TITLE: Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells
AUTHORS: Mackenzie W. Amoroso, Gist F. Croft, Damian J. Williams, Sean O’Keeffe, Monica A. Carrasco, Anne R. Davis, Laurent Roybon, Derek H. Oakley, Tom Maniatis, Christopher E. Henderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Human pluripotent stem cells are a promising source of differentiated 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 within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward 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</span><span style = "vertical-align:super;">+</span><span>/LHX3</span><span style = "vertical-align:super;">-</span><span>). 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.</span></div></div>

[STEPS]
?. [Cell culture maintenance]
Maintain all cell cultures at , 5% CO2.
37 °C
?. [Cell culture maintenance]
Maintain hES and iPS cells [HUES3 (control), male; H9 (control), female; HS001 (ALS-SOD1 N139K), male; LWM002 (ALS-SOD1 A4V), female; MBN007 (ALSSOD1 A4V), female; TM008 (ALS-SOD1 A4V), female; DCM009 (ALSSOD1 V148G), male; 10013.13 (control), male)] on gelatinized tissue-culture plastic on a monolayer of irradiated CF-1mouse embryonic fibroblasts (GlobalStem), in hESC media, consisting of DMEM: nutrient mixture F-12 (DMEM/F:12; Invitrogen) with 20% Knockout Serum Replacer (Invitrogen), 110 uM -mercaptoethanol (BME; Sigma), L-glutamine and nonessential amino acids (NEAA; Invitrogen), and 20 ng/ml basic fibroblast growth factor (bFGF; Invitrogen) (Cowan et al., 2004).
?. [Cell culture maintenance]
Change media every  and passage lines with dispase (Invitrogen, ) in hESC media for – at ).
37 °C
?. [Motor neurons generation]
To generate motor neurons, passage undifferentiated hESCs using dispase () and triturated into small, 50- to 100-cell clumps and placed into ultralow adherent culture dishes (Corning).
?. [Motor neurons generation]
For the first 3 d, keep cells in suspension in hESC medium, supplemented with Rho-associated kinase inhibitor Y27632 (Ascent Scientific) to enhance single cell survival (Watanabe et al., 2007), bFGF (Invitrogen) to enhance growth, and  SB435142 (SB; Sigma) and  LDN193189 (LDN; Stemgent) for neuralization.
?. [Motor neurons generation]
At day 3, switch embryoid bodies (EBs) to neural induction medium (DMEM/F:12 with L-glutamine; NEAA; penicillin/streptomycin; heparin, ; N2 supplement; Invitrogen).
?. [Motor neurons generation]
At day 5, add all-trans retinoic acid (RA;  or ; Sigma), ascorbic acid (; Sigma), and brain-derived neurotrophic factor (; R&D).
?. [Motor neurons generation]
Pursue dual ALK inhibition (SB + LDN) until day 7.
?. [Motor neurons generation]
Initiate Hedgehog signaling on day 7 by application of C25II modified SHH (R&D), at the standard concentration of ; a human Smo agonist (HAG, ; gift from Lee Rubin; Dimos et al., 2008; Boulting et al., 2011); mouse Smo agonist 1.3 (SAG; ; Frank-Kamenetsky et al., 2002; Wichterle et al., 2002; Wada et al., 2009; Boulting et al., 2011); or purmorphamine (PUR, ; Stemgent; Sinha and Chen, 2006; Li et al., 2008).
?. [Motor neurons generation]
At day 17, change basal medium to Neurobasal (Invitrogen), containing all previous factors and with the addition of each of insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) (R&D), plus B27 (Invitrogen).
?. [Motor neurons generation]
At day 20 or 30, disassociate EBs with 0.05% trypsin (Invitrogen) and plate onto poly-lysine/laminin-coated 8-well chamber slides (BD Biosciences) at 0.2-0.5.10 6 cells/well and/or  coverslips at 0.5.106.
15
?. [Motor neurons generation]
Culture plated neurons in the same medium with the addition of BME and  glutamic acid (Sigma) and fix 1 d later.
?. [Immunocytochemistry assay]
Fix cultures for  with 4% paraformaldehyde (PFA) in PBS at , wash three times for  in PBS, quench, and permeabilize in wash buffer (PBS, 0.1% Triton X-100) plus  glycine for .
4 °C
?. [Immunocytochemistry assay]
For the EB outgrowth RALDH2 staining, fix samples for  at  with 4% PFA/10% sucrose prewarmed to .
4 Room temperature
37 °C
?. [Immunocytochemistry assay]
Block samples with wash buffer plus 10% normal donkey serum for  and incubate with primary antibody (Table 1) .
?. [Immunocytochemistry assay]
Wash cells, incubate with DyLight coupled donkey primary anti-secondary antibodies (Jackson ImmunoResearch, 1:1000).
?. [Immunocytochemistry assay]
Finally, wash cells and counterstain with DAPI (Invitrogen).
?. [Quantitative image analysis of differentiated neuronal cultures]
Perform quantitative image analysis of differentiated neuronal cultures using the Multi-Wavelength Cell Scoring module in MetaMorph software (Molecular Devices).
