Dataset View [GSE75140]

TitleHuman cerebral organoids recapitulate gene expression programs of fetal neocortex development.
ArticleTreutlein B,Huttner WB,Pääbo S,Lachmann R,Knoblich JA,Lancaster M,Hevers W,Sykes A,Lewitus E,Wilsch-Bräuninger M,Gerber T,Kanton S,Florio M,Badsha F,Camp JG.Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.Proceedings of the National Academy of Sciences of the United States of America.2015 Dec 22
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304502
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP066834
Overall Desgin734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).
SummaryCerebral organoids – three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells – have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
Experimental ProtocolCells were loaded into the Fluidigm C1 microfluidic platform, where single cells were captured. Lysis of single cells, reverse-transcription of mRNA into cDNA as well as preamplification of cDNA occured within the microfluidic device using reagents provided by Fluidigm as well as the SMARTer Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions at a dilution of 1:40,000.; Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.
Data processingfreeIbis was used as an efficient basecaller with calibrated quality scores for Illumina sequencers. (Bioinformatics. 2013 May 1;29(9):1208-9. doi: 10.1093/bioinformatics/btt117. Epub 2013 Mar 6.); leeHom was used to trim adapters and merge Illumina sequencing reads. (Nucleic Acids Res. 2014 Oct;42(18):e141. doi: 10.1093/nar/gku699. Epub 2014 Aug 6.); deML was used for robust demultiplexing of Illumina sequences using a likelihood-based approach. (Bioinformatics. 2015 Mar 1;31(5):770-2. doi: 10.1093/bioinformatics/btu719. Epub 2014 Oct 30.); Reads were aligned against the human genome (build GRCh38.77 from ENSEMBLE) using TopHat v2.0.14. The genome was indexed using Bowtie v2.2.6; Fragments Per Kilobase of transcript Per Million mapped reads (FPKM) values were computed using cufflinks v2.2.1; Genome_build: GRCh38; Supplementary_files_format_and_content: Master data frame including sample name, gene ID, and FPKM values for each single cell.
Public OnPublic on Dec 07 2015

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