Cell Systems.

A fundamental structural component of extracellular matrix in all connective and interstitial tissue, collagen is the most abundant protein in the human body. To date, mammalian collagens sources represent the golden standard for multiple biomedical applications, while marine-derived collagens have largely been used in industry (food, pharmaceutical, and cosmetic), with little use in research and clinical applications. Herein we demonstrate the effective use Rhizostoma pulmo jellyfish collagen, a source of biocompatible, sustainable collagen for 2D and 3D cell culture, addressing the global drive for technological developments that result in the replacement of animals and their derived products in research. Jellyfish collagen harbors similar structural features mammalian collagen type I, despite differing slightly in amino acid content. Jellyfish collagen supports ovarian cancer (OvCa) cell line proliferation, cellular morphology and expression of epithelial to mesenchymal transition markers, supporting the use of R. pulmo as a non-mammalian collagen cell culture substrate. Furthermore, R. pulmo collagen is effective in 3D device fabrication such as sponges where it mimics tissue architecture complexity. OvCa cells migrated and differentiated within the R. pulmo collagen 3D scaffolds confirming its suitability for advanced cell culturing applications, providing an excellent alternative to mammalian collagen sources for the culture of human cells.

Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency.

Mesenchymal stromal cell (MSC) therapies have been pursued for a broad spectrum of indications but mixed reports on clinical efficacy have given rise to some degree of skepticism regarding the effectiveness of this approach. However, recent reports of successful clinical outcomes and regulatory approvals for graft-versus-host disease, Crohn's disease and critical limb ischemia have prompted a shift in this perspective. With hundreds of clinical trials involving MSCs currently underway and an increasing demand for large-scale manufacturing protocols, there is a critical need to develop standards that can be applied to processing methods and to establish consensus assays for both MSC processing control and MSC product release. Reference materials and validated, uniformly applied tests for quality control of MSC products are needed. Here, we review recent developments in MSC manufacturing technologies, release testing and potency assays. We conclude that, although MSCs hold considerable promise clinically, economies of scale have yet to be achieved although numerous bioreactor technologies for scalable production of MSCs exist. Additionally, rigorous disease-specific product testing and comprehensive understanding of mechanisms of action, which are linked to relevant process and product release potency assays, will be required to ensure that these therapies continue to be successful.

Access to Preventive Oral Health Services for Homebound Populations: A pilot program.

Purpose: Certified Public Health Dental Hygienists (CPHDH) perform traditional dental hygiene scope of practice duties, along with caries stabilization (interim therapeutic restorations) through collaborative agreements with a dentist, in the state of New Hampshire. The purpose of this pilot study was to assess the oral health status, dental needs, including referral and utilization, and satisfaction of care received by homebound individuals in their place of residence when provided by a CPHDH.Methods: A purposive sample of homebound individuals participated in a mixed methods study that included quantitative data from an intake survey, a retrospective chart review, and qualitative in-depth interviews.Results: Study participants (n=15) had an average of 22.4 natural teeth; 44% of participants had not seen a dentist for two or more years. Forty-three percent of participants required a referral to a dentist due to dental needs beyond the scope of the CPHDH. Themes from the interviews included: difficulty in accessing a traditional dental care delivery model despite a high value placed on oral health and a high need for dental care. In general, participants expressed satisfaction with care received by the CPHDH.Conclusion: Participants reported a positive experience and satisfaction with care received from a CPHDH suggesting that this is a viable approach to provide preventive oral health services and caries stabilization to populations with complex access to care challenges.

The contribution of massive fetomaternal hemorrhage to antepartum stillbirth: a 25-year cross-sectional study.

