The potential of stem cell therapy to tackle visual impairment.

Visual impairment adversely impacts quality of life and affects more than 295 million individuals globally. Currently, there is no cure or tissue regenerative approaches in clinical practice for vision loss caused by corneal disease, glaucoma, cataracts, macular degeneration, diabetic retinopathy, and inherited retinal disease. Stem cells-based therapeutic approaches to diseases causing moderate to severe visual impairment have shown encouraging outcomes in animal models and in vitro studies. The goal of this narrative review is to describe and evaluate the potential of stem cell-based treatment, and their advantages and safety concerns in treating conditions causing vision loss.

How mechanisms of stem cell polarity shape the human cerebral cortex.

Apical-basal progenitor cell polarity establishes key features of the radial and laminar architecture of the developing human cortex. The unique diversity of cortical stem cell populations and an expansion of progenitor population size in the human cortex have been mirrored by an increase in the complexity of cellular processes that regulate stem cell morphology and behaviour, including their polarity. The study of human cells in primary tissue samples and human stem cell-derived model systems (such as cortical organoids) has provided insight into these processes, revealing that protein complexes regulate progenitor polarity by controlling cell membrane adherence within appropriate cortical niches and are themselves regulated by cytoskeletal proteins, signalling molecules and receptors, and cellular organelles. Studies exploring how cortical stem cell polarity is established and maintained are key for understanding the features of human brain development and have implications for neurological dysfunction.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Mother's care-seeking behavior for neonatal danger signs from qualified providers in rural Bangladesh: A generalized structural equation modeling and mediation analysis.

Background: Neonatal deaths contribute to nearly half (47%) of under-five mortality globally and 67% in Bangladesh. Despite high neonatal mortality, care-seeking from qualified providers for newborn danger signs remains low. Identification of direct and indirect factors and their pathways affecting care-seeking will help to design a well-targeted intervention. This study assessed the direct, indirect, and total effect of the predictive factors on neonatal care-seeking in Bangladesh. Materials and methods: This was a cross-sectional baseline household survey conducted in 14 districts of Bangladesh in 2019 with 17,251 recently delivered women (RDW) with a live birth outcome in the preceding 15 months. We used a two-stage stratified cluster sampling process to select the samples from 14 districts. We investigated the inter-relationship of maternal background characteristics, maternal health utilizations, child/neonate factors, health service delivery-related factors and newborn danger sign knowledge with newborn care-seeking practices and estimated the direct, indirect, and total effects using Generalized Structural Equation Modeling (GSEM) and mediation analysis. p-value = 0.05 was considered statistically significant. The result of the mediation analysis was reported in Log Odds (LOD). The positive LOD (LOD > 0) implies a positive association. Results: Half of the mothers (50.8%) reported a neonatal illness and among them, only 36.5% mothers of sick neonates sought care from qualified providers. Our mediation analysis showed that maternal health utilization factors, i.e., 4 + antenatal care visits (ANC) from a qualified provider (LOD: 0.63, 95% CI: 0.49, 0.78), facility delivery (LOD: 0.74, 95% CI: 0.30, 1.17) and postnatal care (PNC) from a qualified provider (LOD: 0.50, 95% CI: 0.21, 0.78) showed the highest total effect over other factors domains, and therefore, were the most important modifiable predictors for qualified neonatal care-seeking. Other important factors that directly and/or indirectly increased the chance of newborn care-seeking from qualified providers were household wealth (LOD: 0.86, 95% CI: 0.70, 1.02), maternal education (LOD: 0.48, 95% CI: 0.32, 0.63), distance to nearest health facility (LOD: 0.20, 95% CI: 0.10, 0.30), community health worker's (CHWs) home visits during ANC (LOD: 0.24, 95% CI: 0.13, 0.36), neonatal danger sign counseling after delivery (LOD: 0.20, 95% CI: 0.06, 0.34) and women's knowledge of neonatal danger signs (LOD: 0.37, 95% CI: 0.09, 0.64). Conclusion: The inter-relationship and highest summative effect of ANC, facility delivery, and PNC on newborn care-seeking suggested the maternal care continuum altogether from ANC to facility delivery and PNC to improve care-seeking for the sick newborn. Additionally, referral training for unqualified providers, targeted intervention for poorer households, increasing CHWs home visits and neonatal danger sign counseling at the facility and community should also be considered.

Prevalence and factors associated with skin-to-skin contact (SSC) practice: findings from a population-based cross-sectional survey in 10 selected districts of Bangladesh.

