News Article | November 30, 2016
The following strains of mice were used (see details in following sections): Swiss Webster females and males, C57BL/6J or C57BL6/N males, B6.Cg-Tg(Pou5f1-GFP)1Scho25 males, CD-1 females and males. 6–10-week-old female mice, and 6-week- to 6-month-old male mice were used. Animals were maintained on 12 h light–dark cycle and provided with food and water ad libitum in individually ventilated units (Techniplast at TCP, Laboratory Products at UCSF) in the specific-pathogen-free facilities at UCSF and at TCP. All procedures involving animals were performed in compliance with the protocol approved by the IACUC at UCSF, as part of an AAALAC-accredited care and use program (protocol AN091331-03); and according to the Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care. Animal Care Committee reviewed and approved all procedures conducted on animals at TCP. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. No statistical methods were used to predetermine sample size estimate. Unless otherwise indicated, Swiss Webster females were mated to Swiss Webster males, or to C57BL/6 males homozygous for an Oct4-GFP transgene (B6.Cg-Tg(Pou5f1-GFP)1Scho)25. Preimplantation embryos were collected at indicated time-points after detection of the copulatory plug by flushing oviducts (E1.5–E2.5) or uteri (E3.5) of pregnant females using M2 medium (Zenith Biotech) supplemented with 2% BSA (Sigma). Subsequent embryo culture was performed in 4-well plates in 5% O , 5% CO at 37 °C in KSOMAA Evolve medium (Zenith Biotech) with 2% BSA and the following inhibitors, after optimization of concentrations: 200 nM INK128 (Medchem Express), 2.5 μM 10058-F4 (Sigma), 100 ng ml−1 cycloheximide (Amresco), 50 μM Anacardic Acid (Sigma). Other mTOR inhibitors (AZD2014, Everolimus and Rapamycin (Medchem Express) and RapaLink-1 (gift of K. Shokat)) and autophagy inhibitors chloroquine (Sigma) and SBI-0206965 (Medchem Express) were used at the indicated concentrations under same culture conditions. Diapause was induced as previously described9 after natural mating of Swiss Webster mice. Briefly, pregnant females were injected at E2.5 and EDG5.5 with 10 μg tamoxifen (intra-peritoneally) and at E2.5 only with 3 mg medroxyprogesterone 17-acetate (subcutaneously). Diapaused blastocysts were flushed from uteri in M2 media after 4 days of diapause at EDG8.5. Both surgical and non-surgical embryo transfers (NSET) were performed. For surgical transfers, superovulated CD-1 females were mated to C57BL/6J or C57BL6/N males and embryos were flushed at E3.5. Embryo culture (as described above) and surgical embryo transfer into the uteri of 2.5 days post coitus pseudopregnant CD-1 females previously mated with vasectomized CD-1 males was performed essentially as described26. For NSET, Swiss Webster females were mated to vasectomized CD-1 males and transfer was performed at E2.5 of surrogate according to manufacturer’s instructions (ParaTechs, Lexington). Before embryo transfer, embryos were cultured in KSOMAA, 2% BSA without inhibitor for 1 h. In the cases indicated (Extended Data Fig. 1a), Caesarian delivery was performed at E20, followed by fostering to Swiss Webster females. Coat colour markers (agouti versus albino) were used to distinguish transferred embryos after birth. ES cell derivation was performed as previously described27. Swiss Webster females were naturally mated to Swiss Webster-C57BL/6 males heterozygous for an Oct4-GFP transgene (B6.Cg-Tg(Pou5f1-GFP)1Scho)25. Blastocysts were collected by flushing uteri of pregnant females at E3.5, and were seeded on feeders either immediately or after culturing for 7 days in KSOMAA, 2% BSA, 200 nM INK128. Imaging of fluorescence driven by the Oct4-GFP transgene and alkaline phosphatase activity (VECTOR Red AP Substrate Kit, Vector Laboratories) was performed using a Leica DM IRB microscope. For immunofluorescence stainings, normal (E3.5), in vivo diapaused or ex vivo paused embryos were fixed in 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking in PBS, 2.5% BSA, 5% donkey serum for 1 h, embryos were incubated overnight at 4 °C with the following primary antibodies in blocking solution: phospho-4EBP1 (Thr37/46, clone 236B4), phospho-Akt (Ser473), phospho-Ulk1 (Ser757), Nanog, c-Parp, c-Caspase3 (all from Cell Signaling), H3K4me3, H4K16ac, H4K5/8/12ac, H3K9me3 (all from Millipore), Oct4 and Rex1 (Santa Cruz Biotechnology) and H3K36me2 (Abcam). Embryos were washed in PBS-Tween20, 2.5% BSA, incubated with fluorescence-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature, and mounted in VectaShield mounting medium with DAPI (Vector Laboratories). For labelling nascent transcription or translation, embryos were labelled in their respective culture medium for 20 min with EU (5-ethynyl uridine) or HPG (l-homopropargylglycine) following the manufacturer’s instructions for Click-iT RNA and protein labelling kits (Thermo Fisher Scientific). Imaging was performed using a Leica SP5 confocal microscope with automated z-stacking at 10 μm intervals. Cell Profiler Software28 was used for image quantification and Prism (Graphpad Software) was used for plotting data points. Datasets do not show similar variance between control and paused/diapaused embryos in all cases, therefore we applied Welch’s correction to the statistical analysis. E14 (from B. Skarnes, Sanger Institute), Oct4-GiP (from A. Smith, University of Cambridge) and v6.5 (from R. Blelloch, UCSF) ES cell lines were used. ‘Serum’ cells were cultured in ES-FBS medium: DMEM GlutaMAX with Na Pyruvate (Thermo Fisher Scientific), 15% FBS (Atlanta Biologicals), 0.1 mM non-essential amino acids, 50 U ml−1 penicillin/streptomycin (UCSF Cell Culture Facility), 0.1 mM EmbryoMax 2-Mercaptoethanol (Millipore) and 2,000 U ml−1 ESGRO supplement (LIF, Millipore). ‘2i’ cells were cultured in ES-2i medium: DMEM/F-12, Neurobasal medium, 1× N2/B27 supplements (Thermo Fisher Scientific), 1 μM PD0325901, 3 μM CHIR99021 (Selleck Chemicals), 50 μM Ascorbic acid (Sigma) and 2,000 U ml−1 ESGRO supplement (LIF) (Millipore). ‘Paused’ cells were cultured in ES-FBS medium containing 200 nM INK128 (Medchem). ES cells can also be paused in 2i medium, but the mTOR inhibitor needs to be removed at each passaging and reintroduced after colony formation to avoid major cell death (Extended Data Fig. 6a). The cell lines have not been authenticated. E14 and v6.5 tested negative for mycoplasma contamination. Oct4-GiP was not tested. R1 (129S1×129X1)29 and G4 (129S6×B6N)30 ES cells were used for morula aggregations. ES cells were cultured in DMEM containing 10% FBS (Wisent, lot-tested to support generation of germline chimaeras), 10% KnockOut Serum Replacement, 2 mM GlutaMAX, 1 mM Na Pyruvate, 0.1 mM non-essential amino acids, 0.1 mM 2-Mercaptoethanol (all Thermo Fisher Scientific), 1,000 U ml−1 LIF (Millipore). G4 ES cells were grown on MEF obtained from TgN(DR4)1Jae/J mice at all times except one passage on gelatinized tissue culture plates before aggregation. R1 ES cells were cultured in feeder-free conditions on gelatinized tissue culture plates. CD-1 (ICR) (Charles River) outbred albino stock was used as embryo donors for aggregation with ES cells and as pseudopregnant recipients. Details of morula aggregation can be found in26. Briefly, embryos were collected at E2.5 from superovulated CD-1(ICR) female mice. Zonae pellucidae of embryos were removed by the treatment with acid Tyrode’s solution (Sigma). ES cell colonies were treated with 0.05% Trypsin-EDTA to lift loosely connected clumps. Each zona-free embryo was aggregated with 10-15 ES cells inside depression well made in the plastic dish with an aggregation needle (BLS Ltd, Hungary) and cultured overnight in microdrops of KSOMAA covered by embryo-tested mineral oil (Zenith Biotech) at 37 °C in 94% air/6% CO . The following morning morulae and blastocysts were transferred into the uteri of E2.5 pseudopregnant CD-1(ICR) females previously mated with vasectomized males. Chimaeras were identified at birth by the presence of black eyes and later by the coat pigmentation. Chimeric males with more than 50% coat colour contribution were individually bred with CD-1(ICR) females. Germline transmission of ES cell genome was determined by eye pigmentation of pups at birth and later by the coat pigmentation. 1 × 106 cells were collected and lysed in RIPA buffer containing 1× Protease Inhibitor Cocktail, 1 mM PMSF, 5 mM NaVO and 5 mM NaF. Extracts were loaded into 4–15% Mini-Protean TGX SDS Page gels (Bio-Rad). Proteins were transferred to PVDF membranes. Membranes were blocked in 5% milk/PBS-T buffer for 30 min and incubated either overnight at 4 °C or 1 h at room temperature with the following antibodies: 4EBP1 (total or pThr37/46), S6K1 (total or pThr389), Akt (total or pSer473), mTOR (total or pSer2448) (Cell Signaling Technology), Gapdh (Millipore) and anti-rabbit/mouse secondary antibodies (Jackson Labs). Membranes were incubated with ECL or ECL Plus reagents and exposed to X-ray films (Thermo Fisher Scientific). 4 × 105 cells were seeded on 6-well plates. After overnight culture, cells were incubated for 1 h with 5-ethynyl-2-deoxyuridine (EdU) diluted to 10 μM in the indicated ES cell media. All samples were processed according to the manufacturer’s instructions (Click-iT EdU Alexa Fluor 488 Imaging Kit, Thermo Fisher Scientific). EdU incorporation was detected by Click-iT chemistry with an azide-modified Alexa Fluor 488. Cells were resuspended in EdU permeabilization/wash reagent and incubated for 30 min with FxCycle Violet Stain (Thermo Fisher Scientific). For EdU dilution experiments, ES cells were labelled for 90 min in serum, and afterwards were split into either serum or pause conditions; EdU analysis was done every 12 h for 48 h. Flow cytometric was performed on a LSRII flow cytometer (BD) and analysed using FlowJo v10.0.8. Data sets show similar variance. Total nascent transcription (Ethynyl Uridine, EU) or translation (l-homopropargylglycine, HPG) were assessed in ES cells using the Click-iT RNA Alexa Fluor 488 HCS Assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Samples were analysed on a BD LSRII. Datasets show similar variance. After overnight culture on a 96-well plate, ESCs were washed once with PBS and trypsinized to single cells. They were resuspended in 10 μl of Annexin V diluted 1:100 in Binding Buffer (BioLegend) and incubated for 10 min in the dark. Cells were resuspended in 90 μl of binding buffer with Sytox Blue (Thermo Fisher Scientific) at 1:10,000. Data were collected on a BD LSRII. Datasets show similar variance. Three replicates were used for all samples. Freshly collected single-cell suspensions were sorted on a FACSAriaII cell sorter to collect 105 cells for each sample. Total RNA was isolated using the RNeasy kit (Qiagen). All samples were spiked-in with ERCC control RNAs (Thermo Fisher Scientific) following manufacturer’s recommendations. mRNA isolation and library preparation were performed on 250 ng total RNA from all samples using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs). Samples were sequenced at The Center for Advanced Technology, UCSF on Illumina HiSeq2500. Single-end 50-bp reads were mapped to the mm10 mouse reference genome using Tophat2 (ref. 31) with default parameters. We used Cuffnorm and Cuffdiff with the gtf file from UCSC mm10 (Illumina iGenomes July 17, 2015 version) as transcript annotation to evaluate relative expression level of genes (fragments per kilobase of transcript per million mapped reads (FPKM)) and call differentially expressed genes. The alignment rate exceeded 96% in all of our samples, yielding ~40 million aligned reads per sample. Data from ref. 20 and ref. 6 were downloaded from GEO and ArrayExpress, respectively, and processed with the same pipeline as our data. The absolute abundance of mRNA transcripts was estimated using the ERCC92 RNA spike-in32. ERCC92 contains 92 synthetic sequences with lengths ranging from 250 to 2,000 bp and concentration ranging over several orders of magnitude. ERCC sequences were designed to mimic mammalian mRNA, but are not homologous to the mouse genome, ensuring their unique mappability. We aligned the reads to the 92 reference spike-in sequences and compared the abundance of these sequences between different samples. As ERCC sequence abundances followed a highly linear trend in all pairs of samples across at least 5 orders of magnitude (Pearson correlation coefficient larger than 99.7%, see Extended Data Fig. 7), we assessed the absolute abundance of mRNA as the number of mRNA fragments per kilobase of transcript per 10 thousand mapped reads of ERCC. The overall abundance of ERCC spike-in sequences in our samples varied from 0.3% to 0.5% of aligned reads. To facilitate better comparison between our data and data from ref. 20 and to reduce possible batch effects, in Fig. 4e, we followed the ‘batch mean-centering’ approach widely used in microarray gene expression data analysis for batch effect removal33. Specifically, we separately mean-centred the log (FPKM + 1) value of each gene by subtracting the mean log (FPKM + 1) across all our samples (serum, 2i and paused) and across the samples from ref. 20. The numerical values of the mean-centred expression may not be directly comparable across all samples, because they may still have different dynamic ranges in different batches. We therefore used 1 − Spearman correlation coefficient as distance in the hierarchical clustering. In Fig. 4c, we identified 5,992 genes with robust expression (cell-number-normalized expression value >50 in serum, 2i, or paused states). The cell-number-normalized expression value of each gene was standardized across the 9 samples by subtracting the mean and then dividing by the standard deviation. Hierarchical clustering was performed using the standardized expression values using Euclidean metric and average linkage. In Fig. 4e, in order to compare our samples with those from ref. 20, we used the log (FPKM + 1) value of each gene. Hierarchical clustering was performed using mean-centred (within each batch) expression values of 9,418 genes robustly expressed (FPKM >10) in at least one cell state (serum, 2i, paused, diapause EPI, E2.5 MOR, E3.5 ICM, E4.5 EPI, E4.5 PrE, E5.5 EPI, or ESC 2i/LIF). 1 − Spearman correlation coefficient was used as distance and average linkage was used. For each of the 3,772 gene ontology terms that are associated with at least 10 genes34, we defined the gene ontology term expression as the mean FPKM values of genes associated with the corresponding term. In Fig. 4f, the log fold-change of gene ontology term expressions between paused ES cells and serum ES cells was plotted on the y axis against that between various samples in ref. 20 and E4.5 EPI on the x axis. The Spearman correlation coefficient of the 3,772 gene ontology terms is indicated. Extended Data Figure 10a was generated similarly, but with the log fold-change of gene ontology term expressions between Myc DKO and wild-type cells from ref. 6 on the y axis. For each of the 281 KEGG pathways that contain at least 10 genes35, we defined the pathway expression as the mean FPKM values of genes associated with the corresponding pathway. In Extended Data Fig. 9b, the log fold change of pathway expressions between paused ES cells and serum ES cells was plotted on the y axis against that between various samples in ref. 20 and E4.5 EPI. The Spearman correlation coefficient of the 281 pathways was indicated. Extended Data Fig. 10c was generated similarly, but with the log fold change of pathway expressions between Myc dKO and wild-type cells from ref. 6 on the y axis. Custom codes used for the RNA-seq analysis are available upon request. RNA-seq data have been deposited in Gene Expression Omnibus (GEO) under accession number GSE81285. RNA-seq data from refs 6 and 20 are available under the accession numbers GSE74337 and E-MTAB-2958. The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files.
