RosetteArray® Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk.

Neural organoids have revolutionized how human neurodevelopmental disorders (NDDs) are studied. Yet, their utility for screening complex NDD etiologies and in drug discovery is limited by a lack of scalable and quantifiable derivation formats. Here, we describe the RosetteArray® platform's ability to be used as an off-the-shelf, 96-well plate assay that standardizes incipient forebrain and spinal cord organoid morphogenesis as micropatterned, 3-D, singularly polarized neural rosette tissues (>9000 per plate). RosetteArrays are seeded from cryopreserved human pluripotent stem cells, cultured over 6-8 days, and immunostained images can be quantified using artificial intelligence-based software. We demonstrate the platform's suitability for screening developmental neurotoxicity and genetic and environmental factors known to cause neural tube defect risk. Given the presence of rosette morphogenesis perturbation in neural organoid models of NDDs and neurodegenerative disorders, the RosetteArray platform could enable quantitative high-throughput screening (qHTS) of human neurodevelopmental risk across regulatory and precision medicine applications.

Self-organization of embryonic stem cells into a reproducible embryo model through epigenome editing.

Embryonic stem cells (ESCs) can self-organize in vitro into developmental patterns with spatial organization and molecular similarity to that of early embryonic stages. This self-organization of ESCs requires transmission of signaling cues, via addition of small molecule chemicals or recombinant proteins, to induce distinct embryonic cellular fates and subsequent assembly into structures that can mimic aspects of early embryonic development. During natural embryonic development, different embryonic cell types co-develop together, where each cell type expresses specific fate-inducing transcription factors through activation of non-coding regulatory elements and interactions with neighboring cells. However, previous studies have not fully explored the possibility of engineering endogenous regulatory elements to shape self-organization of ESCs into spatially-ordered embryo models. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize ESCs into early embryonic models. Our results show that CRISPR-based activation (CRISPRa) of only two endogenous regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that the emergence of spatially-ordered embryonic patterns happens through the intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-Programmed Embryo Models (CPEMs) show highly consistent composition of major embryonic cell types that are spatially-organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CPEMs with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings offer a programmable embryo model and demonstrate that minimum intrinsic epigenome editing is sufficient to self-organize ESCs into highly consistent pre-gastrulation embryo models.

Reproducibility and stability of the immature platelet fraction using Sysmex XN-10.

BACKGROUND: The Immature Platelet Fraction (IPF) is an indicator of thrombopoiesis which is a useful parameter in thrombocytopenia. It demonstrates compensatory mechanisms in production of platelets, but currently not implemented in routine clinical practice. The aim of this study was to establish the reproducibility and stability of IPF, for both percentage (%-IPF) and absolute (A-IPF) measurements.Material/methods: A total of 71 samples, of which 45 for reproducibility and 26 for stability analysis, were assayed for full blood count using the Sysmex XN-10 analyser at room temperature (RT:19-25 °C). For reproducibility analysis, IPF measurements were analysed 11 times by different appraisers using the same sample, while for stability analysis, IPF was measured over fourteen hourly-intervals up to 24 h (n = 21) and then separately extended beyond the point of stability to 72 h (n = 5). RESULTS: Reproducibility analysis of %-IPF and A-IPF (n = 45) showed very reliable results, with the range of mean CV% values between 1.25-8.90% and 1.70-9.96%, respectively. On the other hand, overall, stability analysis of %-IPF and A-IPF (n = 21) at RT over 24 h showed reliable results, with pooled mean CV% values of 1.32% and 1.43%, respectively, with no significant difference between %-IPF and A-IPF (p = 0.767 and p = 0.821). All %-IPF and A-IPF values had exceeded the set acceptance criterion of stability (CV% ≥ 10.0%) before 72 h. CONCLUSIONS: Overall, %-IPF and A-IPF reproducibility and storage at RT for 24 h predominantly demonstrates the suitability of their usage for testing on the Sysmex XN-series analysers.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

Tracking and Predicting Human Somatic Cell Reprogramming Using Nuclear Characteristics.

Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) generates valuable resources for disease modeling, toxicology, cell therapy, and regenerative medicine. However, the reprogramming process can be stochastic and inefficient, creating many partially reprogrammed intermediates and non-reprogrammed cells in addition to fully reprogrammed iPSCs. Much of the work to identify, evaluate, and enrich for iPSCs during reprogramming relies on methods that fix, destroy, or singularize cell cultures, thereby disrupting each cell's microenvironment. Here, we develop a micropatterned substrate that allows for dynamic live-cell microscopy of hundreds of cell subpopulations undergoing reprogramming while preserving many of the biophysical and biochemical cues within the cells' microenvironment. On this substrate, we were able to both watch and physically confine cells into discrete islands during the reprogramming of human somatic cells from skin biopsies and blood draws obtained from healthy donors. Using high-content analysis, we identified a combination of eight nuclear characteristics that can be used to generate a computational model to predict the progression of reprogramming and distinguish partially reprogrammed cells from those that are fully reprogrammed. This approach to track reprogramming in situ using micropatterned substrates could aid in biomanufacturing of therapeutically relevant iPSCs and be used to elucidate multiscale cellular changes (cell-cell interactions as well as subcellular changes) that accompany human cell fate transitions.

A multilevel approach to correlates of anaemia in women in the Democratic Republic of Congo: findings from a nationally representative survey.

BACKGROUND/OBJECTIVES: Anaemia accounts for a significant proportion of pre- and post-partum morbidity and mortality in low-income countries with sequelae, including an increased risk of infection. Factors contributing to anaemia need to be addressed through the introduction of evidence-based measures to control and prevent the disease. We aimed to determine the prevalence of anaemia in women of child-bearing age in the Democratic Republic of Congo (DRC) and investigate the associated individual, household and community level factors. SUBJECTS/METHODS: Cross sectional representative population data from the 2013-2014 DRC Demographic and Health Survey (DHS) was used. The primary outcome was anaemia in women, stratified according to pregnancy in those of child-bearing age. A haemoglobin level of below 11 g/dl for pregnant women and 12 g/dl for non-pregnant women was used as the indicator of anaemia. Using a three-level random intercept model this study explored risk factors at individual, household and community levels and quantified the observed and unobserved variations between households and communities. RESULTS: Thirty-eight percent of women in the DRC are anaemic. Anaemia is significantly higher in younger, pregnant and underweight women, as well as those with comorbidities, including HIV and malaria who are living in the capital city Kinshasa. Anaemia varies within and between households and communities in the DRC. CONCLUSIONS: Integrated approaches to reduce anaemia in settings with high malaria and HIV prevalence such as the DRC should target households.

Sex-divided reference intervals for mean platelet volume, platelet large cell ratio and plateletcrit using the Sysmex XN-10 automated haematology analyzer in a UK population

ABSTRACT Background: This study aimed to define the sex-divided reference intervals for platelet indices (mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR) and plateletcrit (PCT)) on the Sysmex XN-10. Methods: A total of 2376 samples were assayed for full blood count on the Sysmex XN-10 haematology analyzer. After removing the outliers, reference intervals were calculated using the mean ± 2SD. The P value 0.05 was adopted to denote statistical significance. Results: There was a statistical significance (α = 0.05) between sex-divided reference intervals for MPV (p = 0.007), P-LCR (p = 0.015) and PCT (p < 0.001), thus separate reference intervals were calculated for these indices, with orientation to the sex-divisions as follows: MPV, 9.1-13.0 fL [males] and 9.2-12.8 fL [females]; P-LCR, 17.6-47.0% [males] and 17.8-47.8% [females]; and PCT, 0.16-0.35% [males] and 0.18-0.37% [females]. No significance was found between sex-divided reference intervals for the PDW (p = 0.838), therefore a reference interval for total individuals was calculated for this platelet measurement as 9.3-17.3 fL. Conclusion: This study showed comparable reference intervals, using the Sysmex XN-10, with the previous literature. It determined the need to define sex-specific reference intervals for the MPV, P-LCR and PCT, but not for the PDW. These reference intervals will allow for low and high values to be facilitated in order to do further research and guide platelet disorder management.

