التفاصيل البيبلوغرافية
العنوان: |
Dynamic 3D Combinatorial Generation of hPSC-Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities |
المؤلفون: |
Dosh Whye, Delaney Wood, Kristina H. Kim, Cidi Chen, Nina Makhortova, Mustafa Sahin, Elizabeth D. Buttermore |
المصدر: |
Current protocols. 2(10) |
سنة النشر: |
2023 |
مصطلحات موضوعية: |
Pluripotent Stem Cells, General Immunology and Microbiology, General Neuroscience, Health Informatics, Poloxamer, General Biochemistry, Genetics and Molecular Biology, Organoids, Fibroblast Growth Factors, Medical Laboratory Technology, Retinoids, Pregnancy, Humans, Female, Hedgehog Proteins, General Pharmacology, Toxicology and Pharmaceutics |
الوصف: |
Neuromesodermal progenitors represent a unique, bipotent population of progenitors residing in the tail bud of the developing embryo, which give rise to the caudal spinal cord cell types of neuroectodermal lineage as well as the adjacent paraxial somite cell types of mesodermal origin. With the advent of stem cell technologies, including induced pluripotent stem cells (iPSCs), the modeling of rare genetic disorders can be accomplished in vitro to interrogate cell-type specific pathological mechanisms in human patient conditions. Stem cell-derived models of neuromesodermal progenitors have been accomplished by several developmental biology groups; however, most employ a 2D monolayer format that does not fully reflect the complexity of cellular differentiation in the developing embryo. This article presents a dynamic 3D combinatorial method to generate robust populations of human pluripotent stem cell-derived neuromesodermal organoids with multi-cellular fates and regional identities. By utilizing a dynamic 3D suspension format for the differentiation process, the organoids differentiated by following this protocol display a hallmark of embryonic development that involves a morphological elongation known as axial extension. Furthermore, by employing a combinatorial screening assay, we dissect essential pathways for optimally directing the patterning of pluripotent stem cells into neuromesodermal organoids. This protocol highlights the influence of timing, duration, and concentration of WNT and fibroblast growth factor (FGF) signaling pathways on enhancing early neuromesodermal identity, and later, downstream cell fate specification through combined synergies of retinoid signaling and sonic hedgehog activation. Finally, through robust inhibition of the Notch signaling pathway, this protocol accelerates the acquisition of terminal cell identities. This enhanced organoid model can serve as a powerful tool for studying normal developmental processes as well as investigating complex neurodevelopmental disorders, such as neural tube defects. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Robust generation of 3D hPSC-derived spheroid populations in dynamic motion settings Support Protocol 1: Pluronic F-127 reagent preparation and coating to generate low-attachment suspension culture dishes Basic Protocol 2: Enhanced specification of hPSCs into NMP organoids Support Protocol 2: Combinatorial pathway assay for NMP organoid protocol optimization Basic Protocol 3: Differentiation of NMP organoids along diverse cellular trajectories and accelerated terminal fate specification into neurons, neural crest, and sclerotome derivatives. |
تدمد: |
2691-1299 |
URL الوصول: |
https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3a7f0a83278d6c6762790131a09dc480 https://pubmed.ncbi.nlm.nih.gov/36264199 |
حقوق: |
CLOSED |
رقم الأكسشن: |
edsair.doi.dedup.....3a7f0a83278d6c6762790131a09dc480 |
قاعدة البيانات: |
OpenAIRE |