News Article | November 30, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Yeast strains are all based on the BY4741 laboratory strain28. Manipulations were performed using a standard PEG/LiAC transformation protocol29. All deletions were verified using primers from within the endogenous open reading frame. Primers for all genetic manipulations were planned either manually or using the Primers-4-Yeast web tool30. All strains, primers and plasmids used in this study28, 31, 32, 33, 34 are listed in Supplementary Table 4. SGA and microscopic screening were performed using an automated microscopy set-up as previously described11, 15, using the RoToR bench-top colony arrayer (Singer Instruments) and automated inverted fluorescent microscopic ScanR system (Olympus). Images were acquired using a 60× air lens with excitation at 490/20 nm and emission at 535/50 nm (GFP) or excitation at 575/35 nm and emission at 632/60 nm (RFP). After acquisition, images were manually reviewed using the ScanR analysis program. Manual microscopy was performed using by one of two apparatuses. (I) Olympus IX71 microscope controlled by the Delta Vision SoftWoRx 3.5.1 software. Images were acquired using a 60× oil lens and captured by PhoetometricsCoolsnap HQ camera with excitation at 490/20 nm and emission at 528/38 nm (GFP/YFP) or excitation at 555/28 nm and emission at 617/73 nm (mCherry/RFP). (II) VisiScope Confocal Cell Explorer system, composed of a Zeiss Yokogawa spinning disk scanning unit (CSU-W1) coupled with an inverted Olympus IX83 microscope. Images were acquired using a 60× oil lens and captured by a connected PCO-Edge sCMOS camera, controlled by VisView software, with wavelength of 488 nm (GFP) or 561 nm (mCherry/RFP). Images were transferred to Adobe Photoshop CS6 for slight adjustments to contrast and brightness. Lysates for immunoprecipitation were prepared from indicated strains in mid-logarithmic growth grown in YPD reach medium. Cells were harvested, washed in distilled water, and resuspended in lysis buffer (50 mM Tris HCl pH 7, 150 mM NaCl) supplemented with protease inhibitors (complete EDTA-free cocktail; Roche) and frozen in a drop-by-drop fashion in liquid nitrogen. Frozen cells were then pulverized in a ball mill (1 min at 30 Hz; Retsch) and thawed with nutation. Samples were thawed in 1 ml lysis buffer supplemented with protease inhibitors and 1% CHAPS (Sigma Aldrich) at 4 °C for 1 h. All samples were then clarified by centrifugation at 14,000g at 4 °C for 15 min. The remaining supernatant was added to GFP-trap (Chromotek) for 1 h followed by centrifugation at 1,000g at 4 °C for 3 min, and the supernatant was set aside as the flow through. Beads were washed three times with lysis buffer supplemented with protease inhibitors, and bound proteins were released from the beads by a 5-min incubation at 95 °C in sample buffer. The total protein lysate, the flow through and the immunoprecipitation (IP) fraction were analysed by western blotting. Yeast proteins were extracted by either NaOH or TCA protocol as previously described9, 35 and resolved on polyacrylamide gels, transferred to nitrocellulose membrane blots, and probed with primary rabbit/mouse antibodies against HA (BioLegend, 901502), GFP (Abcam ab290), RFP (Abcam ab62341), histone H3 (Abcam ab1791), actin (Abcam ab8224), Sec65 (kindly provided by P. Walter) or Sec61 (kindly provided by M. Seedorf). The membranes were then probed with a secondary goat-anti-rabbit/mouse antibody conjugated to IRDye800 or to IRDye680 (LI-COR Biosciences). Membranes were scanned for infrared signal using the Odyssey Imaging System. Images were transferred to Adobe Photoshop CS6 for slight adjustments to contrast and brightness. Newly synthesized yeast proteins were radioactively labelled in vivo by a 7–10 min pulse with [35S]methionine at either 30 °C or 37 °C. Labelling was stopped by adding to the cells ice-cold TCA to a final concentration of 10%. Cells were then lysed and proteins were immunoprecipitated as previously described36 with antibodies against RFP (Abcam, ab62341), HA (BioLegend, 901502), Kar2 (kindly provided by P. Walter) or CPY (Abcam, ab113685). Protease inhibitors (complete EDTA-free cocktail; Roche) were used throughout the extraction and immunoprecipitation process. Immunoprecipitated samples were resolved on polyacrylamide gels, which were then exposed to Phosphor Screen (GE Life Sciences) and scanned by phosphorimager. Translocation efficiency was calculated as . The statistical significance of differences was measured using two-tailed student t-test with unequal variance, as indicated in the figure legends. For the Tetp-repression experiments, doxycycline (Sigma-Aldrich) was added to the overnight culture and to the back-dilution medium at a final concentration of 15 μg/ml. The ribosomal subunits RPL16a/b were conjugated to AVI-tag (biotin acceptor peptide), and Sec63 was conjugated to BirA (biotin ligase), allowing the specific biotinylation and streptavidin pull-down of ribosomes in close physical proximity to the ER membrane. By comparing the ribosomal footprints obtained from the total ribosome fraction and the streptavidin-pulled fraction, we measured ER-localized translation enrichment. Biotin induction was carried out at mid-logarithmic growth phase in the presence of cycloheximide, which was added to the medium 2 min before the addition of biotin, at a final concentration of 100 μg/ml. To induce biotinylation, d-biotin was added to the medium to a final concentration of 10 nM and biotinylation was allowed to proceed for 2 min at the same temperature as growth. Cells were harvested by filtration onto 0.45 μm pore size nitrocellulose filters (Whatman), scraped from the membrane, and immediately submerged in liquid nitrogen. The following steps of monosome isolation, streptavidin pulldown of biotinylated ribosomes, and library generation were done as previously described25. Sequencing reads were demultiplexed and stripped of 3′ cloning adapters using in-house scripts. Reads were mapped sequentially to Bowtie indices composed of rRNAs, tRNAs, and finally all chromosomes using Bowtie 1.1.0. Only uniquely-mapped, zero-mismatch reads from the final genomic alignment were used for subsequent analyses. These alignments were assigned a specific P-site nucleotide using a 15-nt offset from the 3′ end of reads. Gene-level enrichments were computed by taking the log ratio of biotinylated footprint density (reads per million) within a gene coding sequence (CDS) over the corresponding density of matched input ribosome-profiling experiment. Yeast genes were excluded from all analysis if they met any of the following criteria: had fewer than 100 CDS-mapping footprints in the input sample of a particular experiment; were annotated as ‘dubious’ in the SGD database; mapped to the mitochondrial chromosome. Additionally, regions in which a CDS overlapped another same-strand CDS were excluded from enrichment calculations. TMD positions were predicted using the Phobius algorithm. TMD classification was divided based on the start site of the first predicted TMD: N-terminal TMDs start in the first 95 amino acids of the protein; downstream TMDs start after the first 95 amino acids of the protein. Genes that were dependent on SND components were identified by comparing the Sec63-BirA ER enrichment in a wild-type strain (yJW1784) with that in a Δsnd strain (yJW1811, yJW1812, or yJW1813) as previously described25. Briefly, log enrichments were separately normalized by subtracting the mean enrichment and dividing by the standard deviation of enrichments for the corresponding experiment. Genes were then binned by the minimum number of sequencing counts in either wild-type or Δsnd input sample, and the difference between normalized enrichments was compared within each bin. Enriched genes were defined as those genes whose Δsnd log enrichments were greater than 0.3 and whose enrichments increased in the Δsnd sample by at least two standard deviations compared to other genes in that bin. Depleted genes were defined as those genes whose wild type log enrichments were greater than 0.3 and whose enrichments decreased in the Δsnd sample by at least two standard deviations compared to other genes in that bin. Significant depletion of 10–23%, 9–42% and 14–45% was observed in Δsnd1, Δsnd2 and Δsnd3, respectively. Including or excluding SS-bearing proteins had no effect on this trend. Mitochondrial proteins were excluded from the analysis. Lysates for immunoprecipitations were prepared from yeast that expressed GFP-tagged SND genes or a constitutively expressed GFP-negative control, in mid-logarithmic growth grown in YPD reach medium. Cells were harvested, washed in distilled water, and resuspended in lysis buffer (50 mM Tris HCl pH 7, 150 mM NaCl) supplemented with protease inhibitors (complete EDTA-free cocktail; Roche) and frozen in a drop-by-drop fashion in liquid nitrogen. Frozen cells were then pulverized in a ball mill (1 min at 30 Hz; Retsch) and thawed with nutation. Samples were thawed in 1 ml lysis buffer supplemented with protease inhibitors and 1% digitonin (Sigma Aldrich) at 4 °C for 1 h. All samples were then clarified by centrifugation at 14,000g at 4 °C for 15 min. The remaining supernatant was added to GFP-trap (Chromotek) for 1 h followed by three washes with lysis buffer supplemented with protease inhibitors and 1% digitonin. Bound proteins were released from the beads by a 5-min acidic treatment (0.2 M glycine pH 2.5), which was neutralized with 1 M Tris pH 9.4. The eluted proteins were digested with 0.4 μg sequencing grade trypsin for 2 h in the presence of 100 μl of 2 M urea, 50 mM Tris HCl pH 7.5 and 1 mM DTT. The resulting peptides were acidified with trifluoroacetic acid (TFA) and purified on C18 StageTips. LC–MS/MS analysis was performed on an EASY-nLC1000 UHPLC (Thermo Scientific) coupled to a Q-Exactive mass spectrometer (Thermo Scientific). Peptides were loaded onto the column with buffer A (0.5% acetic acid) and separated on a 50-cm PepMap column (75 μm i.d., 2 μm beads; Dionex) using a 4-h gradient of 5–30% buffer B (80% acetonitrile, 0.5% acetic acid). Interactors were extracted by comparing the protein intensities to a GFP control. Yeast microsomes were extracted from the ADHp-SND2–GFP/SND3–HA strain as described37. In brief, spheroplasts of yeast were lysed by dounce homogenization (25 strokes) in lysis buffer (0.1 M sorbitol, 20 mM HEPES pH 7.4, 50 mM potassium acetate, 2 mM EDTA, 1 mM DTT, 1 mM PMSF) at 4 °C. The lysates were centrifuged at 1,000g and the resulting supernatant at 27,000g for 10 min at 4 °C. The crude membrane pellet was re-suspended in lysis buffer and layered onto a discontinuous sucrose density gradient consisting of 1.2 and 1.5 M sucrose. Following centrifugation at 100,000g for 60 min at 4 °C, the membranes at the 1.2–1.5 M sucrose interface were collected and washed twice in lysis buffer. The membrane pellets were re-suspended in membrane storage buffer (50 mM NaCl, 0.32 M sucrose, 20 mM HEPES pH 7.4, 2 mM EDTA containing protease inhibitors) and the protein concentration determined by a standard Bradford assay. Microsomes were solubilized in ComplexioLyte 48 buffer (1 mg/ml, Logopharm) for 30 min at 4 °C38. Solubilized extracts were centrifuged at 100,000g for 30 min at 4 °C, supplemented with glycerol (5%) and coomassie G-250 (0.3%) and loaded on a 3.5–15% linear native polyacrylamide gel. The BN-PAGE gel was prepared as described39. The gel buffer contained 25 mM imidazole and 500 mM 6-aminohexanoic acid. The cathode chamber was first filled with cathode buffer B (50 mM Tricine, 7.5 mM imidazole and 0.02% coomassie) and subsequently replaced by cathode buffer B/10 (containing 0.002% coomassie) after the gel running front had covered a third of the desired distance of electrophoresis. The anode chamber was filled with 25 mM imidazole pH 7.0. A high-molecular-weight calibration kit for native electrophoresis from GE Healthcare was used as a standard. For 2D BN-PAGE, the excised lanes were equilibrated in 2D-dissociation buffer (60 mM Tris/HCl pH 6.8, 10% glycerol, 2% SDS, 5% v/v β-mercaptoethanol, 6 M urea) before separation on the second dimension by SDS–PAGE. After electro-blotting, the nitrocellulose membrane was detected with the indicated antibodies. The HEK293 cell line used was obtained from DSMZ (no. ACC 305). DSMZ supplied verification of authentication of the cells, tested by DSMZ via short tandem repeat loci (STR profile). The cell line is routinely tested for mycoplasma contamination. This cell line was chosen as it is routinely used for fractionation experiments. Rough microsomes from human cells were prepared as described40. Briefly, 30 × 106 HEK293 cells were harvested and washed once with PBS and twice with buffer 1 (50 mM HEPES/KOH pH 7.5; 0.25 M sucrose; 50 mM KOAc; 6 mM MgOAc; 4 mM PMSF; 1 mM EDTA; 1 mM DTT; 0.1 mg/ml cycloheximide; 0.3 U/ml RNAsin (Promega); protease inhibitor cocktail). After homogenization in buffer 1 using a glass/Teflon homogenizer, the suspension was centrifuged at 1,000g for 10 min. The supernatant was centrifuged at 10,000g for 10 min. The new supernatant was layered onto 0.6 M sucrose in buffer 2 (50 mM HEPES/KOH pH 7.5, 0.6 M sucrose, 100 mM KOAc, 5 mM MgOAc, 4 mM DTT, 0.1 mg/ml cycloheximide, 40 U/ml RNAsin) and centrifuged at 230,000g for 90 min. The resulting membrane pellet was previously shown to comprise rough ER. Here, it was resuspended in buffer 2 and adjusted to 2.3 M sucrose, which was overlaid with 1.9 and 0 M sucrose, respectively, in buffer 2. After flotation at 100,000g for 18 h, the interphase between 0 and 1.9 M sucrose, two fractions of the remaining supernatant, and the pellet were collected. After centrifugation of the interphase at 100,000g for 1 h, the membrane pellet corresponded to purified rough ER. All steps after the first washing step were carried out on ice. Western blot analyses employed antibodies against β-actin (Sigma), CAML (Synaptic Systems SA7679), or rabbit antibodies that were raised against the depicted proteins: the C-terminal peptide of hSnd2 (KRQRRQERRQMKRL) plus an N-terminal cysteine; or an internal peptide of SRα (KKFEDSEKAKKPVR) plus a C-terminal cysteine, cross-linked to KLH. The SRα and β-actin antibodies were visualized using ECL Plex goat-anti-rabbit IgG-Cy5-conjugate or ECL Plex goat-anti-mouse IgG-Cy3-conjugate (GE Healthcare) and the Typhoon-Trio imaging system (GE Healthcare) in combination with Image Quant TL software 7.0 (GE Healthcare). The hSnd2 and CAML antibodies were visualized using secondary peroxidase (POD)-coupled anti-rabbit antibody (Sigma) plus ECL (GE Healthcare) and the Fusion SL luminescence-imaging system (Peqlab) in combination with Image Quant TL software 7.0. Ribosome-profiling data have been deposited in Gene Expression Omnibus (GEO) under accession number GSE85686. Gel source images can be found in Supplementary Fig. 1. Other data that support the findings of this study are available from the authors on reasonable request.
News Article | October 25, 2016
Plvaptm1Salm (Plvap−/−), Nt5etm1Lft (Nt5e−/−) and Aoc3tm1Salm (Aoc3−/−) mice have been previously described24, 31, 32. Nt5e−/− and Aoc3−/− mice manifest with altered leukocyte trafficking33. We produced inducible PlvapF/F; CAGGCre-ERTM mice by crossing conditional PlvapF/F mice with a CAGGCre-ERTM (B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (stock 004682 from the Jackson Laboratory) deletor Cre mouse line, in which Cre-ER is ubiquitously expressed after tamoxifen injection34. To achieve selective deletion of Plvap during embryonic development in LYVE-1+ endothelial cells, including yolk-sac endothelium and liver sinusoidal endothelial cells, but not the majority of other blood vessels35, conditional PlvapF/F mice were crossed with Lyve1-Cre mice (Lyve1tm1.1(EGFP/cre)Cys, stock 012601 from the Jackson Laboratory36) to generate PlvapF/F; Lyve1-Cre mice. Cav1tm1Mls/J (Cav1−/−, stock 004585) mice, which lack all caveolae37, were obtained from the Jackson Laboratory. C57BL/6J, C57BL/6N and BALB/c mice were purchased from Charles River and Janvier labs. F1 hybrid C57BL/6;129 (stock 101045) mouse strain was obtained from the Jackson Laboratory. Both genders were used in the experiments (except in mammary gland analyses). Sex-matched wild-type littermate mice were used as controls in each experiment. Embryonic development was estimated considering the day of vaginal plug as embryonic age of 0.5 days (E0.5). The adult mice were 4–5 weeks old, since few Plvap−/− mice survive till early adulthood24, 28, 38. All animal experiments were approved by the Ethical Committee for Animal Experimentation in Finland. They were carried out in adherence with the rules and regulations of the Finnish Act on Animal Experimentation (497/2013) and in accordance to the 3R-principle under Animal License number 5587/04.10.07/2014. Genotyping of Plvap−/−, Cav1−/−, Nt5e−/− and Aoc3−/− mice was performed according to protocols described previously24, 31, 32, 37. PlvapF/F; CAGGCre-ERTM and PlvapF/F; Lyve1-Cre mice genotyping was conducted using the following primers: primers A (3′-GTACATGCAACACCACTGAGC-5′) and B (3′-CCTTGACAGGTGATGTCTGC-5′) detect the wild-type Plvap allele (a 210-bp fragment) and the targeted Plvap allele (a 310-bp fragment; data not shown). Genotyping of CAGGCre-ERtm and Lyve1-Cre was done according to protocols described previously34, 36. Pregnant females were killed by carbon dioxide inhalation and cervical dislocation. Embryos from E10.5–E16.5 were dissected out from uterus and immersed in cold PBS (Invitrogen). The blood was collected after decapitation to heparin-containing tubes. Liver, lungs, spleen and brains were carefully dissected from the embryo and the yolk sac was collected. To obtain single-cell suspensions, the organs were incubated in Hank’s buffered saline (HBS) containing 1 mg ml−1 collagenase D (Roche), 50 μg ml−1 DNase I (Sigma) at 37 °C in 5% CO (30 min for liver, lung, spleen and brain and 2 h for yolk sac), and then passed through a 70-μm cell strainer. Erythrocytes were lysed from the blood and spleen samples as described24. The brain cells were re-suspended in isotonic Percoll and the microglia were isolated as described6. The cells from the adult tissues were isolated by the same method with some modifications. The blood was collected by a cardiac puncture into heparinized tubes. Lymph nodes, lung tissue and mammary fat pads were mechanically dissociated before a 60-min collagenase D and DNase I digestion. Livers were dissociated using Gentle MACS C-tube (Miltenyi Biotech) and immune cells were purified via OptiPrep density gradient centrifugation (Sigma D1556). The bone marrow was isolated by gently crushing the femurs before filtration. Lamina propria cells from the colon were isolated by an enzymatic digestion as described23. Peritoneal cells were collected by flushing the peritoneal cavity with RPMI 1640 supplemented with 2% FBS and 5 IU ml−1 heparin. Total leukocyte numbers in different organs were enumerated by determining the absolute numbers of viable cells in the cell suspensions by an automated cell counter (Cellometer Auto 2000, Nexcelcom) and the percentage of CD45+ cells (and of the various leukocyte subpopulations) by flow cytometry. Absolute leukocyte numbers in the blood were counted using an automated haemocytometer (VetScan HM5, Abaxis). Pregnant heterozygous Plvap−/+ females were transiently treated with anti-CSF-1R monoclonal antibody (clone AFS98, Bio X Cell) or with the rat IgG2a isotype control (clone 2A3, Bio X Cell) at E6.5 using a single intraperitoneal injection (3 mg of antibodies in sterile PBS). This treatment prevents the development of yolk sac macrophages, but does not affect EMP development10, 39. The fluorochrome-conjugated monoclonal antibodies (the antibody clones, fluorochromes, suppliers and catalogue numbers) against mouse molecules that were used for flow cytometry stains are listed in Supplementary Table 2. Before staining, the cell suspensions were incubated with purified anti-CD16/32 (clone 2.4G2, 553142 from Becton Dickinson) for 10 min on ice to block non-specific binding to Fc-receptors. Isotype-matched negative control antibodies conjugated to the appropriate fluorochromes were used (Supplementary Table 2). All FACS analyses were run using an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo (TreeStar) software. The FACS gates used to define each leukocyte subpopulation in different organs and tissues of embryos and adult mice are shown in Extended Data Figs 1, 2b, d, g, 4b, 5c–e, 10d and Supplementary Table 1. EMPs (CD41+CD45+c-KithighF4/80− cells) from E10.5 yolk sac, and yolk-sac-derived macrophages (CD11b+F4/80high cells) and fetal liver-derived macrophages (CD11b+F4/80intermediate cells) from E14.5 (wild-type and Plvap−/−) and E16.5 (wild-type) fetal livers (mechanical dissociation without enzymatic digestions) were sorted from embryos using Sony SH800Z (100-μm nozzle, Sony Biotechnology) and FACS aria II (70-μm nozzle, Becton Dickinson) cell sorters. The purity of the isolated populations was >95%. Approximately 5,000 macrophages sorted from fetal livers (see above) were spun down onto microscopic slides using a cytospin centrifuge (Shandon cytospin III, Tecan). The cells were stained with Diff-Quick (REASTAIN), and photographed using Zeiss AxioVert 200M (Zeiss) using a Plan-Noefluar 40×/0.60 objective. Fetal livers from E12.5–E16.5 embryos were excised from the mice after decapitation. They were embedded in optimal cutting temperature (OCT) compound and snap-frozen. Cryostat sections (6 μm in thickness) were cut and fixed in ice-cold acetone. The sections were overlaid with the following antibodies: Alexa Fluor 488-conjugated rat anti-mouse F4/80 (MF48020, Invitrogen), allophycocyanin-conjugated rat anti-mouse CD31 (102510, BioLegend), rat monoclonal anti-mouse MECA32 (550563, Becton Dickinson), rat anti-mouse MAdCAM-1 (MECA-367; rat IgG2a, a gift from E. Butcher), rabbit polyclonal anti-mouse LYVE-1 (102-PA50AG or 103-PA50; Reliatech), rabbit polyclonal anti-mouse caveolin (SC-894; Santa Cruz) and rabbit polyclonal anti-VEGF (46154, Abcam). Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247, Life Technologies), Alexa Fluor 546-conjugated goat anti-rabbit immunoglobulin (A11035 and A11035, highly cross-absorbed, Invitrogen), Alexa Fluor 633-conjugated goat anti-rabbit immunoglobulin (highly cross-adsorbed A21071, Life Technologies) and Alexa Fluor 488-conjugated donkey anti-rat immunoglobulin (highly cross-adsorbed, A21208, Life Technologies) were used as secondary antibodies as appropriate. The sections were mounted in ProLong Gold with or without DAPI (4′,6-diamidino-2-phenylindole). For visualization of the luminal location of PLVAP in fetal liver sinusoids at E12.5, wild-type C57BL/6N dams were killed at E12.5 and the embryos were excised from the uterine cavity but kept inside the yolk sac in warm PBS. Then 20 μg of unconjugated rat monoclonal anti-mouse MECA-32 (MECA32, custom-made, InVivo BioTech) or isotype-matched control antibody (rat IgG2a 553926 BD) was injected to umbilical and vitelline veins of the yolk sac. After 1 min, the embryos were decapitated, and the livers were collected and processed for ex vivo immunostaining. Acetone-fixed frozen sections were sequentially stained with Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247, Life Technologies) to detect the in vivo bound MECA-32, rabbit anti-mouse LYVE-1 (103-PA50, Reliatech) and Alexa Fluor 546-conjugated goat anti-rabbit immunoglobulin (A11035, Invitrogen). Preliminary analyses verified that species-specific second-stages antibodies showed no cross-reactivity with primary antibodies generated in the other species. Whole-mount immunohistochemistry from optically cleared E8.5 and E10.5 yolk sac and AGM, E9.5 embryos and E14.5 fetal livers was done as described previously24. Primary antibodies were rat monoclonal antibodies against mouse CD117 (c-Kit, 553352, Becton Dickinson), MECA-32 (PLVAP, 550563, Becton Dickinson), CD31 (550274, Becton Dickinson) and rabbit anti-mouse LYVE-1 (103-PA50, Reliatech). Alexa Fluor 488-conjugated donkey anti-rat immunoglobulin (A21208), Alexa Fluor 546-conjugated goat anti-rat immunoglobulin (A11081), Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247), and Alexa Fluor 633-conjugated goat anti-rabbit immunoglobulin (A21071) were used as secondary antibodies (all from Life Technologies). In AGM, c-Kit is expressed in HSCs, and CD31 in endothelial cells and HSCs40. A human fetal liver sample (pregnancy week 18) was cut, acetone-fixed and stained with monoclonal PAL-E (against human PLVAP; Ab8086, Abcam) and Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (highly cross-absorbed, A11029, Invitrogen). After staining, the sections were mounted in Prolong Gold with DAPI. Images were acquired with a LSM 780 confocal microsope (Zeiss) using a c-Apochromat 40×/1.20 W Korr M27 objective or plan-apochromat 20×/0.8 objective (Fig. 4a and Extended Data Fig. 4g) and Zen 2010 software (Zeiss). Using pinhole adjustments, a slice thickness of 4.6 μm and 1.2 μm was used for 20× and 40× objectives, respectively. A background subtraction was used for all images. In certain images, the brightness was linearly changed and noise was reduced using mean filter in ImageJ software. Brightness adjustments and noise reductions were always applied equally to images captured from wild-type and PLVAP-deficient mice. Splenic F4/80high cells were quantified by thresholding the images so that only the F4/80high cells remained visible (the thresholding was applied equally to images captured from wild-type and PLVAP-deficient mice). Thereafter, white pulp areas were excluded (based on MadCAM-1 staining in marginal zone41) and the area fraction in the red pulp containing the F4/80high cells was measured using ImageJ software. A spleen area of at least 2.1 mm2 per mouse was analysed. Z-stacks and images (Extended Data Fig. 4a E8.5 yolk sac) were acquired from the optically cleared samples using a 3i Spinning Disk confocal microscope (Intelligent Imaging Innovations) with a plan-apochromat 20×/0.8, 10×/0.45 or LD c-apochromat 40×/1.1 W objective. Background subtractions, linear brightness adjustments and mean filter noise reductions were done using ImageJ software. To produce maximum projections with SlideBook 6 software (Intelligent Imaging Innovations, Inc.), 15–87 sections with slice thickness of 0.63 μm were used (Extended Data Fig. 4a E10.5 yolk sac and 4g). 