Objectives
Transcriptional profiling by RNA sequencing (RNAseq) enables the sensitive and accurate characterization of the transcriptome1–4. Following enumeration of reads, various methods are available to normalize counts, estimate probability distributions and identify differential gene expression between biological conditions3,5,6. RNAseq has the added advantages of being extremely high-throughput and relatively inexpensive, with a high signal-to-noise ratio and a dynamic range encompassing 4–5 orders of magnitude. This combination of throughput and sensitivity enables the detection of rare transcripts from nanograms of RNA.
We have described a method to produce large quantities of highly enriched, electrically active glutamatergic neurons (ESNs) from suspension-adapted mouse embryonic stem cells (ESCs)7,8. This technique is a modification of the 4/4 method, and is based on the spatiotemporal changes in morphogens that occur during neural induction and patterning9–11. In this method, neuroepithelial stem cells (NESCs) are derived from ESCs by the withdrawal of leukemia inhibitory factor (LIF), then induced to undergo neurogenesis and neural patterning by supplementation with all-trans retinoic acid (RA) in the presence of fetal bovine serum12. We have modified the 4/4 method to include feeder cell-free, suspension culture of ESNs; differentiation under rotary conditions to normalize the intra-aggregate environment; and neuronal induction and neural induction and patterning using 6 µM RA. These refinements have resulted in a facile and economical method to generate large quantities of highly enriched glutamatergic neurons13. Using immunocytochemistry, we have shown that ESNs are composed mostly of glutamatergic neurons (~95%), with about 5% GABAergic neurons, and no evidence of dopaminergic, serotonergic, cholinergic or glycinergic subtypes7. Expression profiling using RNA sequencing has confirmed that derived cultures are primarily glutamatergic, and identified the abundant expression of a wide range of cortical markers, including reelin, Pax6, Otx1, Ctip2 and Cux1/213–19.
Although aspects of corticogenesis have been replicated in vitro by the directed differentiation of pluripotent stem cells, spatiotemporal changes in gene expression responsible for regionalization and apical-to-basal patterning of the cerebral cortex are not well understood20–22. The embryological origin of the cerebral cortex is the telencephalon, which is the most anterior structure of the mammalian neural tube23. Cortical glutamatergic neurons are generated by proliferation and laminar patterning of the dorsal telencephalon, whereas inhibitory GABAergic neurons derive from the ventral telencephalon and migrate tangentially into cortical layers24,25. Our ability to derive predominantly glutamatergic neurons that express markers of the telencephalon and all six cortical layers is consistent with an in vitro model of the developing telencephalon and cortical layer formation23. We propose to apply this model to identify the temporal changes in gene and isoform expression associated with neural patterning and corticogenesis. If successful, we intend to use ESNs to functionally interrogate the roles of individual genes in executing the transcriptional programs underlying the formation of the cerebral cortex.
To evaluate the transcriptional changes associated with differentiation of cortical glutamatergic neurons, we conducted a longitudinal expression profile of the deep transcriptome during neurogenesis from days in vitro (DIV) -8 to 28, where DIV 0 corresponds to the end of differentiation. High quality RNA was isolated from ESCs (DIV -8; n=4); neuroepithelial stem cells (DIV -4; n=3); radial glia (DIV 0; n=3); developmental stage (DS) I/II neurons (DIV 1; n=4); DS III/IV neurons (DIV 7; n=5); and maturing DS IV/V neurons at DIV 16 (n=4); DIV 21 (n=4); and DIV 28 (n=4). The summary data, including quality scores, are presented in Data File 1, and the raw transcript read counts for each biological replicate are presented in Data File 2. The FASTQ files generated for each biological replicate are available at the Sequence Read Archive (SRA), a freely accessible database provided by NCBI (http://www.ncbi.nlm.nih.gov/sra/), under the accession number PRJNA185305.
