Project description:Spinal muscular atrophy (SMA) is characterized by low levels of survival motor neuron (SMN) protein and loss of motor neurons (MN); however, the underlying mechanism that links SMN deficiency to selective motor neuronal dysfunction is still largely unknown. We present here, for the first time, a comprehensive quantitative mass spectrometry study that covers the development of iPSC-derived MNs from both healthy individuals and SMA patients. We show an altered proteomic signature in SMA already at early stages during MN differentiation, associated with ER to Golgi transport, mRNA splicing and protein ubiquitination, in line with known SMA phenotypes. These alterations in the SMA proteome increase further towards later stages of MN differentiation. In addition, we find differences in altered protein expression between SMA patients, which however, have similar biological functions. Finally, we highlight several known SMN-binding partners as well as proteins associated with ubiquitin-mediated proteolysis and evaluate their expression changes during MN differentiation. Altogether, our work provides a rich resource of molecular events during early stages of MN differentiation, containing potentially therapeutically interesting protein expression profiles for SMA.
Project description:The aim of this study is to profile gene expression dynamics during the in vitro differentiation of embryonic stem cells into ventral motor neurons. Expression levels were profiled using Affymetrix microarrays at six timepoints during in vitro differentiation: ES cells (Day 0), embryoid bodies (Day 2), retinoid induction of neurogenesis (Day 2 +8hours of exposure to retinoic acid), neural precursors (Day 3), progenitor motor neurons (Day 4), postmitotic motor neurons (Day 7). The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog agonist. Here, gene expression patterns are profiled at various defined stages during the differentiation process using Affymetrix expression arrays.
Project description:The expression of v5-tagged Hoxc9 is induced and ChIP-seq is used to profile genome-wide occupancy in differentiating motor neurons The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog signaling. Here, ChIP-seq is used to profile the genome-wide occupancy of Hoxc9 after five days of differentiation.
Project description:Progenitor motor neurons can be generated with high-efficiency by differentiating ES cells in vitro in the presence of retinoic acid and hedgehog signalling. Here, we characterize the chromatin landscape associated with progenitor motor neurons (pMNs) in order to assess how histone modification domains shift during the differentiation process. In this study, we characterize the genomic occupancy of H3K27me3, H3K4me3, H3K79me2 and Pol2 using ChIP-seq in progenitor motor neurons that have been differentiated in vitro from ES cells. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included.
Project description:The aim of this study is to profile gene expression dynamics during the in vitro differentiation of embryonic stem cells into ventral motor neurons. Expression levels were profiled using Affymetrix microarrays at six timepoints during in vitro differentiation: ES cells (Day 0), embryoid bodies (Day 2), retinoid induction of neurogenesis (Day 2 +8hours of exposure to retinoic acid), neural precursors (Day 3), progenitor motor neurons (Day 4), postmitotic motor neurons (Day 7).
Project description:Progenitor motor neurons can be generated with high-efficiency by differentiating ES cells in vitro in the presence of retinoic acid and hedgehog signalling. Here, we characterize the chromatin landscape associated with progenitor motor neurons (pMNs) in order to assess how histone modification domains shift during the differentiation process.
Project description:Human pluripotent stem cells are a promising source of diverse cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons, is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that induce up to 50% motor neurons within 3 weeks from human pluripotent stem cells with defined subtype identities that are relevant to neurodegenerative diseases. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3-). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. We analyze 3 samples including 2 positive samples and 1 negative sample. Descriptions are as follow: a) Positive Sample 1: SHH-derived, day 21 GFP-high FACS purified motor neurons.b) Positive Sample 2: S+P-derived, day 21 GFP-high FACS purified motor neurons. c) Negative: S+P condition, day 21 no GFP FACS purified motor neurons
Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.