3D reconstruction was generated from Z-stacks (44 slices with thickness of 2.34 μm (MECA-32 in Supplementary Video 1), 78 slices with slice thickness of 2.34 μm (CD31 in Supplementary Video 1), and 117 slices with thickness of 0.43 μm (MECA-32 in Supplementary Video 2)) and converted to AVI file format with Imaris 8.0 software (Bitplane). Formalin-fixed, paraffin-embedded sections of livers from E11.5–E16.5 wild-type and Plvap−/− mice were cut, deparaffinized and subjected to heat-mediated antigen retrieval in EDTA buffer (Dako S2367). Endogenous peroxide was quenched with 3% H O , non-specific immunoglobulin binding was blocked with rabbit serum, and endogenous biotin and avidin were blocked using DakoCytomation Biotin blocking system (Dako, X0590). The liver sections were incubated overnight at 4 °C with the primary antibody (MECA-32, 1 μg ml−1 in PBS). A secondary antibody (biotinylated anti-rat immunoglobulin) was incubated for 30 min, and then biotin–avidin complexes were formed with using Vectastain ABC kit (PK-6100, Vector Laboratories). Liquid DAB+ substrate Chromogen System (Dako K3468) was used to oxidize and detect the peroxidase complexes. Finally, samples were stained with haematoxylin, dehydrated and mounted. Macrophage-dependent iron recycling was studied in spleen and liver by measuring accumulation of ferric ion42, 43. For Fe3+ stains, the spleens and livers from 5-week-old wild-type and Plvap−/− mice were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0), embedded in paraffin and sectioned. The detection of ferric iron was accomplished using the Prussian blue histological staining method, as described previously25, 43. The slides were counter-stained in nuclear Fast Red for 5 min. The slides were analysed using a Panoramic 250 Flash II slide-scanner (3D Histech). In spleen tissue, the white pulp areas were excluded and the red pulp areas containing the blue-stained Fe3+-containing cells were analysed using image thresholding. Area fractions were measured using ImageJ software. A spleen area of at least 1.5 mm2 per mouse was analysed. In livers, whole sections (at least 21.1 mm2 per mouse) were analysed. The fourth mammary gland was dissected from 4.5-week-old wild-type and Plvap−/− mice, and left to adhere to the object glass. The tissue was fixed by submerging in Carnoy’s medium (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 °C, and rehydrated in decreasing ethanol concentration series. The slides were then stained with carmine alum (0.2% carmine, 0.5% aluminium potassium sulfate dodecahydrate) overnight at room temperature, dehydrated, cleared in xylene for 2–3 days, and mounted in DPX Mountant (Sigma). The samples were imaged with Zeiss SteREO Lumar V12 stereo microscope using NeoLumar 0.8× objective and Zeiss AxioCam ICc3 colour camera. Several images were automatically combined into a mosaic picture using Adobe Photoshop. The area covered by the ductal tree, and the number of ductal branches was tracked manually and quantified using ImageJ with ‘Skeletonize2D/3D’ and ‘AnalyzeSkeleton’ plugins. Livers from E12.5, E14.5 and E16.5 wild-type and Plvap−/− embryos were collected and fixed in 5% glutaraldehyde in 0.16 M s-collidine buffer, pH 7.4. The samples were post-fixed for 2 h with 2% OsO containing 3% potassium ferrocyanide, dehydrated with a series of increasing ethanol concentrations (70%, 96% and twice at 100%) and embedded in 45359 Fluka Epoxy Embedding Medium kit. 70-nm sections were cut with an ultramicrotome, and stained with 1% uranyl acetate and 0.3% lead citrate. The sections were examined with a JEOL JEM-1400 Plus transmission electron microscope. After isolation, 5,000 cells from the yolk sac of E10.5, liver of E12.5 embryos and adult bone marrow of wild-type and Plvap−/− mice were seeded in 1 ml of M3434 Methocult medium (Stem Cell Technologies) into 35-mm culture dishes in duplicates. After a 7-day culture at 37 °C with 5% CO the number of colonies was counted, as described44. Total RNA was isolated from fetal livers of wild-type and PLVAP-deficient mice using the Nucleo-Spin RNA kit (Macherey–Nagel) and from the sorted EMP and macrophages (see above) using the RNAeasy Plus Micro kit (Qiagen). The RNA was reverse-transcribed to cDNA with SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific) according to the manufacturers’ instructions. Quantitative PCR (qPCR) was carried out using Taqman Gene Expression Assays (ThermoFisher Scientific) for Plvap (Mm00453379_m1; target gene), Lyve1 (Mm00475056_m1; target gene) and Actb (Mm00607939_s1; control gene). The expression of reported signature transcripts7 enriched in yolk-sac-derived F4/80high macrophages (Cx3cr1 (Mm00438354_m1), Mrc1 (Mm00485148_m1), Adgre1 (Mm00802529_m1, also known as Emr1 or F4/80), and in F4/80intermediate fetal liver-derived monocytes (Itgam (Mm00434455_m1), Gata2 (Mm00492301_m1), Flt3 (Mm00439016_m1) and Ccr2 (Mm04207877_m1)) in E16.5 fetal liver of wild-type mice was also analysed by quantitative qPCR. The reactions were run using the 7900HT Fast Real-Time PCR System (Applied Biosystems/ ThermoFisher Scientific) or QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems/ ThermoFisher Scientific) at the Finnish Microarray and Sequencing Centre (FMSC), Turku Centre for Biotechnology, Turku, Finland. Relative expression levels were calculated using Sequence Detection System (SDS) Software v2.4.1, QuantStudio 12 K Flex software, and DataAssist software (all from Applied Biosystems/ThermoFisher Scientific). The results were presented as a percentage of the control gene mRNA level from the same samples. A PLVAP–Fc fusion protein expressing the extracellular domain of mouse PLVAP fused to human IgG2 Fc-tail was generated (Extended Data Fig. 9c). The extracellular domain (amino-acid residues 48–438) was PCR-cloned from a full-length cDNA clone for mouse PLVAP (MR206983, Origene) using primers introducing EcoRV and NheI digestion sites. The PCR reaction was carried out using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific). The amplified fragment was purified and annealed to EcoRV and NheI digested pFUSEN-hG2Fc vector (InvivoGen) designed for the production of Fc-chimaeras from type 2 membrane proteins. The intactness of the construct was verified by sequencing, and its reactivity with anti-PLVAP antibody MECA-32 using immunoblotting. The expression plasmid was transfected into HEK293-EBNA cells (CRL-10852, from ATCC) using lipofection (Lipofectamine, Invitrogen), the cells were cultured for 2–3 days in serum-free medium (Pro293A-CDM, Bio-Whittaker). A CD4–Fc chimaera45 was used as a control. For heparin-affinity pull-down assays, agarose beads coupled to heparin (Sigma) or streptavidin (negative-control beads, from GE Healthcare) were washed, and blocked with TBS (pH 7.2) containing 1% BSA. The beads were rocked with clarified 0.5% NP-40 total protein lysates from E14.5 wild-type livers for 2 h at 4 °C in TBS containing 1% BSA. Alternatively, PLVAP–Fc and CD4–Fc fusion proteins were applied to the heparin and control beads. After washing with TBS containing 0.3% NP-40, the bead-bound molecules were eluted in Laemmli’s sample buffer, and subjected to SDS–PAGE separation. In certain experiments, the heparin and control beads were incubated with the fusion proteins and, after washing, the same volumes of the beads were eluted directly in Laemmli’s sample buffer or in 1.0 M NaCl, and the eluted proteins from the supernatants were submitted to SDS–PAGE. In other specificity control experiments, the binding of PLVAP–Fc fusion protein to heparin-beads was analysed in the absence and presence of 100 μg of fibronectin (F1141, Sigma), which binds to heparin46, or 100 μg of collagen (C8919, Sigma). After transfer to nitrocellulose membranes, the bound molecules were visualized using immunoblotting with a horseradish-peroxidase-conjugated anti-human IgG antibody (81-7120, Invitrogen; for the chimaeras) or with anti-PLVAP (MECA-32, BioXCell), anti-neuropilin-1 (AF56615, R&D Systems), and anti-VEGF (sc-152, Santa Cruz) antibodies followed by appropriate HRP-conjugated second-stage reagents (for the liver lysates) using ECL detection. For far-western assays, recombinant mouse neuropilin-1 (R&D Systems, 5994-N1) and recombinant mouse VEGF164 (R&D Systems, 493-MV) were spotted onto filters. Both neuropilin-1 and VEGF are known heparin-binding proteins47, 48. The PLVAP–Fc chimaera (in TBS containing 5% BSA) was allowed to bind to the immobilized proteins in the presence or absence of 50 IU heparin (stock 5,000 IU ml−1; Leo Pharma) for 2 h. The bound chimaera was visualized using HRP-conjugated anti-human IgG antibody and ECL. Detection of VEGF interaction with PLVAP in situ in E14.5 fetal livers was performed using a proximity ligation assay (PLA)49. In brief, primary antibodies were rabbit anti-VEGF (46154, Abcam) or rabbit anti-GFP (A11122, Molecular Probes; as a negative control), and they were detected by Duolink in situ PLA probe anti-rabbit PLUS (DUO92002, Sigma), and rat anti-PLVAP antibody (MECA-32) was directly conjugated to MINUS PLA probe using Duolink in situ probemaker MINUS kit (DUO92010, Sigma). After ligation and amplification, the probes were detected using Detection reagent red (DUO92008, Sigma). During the amplification step, Alexa Fluor 488-conjugated donkey anti-rat IgG (A11035, Invitrogen) was added to detect MECA-32. The samples were stained with DAPI and mounted in Mowiol. Images for PLA were acquired using a 3i Spinning Disk confocal microscope (Intelligent Imaging Innovations) with a plan-apochromat 63×/1.4 oil objective and SlideBook 6 software (Intelligent Imaging Innovations). Background subtractions and linear brightness adjustments were performed using ImageJ. Adjustments were applied equally to images captured from control and anti-VEGF antibody PLVAP PLA stains. For co-immunoprecipitation assays, freshly isolated E14.5 livers of wild-type mice were briefly lysed in a buffer containing 1% NP-40, 150 mM NaCl, 20 mM HEPES (pH 7.5), 2 mM MgCl , 2 mM CaCl , PhosSTOP and Protease inhibitor cocktail (both Roche). After clarification by centrifugation, the supernatants were incubated with a rabbit anti-VEGFA antibody (or with a negative control rabbit antibody) for 5 h at 4 °C. Protein G beads (blocked with 1% BSA) were then added for 1 h at 4 °C, and thereafter the beads were washed 3 times with the lysis buffer. The bound proteins were eluted in non-reducing Laemmli’s sample buffer, separated in SDS–PAGE and immunoblotted for VEGF and PLVAP using IRDye-conjugated second-stage reagents and Odyssey imager. Sample size was empirically determined based on pilot analyses and previous literature. Adult wild-type and Plvap−/− littermates were allocated to experimental groups without specific randomization methods because comparisons involved mice of distinct genotypes. The investigators were blinded to the genotype of the embryos during the experimental procedures. Numerical data are given as mean ± s.e.m. Comparisons between genotypes were performed using Mann–Whitney U-test. SAS 9.4 statistical software and GraphPad Prism software v6 were used for statistical analysis. P < 0.05 was considered to be statistically significant. Each data point (values provided in Source Data) is obtained from a different embryo or mouse, and thus all numeric data and statistical analyses are derived from biological replicates.
News Article | August 22, 2016
The study protocols were approved by University of California San Diego and Salk Institute IRB/ESCRO committees (protocols 141223ZF and 95-0001, respectively). Four TD individuals (ages 8–19 years) and five individuals with WS (ages 8–14 years; Extended Data Fig. 1a) were included in the analysis: four of the latter group had typical WS gene deletions and one (pWS88) had a partial deletion in the WS region. Informed consents were obtained from all participants or their parents as appropriate. Genetic diagnosis of WS was established using fluorescent in situ hybridization probes for elastin (ELN), a gene consistently associated with the deletion in the typical WS region1, 9. All of the participants with WS having confirmed genetic deletion exhibited the medical and clinical characteristics of the WS phenotype, including previously established cognitive, behavioural and physical features associated with the syndrome4. A diagnosis of WS was confirmed on the basis of the Diagnostic Score Sheet (DSS) for WS (American Academy of Paediatrics Committee on Genetics, 2001), with a particular focus on the cardiovascular abnormalities and the characteristic facial features associated with the ELN deletion. The scores for the participants were at the mean for WS (9) or higher, with the individual with partial deletion in the WS chromosomal region (pWS88) scoring lower than the individuals with typical WS deletion. Similarly, pWS88 reported fewer symptoms with connective tissue and growth, his cognitive scores were slightly higher than the typical individuals with WS, and he did not demonstrate the disparity between verbal and visual–spatial abilities typical of WS. However, pWS88 did display behavioural and developmental features consistent with WS, including developmental delay, over-friendliness and anxiousness. The participants were administered standard tests to quantify their non-verbal and verbal abilities, as well as versions of the WS cognitive and social profiles to capture the distinct pattern of strengths and weaknesses both within and across domains associated with the WS cognitive and social phenotype. Details of the tests and the measures tapping into the two profiles are presented in Extended Data Fig. 1. The WS cognitive profiles for the five participants with WS were constructed by calculating the log of predictive likelihood ratios under assumed normality for age-appropriate TD versus WS classifications on the basis of verbal and performance IQ (VIQ and PIQ), Beery Developmental Test of Visual-Motor Integration (VMI) and Peabody Picture Vocabulary Test (PPVT) standard scores, subject to availability. Predictive distributions were based on the published normative mean and s.d. for each of the tests employed, whereas for the WS classification the predictive distributions26 were determined using data from n = 81 (VIQ and PIQ), n = 56 (VMI) and n = 97 (PPVT) participants in a broader WS sample (described in Extended Data Fig. 1d). A tobit model was used to estimate parameters for individuals with WS on the VMI owing to the presence of floor effects. The WS social profiles for the five participants with WS were constructed using measures of social approach behaviour, emotionality/empathy and language use. Quantitative PCR was used to define the breakpoints of deleted regions in DNA isolated from iPSCs, or lymphoblast cell lines for participants with WS, with probes spanning from CALN1 to WBSCR16 and template DNA. Taqman expression assay probes detecting the WS region genes were designed and synthesized with sequences shown in Supplementary Table 11. RNase P (VIC) was used as control. Quantitative PCR was performed on the ABI PRISM 7900HT system and the results were analysed using SDS 3.2. We avoided invasive sample collection methods such as skin biopsy or blood withdrawal by taking advantage of the natural loss of deciduous teeth as a source of somatic cells. We chose to reprogram dental pulp cells (DPCs) because these cells develop from the same set of early progenitors that generate neurons. Furthermore, the neurons derived from iPSCs generated from DPCs express higher levels of forebrain genes compared with those generated from skin fibroblast-derived iPSCs27, serving the purpose of this study. Deciduous teeth were collected when they fell out and were shipped to our laboratory in DMEM 1× (Mediatech) with 4% Pen/Strep (Mediatech). Dental pulp was pulled out, washed in PBS with 4% Pen/Strep and incubated in 5% TrypLE (Gibco) for 15 min. Pulp was partly dissociated using needles and plated in culture medium (DMEM/F12 50:50, 15% FBS, 1%NEAA, 1% fungizone and 2% Pen/Strep). In 1–4 weeks, DPCs migrated out of the pulp and could be passaged and frozen as stock. DPCs in early passage (two to three) were reprogrammed using pMXs retroviruses expressing Yamanaka transcription factors (obtained from Addgene, Cambridge, Massachusetts)12. After 4 days, transduced DPCs were trypsinized, plated on mouse embryonic fibroblasts and cultured using human embryonic stem cell (hESC) medium. After manually picked and clonally expanded, feeder-free iPSCs were grown on matrigel-coated dishes (BD Bioscience, San Jose, California) with mTeSR1 (StemCell Technologies) or iDEAL28. All G-banding karyotyping analyses were performed by Molecular Diagnostics Service (San Diego, California) and Children’s Hospital Los Angeles (Los Angeles, California). Two hundred nanograms of DNA were processed and hybridized to the Illumina Infinium Human Core Exome BeadChip following manufacturer’s instructions. Illumina GenomeStudio V2011.1 with the Genotyping Module version 1.9.4 was used to normalize data and call genotypes using reference data provided by Illumina. Illumina’s cnv Partition and gada R packages were used to automatically detect aberrant copy number region. In addition, the B Allele Frequency (BAF) and Log R Ratio (LRR) distributions were manually checked to determine additional CNVs not detected by the software. Sample identification/relatedness was assessed by comparing called genotypes for each sample. The absolute number of different genotypes was counted and the Euclidean distances were calculated to identify relatedness of the samples. Dissociated iPSC colonies were centrifuged and resuspended in 1:1 matrigel and phosphate buffer saline solution. The cells were injected subcutaneously in nude mice. After 1–2 months, teratomas were dissected, fixed and sliced. Sections were stained with haematoxylin and eosin for further analysis. Protocols were previously approved by the University of California San Diego Institutional Animal Care and Use Committee. iPSCs were cultured on matrigel-coated dishes and fed daily with mTeSR for 7 days. On the next day, mTeSR was substituted by N2 medium (DMEM/F12 supplemented with 0.5× N2-Supplement (Life Technologies), 1 μM dorsomorphin (Tocris) and 1 μM SB431542 (Stemgent)) for 1–2 days. iPSC colonies were lifted off, cultured in suspension on the shaker (95 r.p.m. at 37 °C) for 8 days to form embryoid bodies and fed with N2 media. Embryoid bodies then were mechanically dissociated, plated on a matrigel-coated dish and fed with N2B27 medium (DMEM/F12 supplemented with 0.5× N2-Supplement, 0.5× B27-Supplement (Life Technologies), 1% penicillin/streptomycin and 20 ng/mL FGF-2). The emerging rosettes were picked manually, dissociated completely using accutase and plated on a poly-ornithine/laminin-coated plate. NPCs were expanded in N2B27 medium and fed every other day. To differentiate NPCs into neurons, FGF-2 was withdrawn from the N2B27 medium. NPCs and neurons were characterized for stage-specific markers by immunostaining and flow cytometry (NPCs only), expression profile by single-cell RT–PCR and RNA sequencing and electrophysiological property (neurons). Total RNA of DPCs, iPSCs, NPCs and neurons was extracted using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols. Contaminating DNA in RNA samples was removed using TURBO DNase (Life Technologies) according to the manufacturer’s protocols. Quality and quantity of DNase-treated RNA were assessed using NanoDrop 1000 (Thermo Scientific). RNA was extracted from iPSCs as previously described using Trizol reagent (Life Technologies). cDNA was generated from the RNA using SuperScript III protocol according to the manufacturer’s instructions. PCR was performed using primers listed below at the following cycles: 94 °C for 10 min; 35 repeats of 94 °C for 30 s, 62 °C for 30 s and 72 °C for 1 min; and finally, 72 °C for 7 min. As a positive control, the pMX plasmid of the four vectors used on the reprogramming of the cells was placed along the samples as well as water as a negative template control for amplification. As an additional positive control for the endogenous genes, two hESC lines were used along with our iPSCs: H1 and HUES6 cells. Primers used were as follows. Endo-cMyc: forward, TTG AGG GGC ATC GTC GCG GGA; reverse, GCG TCC TGG GAA GGG AGA TCC. Endo-Klf4: forward, GAA ATT CGC CCG CTC CGA TGA; reverse, CTG TGT GTT TGC GGT AGT GCC. Endo-OCT3/4: forward, TCT TTC CAC CAG GCC CCC GGC TC; reverse, TGC GGG CGG ACA TGG GGA GAT CC. Endo-SOX2: forward, GCC GAG TGG AAA CTT TTG TCG; reverse, GGC AGC GTG TAC TTA TCC TTC T. Exo transgenes pMXs-TgUS: forward, GTG GTG GTA CGG GAA ATC AC. Exo-Oct4 pMXs-Oct3/4-TgDS: reverse, TAG CCA GGT TCG AGA ATC CA. Exo-Sox2 pMXs-Sox2-TgDS: reverse, GGT TCT CCT GGG CCA TCT TA. Exo-Klf4 pMXs-Klf4-TgDS: reverse, GGG AAG TCG CTT CAT GTG AG. Exo-c-Myc pMXs-c-Myc-TgDS: reverse, AGC AGC TCG AAT TTC TTC CA. Partly dissociated iPSCs were re-suspended in embryoid body medium (DMEM/F12 medium, 1× N2 supplement and 1% FBS) and cultured on shaker (95 r.p.m.) at 37 °C. Medium was changed every 3–4 days. After 20 days, total RNA of embryoid bodies was extracted for further gene expression analyses by qPCR. All tissue culture samples were routinely tested for mycoplasma by PCR. One millilitre of media supernatants (with no antibiotics or fungizone) was collected for all cell lines, spun down and resuspended in TE buffer. Ten microlitres of each sample were used in PCR reaction with the following primers: forward, GGC GAA TGG GTG AGT AAC; reverse, CGG ATA ACG CTT GCG ACC T. Any positive sample was immediately discarded. Three hundred nanograms of total extracted RNA from each sample were subjected to microarray by using the Affymatrix GeneChip one-cycle target labelling kit (Affymatrix, Santa Clara, California) according to the manufacturer’s recommended protocols. The resultant biotinylated cRNA was fragmented and then hybridized to the GeneChip Human 1.0 ST Array (764,885 probes, 28,869 genes, 19,734 gene-level probe sets with putative full-length transcript support (GenBank and RefSeq)) on the basis of human genome, Hg18. Arrays were prepared at the University of California DNA Core Facility. Arrays were analysed by the Affy (Affymetrix pre-processing)29 Bioconductor software package for microarray data. Data were then normalized by the RMA (robust multichip averaging) method to background-corrected and normalized probe levels to obtain a summary expression of normalized values for each probe set. Normalized microarray samples were then clustered by a hierarchical approach based on a matrix of distances. Normalized expression data were used to create a distance matrix that was calculated on the basis of Euclidean distance between the transcripts over a pair of samples representing a variation between two samples. Having the distances for all pairs of samples, a linkage method is used to cluster samples in a dendrogram by using calculated distances (sample expression similarities). This method also creates a heat map to graphically show the expression correlation between the samples. RNA samples were reverse transcribed into cDNA using the Super Script III First Strand Synthesis System (Invitrogen, California) according to the manufacturer’s instructions. Reactions were run on the Bio-Rad detection system using Sybr-green master mix (Bio-Rad). Primers were selected from Primerbank; validated database (http://pga.mgh.harvard.edu/primerbank/) and specificities were confirmed by melting curve analysis through a Bio-Rad detection system. Sequences of the primers are described in Supplementary Table 12. Quantitative analysis used the comparative threshold cycle method30. GAPDH was used as housekeeping gene. Each sample was run in triplicate. The RNA-seq analyses were previously described by our group31. Briefly, RNAs were isolated using the RNeasy Mini kit (Qiagen). A total of 1,000 