In addition to providing the basis for a study to characterize transcriptional processes involved in corticogenesis and neuronal maturation, we can foresee several other applications for this data. First, the identification of genes and isoforms that exhibit differential expression in synchronization with known markers is expected to provide novel insight into molecular mechanisms of neurogenesis and neural patterning. Second, a few algorithms are available for statistical determination of differential gene expression within longitudinal data sets. The depth and quality of these data suggests it may be well-suited for the development and validation of such methods. Third, we envision this dataset facilitating inter-specific comparisons of transcription during neurogenesis and corticogenesis, providing insight into transcriptional mechanisms common to and unique in the evolution of the mammalian cerebral cortex. For these reasons, we are making this data publically available for research efforts in bioinformatics, stem cell research and developmental neuroscience studies.
Materials and methods
Suspension adaptation and continuous culture of mouse ESCs
ESCs were adapted to feeder cell-free, suspension culture and maintained as previously described7,13. In brief, aliquots of R1 ESCs (ATCC, Manassas, VA) were thawed and maintained at 37°C at 5% CO2 in 90% relative humidity in 10 cm bacterial plates in ESM (Knockout DMEM supplemented with 100 µM β-mercaptoethanol, 0.1mM nonessential amino acids, 2.0 mM L-glutamine, 5000 units/mL penicillin/streptomycin, 1000 units/mL recombinant mouse LIF [all Life Technologies, Carlsbad, CA] and 15% ES qualified fetal calf serum [ATCC, Manassas, VA])26. Cells were passaged once aggregates first became clearly visible to the naked eye (4–8 days) and maintained for at least 5 passages prior to differentiation. For passaging, aggregates were allowed to settle by gravity, washed once with 0.5 mL PBS and dissociated for 3 min at 37°C with 0.5 ml of TrypLE Express (Life Technologies). Dissociation was terminated by addition of 0.5 mL ESM followed by gently trituration with a P1000 pipette to achieve a single-cell suspension. ESNs were counted manually using a hemocytometer and ~1.5×106 mESCs were transferred to 10 mL ESM in a fresh 10 cm bacterial dish.
Neuronal differentiation
ESCs were differentiated into neurons between 5–30 passages after adaptation to suspension culture. A modified 4/4 protocol was used for neuron differentiation7,27. Following routine sub-passaging, 3.5×106 mouse ESCs were transferred to 30 mL differentiation medium (ESM modified to contain 10% ESC-qualified fetal calf serum and without LIF) in a 10 cm ultra-low attachment suspension culture dish (Corning, Lowell, MA). This was designated as DIV -8. Differentiating aggregates were maintained on a rotary shaker at 45 rpm at 37°C, 5% CO2 and 90% relative humidity. Complete media changes were conducted every 48 h, and media was supplemented with 6 µM retinoic acid (Sigma-Aldrich) at DIV -4 and DIV -2.
On DIV 0, aggregates were dissociated with TrypLE Express for 5 min at 37°C. Trypsinization was halted with 5 mL of 1% soybean trypsin inhibitor (Life Technologies), the aggregates were gently dissociated by trituration with a 10 mL pipet, and the cell suspension was filtered through a 40 µm cell strainer (Thermo Scientific, Waltham, MA). Cells were pelleted for 5 min at 300 x g, washed in N2 medium (Neurobasal-A medium with 1 x N2 vitamins, 2 mM glutamine and antibiotics [Life Technologies]) and counted manually using a hemocytometer. Neuronal progenitors were plated at 1.5×106 cells/cm2 in poly-D coated dishes. Complete washes with N2 medium were conducted at 4 h and 24 h to remove residual serum and non-adherent cells. At DIV 2, N2 was replaced with B27 medium (Neurobasal-A supplemented with antibiotics, 2 mM glutamine and 1 x B27 vitamins [Life Technologies]). Subsequently, ESNs underwent full medium changes with B27 on DIV 4, 8 and 12. On DIV 8 the media was supplemented with 30 µM 5-fluoro-2’-deoxyuridine and 70 µM Uridine (Sigma-Aldrich) to select against any remaining glia. Following DIV 12, ESNs were left undisturbed until RNA harvest. Cultures remained healthy and viable until at least DIV 28 under these conditions.
