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Single cell transcriptome of regional human embryonic stem cell-derived ventral cultures from the spinal cord

ABSTRACT: We generated single RNA-seq data to measure transcriptional profiles of six hESC-derived ventral populations, representing distinct regions of the developing human spinal cord. These cells are differentiated using a protocol that induces collinear activation of region-specific HOX genes during exposure to FGF8 and Wnt signaling (Lippmann et al, 2015 PMID:25843047). By transitioning to media containing retinoic acid, smoothened agonist (SAG), Purmorphamine (Pur), and DAPT after varying durations of Wnt signaling, ventral neuronal cultures are generated with unique rostrocaudal identities. In total we recovered the transcriptomes of 12,540 cells. Analysis verified expression of increasingly caudal HOX paralogs that could be correlated to cervical (HOX1-8; H24-vN, H48-vN, and H72-vN), thoracic (HOX1-9; H120-vN), lumbar (HOX1-11; H168-vN), and lumbosacral (HOX1-13; H216-vN) spinal regions. However, inconsistent differentiation between samples resulted in a mix of neural progenitor, neuron, mesodermal, and neural crest derivatives.

ORGANISM(S): Homo sapiens

PROVIDER: GSE186696 | GEO | 2022/07/28

REPOSITORIES: GEO

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Project description:ALS is a uniformly fatal neurodegenerative disease in which motor neurons in the spinal cord and brain stem are selectively lost. Individual motor - groups of motor neurons innervating single muscles - show widely varying degrees of disease resistance: in the final stages of ALS, nearly all voluntary movement is lost but eye movement and eliminative and sexual functions remain relatively unimpaired. These functions are controlled by motor neurons of the oculomotor (III), trochlear (IV) and abducens (VI) nuclei in the midbrain and brainstem, and by Onuf's nucleus in the lumbosacral spinal cord, respectively. Correspondingly, in ALS autopsies the oculomotor and Onuf's nuclei are almost completely preserved. We used microarray profiling of isolated wildtype mouse motor neurons to identify genes whose expression was characteristic of both oculomotor and Onuf's nuclei but not of vulnerable lumbar spinal neurons, or vice versa. Three wild-type C57BL/6J P7 male animals were perfused with 30% sucrose, lumbosacral spinal cord and midbrain regions were rapidly recovered, embedded in OCT compound, and frozen in liquid nitrogen. 12 um-thick cryosections were mounted on RNAse-free, PEN-foil covered glass slides (Zeiss), fixed for 2 min in 100% EtOH, rinsed in 50% EtOH, stained with 1% cresyl violet for 2 min, rinsed with 50% EtOH, dehydrated in graded solutions of ethanol and air dried prior to LCM using PALM Microbeam system (Zeiss). From each animal, ~200 DL, L5, and oculomotor motor neurons were collected directly in lysis buffer. RNA was purified using Absolutely RNA, NanoPrep kit (Stratagene, La Jolla, CA). RNA integrity was assessed on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). At least 1.5 ng of purified RNA was the starting material used in the WT-Ovation Pico RNA Amplification System (Nugen, San Carlos,CA) with the FL-Ovation cDNA Biotin Module V2 (Nugen) to generate labeled probe. 10 ug of biotinylated cRNA from three independent samples for each motor neuron group isolated by LCM was hybridized to on Affymetrix (Santa Clara, CA) Mouse Genome 430 2.0 Arrays. Gene ontology and pathway analysis was performed using DAVID Bioinformatics Resources 6.7 (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD).

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Single cell transcriptome of regional human embryonic stem cell-derived ventral cultures from the spinal cord

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