ng of RNA was used for library preparation using the Illumina TruSeq RNA Sample Preparation Kit. The RNAs were sequenced on Illumina HiSeq2000 with 50 bp paired-end reads, generating 50 million high-quality sequencing fragments per sample on average. For validation purposes of biological samples subjected to RNA-seq, hESC and iPSC data available from the literature were downloaded and used to compare with our sequenced cell lines. The two hESC lines used are available (HUES-6, referred as ES(HUES), SRR873630, http://www.ncbi.nlm.nih.gov/sra/SRX290739; and H1, referred to here as ES(H1), SRR873631, http://www.ncbi.nlm.nih.gov/sra/SRX290740). The two human iPSC lines used are available under accession codes SRR873619 (referred to here as iPS(TD,1)) and SRR873620 (referred to here as iPS(TD,2)). RNA-seq enrichment used WebGestalt32 and Cytoscape33 software plugins, considering only categories having statistical significance (P < 0.05). Genes tested for differential expression were used as the background for GO annotation and enrichment analysis. NPCs were seeded onto poly-ornithine/laminin-coated six-well plates at a total number of 105 cells per well on day 0. Medium change was done on day 2. Cells were collected and counted on day 4. NPCs were resuspended, dissociated with accutase and fixed using fixation buffer (BioLegend) for 15 min followed by three PBS washes. The cell pellet was incubated and kept in Perm III buffer (BD Biosciences) at −20 °C until needed for the experiment. A total of 106 cells were incubated with antibodies Sox1 (PE), Sox2 (APC) or Nestin (PE) and Pax6 (APC) (Bd Biosciences) for 30 min and then washed three times before being resuspended for cell analyses. Cells were analysed in a plate reader mode using FACS Canto II machine (BD Biosciences). Cells were fixed in 4% paraformaldehyde for 10–20 min, washed with PBS three times (5 min each), permeabilized with 0.1% Triton X-100 for 15 min, incubated in blocking solution (2% BSA) for 1 h at room temperature and then in primary antibodies (goat anti-Nanog, Abcam ab77095, 1:500; rabbit anti-Lin28, Abcam ab46020, 1:500; rabbit anti-Oct4, Abcam ab19857, 1:500; mouse anti-SSEA4, Abcam ab16287, 1:200; mouse anti-Nestin, Abcam ab22035, 1:200; rabbit anti-Musashi1, Abcam ab52865, 1:250; rat anti-CTIP2, Abcam ab18465, 1:250; rabbit anti-SATB2, Abcam ab34735, 1:200; chicken anti-MAP2, Abcam ab5392, 1:1,000; rabbit anti-FZD9, Origene TA314730, 1:150; chicken anti-EGFP, Abcam ab13970, 1:1,000; rabbit anti-Synapsin1, EMD-Millipore AB1543P, 1:500; mouse anti-Vglut1, Synaptic Systems 135311, 1:500; rabbit anti-Homer1, Synaptic Systems 160003, 1:500) overnight at 4 °C. The next day, cells were washed with PBS three times (5 min each), incubated with secondary antibodies (Alexa Fluor 488, 555 and 647, Life Technologies, 1:1,000) for 1 h at room temperature and washed with PBS three times (5 min each). Nuclei were stained using DAPI (1:10,000). Slides or coverslips were mounted using ProLong Gold antifade mountant (Life Technologies). One million NPCs were harvested to single-cell suspension in 1mL PBS, then fixed by addition of 3 mL of 100% ethanol and stored at 4 °C for at least 2 h. NPC pellets were washed once with 5 mL PBS. After removal of PBS, cells were resuspended in 1 mL of propidium iodide (PI) staining solution (0.1% (v/v) Triton X-100, 10 μg/mL PI and 100 μg/mL RNase A in 1× PBS). WS and TD NPC samples were analysed by FACS on a Becton Dickinson LSRI, and gating of subG1 population (cells with fragmented DNA) was examined using FlowJo flow cytometry analysis software. Caspase activity was measured using a Green FLICA Caspases 3 & 7 Assay Kit (ImmunoChemistry Technologies). Briefly, NPCs were harvested, washed and stained with 1× carboxyfluorescein Fluorochrome Inhibitor of Caspase Assay (FAM-FLICA) reagent, 10 μg/mL Hoechst and 10 μg/mL propidium iodide (PI). Samples were analysed on the NC-3000 using the pre-optimized Caspase Assay. The population with caspase activity was used to analyse for apoptosis. NPC proliferation was assessed using BD Pharmingen BrdU Flow Kits (BD Biosciences) according to the manufacturer’s protocol. Briefly, NPCs were incubated with 1 μM BrdU for 45 min at 37 °C and harvested to single-cell suspension. NPCs were then fixed and permeabilized using BD Cytofix/Cytoperm Buffer and stained using FITC-conjugated anti-BrdU antibody and 7-aminoactinomycin D (7-AAD), a fluorescent dye for labelling DNA. Fluorescence-activated cell sorting (FACS) was done on LSRFortessa (BD Biosciences) and, to obtain the percentage of the BrdU-positive population, the cell-cycle profiles were analysed using FlowJo flow cytometry analysis software. Commercially available lentiviral vectors (pLKO.1) expressing short hairpin RNAs (shRNAs) against FZD9 under the control of the U6 promoter (Thermo Scientific) were engineered to express the Discosoma sp. red fluorescent protein (RFP) mCherry under the control of the hPGK (human phosphoglycerate kinase) promoter. The following shRNAs against FZD9 and a non-silencing scrambled control shRNA were selected (Thermo Scientific): shRNA-control, 5′-TTC TCC GAA CGT GTC ACG T-3′; shRNA-FZD9, 5′-ATC TTG CGG ATG TGG AAG AGG-3′. For rescue experiments, FZD9 cDNA was amplified from TD NPC cDNA as template by the following primer pair: 5′-CCG AGA TCT TCG AGG TGT GTG GGG TTC TCC AAA G-3′; 5′-TCT AGA GCC ACC ATG GCC GTA GCG CCT CTG-3′. The reaction was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) according to the manufacturer’s protocol. The FZD9 cDNA was cloned into a lentiviral vector driven by the ubiquitin promoter followed by a self-clevage peptide and GFP sequence. The specificity and efficiency of shRNA-control, shRNA-FZD9, and the FZD9-WT constructs were verified by co-transfection into HEK-293 cells. Cell lysates were collected and analysed by western blot analysis with anti-FZD9 antibodies (Aviva OAEC02415, 1:1,000). CHIR-98014 (Selleckchem) was resuspended according to manufacturer’s instructions into 10 mM stock using DMSO and then diluted to 100 μM. Final concentration used in cells was 100 nM of CHIR-98014, whereas the vehicle cells received only DMSO. For qPCR experiments, NPCs were propagated in six-well plates until 70% confluency and then treated with CHIR-98014 for 6 h to have their RNA collected using Trizol as previously described. For the NPC counting experiment, cells were seeded in six-well plates as described in the presence of CHIR-98014 or DMSO, in triplicates (TD and WS). After 48 h, the culture medium was changed and treatment was repeated. Cells were collected and counted after 96 h of incubation. The TD NPCs were lifted into suspension and maintained on a shaker (95 r.p.m.) to form neurospheres for 3 weeks. For the first week, the spheres were grown with N2B27 medium. The neurospheres were overlaid with the astrocyte medium (Lonza) for the remaining 2 weeks. The neurospheres were plated onto poly-ornithine- and laminin-coated plates and expanded for two to three passages before experimentation. Co-cultures of neurons and astrocytes were prepared for morphometric and functional analyses. NPCs were lysed in RIPA buffer with protease inhibitor. Rabbit anti-FZD9 antibody (Aviva OAEC02415, 1:1,000) and mouse anti-β-actin (Abcam ab8226, 1:3,000) were used as primary antibodies. IRDye 800CW goat anti-rabbit and IRDye 680RD goat anti-mouse (1:10,000) were used as secondary antibodies. The Odyssey system was used for signal detection. Signal intensities were measured using the Odyssey Image Studio and semi- quantitative analysis of FZD9 signal intensity was corrected with respect to β-actin relative quantification. A paired t-test analysis with P < 0.05 was used in the comparison of TD and WS FZD9 signal intensity normalized data. Co-localized Vglut (presynaptic) and Homer1 (postsynaptic) puncta were quantified after three-dimensional reconstruction of z-stack random images for all individuals and from two different experiments. Slides were analysed under a fluorescence microscope (Z1 Axio Observer Apotome, Zeiss). Only puncta in proximity of MAP2-positive processes were scored. Specific target amplification was performed in individual dissociated NPCs or 6-week-old neurons using C1 Single-Cell and BioMark HD Systems (Fluidigm), according to the manufacturer’s protocol and as described previously34, 35, 36. Briefly, single cells were captured on a C1 chip (10- to 17-μm cells) and cell viability was checked using a LIVE/DEAD Cell Viability/Cytotoxicity kit (Life Technologies). After lysis, RNA was reverse transcribed into cDNA with validated amplicon-specific DELTAgene Assays (Supplementary Table 13) using SuperScript III RT Platinum Taq Mix. Specific target amplification was performed by 18 cycles of 95 °C denaturation for 15 s and 60 °C annealing and amplification for 4 min. Each preamplified cDNA was mixed with 2× SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and then pipetted into an individual sample inlet in a 96.96 Dynamic Array IFC chip (Fluidigm). DELTAgene primer pairs (Supplementary Table 13) were diluted and pipetted into individual assay inlets in the same 96.96 Dynamic Array IFC chip. Quantitative PCR results were analysed using Fluidigm’s Real-time PCR Analysis software using the linear (derivative) baseline correction method and the automatic (gene) C threshold method with 0.65 curve quality threshold. Hierarchical clustering heat map, PCA analyses, violin plots of log (expression of C values) (limit of detection = 24) and ANOVA statistical analysis were performed using Singular Analysis Toolset 3.0 (Fluidigm). Neuronal networks derived from human iPSCs were transduced with lentivirus carrying the Syn::RFP reporter construct. Cell cultures were washed with Krebs HEPES buffer (KHB) (10 mM HEPES, 4.2 mM NaHCO , 10 mM dextrose, 1.18 mM MgSO , 1.18 mM KH PO 4.69 mM KCl, 118 mM NaCl, 1.29 mM NaCl ; pH 7.3) and incubated with 2–5 μM Fluo-4AM (Molecular Probes/Invitrogen, Carlsbad, California) in KHB for 40 min. Five thousand frames were acquired at 28 Hz with a region of 256 pixels × 256 pixels (×100 magnification), using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC) filter on an Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical, Japan). Images were acquired with MetaMorph 7.7 (MDS Analytical Technologies, Sunnyvale, California), processed and analysed using individual circular regions of interest (ROI) on ImageJ and Matlab 7.2 (Mathworks, Natick, Massachusetts). Syn::RFP+ neurons were selected after confirmation that calcium transients were blocked with 1 mM of tetrodotoxin (TTX). The amplitude of signals was presented as relative fluorescence changes (ΔF/F) after background subtraction. The threshold for calcium spikes was set at the 95th percentile of the amplitude of all detected events. For whole-cell patch-clamp recordings, individual coverslips containing live 1-month-old neurons were transferred into a heated recording chamber and continuously perfused (1 mL/min) with artificial cerebrospinal fluid bubbled with a mixture of CO (5%) and O (95%) and maintained at 25 °C. Artificial cerebrospinal fluid contained (in mM) 121 NaCl, 4.2 KCl, 1.1 CaCl2, 1 MgSO , 29 NaHCO , 0.45 NaH PO -H O, 0.5 Na HPO and 20 glucose (all chemicals from Sigma). Whole-cell recordings were performed using a digidata 1440A/ Multiclamp 700B and Clampex 10.3 (Molecular devices). Patch electrodes were filled with internal solutions containing 130 mM K-gluconate, 6 mM KCl, 4 mM NaCl, 10 mM Na-HEPES, 0.2 mM K-EGTA; 0.3 mM GTP, 2 mM Mg-ATP, 0.2 mM cAMP, 10 mM d-glucose, 0.15% biocytin and 0.06% rhodamine. The pH and osmolarity were adjusted for physiological conditions. Data were all corrected for liquid junction potentials, electrode capacitances were compensated on-line in cell-attached mode and a low-pass filter at 2 kHz was used. The access resistance of the cells in our sample was around 37 MΩ with resistance of the patch pipettes 3–5 MΩ. Spontaneous synaptic AMPA events were recorded at the reversal potential of Cl− and could be reversibly blocked by AMPA receptor antagonist (10 μM NBQX, Sigma). Spontaneous synaptic GABA events were recorded at the reversal potential of Na+ and could be reversibly blocked with GABA receptor antagonist (10 μM SR95531, Sigma). Using 12-well MEA plates from Axion Biosystems, we plated the same density of NPCs from TD and WS individuals in triplicate. Each well was seeded with 10,000 NPCs that were induced into neuronal differentiation as previously described. Each well was coated with poly-l-ornithine and laminin before cell seeding. Cells were fed once a week and measurements were taken before the medium was changed. Recordings were performed using a Maestro MEA system and AxIS software (Axion Biosystems), using a band-pass filter with 10 Hz and 2.5 kHz cutoff frequencies. Spike detection was performed using an adaptive threshold set to 5.5 times the standard deviation of the estimated noise on each electrode. Each plate first rested for 5 min in the Maestro, and then 5–10 min of data were recorded to calculate the spike rate per well. MEA analysis was performed using the Axion Biosystems Neural Metrics Tool, wherein electrodes that detected at least five spikes per minute were classified as active electrodes. Bursts were identified in the data recorded from each individual electrode using an adaptive Poisson surprise algorithm. Network bursts were identified for each well, using a non-adaptive algorithm requiring a minimum of ten spikes with a maximum inter-spike interval of 100 ms. Only channels that exhibited bursting activity (more than ten spikes in 5 min interval) were included in this analysis. After measurement, neurons were immunostained to check morphology and density. We used six post-mortem brains (two WS and four TD) that were gender-, age- and hemisphere-matched. All brain specimens were harvested within a post-mortem interval of 18–30 h and had been immersed and fixed in 10% formalin for up to 20 years. For the purpose of the present experiments, samples were obtained from anatomically well-identified cortical areas in a consistent manner across specimens. Tissue blocks approximately 5 mm3 were removed from primary somatosensory cortex (Brodmann area 3) and primary motor cortex (Brodmann area 4) in the arm/hand knob region of the pre- and postcentral gyri, respectively, and from the secondary visual area (Brodmann area 18) from approximately 1.4 cm dorsally to the occipital pole and 2 cm from the midline37, 38. We focused specifically on these parts of the cortex because pathologies in dendritic morphology in these areas have been reported in other neurodevelopmental disorders39, 40, 41. In addition, pyramidal neurons in the selected areas reach their mature-like morphology early in development and start displaying dendritic pathologies sooner than high integration areas, such as the prefrontal cortex, allowing comparison of post-mortem findings with iPSC-derived neurons in early stages of development42, 43. Sampled tissue blocks were processed using an adaptation of the Golgi–Kopsch method44, which has been shown to give good results with tissue that has been fixed for long periods45. Briefly, blocks were immersed in a solution of 3% potassium dichromate, 0.5% formalin for 8 days, followed by immersion into 0.75% silver nitrate for 2 days. Blocks were then sectioned on a vibratome, perpendicular to the pial surface, at a thickness of 120 μm. Golgi sections were cut into 100% ethyl alcohol and transferred briefly into methyl salicylate followed by toluene, mounted onto glass slides and coverslipped. Adjacent blocks from each region were sectioned at 60 μm and stained with thionin for visualization of cell bodies and laminar organization, which enabled identification of the position of each neuron within a specific cortical layer. Cytoarchitectonic analysis of histological sections from each block confirmed that tissue was sampled from the ROI and that the Golgi-impregnated pyramidal neurons were located in cortical layers V/VI. Cortical neurons from all six post-mortem brains were used in the study. Neurons included in the morphological analysis did not display degenerative changes46. Only neurons with fully impregnated soma, apical dendrites with present oblique branches and at least two basal dendrites with third-order segments were chosen for the analysis47. To minimize the effects of cutting on dendritic measurements, we included neurons with cell bodies located near the centre of 120-μm-thick histological sections, with natural terminations of higher-order dendritic branches present where possible37, 47. Inclusion of the neurons completely contained within 120-μm sections biases the sample towards smaller neurons, leading to the underestimation of dendritic length48; therefore, we applied the same criteria blinded across all WS and TD specimens, and we thus included the neurons with incomplete endings if they were judged to otherwise fulfil the criteria for successful Golgi impregnation. All neurons were oriented with apical dendrite perpendicular to the pial surface; inverted pyramidal cells as well as magnopyramidal neurons were excluded from the analysis. Neuronal morphology was quantified along x-, y-, and z-coordinates using Neurolucida version 10 software (MBF Bioscience, Williston, Vermont) connected to a Nikon Eclipse 80i microscope, with a ×40 (0.75 numerical aperture) Plan Fluor dry objective. Tracings were conducted on both apical and basal dendrites, and the results reflect summed values for both types of dendrite per neuron. Following the recommendation that the applications of Sholl’s concentric spheres or Eayrs’ concentric circles for the analysis of neuronal morphology are not adequate when neuronal morphology is analysed in three dimensions48, we conducted dendritic tree analysis with the following measurements37, 47: (1) soma area—cross sectional surface area of the cell body; (2) dendritic length—summed total length of all dendrites per neuron; (3) dendrite number—number of dendritic trees emerging directly from the soma per neuron; (4) dendritic segment number—total number of segments per neuron; (5) dendritic spine/protrusion number—total number of dendritic spines per neuron; (6) dendritic spine/protrusion density—average number of spines per 20 μm of dendritic length; and (7) branching point number—number of nodes (points at the dendrite where a dendrite branches into two or more) per neuron. Dendritic segments were defined as parts of the dendrites between two branching points—between the soma and the first branching point in the case of first-order dendritic segments, and between the last branching point and the termination of the dendrite in the case of terminal dendritic segments. Since the long formalin-fixation time could have resulted in degradation of dendritic spines, spine values might be underestimated and are thus reported here with caution. All of the tracings were accomplished blind to brain region and diagnostic status. The iPSC-derived sample consisted of EGFP-positive 8-week-old neurons with pyramidal- or ovoid-shaped soma and at least two branched neurites (dendrites) with visible spines/protrusions. Protrusions from dendritic shaft, which morphologically resembled dendritic spines in post-mortem specimens, were considered and quantified as dendritic spines in iPSC-derived neurons. The neurites were considered dendrites on the basis of the criteria applied in post-mortem studies: (1) thickness that decreased with the distance from the cell body; (2) branches emerging under acute angle; and (3) presence of dendritic spines. In addition, only enhanced-GFP-positive neurons with nuclei co-stained with CTIP2, indicative of layer V/VI neurons, and with the dendrites displaying evenly distributed fluorescent stain along their entire length, were included in the analysis. The morphology of the neurons was quantified along x-, y-, and z-coordinates using Neurolucida version 9 software (MBF Bioscience, Williston, VT) connected to a Nikon Eclipse E600 microscope with a ×40 oil objective. No distinction was made between apical and basal dendrites, and the results reflect summed length values of all neurites/dendrites per neuron, consistent with what was done for the post-mortem neurons. The same set of measurements used in the analysis of Golgi-impregnated neurons was applied to the analysis of iPSC-derived neurons, and all of the tracings were accomplished blind to the diagnostic status and were conducted by the same rater (B.H.-M.). Intra-rater reliability was assessed by having the rater trace the same neuron after a period of time. The average coefficient of variation between the results of retraced neurons was 2% for soma area (SA), total dendritic length (TDL), dendritic segment number (DSN) and branching point number (BPN), and 3% for dendritic spine/protrusion number (DPN); there was no variation in tree/dendrite number (TN) in different tracings of the same neuron. The accuracy was further checked by having three individuals (B.H.-M., B.J. and L.S.) trace the same neuron. MRI scanning was completed in 19 participants with WS (aged 19–43 years; mean 29.0, s.d. 8.8; 11 males, 8 females) and 19 TD comparison participants (aged 16–43 years; mean 26.2, s.d. 7.3; 8 males, 11 females). There was no significant difference between the groups in age (t = 1.0, P < 0.30) or in gender ratio (Pearson’s χ2 = 0.95, P < 0.33). A standardized multiple modality high-resolution structural MRI protocol was implemented, involving three-dimensional T - and T -weighted volumes and a set of diffusion-weighted scans. Imaging data were obtained at the University of California San Diego Radiology Imaging Laboratory on a 1.5 T GE Signa HDx 14.0M5 TwinSpeed system (GE Healthcare, Waukesha, Wisconsin) using an eight-channel phased array head coil. A three-dimensional inversion recovery spoiled gradient echo (IR-SPGR) T -weighted volume was acquired with pulse sequence parameters optimized for maximum grey/white matter contrast (echo time = 3.9 ms, repetition time = 8.7 ms, inversion time = 270 ms, flip angle = 8°, difference in echo times = 750 ms, bandwidth = ± 15.63 kHz, field of view = 24 cm, matrix = 192 × 192, voxel size = 1.25 mm × 1.25 mm × 1.2 mm). All MRI data were collected using prospective motion (PROMO) correction for non-diffusion imaging49. This method has been shown to improve image quality, reduce motion-related artefacts, increase the reliability of quantitative measures and improve the clinical diagnostic utility of MRI data obtained in children and clinical groups50, 51. Standardized quality control procedures were followed for both raw and processed data, including visual inspection ratings by a trained imaging technician and computer algorithms testing general image characteristics as well as aspects specific to each imaging modality, such as contrast properties, registrations and artefacts from motion and other sources. Participants included in the current analyses were only those who passed all raw and processed quality control measures. Image post-processing and analysis were performed using FreeSurfer software suite (http://surfer.nmr.mgh.harvard.edu/). Surface-based cortical reconstruction and subcortical volumetric segmentation procedures have been shown elsewhere52, 53, 54, 55, 56, 57, 58. Briefly, a three-dimensional model of the cortical surface was generated using MRI scans with four attributes: white matter segmentation; tessellation of the grey/white matter boundary; inflation of the folded, tessellated surface; and correction of topological defects53, 54. Cortical thickness was measured using the distances from each point on the white matter surface to the pial surface57. Cortical surface area was measured at the pial surface for the entire cerebrum and for each parcel of the Desikan and Destrieux atlases53, 54, 58, 59. Means ± s.e.m. for each parameter were obtained from samples described in Supplementary Table 1. There were no statistical methods used to predetermine sample size. The experiments were not randomized. All of the tracings were accomplished blind to brain region and diagnostic status. All statistical analyses were done using Prism (Graphpad). Before statistical analysis comparing means between three to five unmatched groups of data, normal distribution was tested using D’Agostino and Pearson omnibus normality test and variance similarity was tested using Bartlett’s test for equal variances. Means of three to five unmatched groups, where normal distribution and equal variances between groups were confirmed, were statistically compared using one-way ANOVA and Tukey’s post hoc test. Otherwise, a Kruskal–Wallis test and Dunn’s multiple comparison test were used. Before statistical analysis comparing means between two unmatched groups of data, normal distribution was tested using D’Agostino and Pearson omnibus normality test and variance similarity was tested using an F test to compare variances. To compare the means of two groups where normal distribution and similar variance between groups were confirmed, Student’s t test was used. Otherwise, a Mann–Whitney test was used. Significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001 or ****P < 0.0001.
