Dynamic 3D Combinatorial Generation of hPSC‐Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities

Abstract

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

A Robust Pipeline for the Multi‐Stage Accelerated Differentiation of Functional 3D Cortical Organoids from Human Pluripotent Stem Cells

“…We have also found that for some challenging disease hPSC lines an additional troubleshooting step to Stage 0 aggregation recipe may call for the early application of the GSK3b inhibitor, CHIR99021 (3 μM final), which acts to stimulate proliferation by canonical Wnt pathway activation. However, it has previously been established that prolonged exposure to FGF2 and Wnt signaling can cause hPSCs to drift towards a caudal neuromesodermal progenitor identity (Whye et al, 2022); therefore, if this troubleshooting approach is utilized then it is suggested that sustained FGF receptor inhibition (by SU-5402) is followed for the full duration of the 7-day neural induction phase in Stage 1. 3D neural spheroids can be characterized at Day 7 by immunostaining for the markers SOX1, SOX2, PAX6, and NESTIN (Table 1).…”

“…An overview of the 35-day accelerated 3D cortical organoid differentiation process is depicted by the schematic in Figure 1. The method for generating 3D aggregate suspensions from pluripotent stem cells is as previously described (Whye et al, 2022); however, the media recipes differ. The initial step for cortical organoid generation requires aggregation of hPSCs in a FGF2-rich stem cell medium for a period of 24 hr to generate robust, homogenous populations of spheroids.…”

“…It has recently been suggested that the culture conditions and growth media for pluripotent stem cell maintenance have a profound effect on the differentiation success into cortical neurons and organoids (Cornacchia et al., 2019; Watanabe et al., 2022); however, we have observed successful cortical organoid and neuronal differentiation with all the culture media proposed herein. For a more detailed method on 3D suspension aggregate generation from human pluripotent stem cells, refer to our previous work (Current Protocols article: Whye et al., 2022). This protocol introduces a method for the in vitro generation of pure populations of cortical neurons; however, long‐term functional maturation may rely on non‐cell autonomous influences by co‐culture with astrocytes (Odawara, Katoh, Matsuda, & Suzuki, 2016; Odawara, Matsuda, Ishibashi, Yokoi, & Suzuki, 2018; Odawara, Saitoh, Alhebshi, Gotoh, & Suzuki, 2014) or by using a physiologically enhanced medium such as BrainPhys (Autar et al., 2022; Bardy et al., 2015).…”

A new era of stem cell and developmental biology: from blastoids to synthetic embryos and beyond

“…3D aggregates that broke symmetry formed extensions along the AP axis with organized germ layers 83 , 84 . Through the development of the technology of generating human gastruloids, specific gastruloids that addressed parts of the early stages of post-implantation human embryonic development were created, such as the processes of primitive streak formation 80 , neurulation 84 – 86 , and somitogenesis 87 .…”

Advances in the design, generation, and application of tissue-engineered myocardial equivalents

“…Yet, few models are available to study pathologies with atria-, conduction tissues-, or ventricles-specific implications ( Lemme et al, 2018 ; Zhao et al, 2019 ; Kerr et al, 2021 ; Williams et al, 2021 ; Krause et al, 2022 ). Moreover, there is nevertheless a paucity of in vitro platforms of innervated cardiac muscle ( Whye et al, 2022 ), which could be essential to study the diseases, in which the impairment of the autonomic nervous system has tremendous effects on heart function, as in the case of Alzheimer syndrome and other neurodegenerative diseases ( Elia and Fossati, 2023 ). The progressive development of specific protocols to enrich CM subpopulations during hiPS differentiation ( Cyganek et al, 2018 ; Kerr et al, 2021 ) will not only solve the issues of graft unfunctional behavior described before but will further help to generate chamber-specific pathology models, also for rare congenital diseases.…”