Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

Wind, Matthew; Gogolou, Antigoni; Manipur, Ichcha; Granata, Ilaria; Butler, Larissa; Andrews, Peter W.; Barbaric, Ivana; Ning, Ke; Guarracino, Mario Rosario; Placzek, Marysia; Tsakiridis, Anestis

doi:10.1242/dev.194415

Cited by 20 publications

(27 citation statements)

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“…Spinal cord developmental principles and corresponding implementation into stem cell research has enabled the generation of cell types that are regionally matched to the site of injury ( Gouti et al., 2014 ; Sagner and Briscoe, 2019 ). Importantly, the caudal neuromesodermal progenitor-derived cells represent the appropriate bifurcated developmental lineage for spinal neurons ( Nedelec and Martinez-Arias, 2021 ; Wind et al., 2021 ). This is a relatively recent finding and may be relevant even for neural stem cell studies in which the past use of a default CNS pathway with anterior telencephalic identity may have had an impact ( Dulin et al., 2018 ; Kumamaru et al., 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury

et al. 2021

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Summary To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment.

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Section: Introductionmentioning

confidence: 99%

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury

et al. 2021

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“…This strategy relies on exposure of hPSC-derived NMPs to TGF-β and BMP inhibitors to promote a posterior neural tube identity, similar to that observed by Verrier et al (2018). Further differentiation in the presence of Shh signals and TGF-β and BMP inhibitors coordinate more efficient ventralization to promote a MN identity, while continued high levels of FGF and Wnt signals are required to promote a posterior axial identity (Mouilleau et al, 2021;Wind et al, 2021).…”

Section: Commentary Background Informationmentioning

confidence: 95%

“…4C,D). Electrophysiological functionality, displayed through whole-current patchclamp recordings, can also be used to demonstrate the functional maturity of the derived motor neurons (Wind et al, 2021).…”

Section: Y-27632 Is Included Only On Day 14 When Cells Are Replatedmentioning

confidence: 99%

“…Similarly, NMP-like cells generated in vitro from hPSCs can also be steered towards posterior neural, mesodermal, and neural crest lineages (Cooper et al, 2021;Frith et al, 2018;Gouti et al, 2014;Lippmann et al, 2015;Mouilleau et al, 2021;Turner, Rué, Mackenzie, Davies, & Martinez Arias, 2014;Verrier, Davidson, Gierliński, Dady, & Storey, 2018). Moreover, we have recently shown that the efficient generation of posterior thoracic HOX PG(6-9) motor neurons is best achieved by differentiating through a neuromesodermal progenitor intermediary state (Wind et al, 2021). This strategy relies on exposure of hPSC-derived NMPs to TGF-β and BMP inhibitors to promote a posterior neural tube identity, similar to that observed by Verrier et al (2018).…”

Section: Commentary Background Informationmentioning

confidence: 99%

“…We recently described an efficient protocol for generation of posterior motor neurons that mainly display a thoracic axial identity (Wind et al., 2021). Our strategy is based on initial induction of a neuromesodermal progenitor (NMP)–like population from hPSCs following treatment with WNT and FGF signaling pathway agonists (Frith et al., 2018; Gouti et al., 2014; Lippman et al., 2015).…”

Section: Introductionmentioning

confidence: 99%

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In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

Wind

Tsakiridis

2021

Current Protocols

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The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron-based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13) + spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity.

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

et al. 2022

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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

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Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

Cited by 20 publications

References 145 publications

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury

In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

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

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