News Article | November 23, 2016
Plasmids containing the 9-kb mouse villin promoter (pBS-Villin)26, 27 and simian diphtheria toxin receptor (HBEGF (‘DTR’)) with the enhanced green fluorescent protein (pDTR–eGFP) fusion gene28 have been previously described. The pDTR–eGFP was PCR amplified with primers harbouring a 5′ BsiWI site and a 3′ MluI site and cloned into pBS-Villin. The pBS-Villin/DTR–eGFP plasmid was verified by sequencing and the transgene was isolated from the plasmid by restriction enzyme digestion and gel purification. The transgene was microinjected into fertilized eggs from C57BL/6J mice (Jackson Laboratory) and transferred into oviducts of ICR foster mothers as previously described26. Identification of the transgenic mice was performed by PCR amplification using the following primers: 5′-ACTGCTCTCACATGCCTTCT-3′ and 5′-CTTCTTCCCTAGTCCCTTGC-3′. For diphtheria toxin administration, mice were injected intraperitoneally with 2 or 10 ng g−1 diphtheria toxin (EMD Chemicals) and humanely killed 1–24 h later29. Control mice were injected with PBS. For dextran sulphate solution (DSS) (MP Biomedicals) studies, mice were supplemented with 3% DSS in the drinking water for five days. On day three, water bottles were refilled with 3% DSS solution and on day five, replaced with fresh drinking water. Mice were weighed and monitored daily for signs of distress, morbidity or mortality during the course of the experiment until they were killed on day 7. Both male and female mice ages 6–8 weeks were used for all studies. All experiments were approved by the institutional animal care and use committee and carried out in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication 86–23, revised 1985). Before isolating professional phagocytes (‘phagocytes’), VDTR and VDTR negative littermate controls were intraperitoneally injected with PBS (vehicle) or diphtheria toxin (EMD Chemicals) at a low (2 ng g−1) or high (10 ng g−1) dose per body weight. Mice were then killed 1–24 h later and phagocytes were isolated from the SILP as previously described with some modifications30. In brief, the small intestine, including the duodenum, jejunum and ileum, was excised and Peyer’s patches removed. Next, the small intestine was opened longitudinally with surgical scissors and flushed with ice-cold PBS to remove the faecal content. Intestines were then cut into 0.5-cm pieces and transferred into 50-ml conical tubes containing 20 ml of PBS. Samples were then vigorously shaken for 30 s using the vortex genie (Scientific Industries) and passed over 100-μm nylon cell strainers (BD Falcon). Fresh PBS was added to the tissue samples and the shaking and filtering process was repeated a total of eight times. To isolate and remove the intestinal epithelial cell layer, samples were washed with 20 ml of warm PBS containing 3 mM EDTA and passed over cell strainers. This was repeated three times. Flow-through was kept as purified for IECs, whereas whole tissues were further processed to isolate dendritic cell and macrophage subsets. Next, samples were washed with ice-cold PBS followed by RPMI 1640 (Sigma) containing 5% FBS to remove the EDTA. Samples were then re-suspended with RPMI 1640 containing 5% FBS, 1 mg ml−1 collagenase D (Roche), and 1 mg ml−1 DNase I (Roche) and incubated in a 37 °C water bath for 60 min. Samples were shaken every 20 min during this time. At the completion of the incubation, samples were washed with FACS buffer to remove the collagenase and then passed through an 18-gauge needle followed by a 21-gauge needle to create a single-cell suspension. Phagocytes were then enriched from samples by using a 1.065 g ml−1 OptiPrep (Sigma) density gradient according to the manufacturer’s protocol. Following centrifugation, phagocytes were isolated from both low- and mid-density bands and finally re-suspended in FACS buffer for flow cytometric analyses. Mouse spleen was digested in parallel with small intestine samples and used for single-colour compensation controls. All samples were pretreated with Fc block for 10 min at 4 °C followed by fluorescently conjugated antibody labelling at 4 °C for 60 min. The following antibodies were used for these studies: Antibodies from BioLegend including Alexa Fluor 647- or 700-conjugated anti-CD11c (clone N418), PerCP/Cy5.5-conjugated anti-CD24 (clone M1/69), APC/Cy7-conjugated anti-CD45 (clone 30-F11), APC-conjugated anti-CD64 (clone X54-5/7.1), APC-conjugated anti-CD274 (clone 10F.9G2), PerCP/Cy5.5-conjugated anti-F4/80 (clone BM8), Alexa Fluor 700-conjugated anti-Ly-6c (clone HK1.4), and Phycoerythrin (PE) or Brilliant Violet 421-conjugated anti-MHCII I-A/I-E (clone M5/114.15.2); antibodies from eBioscience including FITC-conjugated anti-CD4 (clone RM4-5), PE/Cy7-conjugated anti-CD11b (clone M1/70) and PE-conjugated anti-CD103 (clone 2E7); and TxRed-conjugated anti-CD45 (clone 30-F11) from Invitrogen. Live/Dead Aqua (Life Technologies) was used to discriminate viable cells. Phagocytes isolated from the SILP were surface stained for: APC/Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD11c, Brilliant Violet 421-conjugated anti-MHCII I-A/I-E, PE/Cy7-conjugated anti-CD11b, PE-conjugated anti-CD103, PerCP/Cy5.5-conjugated anti-CD24, and APC-conjugated anti-CD64. The identification of phagocytes from VDTR mice with IEC cargo was determined by the presence of eGFP and this gate was defined on the basis of C57BL/6J and VDTR− littermate controls that were eGFP−. Sample acquisition was performed using the LSRFortessa (BD Biosciences) and data analyses were performed using the FlowJo analytical software (Tree Star). To sort phagocytes with and without apoptotic IEC cargo, the following surface markers were used: APC/Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD11c, Brilliant Violet 421-conjugated anti-MHCII I-A/I-E, PE/Cy7-conjugated anti-CD11b, PE-conjugated anti-CD103, PerCP/Cy5.5-conjugated anti-CD24, and APC-conjugated anti-CD64. The identification of phagocytes from VDTR mice with IEC cargo was determined by the presence of eGFP and this gate was defined on the basis of C57BL/6J and VDTR− littermate controls that were eGFP−. Sorted populations were live, CD45+MHCII+CD11c+ phagocytes that were either eGFP− or eGFP+ including (i) CD103+CD11b−CD24+CD64− (hereafter CD103), (ii) CD103+CD11b+ CD24−CD64+ (hereafter CD103 CD11b), and (iii) CD103−CD11b+CD24− CD64+ (hereafter CD11b) for a total of six populations. Owing to the four-sample sort-maximum of the instrument, the three eGFP+ populations were collected first and then fresh collection tubes were added for the three eGFP− populations. Cells were sorted directly into 0.5 ml TRIzol LS reagent (Life Technologies) for microarray processing (see below). Each sort was performed at 4 h following diphtheria toxin administration and consisted of 3–4 pooled VDTR mice. The following are the cell yield ranges for each subset: 1,000–5,000 eGFP+CD103+; 3,000–9,000 eGFP+CD103+CD11b+; 10,000–40,000 eGFP+CD11b+; 4,500–10,000 eGFP−CD103+; 40,000–80,000 eGFP−CD103+CD11b+; and 30,000–100,000 eGFP−CD11b+. FACS was conducted on the FACSAria IIu SORP (BD Biosciences). The following are the RNA yield ranges for each subset: 200–2,400 pg eGFP+CD103+; 200–3,000 pg eGFP+CD103+CD11b+; 600–3,000 pg eGFP+CD11b+; 200–4,600 pg eGFP−CD103+; 600–5000 pg eGFP−CD103+CD11b+; and 450–4,000 pg eGFP−CD11b+. The purity and identity of each subset was validated as indicated in Extended Data Fig. 5 and according to markers as previously reported31. For analysis of IEC engulfment by CD11c+ phagocytes, single-cell suspensions were prepared as described for flow cytometric analyses and acquired using the IS 100 Imaging flow cytometer (Amnis Corp). Phagocytes with eGFP+ cargo were identified as those that contained single nuclei and were CD45+, CD11c+ and MHCII+. Data were analysed using IDEAS software (Amnis Corp) and spectrally compensated using a compensation matrix generated from the following single-colour controls; FITC-conjugated CD4, PE-conjugated MHCII, Alexa Fluor647-conjugated CD11c, TxRed-conjugated CD45, and Hoechst stain. Total RNA was isolated from mouse small intestine using RNeasy mini-kit (Qiagen) and quantified by a spectrophotometer. Reverse transcription was performed with Superscript III (Invitrogen) and cDNA was synthesized using the Mastercycler ep (Eppendorf). Real-time quantitative RT–PCR was conducted in duplicate on a ViiA 7 Real-time PCR System (Life Technologies) using TaqMan quantitative PCR Master Mix at a concentration of 1× (Applied Biosystems) or SYBR Green Real-Time PCR Master Mixes for the eGFP and HBEGF (‘DTR’) transgenes. Samples were normalized to β-actin and relative expression was determined by 2-ΔΔC method. Forward (FW) and reverse (RV) primers for SYBR Green include: All probe sequences are in the format: 5′ FAM-sequence-BHQ-1 3′ and together with forward (FW) and reverse (RV) primer pairs were synthesized by Biosearch Technologies. 5′-AGCCACCCCCACTCCTAAGAGGAGG-3′ Actb probe, 5′-GAAGTCCCTCACCCTCCCAA-3′ Actb FW, 5′-GGCATGGACGCGACCA-3′ Actb RV; 5′-AAATCGGTGATCCAGGGATTGTTCCA-3′ Acadsb probe, 5′-CCTCTGGTTTCCTCTATGGATGA-3′, Acadsb FW, 5′-TCCCTCCATATTGTGCTTCAAC-3′ Acadsb RV; 5′-CGGGACAGGGCAACTCTTGCAA-3′ Aldh1a2 probe, 5′-GCTTGCAGACTTGGTGGAA-3′ Aldh1a2 FW, 5′-GCTTGCAGGAATGGCTTACC-3′ Aldh1a2 RV; 5′-CCCACTTTCCTTGTGGTACTCTGGAC-3′ Alox5ap probe, 5′-CAACCAGAACTGCGTAGATGC-3′ Alox5ap FW, 5′-GAAGGCGGCAGGGACTTG-3′ Alox5ap RV; 5′-TGCCTTTAGTGGCCTCATTGTTCC-3′ Atrn probe, 5′-GGACTCAATCTACGCACCTCTGAT-3′ Atrn FW, 5′-GCCGTCTCATTGCCATCTCTT-3′ Atrn RV; 5′-TTGGCATCAATCTGAGCTGTTGGTG-3′ Axl probe, 5′-GCCCATCAACTTCGGAAGAAAG-3′ Axl FW, 5′-CCTCTGGCACCTGTGATATTCC-3′ Axl RV; 5′-AGTGAAGGAGTTCTTCTGGACCTCAA-3′ Ccl22 probe, 5′-CACCCTCTGCCATCACGTT-3′ Ccl22 FW, 5′-ATCTCGGTTCTTGACGGTTATCA-3′ Ccl22 RV; 5′-CCACTGCTCATGGATATGTTGAACAATAGAGACC-3′ Ccr2 probe, 5′-AGGGTCACAGGATTAGGAAGGTT-3′ Ccr2 FW, 5′-CGTTCTGGGCACCTGATTTAA-3′ Ccr2 RV; 5′-CAGTGCCCAAGTGGAGGCCTTGATC-3′ Ccr7 probe, 5′-CACGCTGAGATGCTCACTGG-3′ Ccr7 FW, 5′-ATCTGGGCCACTTGGATGG-3′ Ccr7 RV; 5′-AGATTCGCTGTCACCAGCACAGACA-3′ Cd40 probe, 5′-TCTCAGCCCAGTGGAACA-3′ Cd40 FW, 5′-CGGTGCCCTCCTTCTTAACC-3′ Cd40 RV; 5′-CGAATCACGCTGAAAGTCAATGCCC-3′ Cd274 probe, 5′-CGGTGGTGCGGACTACAAG-3′ Cd274 FW, 5′-CCCTCGGCCTGACATATTAGTTC-3′ Cd274 RV; 5′-TTCCCAGGGCTTGAGGCTCCC-3′ Cd300a probe, 5′-GGCCACCGTGAACATGACTA-3′ Cd300a FW, 5′-GCAGGAGAGCTAACACAGACAAC-3′ Cd300a RV; 5′-ATGGAAAATGGGTGGCGTCTAACCCA-3′ Cfh probe, 5′-CCGAACACTTGGCACTATTGTAA-3′ Cfh FW, 5′-CTCCGGGATGCCCACAAG-3′ Cfh RV; 5′-CCCTGAACAACCAACAGATGACACTGG-3′ Elf3 probe, 5′-GGCACTGAAGACTTGGTGTTG-3′ Elf3 FW, 5′-CCCTGAACAACCAACAGATGACACTGG-3′ Elf3 RV; 5′-AGCTGACAGATACACTCCAAGCGGA-3′ Fos probe, 5′-AGTGCCGGAATCGGAGGA-3′ Fos FW, 5′-TGCAACGCAGACTTCTCATC-3′ Fos RV; 5′-CTGCTCCTGCTGGCTTCCGAGT-3′ Gas6 probe, 5′-CTGGGCACTGCGCTTCTG-3′ Gas6 FW, 5′-CGCAACAGCACAGTGTGA-3′ Gas6 RV; 5′-TCTTATGCAGACTGTGTCCTGGCA-3′ Ido1 probe, 5′-GGGCCTGCCTCCTATTCTG-3′ Ido1 FW, 5′-CCCACCAGGAAATGAGAACAGA-3′ Ido1 RV; 5′-TCACAAGCAGAGCACAAGCCTGTC-3′ Il1b probe, 5′-AAAGACGGCACACCCACCCTGC-3′ Il1b FW, 5′-TGTCCTGACCACTGTTGTTTCCCAG-3′ Il1b RV; 5′-TCTGCAAGAGACTTCCATCCAGTTGCCT-3′ Il6 probe, 5′-CCAGAAACCGCTATGAAGTTCC-3′ Il6 FW, 5′-TCACCAGCATCAGTCCCAAG-3′ Il6 RV; 5′-TTCAAACAAAGGACCAGCTGGACA-3′ Il10 probe, 5′-TCAGCCAGGTGAAGACTTTC-3′ Il10 FW, 5′-GGCAACCCAAGTAACCCTTA-3′ Il10 RV; 5′-TAACTGGGATCCAGGCACGCC-3′ Ly75 probe, 5′-GTCAGACTTCAGGCCACTCAA-3′ Ly75 FW, 5′-TGACCCACCAATCACAGGT-3′ Ly75 RV; 5′-TCCCTTACTTTATTAAGCAGCCTGAGAGTG-3′ Mertk probe, 5′-TGATCCCATATACGTGGAAGTTCA-3′ Mertk FW, 5′-CCTGGCAGGTGAGGTTGAAG-3′ Mertk RV; 5′-TTTGCGTCTGACTGCCGAGACTC-3′ Muc2 probe, 5′-CCTGGCCTCTGTGATTACAAC-3′ Muc2 FW, 5′-GGTGCACAGCAAATTCCTTGTAG-3′ Muc2 RV; 5′-TCGCAACCAGATCGGAGATGTGG-3′ Nlrc5 probe, 5′-CCAGAACTCAGGAAATTTGACTTGA-3′ Nlrc5 FW, 5′-TTTGGCAAGATGGCAGCTAA-3′ Nlrc5 RV; 5′-CTGCTGCCTCACTTCTAGCTTCTGC-3′ Nlrp3 probe, 5′-GTTGCCTGTTCTTCCAGACT-3′ Nlrp3 FW, 5′-GGCTCCGGTTGGTGCTTAG-3′ Nlrp3 RV; 5′-TAGGCTGCTTTGGGAATGGCACC-3′ Oasl1 probe, 5′-CGCGTGCTCAAGGTACTCAAG-3′ Oasl1 FW, 5′-GACCAGCTCCACGTCTGTAG-3′ Oasl1 RV; 5′-TTGTGATGACTACATGGTCACACTCTTC-3′ Plac8 probe, 5′-GAACCCGATACGGCATTCCT-3′ Plac8 FW, 5′-TCTTGCCATCCAGCTCCTTAG-3′ Plac8 RV; 5′-ACCAACACATCGGAGCTGCGGA-3′ Relb probe, 5′-GAGCCTGTCTACGACAAGAAGTC-3′ Relb FW, 5′-GCCCGCTCTCCTTGTTGATTC-3′ Relb RV; 5′-AGTTATGCACGAGTGCGAGCTGT-3′ Spred1 probe, 5′-CGGCGACTTCTGACAACGATA-3′ Spred1 FW, 5′-GGTAGCCATCCACCACTTGAG-3′ Spred1 RV; 5′-AGAGGTCACCCGCGTGCTAATGGTG-3′ Tgfb1 probe, 5′-CCCGAAGCGGACTACTATGC-3′ Tgfb1 FW, 5′-ATAGATGGCGTTGTTGCGGT-3′ Tgfb1 RV; 5′-CTCTGCCTGCATCCAATCACTCTCA-3′ Timd4 probe, 5′-GGTCCGCCTTCACTACAGAATC-3′ Timd4 FW, 5′-GGCCTGAGTACGGCTATGTC-3′ Timd4 RV; 5′-TGGGCTTTCCGAATTCACTGGAGC-3′ Tnf probe, 5′-ATGCACCACCATCAAGGACTCAA-3′ Tnf FW, 5′-ACCACTCTCCCTTTGCAGAACTC-3′ Tnf RV; 5′-TCAACTGGTGTCGTGAAGTCAGGA-3′ Tnfaip3 probe, 5′-TCCCTGGAAAGCCAGAAGAAG-3′ Tnfaip3 FW, 5′-GAGGCAGTTTCCATCACCATTG-3′ Tnfaip3 RV; 5′-TCCGGAGCTACTTCAAGCAAGGC-3′ Vil1 probe, 5′-GGCAACGAGAGCGAGACTT-3′ Vil1 FW, 5′-CGCTGGACATCACAGGAGTT-3′ Vil1 RW. A total of five sorting experiments with a pool of 3–4 mice were performed for the cDNA microarrays. Following cell sorting into TRIzol LS reagent, samples were shipped on dry ice to the Center for Functional Genomics and the Microarray & HT Sequencing Core Facility at the University at Albany (Rensselaer). A sample clean-up step was performed using RNeasy columns (Qiagen) that included DNase treatment. The isolated RNA was checked for quality using NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent), following which 1 ng of total RNA was processed using WT-Ovation Pico RNA Amplification System (NuGEN). A total of three biological replicates were used for the microarray. When required, RNA was pooled from additional sorts to achieve the 1 ng of total RNA needed for the amplification system. The following are the sort experiments used for each sample: (2, 2 and 5, 2 and 5) eGFP+CD103+; (2, 3, 2 and 5) eGFP+CD103+CD11b+; (2, 4, 5) eGFP+CD11b+; (3, 2 and 4 and 5, 2 and 4 and 5) eGFP−CD103+; (2, 3, 4) eGFP−CD103+CD11b+; and (2, 3, 5) eGFP−CD11b+. RNA was reverse-transcribed and sense-target cDNAs were targeted using the standard NuGEN protocol and hybridized to Affymetrix mouse Gene 2.0 ST arrays. These arrays were then washed, stained on a FS 450 station, and scanned on a GeneChip 3000 7G scanner using Affymetric GeneChip Command Console Software (AGCC). The Affymetrix microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO)32 and are accessible with the GEO series accession number GSE85682. Fold changes and statistical significance were identified as those genes that were differentially expressed between eGFP+ and eGFP− subsets by at least 1.2 fold (ANOVA (Benjamini–Hochberg false discovery rate correction Q < 0.05) and Tukey’s HSD post-hoc test (P < 0.05; -1.2> fold >1.2) and determined using R software (version 3.2.0). Hierarchical clustering of differentially expressed genes meeting the aforementioned criteria were Z-scored and plotted with heatmap.2 (gplots version 2.17.0, CRAN/R). Principal component analyses of the 1,534 genes (ANOVA (Benjamini–Hochberg false discovery rate correction Q < 0.05); 4.8% of total) with the most variable expression in each CD11c+ subset with and without eGFP cargo were generated using R software which are freely available online. Small and large intestine were dissected and fixed in 10% formalin (Fisher Scientific) for 24 h and then processed for paraffin embedding. Tissue blocks were then cut into 5-mm sections, de-paraffinized by xylene immersion, and hydrated by serial immersion in 100%, 90%, 80%, 70% ethanol and PBS. Antigen retrieval was performed by heating samples in a pressure cooker (Cuisinart) in citrate buffer solution (10 mM citric acid monohydrate, 0.05% Tween 20 and PBS). Sections were then washed twice in PBS, blocked for 30 min in blocking buffer (10% BSA, 0.3% Triton X-100 (Sigma) and TBS), and prepared for labelling. TdT-mediated dUTP nick end labelling (TUNEL) was performed using the in situ cell-death detection kit, TMR red (Roche), per the manufacturer’s instructions, stained with DAPI, and mounted using Fluoromount-G (Southern Biotech). For cleaved caspase-3 (Cell Signaling), samples were labelled for 60 min at room temperature, stained with DAPI, and mounted using Fluoromount-G. For paraffin images, eGFP signal was not present owing to sample quenching following paraffin embedding and processing. Small and large intestine were dissected and fixed overnight in 1.6% paraformaldehyde (Thermo Scientific) containing 20% sucrose at 4 °C. Samples were then placed in OCT (Tissue-Tek) and snap-frozen over dry ice. Tissue sections of 8-mm thickness were cut, air-dried and blocked using blocking solution. Tissues were then labelled using an Alexa Fluor 594-conjugated phalloidin (Invitrogen) or a primary mouse anti-mouse pan-cytokeratin antibody (clone PCK-26) (Abcam) for 60 min in a humidified atmosphere followed by a secondary goat anti-mouse Alexa Fluor 594 (Thermo Fisher Scientific) for 30 min, then stained with DAPI, and mounted using Fluoromount-G. For fluorescent in situ hybridization, small intestine and large intestine were dissected and prepared as described for frozen sections33. Following tissue blocking, sections were incubated with 0.45 pmol μl−1 eubacterial oligonucleotide probe (AminoC6 + Alexa Fluor 594) 5′-GCTGCCTCCCGTAGGAGT-3′; (Operon)33 in a pre-chilled hybridization buffer (Sigma) overnight at 4 °C. Sections were counterstained with DAPI and mounted with Fluoromount-G. To label small intestine tissues, the whole-mount histology protocol was modified from previously described methods34. In brief, small intestine samples were excised, opened longitudinally, and washed in ice-cold PBS. Samples were then cut to 1 cm in length and placed in 6-ml polypropylene tubes (BD Biosciences). Next, samples were incubated with Fc block at 10 μg ml−1 in 200 μl of 2% paraformaldehyde with 1% FBS, 0.3% Triton X-100 in PBS for 3 h at 4 °C with gentle rocking. After blocking and fixing, samples were put into new polypropylene tubes and labelled using 3 μg ml−1 of the following antibodies: PE-conjugated anti-CD11c (clone N418) (eBioscience), APC-conjugated anti-CD31 (clone 390) (eBioscience) and anti-cleaved caspase-3 at 1:100. All labelling was conducted in the dark at 4 °C with gentle rocking for 3 h. Finally, samples were washed for 30 min in the dark at 4 °C with fresh PBS and mounted for imaging. Conventional microscopy was performed using the Eclipse Ni-E motorized upright microscope (Nikon) and images were acquired from paraffin, frozen, and whole mount tissue sections using a Nikon DS-Qi1 Mc camera. Cell quantification was calculated using NIS Elements imaging software (Nikon) and the object count application including intensity of stain thresholds and area restriction filters. Confocal microscopy was performed at the Microscope CORE at the Icahn School of Medicine at Mount Sinai using the Leica SP5 DM upright microscope and Leica LAS AF software. Naive mouse splenic CD4+ T cells were isolated by sorting with MACS CD4+ beads (Miltenyi Biotech) according to the manufacturer’s instructions and then by FACS using the FACSAria IIu SORP. T cells were sorted on the basis of the following criteria: live, CD45+CD3+CD4+ CD25−CD44−/lowCD62L+/high. Surface antibodies for sorting included: APC/Cy7-conjugated anti-CD45, eFluor 450-conjugated anti-CD3 (clone 145-2c11), PE-conjugated anti-CD4, APC-conjugated anti-CD25 (clone PC61.5), FITC-conjugated CD62L (clone MEL-14), and Alexa 700-conjugated anti-CD44 (clone IM7) (all eBiosciences). 1 × 105 T cells were then cultured with 1 × 104 eGFP+ or eGFP− CD103 dendritic cells sorted from the MLN which were identified as: live, CD45+MHCIIhiCD11c+, eGFP+ or eGFP−, CD103+CD11b− using the aforementioned antibodies for flow cytometry. These cells were cultured in round-bottom 96-well plates (Falcon) with complete IMDM (Gibco) supplemented with 10% FBS, 100 μg ml−1 penicillin, 100 μg ml−1 streptomycin, 2 mM l-glutamine, 10 mM HEPES and 1 nM sodium pyruvate for 5 days. Additionally, all cultures were supplemented with 1 μg ml−1 of soluble anti-CD3 (clone 2C11) as well as 5 ng ml−1 of recombinant human anti-IL-2 (Pepro Tech) on days 2 and 4. A total of 2 ng ml−1 of recombinant human anti-TGFβ1 (clone 1D11 R&D systems) was added to culture wells where indicated on days 1 and 4. On day 5, cells were first surface stained with FITC-conjugated anti-CD25, PE/Cy7-conjugated anti-CD4, Alexa Fluor 700-conjugated anti-CD3, and APC/Cy7-conjugated anti-CD45, followed by fixation and permeabilization (using the concentrate and diluent provided by eBioscience), and finally intracellular staining for eFluor 450-conjugated anti-FOXP3 (clone FJK-16 s), PE-conjugated anti-RORγ(t) (clone B2D), PerCP-eFluor 710-conjugated anti-GATA-3 (clone TWAJ), and APC-conjugated anti-T-bet (clone eBio4B10) (all eBioscience). Data are presented as mean ± s.e.m. Statistical significances were determined by a one-way ANOVA with Dunnett’s and Newman–Keuls post-tests or unpaired two-tailed t-test with Welch correction where specified. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not statistically significant (P > 0.05). No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The Affymetrix microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under GEO series accession number GSE85682.
