Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns

Abstract

Our inability to derive the neuronal diversity that comprises the posterior central nervous system (pCNS) using human pluripotent stem cells (hPSCs) poses an impediment to understanding human neurodevelopment and disease in the hindbrain and spinal cord. Here, we establish a modular, monolayer differentiation paradigm that recapitulates both rostrocaudal (R/C) and dorsoventral (D/V) patterning, enabling derivation of diverse pCNS neurons with discrete regional specificity. First, neuromesodermal progenitors (NMPs) with discrete HOX profiles are converted to pCNS progenitors (pCNSPs). Then, by tuning D/V signaling, pCNSPs are directed to locomotor or somatosensory neurons. Expansive single-cell RNA-sequencing (scRNA-seq) analysis coupled with a novel computational pipeline allowed us to detect hundreds of transcriptional markers within region-specific phenotypes, enabling discovery of gene expression patterns across R/C and D/V developmental axes. These findings highlight the potential of these resources to advance a mechanistic understanding of pCNS development, enhance in vitro models, and inform therapeutic strategies.

RosetteArray®Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk

“…Previously, we demonstrated that seeding WA09 hESCs 29 or cervical spinal NMP progeny 22,28 from culture onto micropatterned substrates could be used to generate FB and cervical SC RosetteArrays 21 . However, for scalable applications, an off-the-shelf protocol that uses direct seeding of cryopreserved cells is required to avoid errors caused by either genetic drift during long-term cell culture 39 and/or differences in hPSC culturing or NMP derivation between experiments.…”

“…Thus, we cryopreserved banks of WA09 hESCs and cervical spinal NMP progeny, and tested whether their direct seeding onto micropatterned substrates could also generate FB and SC RosetteArrays in a 12-well format ( Figure 1A, B ). After 24-hrs post seeding in E8 media with 10 μm Rock inhibitor (R), prospective FB and SC RosetteArrays were cultured for an additional 5 days in E6 media without 29 or with retinoic acid (RA) 22,28 , respectively, to permit differentiation into Pax6 + /N-cadherin + NECs with subsequent rosette emergence ( Figure 1C ). As previously optimized 21 , FB and SC RosetteArrays were derived using micropatterned culture substrates presenting an array of 250 μm and 150 μm diameter circles, respectively, to preferentially induce single rosette emergence with regionally distinct tissue morphologies ( Figure 1D ).…”

“…Here, we present development and validation of a hPSC-derived RosetteArray platform for scalable, off-the-shelf, qHTS of environmental and genetic factors, including clinically relevant multifactorial scenarios, known to cause neurodevelopmental risk. First, reproducible derivation of forebrain (FB) and spinal cord (SC) RosetteArrays from direct seeding of cryopreserved hPSCs or neuromesodermal progenitors 22,28 (NMPs), respectively, was demonstrated. Second, the RosetteArray's capability to detect a risk associated with pharmaceuticals and agrochemicals known to cause NTDs was shown.…”

Fully Integrated 3D Microelectrode Arrays with Polydopamine-Mediated Silicon Dioxide Insulation for Electrophysiological Interrogation of a Novel 3D Human, Neural Microphysiological Construct

“…Cryogenically preserved human stem-cell-derived spinal cord dorsal horn (SCDH) precursor cells were provided by the laboratory of Prof. Randolph Ashton at the University of Wisconsin-Madison (UWSCDH). 59 Upon receipt, the cells were rapidly thawed and diluted 1:4 in Neurobasal Medium (Thermo-Fisher Scientific, MA). The cells were centrifuged at 300g for 4 min and the supernatant was removed.…”

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

“…29,30 At present, the challenges of forced expression of neurogenic transcription factors and cellular heterogeneity obstruct the detailed study of the progenitor intermediates and lineages in the human neural tube during their differentiation from NMPs. 29,31 As such, we aimed to capture pure NMP- and posterior neural tube-like stem cells to dissect lineages, elucidate mechanisms underlying human neural tube development, and enhance cellular modelling capabilities.…”