?. [Quantitative image analysis of differentiated neuronal cultures]
Briefly, dissociate EBs enzymatically and plate in the presence of neurotrophic factors at densities for which cell overlap was minimal.
?. [Quantitative image analysis of differentiated neuronal cultures]
Following immunostaining, capture images of at least nine randomly selected fields (15,000 cells in total) for each condition using a preprogrammed automated microscope stage.
?. [Quantitative image analysis of differentiated neuronal cultures]
Analyze images using the Multi-Wavelength Cell Scoring module of the MetaMorph software, using parameters predefined to count only unambiguous bright labeling for each antigen.
?. [Quantitative image analysis of differentiated neuronal cultures]
Set intensity thresholds while blinded to sample identity to selectively identify positive cells that displayed unambiguous signal intensity above local background.
These parameters are used on all samples in a given experiment, and only minimally adjusted for different staining batches as necessary. Script and Parameter files are available upon request (typically, a cell is approximately 5000 gray levels above background to be called positive for any nuclear marker and is approximately 10,000 for cytoplasmic markers).
?. [Quantitative image analysis of differentiated neuronal cultures]
Analyze a minimum of 15,000 cells per sample.
?. [Quantitative image analysis of differentiated neuronal cultures]
Image all samples using 10× or 20× objectives on a Zeiss AxioObserver with a Coolsnap HQ2 camera (Photometrics).
?. [Quantitative image analysis of differentiated neuronal cultures]
Acquire some images using a structured illumination technique using an Apotome module (Zeiss) to achieve  optical sections to ensure colocalization of labeling.
1.9
?. [Quantitative image analysis of differentiated neuronal cultures]
For the figures, adjust the brightness and contrast of each channel of an image in an appropriate manner to improve clarity.
?. [Quantitative image analysis of differentiated neuronal cultures]
For Ca 2+ imaging experiments using the Hb9::GFP reporter, differentiate stem cells under the motor neuron differentiation protocol described above, dissociate at day 21 or day 31, and sort FACS based on green fluorescent protein (GFP) intensity with a five laser ARIA-IIu ROU Cell Sorter configured with a  ceramic nozzle and operating at 20 psi (BD BioSciences).
100
?. [Quantitative image analysis of differentiated neuronal cultures]
Comprise the H9 assays of mixed neuronal cultures, for which a parallel coverslip is stained and quantified to have 53% HB9/ISL1+ motor neurons.
?. [Quantitative image analysis of differentiated neuronal cultures]
Plate all cultures onto – diameter coverslips at a density of 100,000–150,000 cells per coverslip in day 17+ Neurobasal media with factors described above, with the addition of  EdU, and mature 6 d before Ca 2+ imaging.
15
25
?. [Quantitative image analysis of differentiated neuronal cultures]
Load cells with  Fluo-4 AM (Invitrogen) dissolved in 0.2% dimethylsulfoxide/0.04% pluronic acid (Sigma) in HEPES-buffered physiological salt solution (PSS) for  at .
37 Room temperature
PSS contained the following (in mM): 145 NaCl, 5 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, and 5.5 glucose (pH 7.4).
?. [Quantitative image analysis of differentiated neuronal cultures]
Superfuse cultures continuously with PSS at a rate of approximately .
0.5
?. [Quantitative image analysis of differentiated neuronal cultures]
Image the cultures using a 10× objective on an inverted epifluorescent Zeiss AxioObserver microscope, equipped with a Coolsnap HQ2 camera (Photometrics).
?. [Quantitative image analysis of differentiated neuronal cultures]
For imaging spontaneous Ca 2+ transients, acquire single sets of 200–300 images at a rate of approximately 2 Hz from each coverslip.
?. [Quantitative image analysis of differentiated neuronal cultures]
For the kainate (KA) experiments, acquire 36 images at a rate of 0.033 Hz and replace the superfusing PSS with PSS-containing KA () for .
?. [Quantitative image analysis of differentiated neuronal cultures]
Perform image analysis using ImageJ (http://rsb.info.nih.gov/ij/) or AxioVision 4.7 (Zeiss).
?. [Quantitative image analysis of differentiated neuronal cultures]
Determine Ca 2+ transients from regions of interest encompassing the soma of individual cells.
?. [Quantitative image analysis of differentiated neuronal cultures]
Obtain a minimum of two cultures from a single differentiation of each cell line and use each time point for the KA and all Ca 2+ imaging experiments.
?. [Whole-cell patch-clamp recordings]
For whole-cell patch-clamp recordings, Plate S + P differentiated HUES3 Hb9::GFP cells on polyornithine/laminin-coated  diameter coverglass at density of 50,000 per coverslip and culture for  in the presence of EdU before recording (i.e., 21 + 7 days in vitro; DIV).
25
?. [Whole-cell patch-clamp recordings]
Perform current-clamp recordings using an Axopatch 2B amplifier.
?. [Whole-cell patch-clamp recordings]
Digitize data using a Digidata 1322A digital to analog converter and record at a 10 kHz sample rate using pClamp 10 software (all equipment from Molecular Devices).
?. [Whole-cell patch-clamp recordings]
Fabricate patch pipettes using a P-97 pipette puller (Sutter Instruments).