News Article | November 16, 2016
AAV vector plasmids were cloned in the pAAV-MCS plasmid (Agilent Technologies) containing inverted terminal repeats from AAV serotype 2. The HBB rAAV6 GFP and tNGFR donor contained promoter, MaxGFP or tNGFR, and BGH polyA. The left and right homology arms for the GFP and tNGFR HBB donors were 540 bp and 420 bp, respectively. The Glu6Val rAAV6 donor contained 2.2 kb of sequence homologous to the sequence upstream of Glu6Val. The nucleotide changes are depicted in Extended Data Fig. 2. Immediately downstream of the last nucleotide change was 2.2 kb of homologous HBB sequence. HBB cDNA contained same homology arms as GFP and tNGFR donors above except the left homology arm was shortened to end at the sickle mutation. Sequence of full HBB cDNA is depicted in (Extended Data Fig. 9b). The sickle corrective donor used in the SCD-derived HSPCs in Fig. 4 had a total of 2.4 kb sequence homology to HBB with the SNPs shown in Extended Data Fig. 8a in the centre. scAAV6 carrying the SFFV promoter driving GFP was provided by H.-P. Kiem. AAV6 vectors were produced as described with a few modifications43. In brief, 293FT cells (Life Technologies) were seeded at 13 × 106 cells per dish in ten 15-cm dishes one day before transfection. One 15-cm dish was transfected using standard PEI transfection with 6 μg ITR-containing plasmid and 22 μg pDGM6 (a gift from D. Russell), which contains the AAV6 cap genes, AAV2 rep genes, and adenovirus helper genes. Cells were incubated for 72 h until collection of AAV6 from cells by three freeze–thaw cycles followed by a 45 min incubation with TurboNuclease at 250 U ml−1 (Abnova). AAV vectors were purified on an iodixanol density gradient by ultracentrifugation at 237,000g for 2 h at 18 °C. AAV vectors were extracted at the 60–40% iodixanol interface and dialysed three times in PBS with 5% sorbitol in the last dialysis using a 10K MWCO Slide-A-Lyzer G2 Dialysis Cassette (Thermo Fisher Scientific). Vectors were added pluronic acid to a final concentration of 0.001%, aliquoted, and stored at −80 °C until use. AAV6 vectors were titred using quantitative PCR to measure number of vector genomes as described previously44. Frozen CD34+ HSPCs derived from bone marrow or mobilized peripheral blood were purchased from AllCells and thawed according to manufacturer’s instructions. CD34+ HSPCs from cord blood were either purchased frozen from AllCells or acquired from donors under informed consent via the Binns Program for Cord Blood Research at Stanford University and used fresh without freezing. CD34+ HSPCs from patients with SCD were purified within 24 h of the scheduled apheresis. For volume reduction via induced rouleaux formation, whole blood was added 6% Hetastarch in 0.9% sodium chloride injection (Hospira, Inc.) in a proportion of 5:1 (v/v). Following a 60–90-min incubation at room temperature, the top layer, enriched for HSPCs and mature leukocytes, was carefully isolated with minimal disruption of the underlying fraction. Cells were pelleted, combined, and resuspended in a volume of PBS with 2 mM EDTA and 0.5% BGS directly proportional to the fraction of residual erythrocytes—typically 200–400 ml. Mononuclear cells (MNCs) were obtained by density gradient separation using Ficoll and CD34+ HSPCs were purified using the CD34+ Microbead Kit Ultrapure (Miltenyi Biotec) according to manufacturer’s protocol. Cells were cultured overnight and then stained for CD34 and CD45 using APC anti-human CD34 (clone 561; Biolegend) and BD Horizon V450 anti-human CD45 (clone HI30; BD Biosciences), and a pure population of HSPCs defined as CD34bright/CD45dim were obtained by cell sorting on a FACS Aria II cell sorter (BD Biosciences). All CD34+ HSPCs were cultured in StemSpan SFEM II (StemCell Technologies) supplemented with SCF (100 ng ml−1), TPO (100 ng ml−1), Flt3 ligand (100 ng ml−1), IL-6 (100 ng ml−1), and StemRegenin1 (0.75 mM). Cells were cultured at 37 °C, 5% CO and 5% O . The HBB and IL2RG synthetic sgRNAs used were purchased from TriLink BioTechnologies with chemically modified nucleotides at the three terminal positions at both the 5′ and 3′ ends. Modified nucleotides contained 2′-O-methyl-3′-phosphorothioate and the sgRNAs were HPLC-purified. The genomic sgRNA target sequences, with PAM in bold, are: HBB: 5′-CTTGCCCCACAGGGCAGTAACGG-3′ (refs 45, 46); IL2RG: 5′-TGGTAATGATGGCTTCAACATGG-3′. Cas9 mRNA containing 5-methylcytidine and pseudouridine was purchased from TriLink BioTechnologies. Cas9 protein was purchased from Life Technologies. Cas9 RNP was made by incubating protein with sgRNA at a molar ratio of 1:2.5 at 25 °C for 10 min immediately before electroporation. CD34+ HSPCs were electroporated 1–2 days after thawing or isolation. CD34+ HSPCs were electroporated using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofection Kit (VPA-1002, Lonza) as we have found this combination to be superior in optimization studies. The following conditions were used: 5 × 106 cells ml−1, 300 μg ml−1 Cas9 protein complexed with sgRNA at 1:2.5 molar ratio, or 100 μg ml−1 synthetic chemically modified sgRNA with 150 μg ml−1 Cas9 mRNA (TriLink BioTechnologies, non-HPLC purified). Following electroporation, cells were incubated for 15 min at 37 °C after which they were added AAV6 donor vectors at an MOI (vector genomes/cell) of 50,000–100,000 and then incubated at 30 °C or 37 °C overnight (if incubated at 30 °C, plates were then transferred to 37 °C) or targeting experiments of freshly sorted HSCs (Extended Data Fig. 5g), cells were electroporated using the Lonza Nucleofector 4D (program EO-100) and the P3 Primary Cell Nucleofection Kit (V4XP-3024). For the electroporation of 80 million CD34+ HSPCs, the Lonza 4D-Nucleofector LV unit (program DZ-100) and P3 Primary Cell Kit were used. Subsequently, we have found no benefit to the 30 °C incubation and now perform all of our manufacturing at 37 °C. Rates of targeted integration of GFP and tNGFR donors were measured by flow cytometry at least 18 days after electroporation. Targeted integration of a tNGFR expression cassette was measured by flow cytometry of cells stained with APC-conjugated anti-human CD271 (NGFR) antibody (BioLegend, clone: ME20.4). For sorting of GFPhigh or tNGFRhigh populations, cells were sorted on a FACS Aria II SORP using the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies) to discriminate live and dead cells according to manufacturer’s instructions. Positive selection of targeted HSPCs was performed using the CD271 (tNGFR) Microbead Kit (Miltenyi Biotech), according to the manufacturer’s instructions 72 h after electroporation. In brief, tNGFR+ cells were magnetically labelled with CD271 Microbeads after which the cell suspension was loaded onto an equilibrated MACS column inserted in the magnetic field of a MACS separator. The columns were washed three times, and enriched cells were eluted by removing the column from the magnetic field and eluting with PBS. Enrichment was determined by flow cytometry during culture for 2–3 weeks by FACS analysis every 3 days. Collected wells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies) and then with anti-human CD34 PE-Cy7 (581, BioLegend), CD38 Alexa Fluor 647 (AT1, Santa Cruz Biotechnologies), CD45RA BV 421 (HI100, BD Biosciences), and CD90 BV605 (5E10, BioLegend) and analysed by flow cytometry. For sorting of CD34+ or CD34+ CD38− CD90+ cells, cord-blood-derived CD34+ HSPCs were stained directly after isolation from blood with anti-human CD34 FITC (8G12, BD Biosciences), CD90 PE (5E10, BD Biosciences), CD38 APC (HIT2, BD Bioscience), and cells were sorted on a FACS Aria II (BD Bioscience), cultured overnight, and then electroporated with HBB RNP and transduced with HBB GFP rAAV6 using our optimized parameters. For assessing the allele modification frequencies in samples with targeted integration of the Glu6Val rAAV6 donor, PCR amplicons spanning the targeted region (see Extended Data Fig. 2a) were created using one primer outside the donor homology arm and one inside: HBB_outside 5′-GGTGACAATTTCTGCCAATCAGG-3′ and HBB_inside: 5′-GAATGGTAGCTGGATTGTAGCTGC-3′. The PCR product was gel-purified and re-amplified using a nested primer set (HBB_nested_fw: 5′-GAAGATATGCTTAGAACCGAGG-3′ and HBB_nested_rv: 5′-CCACATGCCCAGTTTCTATTGG-3′) to create a 685-bp PCR amplicon (see Extended Data Fig. 2a) that was gel-purified and cloned into a TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (Life Technologies) according to the manufacturer’s protocol. TOPO reactions were transformed into XL-1 Blue competent cells, plated on kanamycin-containing agar plates, and single colonies were sequenced by McLab by rolling circle amplification followed by sequencing using the following primer: 5′-GAAGATATGCTTAGAACCGAGG-3′. For each of the six unique CD34+ donors used in this experiment, 100 colonies were sequenced. Additionally, 100 colonies derived from an AAV-only sample were sequenced and detected no integration events. INDEL frequencies were quantified using the TIDE software47 (tracking of indels by decomposition) and sequenced PCR products obtained by PCR of genomic DNA extracted at least 4 days after electroporation as previously described14. The CFU assay was performed by FACS sorting of single cells into 96-well plates containing MethoCult Optimum (StemCell Technologies) 4 days after electroporation and transduction. After 12–16 days, colonies were counted and scored based on their morphological appearance in a blinded fashion. DNA was extracted from colonies formed in methylcellulose from FACS sorting of single cells into 96-well plates. In brief, PBS was added to wells with colonies, and the contents were mixed and transferred to a U-bottomed 96-well plate. Cells were pelleted by centrifugation at 300g for 5 min followed by a wash with PBS. Finally, cells were resuspended in 25 μl QuickExtract DNA Extraction Solution (Epicentre) and transferred to PCR plates, which were incubated at 65 °C for 10 min followed by 100 °C for 2 min. Integrated or non-integrated alleles were detected by PCR. For detecting HBB GFP integrations at the 3′ end, two different PCRs were set up to detect integrated (one primer in insert and one primer outside right homology arm) and non-integrated alleles (primer in each homology arm), respectively (see Extended Data Fig. 4a). HBB_int_fw: 5′-GTACCAGCACGCCTTCAAGACC-3′, HBB_int_rv: 5′-GATCCTGAGACTTCCACACTGATGC-3′, HBB_no_int_fw: 5′-GAAGATATGCTTAGAACCGAGG-3′, HBB_no_int_rv: 5′-CCACATGCCCAGTTTCTATTGG-3′. For detecting HBB tNGFR integrations at the 5′ end, a 3-primer PCR methodology was used to detect the integrated and non-integrated allele simultaneously (see Extended Data Fig. 4d). HBB_outside_5′Arm_fw: 5′-GAAGATATGCTTAGAACCGAGG-3′, SFFV_rev: 5′-ACCGCAGATATCCTGTTTGG-3′, HBB_inside_3′Arm_rev: 5′-CCACATGCCCAGTTTCTATTGG-3′. Note that for the primers assessing non-integrated alleles, the Cas9 cut site is at least 90 bp away from the primer-binding sites and since CRISPR/Cas9 generally introduces INDELs of small sizes, the primer-binding sites should only very rarely be disrupted by an INDEL. For in vivo studies, 6 to 8 week-old NSG mice were purchased from the Jackson laboratory (Bar Harbour). The experimental protocol was approved by Stanford University’s Administrative Panel on Laboratory Animal Care. For transplant data in Fig. 3a–c, sample sizes were not chosen to ensure adequate power to detect a pre-specified effect size. Four days after electroporation/transduction or directly after sorting, 500,000 cells (or 100,000–500,000 cells for the GFPhigh group) were administered by tail-vein injection into the mice after sub-lethal irradiation (200 cGy) using an insulin syringe with a 27 gauge × 0.5 inch (12.7 mm) needle. For transplant data in Fig. 3f, g, three days after electroporation, 400,000–700,000 bulk HSPCs or HSPCs enriched for targeting (FACS or bead-enrichment) were transplanted as described above. Mice were randomly assigned to each experimental group and evaluated in a blinded fashion. For secondary transplants, human cells from the RNP plus AAV group were pooled and CD34+ cells were selected using a CD34 bead enrichment kit (MACS CD34 MicroBead Kit UltraPure, human, Miltenyi Biotec), and finally cells were injected into the femurs of female secondary recipients (3 mice total). Because GFPhigh mice had low engraftment, they were not CD34+-selected, but total mononuclear cells were filtered, pooled, and finally injected into the femur of two secondary recipients. At week 16 after transplantation, mice were euthanized, mouse bones (2× femur, 2× tibia, 2× humerus, sternum, 2× pelvis, spine) were collected and crushed using mortar and pestle. MNCs were enriched using Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare) for 25 min at 2,000g, room temperature. Cells were blocked for nonspecific antibody binding (10% v/v, TruStain FcX, BioLegend) and stained (30 min, 4 °C, dark) with monoclonal anti-human CD45 V450 (HI30, BD Biosciences), CD19 APC (HIB19, BD Biosciences), CD33 PE (WM53, BD Biosciences), HLA-ABC APC-Cy7 (W6/32, BioLegend), anti-mouse CD45.1 PE-Cy7 (A20, eBioScience), anti-mouse PE-Cy5 mTer119 (TER-119, eBioscience) antibodies. Normal multi-lineage engraftment was defined by the presence of myeloid cells (CD33+) and B-cells (CD19+) within engrafted human CD45+ HLA-ABC+ cells. Parts of the mouse bone marrow were used for CD34-enrichment (MACS CD34 MicroBead Kit UltraPure, human, Miltenyi Biotec) and the presence of human HSPCs was assessed by staining with anti-human CD34 APC (8G12, BD Biosciences), CD38 PE-Cy7 (HB7, BD Biosciences), CD10 APC-Cy7 (HI10a, BioLegend), and anti-mouse CD45.1 PE-Cy5 (A20, eBioScience) and analysed by flow cytometry. The estimation of the total number of modified human cells in the bone marrow at week 16 after transplant was calculated by multiplying the percentage engraftment with the percentage GFP+ cells among engrafted cells. This number was multiplied by the total number of MNCs in the bone marrow of a NSG mouse (1.1 × 108 per mouse) to give the total number of GFP+ human cells in the total bone marrow of the transplanted mice. The total number of MNCs in the bone marrow of a NSG mouse was calculated by counting the total number of MNCs in one femur in four NSG mice. The total number of MNCs in one mouse was then calculated assuming one femur is 6.1% of the total marrow as found previously48. SCD patient-derived HSPCs were cultured in three phases following targeting at 37 °C and 5% CO in SFEM II media according to previously established protocols39, 40. Media was supplemented with 100 U ml−1 penicillin/streptomycin, 2 mM l-glutamine, 40 μg ml−1 lipids, 100 ng ml−1 SCF, 10 ng ml−1 IL-3 (PeproTech), 0.5 U ml−1 erythropoietin (eBiosciences), and 200 μg ml−1 transferrin (Sigma Aldrich). In the first phase, corresponding to days 0–7 (day 0 being day 4 after electroporation), cells were cultured at 105 cells ml−1. In the second phase, corresponding to days 7–11, cells were maintained at 105 cells ml−1 and erythropoietin was increased to 3 U ml−1. In the third and final phase, days 11–21, cells were cultured at 106 cells ml−1 with 3 U ml−1 of erythropoietin and 1 mg ml−1 of transferrin. Erythrocyte differentiation of edited and non-edited HSPCs was assessed by flow cytometry using the following antibodies: hCD45 V450 (HI30, BD Biosciences), CD34 FITC (8G12, BD Biosciences), CD71 PE-Cy7 (OKT9, Affymetrix), and CD235a PE (GPA) (GA-R2, BD Biosciences). RNA was extracted from 100,000–250,000 differentiated erythrocytes between days 16–21 of erythroid differentiation using the RNeasy Mini Kit (Qiagen) and was DNase-treated with RNase-Free DNase Set (Qiagen). cDNA was made from 100 ng RNA using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). Levels of HbS, HbA (from corrective SNP donor), and HbA-AS3 (anti-sickling HBB cDNA donor) were quantified by qPCR using the following primers and FAM/ZEN/IBFQ-labelled hydrolysis probes purchased as custom-designed PrimeTime qPCR Assays from IDT: HbS primer (fw): 5′-TCACTAGCAACCTCAAACAGAC-3′, HbS primer (rv): 5′-ATCCACGTTCACCTTGCC-3′, HbS probe: 5′-TAACGGCAGACTTCTCCACAGGAGTCA-3′, HbA primer (fw): 5′-TCACTAGCAACCTCAAACAGAC-3′, HbA primer (rv): 5′-ATCCACGTTCACCTTGCC-3′, HbA probe: 5′-TGACTGCGGATTTTTCCTCAGGAGTCA-3′, HbAS3 primer fw: 5′-GTGTATCCCTGGACACAAAGAT-3′, HbAS3 primer (rv): 5′-GGGCTTTGACTTTGGGATTTC-3′, HbAS3 probe: 5′-TTCGAAAGCTTCGGCGACCTCA-3′. Primers for HbA and HbS are identical, but probes differ by six nucleotides, and therefore it was experimentally confirmed that these two assays do not cross-react with targets. To normalize for RNA input, levels of the reference gene RPLP0 was determined in each sample using the IDT predesigned RPLP0 assay (Hs.PT.58.20222060). qPCR reactions were carried out on a LightCycler 480 II (Roche) using the SsoAdvanced Universal Probes Supermix (BioRad) following manufacturer’s protocol and PCR conditions of 10 min at 95 °C, 50 cycles of 15 s at 95 °C and 60 s at 58 °C. Relative mRNA levels were determined using the relative standard curve method, in which a standard curve for each gene was made from serial dilutions of the cDNA. The standard curve was used to calculate relative amounts of target mRNA in the samples relative to levels of RPLP0. The authors declare that the data supporting the findings of this study are available within the paper.