INTRODUCTION: Fatal antepartum fetomaternal hemorrhage is a relatively uncommon clinical presentation, though one that appears quickly and without warning. The pathophysiology of this disease is unclear, and the incidence does not appear to be decreasing in line with overall antepartum mortality. This study was undertaken to analyse trends in antepartum fetal death from fetomaternal hemorrhage over a 25-year period in a single maternity hospital in Dublin, Ireland. MATERIAL AND METHODS: A cross-sectional study of 192 132 nonanomalous infants weighing 500 g or more, delivered in a single tertiary-referral university institution between 1987 and 2011. Data was compared using Fisher's exact test, univariate analysis, and Cuzick's test for trend. RESULTS: There was no decrease in the rate of fatal fetomaternal hemorrhage over the past 25 years (p = 0.29), despite a decline in overall antepartum deaths (p = 0.0049). Fetomaternal hemorrhage accounted for 4.1% (34/828) of antepartum stillbirths. A higher proportion of these stillbirths occurred at term gestations (74%; 25/34) compared with other causes (40%; 321/794; p = 0.0003). Female infants were statistically more likely to be involved than males [odds ratio (OR) 2.33, 95% confidence interval (CI) 1.08-5.47, p = 0.02). Multiple gestations were up to six times as likely to be affected as singleton pregnancies (OR 6.52, 95% CI 1.67-18.50, p = 0.005). CONCLUSIONS: Over the past 25 years there has been no reduction in rates of fatal fetomaternal hemorrhage. Female infants and multiple gestations remain at higher risk of antepartum death from fatal fetomaternal hemorrhage.

A V740L mutation in glycoprotein IIb defines a novel epitope (War) associated with fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Most recently described human platelet antigens (HPAs) have been detected in cases of fetomaternal alloimmune thrombocytopenia (FMAIT) where the mother has been immunized against a low-frequency antigen that the fetus has inherited from the father. Low-frequency antigens are not represented in normal panel platelets (PLTs) and antibody detection and identification in such cases requires incubation of maternal serum with paternal PLTs and definition of the causative mutation. STUDY DESIGN AND METHODS: A suspected case of FMAIT was investigated for PLT-specific antibodies using a panel of both HPA-typed and paternal PLTs. HPA typing was performed by polymerase chain reaction with sequence-specific primers and further DNA analysis was performed using direct sequencing of the coding regions of the ITGA2B and ITGB3 genes. RESULTS: Maternal antibodies reactive only with paternal PLTs were localized to glycoprotein (GP)IIb/IIIa using the monoclonal antibody immobilization of PLT antibody assay. A single-nucleotide polymorphism was detected in Exon 23 of ITGA2B in the father and affected child, which predicted a V740L substitution in the mature protein. Recombinant V740L mutated GPIIb expressed in HEK293 cells was specifically recognized by maternal antibodies. The polymorphism was not detected either in the mother or in a cohort of 100 donors. CONCLUSION: The V740L polymorphism defines a new low-frequency antigen implicated in two cases of FMAIT in a single family. Low-frequency HPAs are clinically important and their elucidation requires both crossmatch studies and gene sequencing in cases where there is strong clinical evidence of FMAIT but initial laboratory investigations do not support the diagnosis.

Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Enzyme-linked immunosorbent assay for the detection of yessotoxin and its analogues.

Polyclonal antibodies were produced for the development of competitive enzyme-linked immunoassays for use in quantifying yessotoxins in shellfish, algal cells, and culture supernatants. Immunizing and plate coating antigens were prepared by derivatization of yessotoxin either by ozonolysis or bromination and conjugation to proteins. Two assays that were the most sensitive for yessotoxin were optimized and characterized. Cross-reactivity studies indicated that the antibodies raised have broad specificity and that binding to analogues was strongly affected by changes to the A-ring and, to a lesser extent, the K-ring regions of the toxin molecule. ELISA provides a sensitive and rapid analytical method that is suitable for screening large numbers of samples and detects all the yessotoxin analogues that the European Commission currently requires shellfish to be tested for. The assay limit of quantitation for yessotoxin in whole shellfish flesh is 75 microg/kg; therefore, assay sensitivity is sufficient to measure toxin levels well below the maximum permitted level set by the European Commission. The antibodies produced can be used in additional applications such as the immunolocalization of yessotoxins in shellfish and preparation of immunoaffinity columns.