The external recording solution contained the following (in mM): 145 NaCl, 5 KCl, 10 HEPES, 10 glucose, 2 CaCl2, and 2 MgCl2.
?. Adjust the  using NaOH and adjust the osmolality to with sucrose.
325
The pipette solution contained the following (in mM): 130 CH3KO3S, 10 CH3NaO3S, 1 CaCl2, 10 EGTA, 10 HEPES, 5 MgATP, and 0.5 Na2GTP (pH 7.3, 305 mOsm).
?. [Whole-cell patch-clamp recordings]
Perform experiments at  (–).
37 Room temperature
21 °C
23 °C
?. [Whole-cell patch-clamp recordings]
During recordings, inject current to hold the cells at approximately -60 mV.
?. [Whole-cell patch-clamp recordings]
Evoke action potentials (APs) using incrementally increasing current steps 1 s in duration.
?. [Whole-cell patch-clamp recordings]
Calculate the maximum amplitude of the current step (20–50 pA) and the size of the increment based on the input resistance of the cell.
?. [Xenotransplantations]
To perform xenotransplantations day 21, collect EBs from HUES3 Hb9::GFP under ventralization with SAG + PUR and place into L-15 media (Invitrogen) containing penicillin/streptomycin (Invitrogen).
?. [Xenotransplantations]
Perform transplantation as previously described (Wichterle et al., 2002).
?. [Xenotransplantations]
Briefly, load lightly triturated EBs into a handheld micro-injector, after a small suction lesion at the prospective intraspinal site was created in a chick embryo at stage 15–18 at somites 15–20.
?. [Xenotransplantations]
Place the EBs into the lesion.
?. [Xenotransplantations]
After 48 h, sacrifices the chicks, fix with 4% PFA for  at , and access neurite outgrowth and cell body placement by cutting   vibratome sections (n = 2) or by cutting  sections along the spinal cord (n = 5).
4 °C
200
30
?. [Xenotransplantations]
Collect human embryonic spinal cords in accordance with the national guidelines of the United States (National Institutes of Health, U.S. Food and Drug Administration) and the State of New York and under Columbia University institutionally approved ethical guidelines relating to anonymous tissue.
?. [Xenotransplantations]
Obtain the material after elective abortions, and classify on the basis of external morphology according to the Carnegie stages.
?. [Xenotransplantations]
Determine gestational age by last menstrual period of the patient or by ultrasound, if the ultrasound estimate differed by >1 week as indicated by the obstetrician.
?. [Xenotransplantations]
Remove the spinal cord as intact as possible before fixation with fresh, cold 4% PFA for .
on ice
?. [Xenotransplantations]
Postfixation, measure the cord and cut into three anatomical sections to accommodate embedding in OCT Compound (Tissue-Tek) and store at  before cutting on a microtome.
-80 °C
?. [Xenotransplantations]
Cut sections () along the full length of the cord, taking care to have all three sections on each slide in seven independent sections.
12
This allowed for full analysis and internal staining controls since each slide had cervical, brachial, thoracic, and lumbar sections that clearly showed staining within the various otor columns present at different rostrocaudal levels of the spinal cord.
?. [Xenotransplantations]
Obtain cDNA from 50,000 FACS-purified motor neurons from either day 21 S + P (methods described above), or from RA/SHH motor neurons at day 31.
?. [Xenotransplantations]
Perform cDNA preparation using commercially available kits following the manufacturer’s instructions: RNA isolation (Trizol LS; Invitrogen) and cDNA by Brilliant II SYBR green (Stratagene) without amplification.
?. [Xenotransplantations]
Process all samples in parallel on the same quantitative PCR (qPCR) plate (Table 2).
?. [Paired-end RNA-Seq experiments]
For paired-end RNA-Seq experiments, Prepare of total RNA after FACS purification of 500,000 GFP+ or GFP-  cells.
400 ng
?. [Paired-end RNA-Seq experiments]
Amplify the RNA samples using a NuGEN RNA kit for genomic sample amplification, and sequence to a depth of 21 (S + P) and 35 (SHH) million paired-end reads on an Illumina HiSeq instrument at the HudsonAlpha Institute of Biotechnology.
?. [Paired-end RNA-Seq experiments]
Align the reads to the reference transcriptome as well as a library of exon junctions using Bowtie (Version 1) (Langmead et al., 2009).
?. [Paired-end RNA-Seq experiments]
Analyze data using Expression Plot (Friedman and Maniatis, 2011) using a p value of 0.001 and a two fold change threshold.
?. [Paired-end RNA-Seq experiments]
Perform gene ontology using DAVID (Huang da et al., 2009a, b) with enrichment sets from Expression Plot.
The RNA-seq data are available in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE41795.
?. [Paired-end RNA-Seq experiments]
Analyze all quantitative data using Sigma Plot 11 or Microsoft Excel.
?. [Paired-end RNA-Seq experiments]
Subject sample groups to Student’s t-test or where appropriate a one-way ANOVA with Holm–Sidak post hoc pairwise comparisons was performed.
All experimental data passed an equal variance and normality test (Shapiro–Wilk).