News Article | December 7, 2016
Wild-type male C57BL/6 mice and B6.129S4–PDGFRαtm11(EGFP)Sor/J mice (Jackson strain number 007669), which contain an H2B–eGFP fusion protein knocked into the Pdgfra locus, were obtained from Jackson Laboratories. Young adult mice were 6–8 weeks of age; aged mice were 22–24 months of age. Mice were housed and maintained in the Veterinary Medical Unit at the Veterans Affairs Palo Alto Health Care System. Animal protocols were performed in accordance with the policies of the Administrative Panel on Laboratory Animal Care of Stanford University. Mice were anaesthetized using isoflurane. To assess muscle regeneration, 50 μl of a 1.2% barium chloride (BaCl ) solution (Sigma-Aldrich) was injected into tibialis anterior muscles as described previously5. To isolate activated FAPs for western blot analysis and FACS analysis, 50 μl of 1.2% BaCl or 50% (v/v) glycerol/water was injected throughout the lower hindlimb muscles. For induction of fibrosis, 30 μl of 50% (v/v) glycerol or 30 μl 1.2% BaCl solution was injected into tibialis anterior muscles. Muscles were dissected from mice and dissociated mechanically. All hindlimb muscles were used except in experiments where FAPs were isolated from VMOs injected into tibialis anterior muscles. In this case, only the tibialis anterior muscle was dissected. The muscle suspension was digested using collagenase II (760 U ml−1; Worthington Biochemical Corporation) in Ham’s F10 medium (Invitrogen) with 10% horse serum (Invitrogen) for 90 min at 37 °C with agitation. The suspension was then washed and digested in collagenase II (152 U ml−1; Worthington Biochemical Corporation) and dispase (2 U ml−1; Invitrogen) for 30 min at 37 °C with agitation. The resultant mononuclear cells were then stained with the following antibodies: VCAM-1-biotin (clone 429; BioLegend, 105704), CD31-APC (clone MEC 13.3; BioLegend, 102510), CD45-APC (clone 30-F11; BioLegend, 103112) and Sca-1-Pacific Blue (clone D7; BioLegend, 108120) at 1:75. Streptavidin-PE-Cy7 (BioLegend, 405206) at 1:75 was used to amplify the VCAM-1 signal. FAPs were collected according to the following sorting criteria: CD31−CD45−Sca-1+. FACS was performed using BD-FACS Aria II and BD-FACS Aria III cell sorters equipped with 488 nm, 633 nm and 405 nm lasers. The cell sorters were carefully optimized for purity and viability and sorted cells were subjected to FACS analysis immediately after sorting to confirm FAP purity. FAPs were isolated from uninjured C57BL/6 mice as described above and lysed. RNA was prepared with the RNeasy Mini Kit as per the manufacturer’s instructions (Qiagen). A 3′ blocking reaction was performed using a poly(A) tailing kit (Ambion) and 3′-dATP (Jena Bioscience) and the reaction mixture was incubated at 37 °C for 30 min. RNA was hybridized to flow cell surfaces for direct RNA sequencing as previously described18. Raw direct RNA sequencing reads were filtered using the Helicos-developed pipeline, Helisphere, to eliminate reads less than 25 nucleotides long or of low quality. These reads were then mapped to the mouse genome (NCBI37/mm9) using an IndexDPgenomic module and reads with a score above 4.3 were allowed. To avoid artefacts from mispriming, reads mapping to regions in the genome where more than four consecutive adenines were coded immediately 3′ to the mapping sequence were excluded from further analysis. Reads were viewed using the Integrative Genomics Viewer32, 33. Total RNA was extracted from FAPs isolated from uninjured C57BL/6 mice using TRIzol (Invitrogen) as per the manufacturer’s instructions. To identify the polyadenylation sites, the sample was reverse transcribed using the SMARTer RACE cDNA amplification kit (Clontech) according to the manufacturer’s instructions using the primers listed in Extended Data Table 1. The amplified fragments were subcloned into pGEM-T-Easy (Promega) and sequenced. Sequencing data were visualized with 4Peaks. To assess levels of the intronic variant and UTR variants, primers were designed to span the Pdgfra transcript (Extended Data Table 2). Variant expression was normalized to Gapdh using the comparative C method27 and reported relative to the average of control-treated samples. A construct corresponding to In-PDGFRα (DNAFORM, AK035501, RIKEN clone 9530057A20) was obtained. This construct was subcloned into the pMXs-IRES-GFP retroviral backbone (Cell BioLabs, Inc.) to generate pMXs-I-Pα. Replication-incompetent retroviral particles were generated by transfection of the 293T human embryonic kidney cell-derived Phoenix helper cell line (gift from G. Nolan). Viral supernatant was filtered through 0.45-μm polyethersulfone filters, concentrated using PEG precipitation and stored at −80 °C. FAPs were plated in 6-well plates and grown in DMEM supplemented with 10% fetal bovine serum (FBS). When cells reached 70% confluency, viral supernatant and polybrene (at a final concentration of 4 μg ml−1) were added to the medium. For overexpression experiments, FAPs were incubated with the viral supernatant for 48 h before analysis. For signalling assays, FAPs were incubated with the viral supernatant for 24 h. Afterwards the medium was changed to serum-free DMEM containing viral supernatant and the cells were incubated for an additional 24 h. The FAPs were then treated with 1 ng ml−1 PDGF-AA for 15 min, after which the cells were used for western blot analysis. A peptide with the sequence GKSAHAHSGKYDLSVV, which represents the unique C-terminal region of In-PDGFRα protein, was generated (Thermo Scientific Pierce, OE0726). To generate In-PDGFRα rabbit polyclonal antibodies directed against In-PDGFRα, New Zealand white rabbits that were specific pathogen free were immunized with 0.25 mg of the peptide in Complete Freund’s Adjuvant. The rabbits received three boosters of antigen consisting of 0.10 mg in Incomplete Freund’s Adjuvant at days 14, 42 and 56 after immunization. Serum was collected at days 70 and 72 (Thermo Scientific Pierce). Cells and homogenized tissues were lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche). The lysates were run on Criterion SDS–PAGE gels (Bio-Rad), transferred to nitrocellulose membranes (Fisher Scientific), and analysed by western blot using the following rabbit antibodies: PDGFRα polyclonal (1:1,000, Cell Signaling, 3174), PDGFRα centre (1:100, Abgent, AP14254c), In-PDGFRα custom (1:1,000), pPDGFRαTyr754 polyclonal (1:1,000, Cell Signaling, 4547), Akt polyclonal (1:1,000, Cell Signaling, 9272), pAkt polyclonal (1:1,000, Cell Signaling, 9271), PLCγ polyclonal (1:1,000, Cell Signaling, 5690), pPLCγ polyclonal (1:1,000, Cell Signaling, 2821), ERK polyclonal (1:2,000, Cell Signaling, 4695), pERK polyclonal (1:2,000, Cell Signaling, 4370), SMAD2/3 monoclonal (1:1,000, Cell Signaling, 8685), and pSMAD2Ser465/Ser467/SMAD3Ser423/Ser425 monoclonal (1:1,000, Cell Signaling, 8828). Membranes were incubated in horseradish-peroxidase-labelled secondary antibodies and bands were visualized with enhanced chemiluminescence (Advansta). siRNAs were designed using the Dharmacon siDESIGN Center for knockdown of In-PDGFRα and FL-PDGFRα (Extended Data Table 2). To knockdown either In-PDGFRα or FL-PDGFRα in FAPs, approximately 8 × 104 cells were plated in a 12-well plate containing DMEM supplemented with 10% FBS and grown to 70–80% confluence. Cells were incubated in 200 nM of either PDGFRα or control siRNAs using Lipofectamine 2000 (Invitrogen). To assess knockdown, cells were collected at 24 h for qPCR analysis. For western blot analyses, 3 × 105 cells were plated in 6-well plates and incubated in Ham’s F10 medium (Invitrogen) supplemented with 10% horse serum (Invitrogen) for 24 h. The medium was then replaced with serum-free Ham’s F10 (Invitrogen) supplemented with 200 nM siRNA and incubated for an additional 24 h. Morpholinos were designed to target two polyadenylation sites on the intronic variant (pA : 5′-TGATTACATTATATCTGTCTTTATT-3′ and pA : 5′-AGCAAAGACCATCATAGCAGAATGA-3′) and the upstream 5′ splice site of the intron (5′ss: 5′-ATGGGCACTTTTACCTAGCATGGAT-3′) (Gene Tools, LLC). For in vitro treatment, cells were grown to 70–80% confluency in DMEM (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals). Cells were incubated in 10 μM of the indicated morpholino using the Endo-Porter transfection reagent (Gene Tools, LLC). Cells were collected at 24 h for qPCR analysis with RNA isolated using the RNeasy Plus Mini kit with on-column DNase digestion as per manufacturer’s instructions (Qiagen). For western blot analysis, cells were transfected for 24 h in Ham’s F10 medium (Invitrogen) supplemented with 10% horse serum (Invitrogen). The medium was then replaced with serum-free Ham’s F10 (Invitrogen) and incubated for an additional 24 h. For signalling assays, cells were then incubated for 15 min with PDGF-AA (Peprotech) at 0.1 ng ml−1 or 20 ng ml−1 for cells treated with pA-AMOs or 5′ss-AMO, respectively, and lysed for western blot analysis as described above. For AMO treatment, FAPs were isolated from the uninjured hindlimb muscles of C57BL/6 mice and seeded at 1 × 105 cells per well in poly-d-lysine-coated 8-well chamber slides (BD Biosciences) coated with ECM gel (Sigma-Aldrich). Cells were transfected with 10 μM AMO using Endoporter (Gene Tools) and expanded for 2 days in Ham’s F10 (Invitrogen) supplemented with 10% horse serum (Invitrogen). The medium was then replaced with Opti-MEM supplemented with 2 ng ml−1 PDGF-AA ligand and 10 μm EdU (Invitrogen). Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) after 24 h. For siRNA treatment, FAPs were isolated from the uninjured hindlimb muscles of C57BL/6 mice and seeded at 2 × 105 cells per well in poly-d-lysine coated 8-well chamber slides (BD Biosciences) coated with ECM gel (Sigma-Aldrich). The medium was supplemented with 200 nM siRNA and transfected using Lipofectamine 2000 (Invitrogen). After 24 h, the medium was replaced with Opti-Mem and the cells were re-transfected with 200 nM siRNA and 50 ng ml−1 PDGF-AA. In siRNA-treated samples, EdU was not included in this medium. Rather, after 20 h the medium was replaced with Opti-Mem containing 10 μm EdU (Invitrogen). Cells were fixed 4 h later. For retroviral overexpression of In-PDGFRα, FAPs were isolated from uninjured hindlimbs of C57BL/6 mice and seeded at 2 × 105 cells per well in poly-d-lysine coated 8-well chamber slides (BD Biosciences) coated with ECM gel (Sigma-Aldrich). FAPs were cultured in DMEM supplemented with 10% FBS along with viral supernatant and 4 μg ml−1 polybrene. After 24 h, the medium was replaced with serum-free DMEM containing viral supernatant and 20 ng ml−1 PDGF-AA. Twenty hours later, the medium was replaced with Opti-MEM containing 10 μM EdU. Cells were fixed after 4 h. For EdU incorporation experiments, cells were stained using the Click-iT EdU Imaging Kit (Invitrogen). Cells were analysed on a Zeiss Observer Z1 fluorescent microscope (Carl Zeiss) equipped with a Hamamatsu Orca-ER camera (Hamamatsu) and Improvision Volocity software (Perkin Elmer). Cells isolated by FACS from uninjured hindlimb muscles were seeded at a density of 3.5 × 104 cells per well in 96-well plates in Ham’s F10 medium supplemented with 2% horse serum. After 48 h, cells were nearly confluent and the medium was changed to Ham’s F10 with 2% horse serum and 20 ng ml−1 PDGF-AA. A wound was made by scratching a 200-μl pipette tip across the monolayer of cells. The initial scratch area was determined immediately and set to 100%. Images were taken at regular intervals and the scratch area at each time point was measured and calculated as a percentage of the initial scratch area. Scratch closure is defined as the inverse of the cell-free area as a percentage of total area. For in vitro microarray analysis, FAPs were isolated from the uninjured hindlimb muscles of C57BL/6 mice. Cells were plated at 1 × 106 cells per well in 12-well plates. Cells were grown for 2.5 days in DMEM supplemented with 10% FBS. The medium was switched to Ham’s F10 supplemented with 10% horse serum and transfected with 10 μM AMO as indicated for 48 h. The medium was then replaced with Opti-Mem and cells were re-transfected with 10 μM AMO. After 48 h, the cells were lysed and RNA was prepared with the RNeasy Mini Kit as per the manufacturer’s instructions (Qiagen). For in vivo microarray analysis, tibialis anterior muscles were injured with 30 μl of glycerol each and injected with the indicated VMO after 3 days. FAPs were then isolated from the muscles 2 days after VMO injection. Cells were pelleted and RNA prepared from samples as indicated above. The microarray data were obtained using Affymetrix Mouse 1.0 ST. For gene set enrichment analysis (GSEA), the samples were normalized and processed using GenePattern ExpressionFileCreator and PreProcessData set modules. Expression data were analysed and visualized with GSEA28 and GENE-E (http://www.broadinstitute.org/cancer/software/GENE-E/). For ingenuity pathway analysis, including causal network analysis, the samples were normalized using Affymetrix Expression Console Software and analysed for enrichment using IPA (Ingenuity Systems, http://www.ingenuity.com). Array data were deposited into Gene Expression Omnibus (Accessions GSE60099 and GSE81744). Vivo-morpholinos were designed to target two polyadenylation sites on the intronic variant (pA -VMO: 5′-TGATTACATTATATCTGTCTTTATT-3′ and pA -VMO: 5′-AGCAAAGACCATCATAGCAGAATGA-3′) and the upstream 5′ splice site of the intron (5′ss-VMO: 5′-ATGGGCACTTTTACCTAGCATGGAT-3′) (Gene Tools, LLC). For treatment in vitro, cells were isolated from hindlimb muscles of C57BL/6 mice and grown to 70–80% confluency in DMEM (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals). Cells were incubated in the 10 μM of the indicated morpholino (Gene Tools, LLC). Cells were collected at 24 h for qPCR analysis. For in vivo qPCR analysis, tibialis anterior muscles were injured with glycerol as described above and injected with 250 ng of the indicated VMO at the site of injury 3 days later. FAPs were sorted by FACS 7 days after VMO injection for qPCR analysis. For ex vivo proliferation and scratch assays, tibialis anterior muscles were injured with glycerol and injected with 250 ng of the indicated VMO 3 days after injury. FAPs were isolated 2 days later by FACS. In EdU incorporation studies, cells were seeded at 4 × 104 cells per well in poly-d-lysine-coated 8-well chamber slides (BD Biosciences) coated with ECM gel (Sigma-Aldrich). Cells were incubated in 10 ng ml−1 PDGF-AA (Peprotech) and 10 μM EdU (Invitrogen) for 24 h. The cells were fixed and stained. In the ex vivo proliferation studies as well as the in vivo proliferation studies described below, the proliferation index was used to denote the percentage EdU incorporation normalized to control. In the scratch assays, cells were seeded and treated as described above. For in vivo proliferation studies, tibialis anterior muscles were injected with 150 ng of the indicated VMO at 0 and 24 h. FAPs were isolated at 48 h via FACS. To assess in vivo proliferation, the cells were exposed to 10 μM EdU immediately after muscle isolation and incubated in 10 μM EdU ex vivo during the collagenase, collagenase/dispase, and antibody incubations as described above. The cells were plated in poly-d-lysine-coated 8-well chamber slides (BD Biosciences) coated with ECM gel (Sigma-Aldrich), fixed 1 h after plating, and stained using the Click-iT EdU Imaging Kit (Invitrogen). For histological analysis, tibialis anterior muscles were injured with glycerol or BaCl and injected at the site of injury with 250 ng of the indicated VMO. After 7 days, the muscles were snap frozen in isopentane cooled in liquid nitrogen immediately after dissection. Muscles sections were stained with Gomori-trichrome (Richard-Allan Scientific) per manufacturer’s instructions or oil red O (Sigma-Aldrich) as previously described29. The fibrotic index was calculated as the area of fibrosis divided by total area of muscle normalized to control-treated muscle. The fibro–adipose index was defined as the area of fibrosis plus the area of adiposis (as detected by oil red O staining) divided by total area of muscle, normalized to control. Major factors in determining sample size included the level of the effect and the inherent variability in measurements obtained. No statistical methods were used to predetermine sample size. Animals were excluded from the study only if their health status was compromised, such as occurred when animals had visible wounds from fighting. Samples were not specifically randomized or blinded. However, mouse identifiers were used when possible to blind evaluators to experimental conditions, and all samples within experiments were processed identically for measurement quantification using automated tools as specified. The sequencing data were deposited into the NCBI Sequence Read Archive (accession number SRP079186). Array data were deposited into Gene Expression Omnibus (accession numbers GSE60099 and GSE81744).
