Abstract
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. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Differentiation of neuromesodermal progenitors Basic Protocol 2: Posterior ventral spinal cord progenitor differentiation Basic Protocol 3: Posterior motor neuron differentiation.
Highlights
During embryonic development, motor neurons (MNs) arise from a set of progenitors within the ventral spinal cord, from which they mature and project axons to innervate target muscles
Wind and Tsakiridis across four distinct chromosomal clusters (A-D) and are expressed along the post-cranial A-P axis in a strict spatiotemporal manner reflecting their 3′-to-5′ genomic order: hindbrain/cervical MNs are marked by Hox paralogous groups (PG)(1-5), whereas more posterior thoracic and lumbosacral MNs are marked by Hox PG(6-9) and Hox PG(10-13), respectively
Most conventional human pluripotent stem cells (hPSCs) differentiation protocols for generation of MNs rely predominantly on initial induction of an anterior neural identity that is successively patterned to a ventral spinal cord/MN fate through exposure to sonic hedgehog (SHH) and retinoic acid signals (Amoroso et al, 2013; Lee et al, 2007; Li et al, 2008; Peljto, Dasen, Mazzoni, Jessell, & Wichterle, 2010; Wichterle, Lieberam, Porter, & Jessell, 2002)
Summary
Motor neurons (MNs) arise from a set of progenitors within the ventral spinal cord, from which they mature and project axons to innervate target muscles. Multiple MN subtypes are specified across the anterior-posterior (A-P) axis of the spinal cord, allowing for innervation of the diverse axial-level-dependent muscle targets (reviewed in Sagner & Briscoe, 2019). The specification of MNs across the A-P axis of the spinal cord is largely regulated by a family of homeobox genes known as HOX genes (Dasen, Liu, & Jessell, 2003; Dasen, Tice, Brenner-Morton, & Jessell, 2005, 2008; Jung et al, 2010). Hox genes are arranged as paralogous groups (PG) 1-13
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