Sex-divided reference intervals for mean platelet volume, platelet large cell ratio and plateletcrit using the Sysmex XN-10 automated haematology analyzer in a UK population.

BACKGROUND: This study aimed to define the sex-divided reference intervals for platelet indices (mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR) and plateletcrit (PCT)) on the Sysmex XN-10. METHODS: A total of 2376 samples were assayed for full blood count on the Sysmex XN-10 haematology analyzer. After removing the outliers, reference intervals were calculated using the mean±2SD. The P value 0.05 was adopted to denote statistical significance. RESULTS: There was a statistical significance (α=0.05) between sex-divided reference intervals for MPV (p=0.007), P-LCR (p=0.015) and PCT (p<0.001), thus separate reference intervals were calculated for these indices, with orientation to the sex-divisions as follows: MPV, 9.1-13.0fL [males] and 9.2-12.8fL [females]; P-LCR, 17.6-47.0% [males] and 17.8-47.8% [females]; and PCT, 0.16-0.35% [males] and 0.18-0.37% [females]. No significance was found between sex-divided reference intervals for the PDW (p=0.838), therefore a reference interval for total individuals was calculated for this platelet measurement as 9.3-17.3fL. CONCLUSION: This study showed comparable reference intervals, using the Sysmex XN-10, with the previous literature. It determined the need to define sex-specific reference intervals for the MPV, P-LCR and PCT, but not for the PDW. These reference intervals will allow for low and high values to be facilitated in order to do further research and guide platelet disorder management.

Engineering induction of singular neural rosette emergence within hPSC-derived tissues.

Human pluripotent stem cell (hPSC)-derived neural organoids display unprecedented emergent properties. Yet in contrast to the singular neuroepithelial tube from which the entire central nervous system (CNS) develops in vivo, current organoid protocols yield tissues with multiple neuroepithelial units, a.k.a. neural rosettes, each acting as independent morphogenesis centers and thereby confounding coordinated, reproducible tissue development. Here, we discover that controlling initial tissue morphology can effectively (>80%) induce single neural rosette emergence within hPSC-derived forebrain and spinal tissues. Notably, the optimal tissue morphology for observing singular rosette emergence was distinct for forebrain versus spinal tissues due to previously unknown differences in ROCK-mediated cell contractility. Following release of geometric confinement, the tissues displayed radial outgrowth with maintenance of a singular neuroepithelium and peripheral neuronal differentiation. Thus, we have identified neural tissue morphology as a critical biophysical parameter for controlling in vitro neural tissue morphogenesis furthering advancement towards biomanufacture of CNS tissues with biomimetic anatomy and physiology.

Reference intervals for absolute and percentage immature platelet fraction using the Sysmex XN-10 automated haematology analyser in a UK population.

BACKGROUND: Immature platelet fraction (IPF) estimation is a non-invasive and sensitive test that is available on recently introduced Sysmex XN-series of automated haematology analysers. It is a direct cellular indicator of thrombopoiesis. The aim of this study was to establish reference intervals for IPF, for both absolute (A-IPF) and percentage (%-IPF) measurements. MATERIAL AND METHODS: A total of 2366 samples that met the inclusion criteria were assayed for full blood count on the Sysmex XN-10 and a non-parametric percentile method was used for calculating the reference intervals. RESULTS: After the outliers were excluded, the reference interval for %-IPF and A-IPF on Sysmex XN-10 were 1.6-10.1% and 4.37-23.21 × 109/L in total individuals, respectively. There was a statistical significance noted between the sexes (p = .004) for %-IPF, therefore a sex-specific reference interval was established, which was 1.8-10.0% for the males and 1.5-10.1% for females. No significant difference in sex status for A-IPF and age status for both %-IPF and A-IPF was observed. A very poor correlation was estimated between age versus %-IPF, ρ = 0.0156, and age versus A-IPF, ρ = -0.0023, indicating that there is no overall biological relationship between age and these parameters. As expected, a strong correlation between %-IPF and A-IPF was noted which could be attributed to their inter-relatedness. CONCLUSIONS: This large-scale study showed comparable reference intervals with the previous studies for %-IPF and A-IPF in a UK population. It found the need to establish sex-specific reference intervals for %-IPF, but not for A-IPF, whereas reference intervals were found to be stable across the age range.

TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

Multifunctional drug nanocarriers formed by cRGD-conjugated ßCD-PAMAM-PEG for targeted cancer therapy.

Polyamidoamine (PAMAM) dendrimer was conjugated with both carboxymethyl-ß-cyclodextrin (ßCD) and poly(ethylene glycol) (PEG). Cyclic RGD peptide, used as a tumor targeting ligand, was then selectively conjugated onto the distal ends of the PEG arms. The resulting ßCD-PAMAM-PEG-cRGD polymer was able to form stable and uniform nanoparticles (NPs) in aqueous solution. Doxorubicin (Dox), a model hydrophobic anticancer drug, was effectively encapsulated in the NPs via an inclusion complex formed between the drug and ßCD. The Dox loading level was 16.8 wt%. The cellular uptake of cRGD-conjugated Dox-loaded NPs in the U87MG cell line was much higher than that of non-targeted NPs. Furthermore, the anti-proliferative effect of the cRGD-conjugated NPs was superior to that of free drug and non-targeted NPs. These results suggest that NPs formed by ßCD-PAMAM-PEG-cRGD with a high drug payload may significantly improve the anticancer efficacy by tumor-targeted delivery and enhanced cellular uptake.

Fabricating complex culture substrates using robotic microcontact printing (R-µCP) and sequential nucleophilic substitution.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (µCP) is a versatile technique to pattern such grafted PEG brushes, but manual µCP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-µCP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal 'click' chemistries. Here, we describe in detail the workflow to manufacture such substrates.

High-precision robotic microcontact printing (R-µCP) utilizing a vision guided selectively compliant articulated robotic arm.

Increased realization of the spatial heterogeneity found within in vivo tissue microenvironments has prompted the desire to engineer similar complexities into in vitro culture substrates. Microcontact printing (µCP) is a versatile technique for engineering such complexities onto cell culture substrates because it permits microscale control of the relative positioning of molecules and cells over large surface areas. However, challenges associated with precisely aligning and superimposing multiple µCP steps severely limits the extent of substrate modification that can be achieved using this method. Thus, we investigated the feasibility of using a vision guided selectively compliant articulated robotic arm (SCARA) for µCP applications. SCARAs are routinely used to perform high precision, repetitive tasks in manufacturing, and even low-end models are capable of achieving microscale precision. Here, we present customization of a SCARA to execute robotic-µCP (R-µCP) onto gold-coated microscope coverslips. The system not only possesses the ability to align multiple polydimethylsiloxane (PDMS) stamps but also has the capability to do so even after the substrates have been removed, reacted to graft polymer brushes, and replaced back into the system. Plus, non-biased computerized analysis shows that the system performs such sequential patterning with <10 µm precision and accuracy, which is equivalent to the repeatability specifications of the employed SCARA model. R-µCP should facilitate the engineering of complex in vivo-like complexities onto culture substrates and their integration with microfluidic devices.

Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes.

In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 µm(2) to 160,000 µm(2). The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 µm and 80 µm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.

Hemoglobinopathies in the Sudan.

In a prospective study, we evaluated hematological parameters in freshly obtained venous blood samples from 632 Sudanese patients attending the outpatient department at Khartoum Teaching Hospital, Khartoum, Sudan, in the period between March and July 2005. The patients were surveyed for full blood count (FBC) and hemoglobin (Hb) electrophoresis using a cellulose acetate method. Hb S [beta6(A3)Glu-->Val] was the most common abnormal Hb, which was not unexpected because the subjects live in the center of a malaria-affected area. The study showed low hematological parameters due to various causes including poor nutrition as well as infections and hemolytic processes.