Title: Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns
Legend: Novel, regionally discrete patterns of neurodevelopmental gene expression. A) Schematic of differentiation and experimental protocol for deriving a spectrum of region-specific posterior CNS (pCNS) neuronal cultures. B) t-SNE plot of RNA-seq data (n= 49,959 cells) showing HOX profile clusters. C) t-SNE plot with 25 primary clusters (Hindbrain- HB; Spinal- SC) broadly dividing the dataset by pCNS cardinal neuron identity. D) Comparison of gene expression states in V2a/V3 interneuron clusters across rostrocaudal regions highlights potential region-specific gene expression patterns.
Citation: Iyer, N. R., Shin, J., Cuskey, S., Tian, Y., Nicol, N. R., Doersch, T. E., Seipel, F., McCalla, S. G., Roy, S., & Ashton, R. S. (2022). Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns. Science advances, 8(39), eabn7430. https://doi.org/10.1126/sciadv.abn7430
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.
About the Lab: The goal of the Ashton Lab is to understand, model, and recapitulate in vitro the instructive signals utilized by human embryos to pattern tissue-specific differentiation of pluripotent stem cells, and apply this knowledge towards the rational design of tissue engineered scaffolds and other regenerative therapeutic strategies. Currently, the lab is primarily focusing on generating tissues and therapies for the central nervous system.
Investigator: Randolph Ashton, PhD