Embryonic Spinal Cord Innervation in Human Trunk Organogenesis Gastruloids: Cardiac Versus Enteric Customization and Beyond.

Trunk-biased human gastruloids provide the ability to couple developmentally relevant spinal neurogenesis and organ morphogenesis via spatiotemporal self-organization events from derivatives of the three germ layers. The multi-lineage nature of gastruloids provides the full complexity of regulatory signaling cues that surpasses directed organoids and lays the foundation for an ex vivo self-evolving system. Here we detail two distinct protocols for trunk-biased gastruloids from an elongated, polarized structure with coordinated organ-specific neural patterning. Following an induction phase to caudalize iPSCs to trunk phenotype, divergent features of organogenesis and end-organ innervation yield separate models of enteric and cardiac nervous system formation. Both protocols are permissive to multi-lineage development and allow the study of neural integration events within a native, embryo-like context. We discuss the customizability of human gastruloids and the optimization of initial and extended conditions that maintain a permissive environment for multi-lineage differentiation and integration.

Massive testing in the Galapagos Islands and low positivity rate to control SARS-CoV-2 spread during the first semester of the COVID-19 pandemic: a story of success for Ecuador and South America.

INTRODUCTION: During the first months of the COVID-19 pandemic in Latin America, countries like Ecuador, Peru and Colombia experienced chaotic scenarios with public health systems collapsing and lack of testing capacity to control the spread of the virus. In main cities like Guayaquil in Ecuador, dramatic situations such as corpses in the streets were internationally broadcasted. METHODS: While the COVID-19 pandemic was devastating South America, SARS-CoV-2 transmission was successfully managed in the Galapagos Islands due to the implementation of a massive screening strategy including hospitalized and community-dwelling populations, and travel restrictions facilitated by its geographical location (972 km from the Ecuadorian continental territory). Floreana Island was one of the few locations in the world that remained COVID-19 free during 2020. RESULTS: In this study, we retrospectively analyzed the data related to SARS-CoV-2 massive testing campaigns from April to September 2020 in the Galapagos Islands, and found this territory to have the lowest positivity rate in South America (4.8-6.7%) and the highest testing ratio among Ecuadorian provinces (9.87% of the population, which is 2480 out of 25 124 inhabitants) during the first wave of the COVID-19 pandemic. CONCLUSION: This story of success was possible because of the interinstitutional collaboration between the regional government of Galapagos Islands (Consejo de Gobierno), the local authorities (Gobiernos Autonomos Descentralizados de Santa Cruz, San Cristobal and Isabela), the regional authorities from Ecuadorian Ministry of Health, the Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos and Universidad de Las Américas.

High SARS-CoV-2 infection rates and viral loads in community-dwelling individuals from rural indigenous and mestizo communities from the Andes during the first wave of the COVID-19 pandemic in Ecuador.

Background: Neglected indigenous groups and underserved rural populations in Latin America are highly vulnerable to COVID-19 due to poor health infrastructure and limited access to SARS-CoV-2 diagnosis. The Andean region in Ecuador includes a large number of isolated rural mestizo and indigenous communities living under poverty conditions. Objective: We herein present a retrospective analysis of the surveillance SARS-CoV-2 testing in community-dwelling populations from four provinces in the Ecuadorian Andes, carried out during the first weeks after the national lockdown was lifted in June 2020. Results: A total number of 1,021 people were tested for SARS-CoV-2 by RT-qPCR, resulting in an overall high infection rate of 26.2% (268/1,021, 95% CI: 23.6-29%), which was over 50% in several communities. Interestingly, community-dwelling super spreaders with viral loads over 108 copies/mL represented 7.46% (20/268, 95% CI: 4.8-11.1%) of the SARS-CoV-2 infected population. Conclusion: These results support that COVID-19 community transmission in rural communities from the Andean region was happening at the early stages of the COVID-19 pandemic in Ecuador and point out the weakness of the COVID-19 control program. Community-dwelling individuals in neglected rural and indigenous communities should be considered for a successful control and surveillance program in future pandemics in low- and middle-income countries.

Sustained COVID-19 community transmission and potential super spreading events at neglected afro-ecuadorian communities assessed by massive RT-qPCR and serological testing of community dwelling population.