Expression profiling
Neurons were harvested from 6 cm dishes at DIV -8, -4, 0, 1, 7, 16, 21 and 28 (n=3 to 5) and RNA was isolated by QIAcube (Qiagen, Valencia, CA) using the RNeasy mini kit protocol (Qiagen) and submitted to Expression Analysis, Inc. (Durham, NC) for library preparation and sequencing. Typical recovery was ~3 µg per 6 cm dish, with an RNA integrity number exceeding 8 and a 260:280 of 1.8 – 2.2. PolyA+ RNA was purified from 500 ng of total RNA using polyA selection, chemically fragmented and reverse transcribed using random hexamers. This was followed by second strand synthesis and end repair. Libraries were prepared for paired-end sequencing using the TruSeq™ RNA sample prep kits (Illumina, San Diego, CA) per manufacturer’s instructions, and library size and integrity were determined using the Agilent Bioanalyzer 2100 (Santa Clara, CA). Libraries were bound to flow cell surfaces using the Standard Cluster Generation Kit v5 (Illumina). Flow cells were transferred to the Illumina HiSeq 2000 and run using TruSeq SBS Kits (Illumina). Paired-end sequencing data were generated over 2x50 sequencing cycles. Sequence information and quality scores are available at SRA in FASTQ format, with the forward and reverse reads appended with an "F" or "R" (e.g., DIV7.1F and DIV7.1R). Raw sequence reads were aligned using the University of California, Santa Cruz’s mouse knownGENE track, and transcriptome abundance estimation was performed on completed alignments using RSEM (RNA-Seq by Expectation Maximization)28. A summary flatfile containing the total raw reads for each gene in each biological replicate is presented in Data File 2. Genes were excluded if no reads occurred in any biological sample.
Author contributions
KSH, IMG and PMM conceived the study. IMG, PMM and MEL conducted ESC culture, produced neurons and extracted the RNA. KSH, IMG and PMM organized the data sets and prepared the manuscript. All authors approved the final manuscript for publication.
Competing interests
No competing interests were disclosed.
Grant information
This work was funded by the National Institutes of Health National Institute of Allergy and Infectious Diseases (IAA number AOD12058-0001-0000) and the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division (grant number CBM.THRTOX.01.10.RC.021). This research was performed while IMG held a Defense Threat Reduction Agency-National Research Council Research Fellowship Award and KSH held a National Research Council Research Associateship Award.
Acknowledgements
We thank Angela Adkins, Cindy Kronman and Marian Nelson (USAMRICD, MD) for administrative, logistical and technical assistance. We also thank Expression Analysis, Inc. (Durham, NC) for assistance with RNA sequence generation. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.
F1000 recommendedReferences
- 1.
Bullard JH, Purdom E, Hansen KD, et al.:
Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments.
BMC Bioinformatics.
2010; 11: 94.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 2.
Griffith M, Griffith OL, Mwenifumbo J, et al.:
Alternative expression analysis by RNA sequencing.
Nat Methods.
2010; 7(10): 843–847.PubMed Abstract
| Publisher Full Text
- 3.
Marioni JC, Mason CE, Mane SM, et al.:
RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.
Genome Res.
2008; 18(9): 1509–1517.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 4.
Hebenstreit D, Fang M, Gu M, et al.:
RNA sequencing reveals two major classes of gene expression levels in metazoan cells.
Mol Syst Biol.
2011; 7: 497.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 5.
Sultan M, Schulz MH, Richard H, et al.:
A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome.
Science.
2008; 321(5891): 956–960.PubMed Abstract
| Publisher Full Text
- 6.
Robinson MD, Smyth GK:
Moderated statistical tests for assessing differences in tag abundance.
Bioinformatics.
2007; 23(21): 2881–2887.PubMed Abstract
| Publisher Full Text
- 7.
McNutt P, Celver J, Hamilton T, et al.:
Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.
Biochem Biophys Res Commun.
2011; 405(1): 85–90.PubMed Abstract
| Publisher Full Text
- 8.
Hubbard KS, Gut IM, Lyman ME, et al.:
High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.
BMC Neurosci.
2012; 13: 127.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 9.