BACKGROUND: Skin-to-skin contact (SSC) practice improves newborn survival and child development through preventing hypothermia in newborns, improving early initiation of breastfeeding practice, and strengthening mother-child bonding. Despite having numerous benefits, it is one of the least practiced interventions in low and middle-income countries (1 to 74%). In Bangladesh, the prevalence of SSC was 26% in 2014. In this study, we aimed to estimate the prevalence of SSC in the study districts and identify factors that facilitate or inhibit SSC practice so that context-specific recommendations can be made to advance the use of this intervention. METHODS: We used baseline household survey data of USAID's MaMoni MNCSP project conducted in 10 districts of Bangladesh in 2019. Our analysis included 13,695 recently delivered women (RDW) with a live birth outcome. Our primary outcome was the mother's reported practice of SSC. We examined various antepartum, intrapartum, newborn, and sociodemographic factors associated with SSC using a multivariable generalized linear model. Our findings were reported using adjusted Prevalence Risk Ratios (aPRRs) and 95% Confidence Intervals (CIs). RESULTS: Overall, 28% of RDW reported practicing SSC across the 10 surveyed districts. Our multivariable analysis showed that public facility delivery (aPRR 2.01; 95%CI: 1.80, 2.26), private facility delivery (aPRR 1.23; 95%CI: 1.06, 1.42) and ≥ 4 antenatal care (ANC) visits at least one from a medically trained provider (MTP) (aPRR 1.17; 95%CI: 1.03, 1.26) had a significant positive association with SSC practice. Caesarean section (aPRR 0.64; 95%CI: 0.56, 0.73) had a significant negative association with SSC practice compared to vaginal births. We also found a significant positive association of SSC practice with mothers' who perceived the birth size of their baby to be small, mothers with a higher education level (≥10 years), and mothers from households in the highest wealth quintile. CONCLUSIONS: The prevalence of SSC is very low in the surveyed districts of Bangladesh. Considering the factors associated with SSC, relevant stakeholders need to increase their efforts on improving ANC and facility delivery coverages as well as improving SSC practice in the facilities especially after caesarean deliveries. Countries with a high burden of home deliveries, also need to emphasize community-based interventions and increasing coverage of skilled birth attendance for improving this life-saving intervention.

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

Cell stress in cortical organoids impairs molecular subtype specification.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3-5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

NRXN1 Deletion and Exposure to Methylmercury Increase Astrocyte Differentiation by Different Notch-Dependent Transcriptional Mechanisms.

Controversial evidence points to a possible involvement of methylmercury (MeHg) in the etiopathogenesis of autism spectrum disorders (ASD). In the present study, we used human neuroepithelial stem cells from healthy donors and from an autistic patient bearing a bi-allelic deletion in the gene encoding for NRXN1 to evaluate whether MeHg would induce cellular changes comparable to those seen in cells derived from the ASD patient. In healthy cells, a subcytotoxic concentration of MeHg enhanced astroglial differentiation similarly to what observed in the diseased cells (N1), as shown by the number of GFAP positive cells and immunofluorescence signal intensity. In both healthy MeHg-treated and N1 untreated cells, aberrations in Notch pathway activity seemed to play a critical role in promoting the differentiation toward glia. Accordingly, treatment with the established Notch inhibitor DAPT reversed the altered differentiation. Although our data are not conclusive since only one of the genes involved in ASD is considered, the results provide novel evidence suggesting that developmental exposure to MeHg, even at subcytotoxic concentrations, induces alterations in astroglial differentiation similar to those observed in ASD.

A cross-sectional study of partograph utilization as a decision making tool for referral of abnormal labour in primary health care facilities of Bangladesh.

BACKGROUND: In Bangladesh, female paramedics known as Family Welfare Visitors (FWVs), conduct normal deliveries in first-level primary care facilities, or Union Health and Family Welfare Centres (UH&FWC). Utilization of partographs allow for early identification of abnormal labour and referral for advanced care to Emergency Obstetric Care (EmOC) facilities. A systematic assessment of the quality of partograph utilization in clinical-decision making will contribute to understanding the use of the tool by health workers. METHODS: In 2013, the USAID supported MaMoni HSS project, led in country by Save the Children, trained FWVs on the use of partographs in five UH&FWCs in Habiganj district. As part of the follow-up after training, intrapartum case record forms, accompanying partographs, and referral registers for all obstetric cases managed in these five facilities from July 2013 to June 2014 were reviewed. Partographs were reviewed to identify abnormal labour cases based on pre-defined indications. All referred cases were ascertained from the case records in the referral registers. Five health workers were interviewed to assess their knowledge, attitude and experience in partograph use and to explore the challenges for referral decision making associated with the tool. RESULTS: A total of 1,198 deliveries were managed at the study sites, of which 663 presented with cervical dilatation of 8 cm or less. Partographs were initiated in 98% of these cases. Indication of abnormal labour was found in 71 partographs (11%) and among them, only 1 was referred to a higher-level facility. Foetal heart rate and cervical dilatation were appropriately recorded in 61% and 70% of the partographs, respectively. Interviews with health workers revealed poor interpretation of referral indications from the partographs. Limited accessibility to the nearest EmOC facility, inadequate time for referral, and non-compliance to referral by clients were identified by the interviewed health workers as the key barriers for referral decision making. CONCLUSIONS: Supporting the health workers at first-level primary care facilities to better interpret and act on partograph data in a timely manner, and strengthening the referral systems are needed to ensure that women in labour receive the prompt quality care they and their babies require to survive.

Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFß/Smad3 signaling.

BACKGROUND: Krüppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGFß have been shown to regulate each other's expression in non-myogenic cell types. Since MEF2D and TGFß also fulfill crucial roles in skeletal myogenesis, we wanted to identify whether KLF6 functions in a myogenic context. METHODS: KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture. RESULTS: We found that KLF6 and MEF2D are co-localized in the nuclei of mononucleated but not multinucleated myogenic cells and, that the MEF2 cis element is a key component of the KLF6 promoter region. In addition, TGFß potently enhanced KLF6 protein levels and this effect was repressed by pharmacological inhibition of Smad3. Interestingly, pharmacological inhibition of MEK/ERK (1/2) signaling resulted in re-activation of the differentiation program in myoblasts treated with TGFß, which is ordinarily repressed by TGFß treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGFß-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGFß signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGFß stimulation of myoblast proliferation was reduced in KLF6 depleted cells. CONCLUSIONS: Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGFß with consequences for our understanding of muscle development and a variety of muscle pathologies.

Suppression of a MEF2-KLF6 survival pathway by PKA signaling promotes apoptosis in embryonic hippocampal neurons.

In the mammalian nervous system, regulation of transcription factor activity is a crucial determinant of neuronal cell survival, differentiation, and death. The myocyte enhancer factor 2 (MEF2) transcription factors have been implicated in cellular processes underlying neuronal survival and differentiation. A core component of the MEF2 complex is the MEF2D subunit. Recently, we reported that cAMP-dependent protein kinase (cAMP/PKA) signaling negatively regulates MEF2D function in myogenic cells. Here, we assessed whether cAMP signaling converges on the prosurvival role of MEF2D in Sprague Dawley rat embryonic (E18) hippocampal neurons. Initially, we observed that experimental induction of cAMP/PKA signaling promotes apoptosis in primary hippocampal neurons as indicated by TUNEL and FACS analysis. Luciferase reporter gene assays revealed that PKA potently represses MEF2D trans-activation properties in neurons. This effect was largely reversed by engineered neutralizing mutations of PKA phospho-acceptor sites on MEF2D (S121/190A). Krüppel-like factor 6 (KLF6) was identified as a key transcriptional target of MEF2 in hippocampal neurons, and siRNA-mediated knockdown of KLF6 expression promotes neuronal cell death and also antagonizes the prosurvival role of MEF2D. These observations have important implications for understanding the pathways controlling cell survival and death in the mammalian nervous system.

Protein kinase A-regulated assembly of a MEF2{middle dot}HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) maintain the ability to modulate their phenotype in response to changing environmental stimuli. This phenotype modulation plays a critical role in the development of most vascular disease states. In these studies, stimulation of cultured vascular smooth muscle cells with platelet-derived growth factor resulted in marked induction of c-jun expression, which was attenuated by protein kinase Cdelta and calcium/calmodulin-dependent protein kinase inhibition. Given that these signaling pathways have been shown to relieve the repressive effects of class II histone deacetylases (HDACs) on myocyte enhancer factor (MEF) 2 proteins, we ectopically expressed HDAC4 and observed repression of c-jun expression. Congruently, suppression of HDAC4 by RNA interference resulted in enhanced c-jun expression. Consistent with these findings, mutation of the MEF2 cis-element in the c-jun promoter resulted in promoter activation during quiescent conditions, suggesting that the MEF2 cis-element functions as a repressor in this context. Furthermore, we demonstrate that protein kinase A attenuates c-Jun expression by promoting the formation of a MEF2.HDAC4 repressor complex by inhibiting salt-inducible kinase 1. Finally, we document a physical interaction between c-Jun and myocardin, and we document that forced expression of c-Jun represses the ability of myocardin to activate smooth muscle gene expression. Thus, MEF2 and HDAC4 act to repress c-Jun expression in quiescent VSMCs, protein kinase A enhances this repression, and platelet-derived growth factor derepresses c-Jun expression through calcium/calmodulin-dependent protein kinases and novel protein kinase Cs. Regulation of this molecular "switch" on the c-jun promoter may thus prove critical for toggling between the activated and quiescent VSMC phenotypes.

Protein kinase A represses skeletal myogenesis by targeting myocyte enhancer factor 2D.

Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.