Background: Neglected ethnic minorities from underserved rural populations in Latin America are highly vulnerable to coronavirus disease 2019 (COVID-19) due to poor health infrastructure and limited access to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. Esmeraldas is a mainly rural province of the Coastal Region of Ecuador characterized by a high presence of Afro-Ecuadorian population living under poverty conditions. Objective: We herein present a retrospective analysis of the surveillance SARS-CoV-2 testing in community-dwelling population from Esmeraldas carried out by our university laboratory in collaboration with regional health authorities during the first week of October 2020, in a region where no public SARS-CoV-2 detection laboratory was available at that time. Results: A total number of 1,259 people were tested for SARS-CoV-2 by Reverse Transcription quantitative Polimerasa Chain Reaction (RT-qPCR), resulting in an overall infection rate of 7.7% (97/1259, 95% CI: [6.32-9.35%]) for SARS-CoV-2, up to 12.1% in some communities. Interestingly, community-dwelling super spreaders with viral loads over 108 copies/ml represented 6.2% of the SARS-CoV-2-infected population. Furthermore, anti-SARS-CoV-2 IgG serological tests were applied to the same study group, yielding an overall seroprevalence of 11.68% (95% CI: [9.98-13.62%]) but as high as 24.47% at some communities. Conclusion: These results support active COVID-19 community transmission in Esmeraldas province during the first semester of the COVID-19 pandemic as it has been shown for other rural communities in the Ecuadorian Coastal Region.

Human stem cell-derived neurons and neural circuitry therapeutics: Next frontier in spinal cord injury repair.

Spinal cord injury (SCI) remains a life-altering event that devastates those injured and the families that support them. Numerous laboratories are engaged in preclinical and clinical trials to repair the injured spinal cord with stem cell-derived therapeutics. A new developmental paradigm reveals early bifurcation of brain or trunk neurons in mammals via neuromesodermal progenitors (NMPs) relevant to therapies requiring homotypic spinal cord neural populations. Human-induced pluripotent stem cell (hiPSC) NMP-derived spinal motor neurons generated ex vivo following this natural developmental route demonstrate robust survival in vivo when delivered as suspension grafts or as in vitro preformed encapsulated neuronal circuitry when transplanted into a rat C4-C5 hemicontusion injury site. Use of in vitro matured neurons avoids in vivo differentiation challenges of using pluripotent hiPSC or multipotent neural stem cell (NSC) or mesenchymal stem cell therapeutics. In this review, we provide an injury to therapeutics overview focusing on how stem cell and developmental fields are merging to generate exquisitely matched spinal motor neurons for SCI therapeutic studies. The complexity of the SCI microenvironment generated by trauma to neurons and vasculature, along with infiltrating inflammatory cells and scarring, underlies the challenging cytokine microenvironment that therapeutic cells encounter. An overview of evolving but limited stem cell-based SCI therapies that have progressed from preclinical to clinical trials illustrates the challenges and need for additional stem cell-based therapeutic approaches. The focus here on neurons describes how NMP-based neurotechnologies are advancing parallel strategies such as transplantation of preformed neuronal circuitry as well as human in vitro gastruloid multicellular models of trunk central and peripheral nervous system integration with organs. NMP-derived neurons are expected to be powerful drivers of the next generation of SCI therapeutics and integrate well with combination therapies that may utilize alternate biomimetic scaffolds for bridging injuries or flexible biodegradable electronics for electrostimulation.

Generation of human elongating multi-lineage organized cardiac gastruloids.

Human elongating multi-lineage organized (EMLOC) gastruloid technology captures key aspects of trunk neurodevelopment including neural integration with cardiogenesis. We generate multi-chambered, contractile EMLOC gastruloids with integrated central and peripheral neurons using defined culture conditions and signaling factors. hiPSC colonies are primed by activating FGF and Wnt signaling pathways for co-induced lineages. EMLOC gastruloids are then initialized with primed cells in suspension culture using timed exposure to FGF2, HGF, IGF1, and Y-27632. Cardiogenesis is stimulated by FGF2, VEGF, and ascorbic acid. For complete details on the use and execution of this protocol, please refer to Olmsted and Paluh (2022).1.

Analytical and clinical evaluation of a heat shock SARS-CoV-2 detection method without RNA extraction for N and E genes RT-qPCR.

BACKGROUND: The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. OBJECTIVE: The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. RESULTS: 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68.8% (232/337) and 89.4% (202/226) of SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98.6%, 93.3% and 84.8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. CONCLUSIONS: Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients at the time of testing. This affordable protocol can help overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

Crucial contribution of the universities to SARS-CoV-2 surveillance in Ecuador: Lessons for developing countries.

COVID-19 pandemic has challenged public health systems worldwide, particularly affecting developing countries in Latin America like Ecuador. In this report, we exposed the fundamental role of the Ecuadorian universities to improve COVID-19 surveillance in the country, with an overall contribution over 15% of the total SARS-CoV-2 RT-PCR tests done. We highlight the role of our university during the first semester of the COVID-19 pandemic, contributing to a massive free SARS-CoV-2 testing up to almost 10% of the total diagnosis completed in the country, mainly focus on underserved urban, rural and indigenous communities. Finally, we described our contribution to a high quality and low-cost SARS-CoV-2 RT-PCR diagnostic in Ecuador.