Bain G, Kitchens D, Yao M, et al.:
Embryonic stem cells express neuronal properties in vitro.
Dev Biol.
1995; 168(2): 342–357.PubMed Abstract
| Publisher Full Text
- 10.
Bibel M, Richter J, Lacroix E:
Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells.
Nat Protoc.
2007; 2(5): 1034–1043.PubMed Abstract
| Publisher Full Text
- 11.
Glaser T, Brustle O:
Retinoic acid induction of ES-cell-derived neurons: the radial glia connection.
Trends Neurosci.
2005; 28(8): 397–400.PubMed Abstract
| Publisher Full Text
- 12.
Levine AJ, Brivanlou AH:
Proposal of a model of mammalian neural induction.
Dev Biol.
2007; 308(2): 247–256.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 13.
Hubbard KS, Gut IM, Lyman ME, et al.:
High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.
BMC Neurosci.
2012; 13: 127.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 14.
Germain N, Banda E, Grabel L:
Embryonic stem cell neurogenesis and neural specification.
J Cell Biochem.
2010; 111(3): 535–542.PubMed Abstract
| Publisher Full Text
- 15.
Weimann JM, Zhang YA, Levin ME, et al.:
Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets.
Neuron.
1999; 24(4): 819–831.PubMed Abstract
| Publisher Full Text
- 16.
Chen B, Schaevitz LR, McConnell SK:
Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex.
Proc Natl Acad Sci U S A.
2005; 102(47): 17184–17189.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 17.
Meyer G, Goffinet AM:
Prenatal development of reelin-immunoreactive neurons in the human neocortex.
J Comp Neurol.
1998; 397(1): 29–40.PubMed Abstract
| Publisher Full Text
- 18.
Georgala PA, Manuel M, Price DJ:
The generation of superficial cortical layers is regulated by levels of the transcription factor Pax6.
Cereb Cortex.
2011; 21(1): 81–94.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 19.
Li N, Zhao CT, Wang Y, et al.:
The transcription factor Cux1 regulates dendritic morphology of cortical pyramidal neurons.
PLoS One.
2010; 5(5): e10596.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 20.
Gaspard N, Bouschet T, Hourez R, et al.:
An intrinsic mechanism of corticogenesis from embryonic stem cells.
Nature.
2008; 455(7211): 351–357.PubMed Abstract
| Publisher Full Text
- 21.
Hansen DV, Rubenstein JL, Kriegstein AR:
Deriving excitatory neurons of the neocortex from pluripotent stem cells.
Neuron.
2011; 70(4): 645–660.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 22.
Jing Y, Machon O, Hampl A, et al.:
In vitro differentiation of mouse embryonic stem cells into neurons of the dorsal forebrain.
Cell Mol Neurobiol.
2011; 31(5): 715–727.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 23.
Sansom SN, Livesey FJ:
Gradients in the brain: the control of the development of form and function in the cerebral cortex.
Cold Spring Harb Perspect Biol.
2009; 1(2): a002519.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 24.
Butler AB:
The evolution of the dorsal pallium in the telencephalon of amniotes: cladistic analysis and a new hypothesis.
Brain Res Rev.
1994; 19(1): 66–101.PubMed Abstract
| Publisher Full Text
- 25.
Flames N, Marin O:
Developmental mechanisms underlying the generation of cortical interneuron diversity.
Neuron.
2005; 46(3): 377–381.PubMed Abstract
| Publisher Full Text
- 26.
Nagy A, Rossant J, Nagy R, et al.:
Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.
Proc Natl Acad Sci U S A.
1993; 90(18): 8424–8428.PubMed Abstract
| Publisher Full Text
| Free Full Text
- 27.
Bibel M, Richter J, Schrenk K, et al.:
Differentiation of mouse embryonic stem cells into a defined neuronal lineage.
Nat Neurosci.
2004; 7(9): 1003–1009.PubMed Abstract
| Publisher Full Text
- 28.
Li B, Dewey CN:
RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.
BMC Bioinformatics.
2011; 12: 323.PubMed Abstract
| Publisher Full Text
| Free Full Text
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