Title: 4 Steps to Derive the Human Spinal Neuron Spectrum
Legend: During development, the central nervous system is patterned along two axes, as shown in panel A. The rostrocaudal axis denotes “where” the cell belongs along the body axis, with positional identity defined by colinear, combinatorial expression of Hox genes. The dorsoventral axis denotes “what” type of cell to become. Somatosensory neurons are typically derived from the dorsal part of the spinal cord, while locomotor interneurons and motorneurons are derived from the ventral part of the spinal cord. Panel B shows our differentiation schema, whereby we first differentiate human pluripotent stem cells (hPSCs) to neuromesodermal (NMP) cells from distinct anatomical regions. We then direct them to dorsal or ventral progenitor fates and mature them into post-mitotic neurons. Using this strategy, we have been able to generate cells from diverse regions of the hindbrain (HB) through lumbar spinal cord (SC). Panel C is a TSNE plot of single cell-sequencing data of 46,959 cells compiled from 14 samples comprising of dorsal and ventral populations from 6 region-specific differentiations. Non-negative matrix factorization clustering roughly separates the dataset into cardinal cell types, with some separation also induced by differential Hox gene expression as shown in Panel D.
Citation: Iyer, N.*, Shin, JS.*, Roy, S., Ashton, RS. Generation of Human Spinal Neuron Diversity by Directed Differentiation along Rostrocaudal and Dorsoventral Axes (In Preparation).
Abstract: The spinal cord contains billions of neurons, with a huge diversity of subtypes enabling sensory, proprioceptive, and motor function. However, current human stem cell-based in vitro models and prospective cell transplantation therapies fail to reflect the significant regional specificity of spinal cells. Here we recapitulate the diversity of spinal cell types along both the rostrocaudal (R/C) and dorsoventral (D/V) axes with chemically defined, scalable protocols using human pluripotent stem cells (hPSCs). We first induce R/C patterning to generate neuromesodermal (NMPs) cells from a defined anatomical level, then instruct these cells to become early spinal progenitors. By providing appropriate D/V signaling, spinal progenitors can be sub-specified to generate tunable ratios of motor neurons (MNs) and locomotor interneurons (INs) from the ventral spinal cord, or TGF-β-dependent proprioceptive INs and TGF-β-independent sensory INs from the dorsal spinal cord. Cultures with over 95% neuronal yield can be generated in as little as 19 days, and these protocols can be used modularly to generate phenotypes from different anatomical levels of the posterior CNS. Single-cell RNA-sequencing reveals regionally specified neurons with discrete Hox gene profiles, representation of all major motor and somatosensory spinal cell types, and the presence of human-specific cell populations. We applied the computational alrogithm Arborteum to infer modules of coexpressed genes between subpopulations, facilitating the discovery of novel markers defining region-specific neuronal types. Altogether, this dataset enables an unprecedented characterization of the diversity of human spinal cells. We anticipate that access to these cells will advance a mechanistic understanding of spinal development, expand the potential and accuracy of in vitro models, provide insight into novel therapeutic targets, and represent clinically relevant populations for cell transplantation.
About the Lab: Our goal 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, we primarily focus on generating tissues and therapies for the central nervous system. For more information visit the Ashton Lab website.