News Article | April 20, 2016
No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation during experiments and outcome assessment C57BL/6 (CD45.2) mice were purchased from Harlan Laboratories (Rehovot, Israel). B6.SJL (CD45.1) mice were bred in-house. Transgenic Ly6a(Sca-1)-EGFP mice and transgenic ROSA26-eYFP (EndoYFP) reporter mice were purchased from Jackson Laboratories. Transgenic nestin-GFP mice were kindly provided by G. N. Enikolopov (Cold Spring Harbour Laboratory, USA). Transgenic c-Kit-EGFP mice were kindly provided by S. Ottolenghi (University of Milano-Bicocca, Italy). Transgenic VE-cadherin (Cdh5, PAC)-CreERT2 mice were kindly provided by R. H. Adams (Max Planck Institute for Molecular Biomedicine, Germany). Conditional mutants carrying loxP-flanked Cxcr4 were provided by D. Scadden (Harvard University, Cambridge, USA). Conditional mutants carrying loxP-flanked Fgfr1 and Fgfr2 (Fgfr1/Fgfr2lox/lox) mice were provided by S. Werner (Institute of Cell Biology, Switzerland) and by D. Ornitz (Washington University School of Medicine, USA). To induce endothelial-specific Cre activity and gene inactivation/expression, adult VE-cadherin(Cdh5, PAC)-CreERT2 mice interbred with Cxcr4lox/lox (EndoΔCxcr4) or Fgfr1/2lox/lox (EndoΔFgfr1/2) or with ROSA26-eYFP mice (EndoYFP) were injected intraperitoneally (i.p.) with Tamoxifen (Sigma, T5648) at 1 mg per mouse per day for 5 days. Mice were allowed to recover for 4 weeks after tamoxifen injections, before euthanasia and experimental analysis. Mice carrying only VE-cadherin (Cdh5, PAC)-CreERT2 transgene or the Cxcr4lox/lox/Fgfr1/2lox/lox mutations were used as wild-typecontrols to exclude non-specific effects of Cre activation or of floxed alleles mutation. The endothelial Fgfr1/2 deletion was confirmed by qRT–PCR measurements of Cxcr4 and Fgfr1/2 mRNA from isolated BMECs. Male and female mice at 8–12 weeks of age were used for all experiments. All mouse offspring from all strains were routinely genotyped using standard PCR protocols. Sample size was limited by ethical considerations and background experience in stem cell transplantation (bone marrow transplantation) which exists in the laboratory for many years and other published manuscripts in the stem cell field, confirming a significant difference between means. No randomization or blinding was used to allocate experimental groups and no animals were excluded from analysis. All mutated or transgenic mouse strains had a C57BL/6 background. All experiments were done with approval from the Weizmann Institute Animal Care and Use Committee. Mice that were maintained at the Weizmann Institute of Science were bred under defined flora conditions. Two-photon in vivo microscopy procedures that were performed in Harvard Medical School were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. AMD3100 (Sigma-Aldrich) 5 mg per kg was used to treat mice by subcutaneous (s.c.) injection. Mice were euthanized 30 min later. Recombinant murine FGF-2 (ProSpec) 200 μg per kg was used to treat mice by i.p. injections for seven consecutive days. Neutralizing rat anti-VE-cadherin antibodies or rat IgG (eBioscience) at 50 μg per mouse per day were used to treat mice by intravenous (i.v.) injections for 2 or 5 days. Neutralizing mouse anti-CXCR4 antibodies (12G5 clone) or mouse IgG (eBioscience) at 50 μg per mouse were administered twice, with a 30 min interval, by intravenous (i.v.) injections. To inhibit ROS production, the antioxidant N-acetyl-l-cysteine (NAC; Sigma-Aldrich) was administered by i.p. injection of 130 mg per kg for 2, 5 or 7 days. Mice were euthanized 2–4 h following the final injection. For standard and confocal fluorescent microscopy, femurs were fixed for 2 h in 4% paraformaldehyde, which was replaced and then the samples were washed with 30% sucrose, embedded in optimum cutting temperature compound, and then snap-frozen in N-methylbutane chilled in liquid nitrogen. Sections (5–10 μm) were generated with a CM1850 Cryostat (Leica) at −25 °C with a tungsten carbide blade (Leica) and a CryoJane tape transfer system (Instrumedics), and were mounted on adhesive-coated slides (Leica), fixed in acetone and air-dried. Sections were stained by incubation overnight at 4 °C with primary antibodies, followed by 1 h incubation of secondary antibody at room temperature and in some cases also nuclei labelling by Hoechst 33342 (Molecular Probes) for 5 min at room temperature. Standard analysis (5–6 μm sections) was performed with Olympus BX51 microscope and Olympus DP71 camera. Confocal analysis (10 μm sections) was performed using a Zeiss LSM-710 microscope. In some cases, for BMBV morphological and phenotypical confocal analysis, femurs and tibias were fixed for 2 h in 4% paraformaldehyde, decalcified with 0.5 M EDTA at 4 °C with constant shaking, immersed into 20% sucrose and 2% polyvinylpyrrolidone (PVP) solution for 24 hours, then embedded and frozen in 8% gelatin (porcine) in presence of 20% sucrose and 2% PVP. Sections (80–300 μm) were generated using low-profile blades on a CM3050 cryostat (Leica). Bone sections were air-dried, permeabilized for 10 min in 0.3% Triton X-100, blocked in 5% donkey serum at room temperature for 30 min, and incubated overnight at 4 °C with primary antibodies. Confocal analysis was performed using a Zeiss LSM-780 microscope. Z-stacks of images were processed and 3D-reconstructed with Imaris software (version 7.00, Bitplane). As previously described4, tile scan images were produced by combining the signal of multiple planes along the Z-stalk of bone sections to allow visualization of the distinct types of bone marrow blood vessels and the cells in their surroundings. For the quantifications of blood vessel diameters, a region of 600–700 μm from the growth plate towards the caudal region was selected and diameters for arterial and sinusoidal blood vessels were calculated using ImageJ software on the high-resolution confocal images. Primary and secondary antibodies and relevant information about them are indicated in Supplementary Table 1. For in vivo ROS detection in bone marrow sections, mice were injected i.p. with hydroethidine (Life Technologies) 10 mg per kg, 30 min before euthanasia. For in vivo LDL-uptake detection in bone marrow sections, mice were i.v. injected with Dil-Ac-LDL (BTI) 20 μg per mouse, 4 h before euthanasia. Femurs were immediately collected and processed as mentioned earlier. Bone marrow section analysis for scoring ROShigh cells was performed using ImageJ software (Extended Data Fig. 1). Multiple sections (>16 per mouse) were generated and analysed from at least 4 mice per group of experimental procedure, in order to confirm biological repeats of the observed data. In some cases, images were processed to enhance the contrast in order to allow better evaluation of co-localization of cellular borders and markers. Imaris, Volocity (Perkin Elmer), Photoshop and Illustrator (Adobe) software were used for image processing. For blood vessel imaging in the calvarium of Sca-1-EGFP and nestin-GFP mice, we used a microscope (Ultima Multiphoton; Prairie Technologies) incorporating a pulsed laser (Mai Tai Ti-sapphire; Newport Corp.). A water-immersed 20× (NA 0.95) or 40× (NA 0.8) objective (Olympus) was used. The excitation wavelength was set at 850–910 nm. For intravital imaging, mice were anaesthetized with 100 mg ketamine, 15 mg xylazine and 2.5 mg acepromazine per kg. During imaging, mice were supplied with oxygen and their core temperature was maintained at 37 °C with a warmed plate. The hair on the skullcap was trimmed and further removed using urea-containing lotion and the scalp was incised at the midline. The skull was then exposed and a small steel plate with a cut-through hole was centred on the frontoparietal suture, glued to the skull using cyanoacrylate-based glue and bolted to the warmed plate. To visualize blood vessels, mice were injected i.v. with 2 μl of a 2 μM non-targeted nanoparticles solution (Qtracker 655, Molecular Probes). In some cases, mice were i.v. injected with Dil-Ac-LDL (BTI) 40 μg per mouse, 2 h before their imaging. We typically scanned a 50 μm-thick volume of tissue at 4 μm Z-steps. Movies and figures based on two-photon microscopy were produced using Volocity software (Perkin Elmer). For live imaging of blood vessels permeability and leukocyte bone marrow trafficking, a previously described experimental procedures and a home built laser-scanning multiphoton imaging system29, were used with some modifications. Anaesthesia was slowly induced in mice via inhalation of a mixture of 1.5–2% isoflurane and O . Once induced, the mixture was reduced to 1.35% isoflurane. By making a U-shaped incision on the scalp, calvarial bone was exposed for imaging and 2% methocellulose gel placed on it for refractive index matching. For bone marrow blood vessel permeability studies, mice were positioned in heated skull stabilization mount which allowed access to the eye for on-stage retro-orbital injection of 40–60 μl of 10 mg ml−1 70 kDa rhodamine-dextran (Life Technologies). Nestin-GFP (excited at 840 nm) and confocal reflectance (at 840 nm) signals were used to determine a region of interest within the mouse calvarial bone marrow for measurement of permeability. Rhodamine-dextran was injected and was continuously recorded (30 frames per second) for the first 10 min after injection. After video acquisition, mice were removed from the microscope and euthanasized with CO . In some cases, following dextran clearance, the same mice were used for homing experiments to monitor leukocyte cell trafficking in regions and blood vessels that were defined as less or more permeable. For cell homing studies, mice were injected with 2 × 106 DiD-labelled (Life Technologies) lineage depleted immature haematopoietic progenitor cells (Miltenyi depletion) and with 2 × 106 DiI-labelled (Life Technologies) bone marrow MNC isolated from age matched C57BL/6 mice along with 150 μl of 2 nmol per 100 μl Angiosense 750EX (Perkin Elmer) fluorescent blood pool imaging agent, immediately before mounting the mice on a heated stage of a separate confocal/multiphoton microscope. Intravital images of the mouse bone marrow were collected for up to the first 3 h after injection of the cells. After imaging, the mice were removed from the microscope and euthanized with CO . Permeability, blood flow/shear rates and homing experiments were repeated, n = 3 mice each, measuring multiple blood vessels and events, each mouse regarded as an independent experiment, in order to confirm biological repeats of the observed data. The contrast and brightness settings of the images in the figures were adjusted for display purposes only. For permeability studies, the RGB movies were separated into red (Rhodamine-Dextran), green (nestin-GFP), and blue (reflectance) grayscale image stacks. An image registration algorithm (Normalized Correlation Coefficient, Template Matching) was performed on the red stack using ImageJ (v. 1.47p) to minimize movement artefacts within the image stack. Manual selection of regions of interest (ROI) was performed immediately next to individual vessels within the focus. Permeability of the vessels was calculated using the following equation: P is the permeability of the vessel, V is the volume of the ROI next to the vessel, A is the fractional surface area of the vessel corresponding to the ROI, dI/dt is the intensity of the dye in the ROI as a function of time, I is the intensity of the dye inside the corresponding vessel at the beginning of measurement, and I is the intensity of the dye in the ROI at the beginning of measurement. To calculate dI/dt for a given vessel, the change in intensity was measured within the ROI over time and linearly fit the first ~5–40 s of the data. The slope of this linear fit is dI/dt. The ROI intensity curve is only linear for the first 30–40 s, after which it begins to plateau. For cell homing, the number of stationary cells from the calvarial bone marrow images was counted and categorized into two groups: adherent and extravasated. We categorized both cells within the lumen of the vessel and cells in the process of transmigration in the adherent category. Maximum intensity projections of multiple z-stacks of images were used to count the number of cells in the two categories. When there was a gap between cells and vessels in the two-dimensional projection image, those cells were categorized as extravasated. If any part of a cell overlapped a vessel in the projection image, the corresponding three dimensional z-stack was viewed to determine if the cell had undergone extravasation. When it was unclear if a cell had extravasated, it was always categorized as adherent. For the flow speed measurement, red blood cells (RBCs) were labelled with 15 μM CFSE for 12 min at 37 °C in PBS supplemented with 1 g per litre of glucose and 0.1% BSA. About 0.6 billion RBCs were injected (i.v). 40 μl of rhodamineB-dextran 70 kDa (10 mg ml−1) was retro-orbitally injected immediately before imaging for visualizing bone marrow vasculature. Videos of confocal images of blood vessel (RhodamineB, excitation: 561 nm, emission: 573–613 nm) and labelled RBCs (CFDA-SE, excitation: 491 nm, emission: 509–547 nm) were taken with the speed of 120 frames per second. Individual RBCs were traced over a couple of frames. Total displacement of the RBCs was measured by ImageJ and the speed of blood flow was calculated by: To calculate the shear rate, we assumed that the vessels were straight (straight cylinder) and the blood is an ideal Newtonian fluid with constant viscosity. Under these conditions, the shear rate (du/dr) can be calculated by du/dr = 8×u/d (u is the average blood flow speed which was measured by tracing labelled RBCs and d is the diameter of the blood vessel as measured using ImageJ). Immunostaining signal intensity was analysed with MacsQuant (Miltenyi, Germany) or with a FACS LSRII (BD Biosciences) with FACSDiva software, data were analysed with FlowJo (Tree Star). Data of the expression of molecules by cells was analysed and presented as MFI (mean fluorescent intensity). To acquire single bone marrow cell suspensions, freshly isolated bones were cleaned, flushed and crushed using liver digestion medium (LDM, Invitrogen) supplemented with 0.1% DNaseI (Roche) and further digested for 30 min at 37 °C, under shaking conditions. Following the incubation time, cells were filtered and washed extensively. To isolate and acquire mononuclear cells (MNC) from the peripheral blood PB, blood was collected from the heart using heparinized syringes and MNC were separated using Lymphoprep (Axis-Shield) according to the manufacturer’s instructions. Isolated bone marrow and peripheral blood MNC cells underwent red blood cell lysis (Sigma) before staining. Cells were stained for 30 min at 4 °C in standard flow cytometry buffer with primary antibodies and, where indicated, with secondary antibodies. Information about the primary and secondary antibodies can be found in the antibody information (Supplementary Table 1). For antigens that required intracellular staining, cell surface staining was followed by cell fixation and permeabilization with the Cytofix/Cytoperm kit following the manufacturer’s instructions (BD Biosciences). In case of internal GFP labelled cells, cells were fixed for 20 min with 4% PFA at room temperature, washed and incubated at room temperature for 1 h in 30% sucrose. Cells were washed with flow cytometry buffer and further permeabilized. For intracellular ROS detection, cells were incubated for 10 min at 37 °C with 2 μM hydroethidine (Life Technologies). For glucose uptake detection, cells were incubated for 30 min at 37 °C with the glucose analogue 2-NBDG (Life Technologies). For detection of apoptotic cells, cells were resuspended in annexinV binding buffer (BioLegend) and stained with Pacific Blue AnnexinV (BioLegend). Bone marrow cells were enriched for the lineage negative population, prepared as indicated for flow cytometry and analysed using an ImageStreamX (Amnis) machine. Samples were visualized and analysed for the expression of markers and antigens with IDEAS 4.0 software (Amnis). Single-stained control cells were used to compensate fluorescence between channel images. Cells were gated for single cells with the area and aspect ratio features or, for focused cells, with the Gradient RMS feature. Cells were then gated for the selection of positively stained cells only with their pixel intensity, as set by the cutoff with IgG and secondary antibody control staining. At least 5 samples from 5 mice were analysed to confirm biological repeats of observed data. Detection of mouse calcitonin (Cusabio) and mouse PTH (Cloud-Clone Corp.) levels in bone marrow supernatants was performed according to the manufacturer’s instructions. CFU-GM and CFU-F assays were previously described34. For CFU-Ob assay (also known as mineralized nodule formation assay), CFU-F medium was supplemented with 50 μg ml−1 ascorbic acid and with 10 mM β-glycerophosphate. After 3 weeks, cultures were washed, fixed and stained using Alizarin red for mineralized matrix. The area of mineralized nodules per cultured well was quantified based on image analysis using ImageJ. Bone marrow cells were isolated after sterile bone flushing, crushing and digestion (as previously described). After washing, total bone marrow cells were incubated in medium supplemented with or without 25% blood plasma or supplemented with 20 ng ml−1 TGF-β1 (ProSpec) for 2 h. Plasma was isolated and collected from the upper fraction acquired from the peripheral blood after 5 min centrifugation at 1,500 r.p.m. Bone marrow vascular endothelial barrier function was assessed using the Evans Blue Dye (EBD) assay. Evans Blue (Sigma-Aldrich) 20 mg per kg was injected i.v. 4 h before mice were euthanized. In each experiment, a non-injected mouse was used for control blank measurements. Subsequently, mice were perfused with PBS via the left ventricle to remove intravascular dye. Femurs were removed and formamide was used for bone flushing, crushing and chopping. EBD was extracted in formamide by incubation and shaking of flushed and crushed fractions, overnight at 60 °C. After 30 min centrifugation at 2,000g, EBD in bone marrow supernatants was quantitated by dual-wavelength spectrophotometric analysis at 620 nm and 740 nm. This method corrects the specimen’s absorbance at 620 nm for the absorbance of contaminating haem pigments, using the following formula: corrected absorbance at 620 nm = actual absorbance at 620 nm – (1.426(absorbance at 740) + 0.03). Samples were normalized by subtracting control measurements. Levels of EBD bone marrow penetration per femur were expressed as OD /femur and the fold change in EBD bone marrow penetration was calculated by dividing the controls OD /femur from the treated OD /femur in each experiment. Finally, values were normalized per total protein extract as determined by Bradford assay per sample. For competitive LTR assay, B6.SJL (CD45.1) recipient mice were lethally irradiated (1,000 cGy from a caesium source) and injected 5 h later with 2 × 105 donor-derived (C57BL/6 background, CD45.2) bone marrow cells or with 500 μl of donor-derived whole blood together with 4 × 105 recipient derived (CD45.1) bone marrow cells. Recipient mice were euthanized 24 weeks after transplantation to determine chimaerism levels using flow cytometry analysis. For calculation of competitive repopulating units (CRU), recipient mice were transplanted with limiting dilutions of donor derived bone marrow cells (2.5 × 104 to 2 × 105) together with 2 × 105 recipient derived bone marrow cells. Mice were euthanized after 24 weeks and multi-lineage myelo-lymphoid donor derived contribution in the peripheral blood was assessed using flow cytometry analysis. HSC-CRU frequency and statistical significance was determined using ELDA software (http://bioinf.wehi.edu.au/software/elda/). Lineage negative cells were enriched from total bone marrow cells, taken from c-Kit-EGFP mice, using mouse lineage depletion kit (BD) according to the manufacturer’s instructions. Non-irradiated recipient mice were transplanted by i.v. injection with 2 × 106 c-Kit-EGFP-labelled Lin− cells. Recipient mice were euthanized 4 h after transplantation. Bone marrow cells were isolated from femurs and stained for flow cytometry as described above. Femur cellularity was determined in order to calculate the number of homed CD34−/LSK HSPC per femur. For magnetic isolation of BMECs, freshly recovered bones were processed under sterile conditions as described for BMECs flow cytometry analysis, and post-digestion incubated with biotin rat anti-mouse CD31 antibodies (BD pharmigen) for 30 min at 4 °C. Next, cells were washed and incubated with streptavidin particles plus (BD IMag) for 30 min at 4 °C. Positive selection was performed using BD IMagnet (BD) according to the manufacturer’s instructions (BD Biosciences). BD IMag buffer (BD) was used for washing and for antibodies dilution. Isolated cells were seeded on fibronectin (Sigma-Aldrich) coated wells and cultured overnight in EBM-2 medium (Lonza) supplemented with EGM-2 SingleQuots (Lonza) at 37 °C 5% CO . Non-adhesive cells were removed and adherent cells were collected using accutase (eBioscience). Flow cytometry was applied to confirm endothelial identity and >90% purity of recovered cells. BMEC were further processed to isolate RNA. Total RNA was isolated using TRI-Reagent (Sigma-Aldrich) according to the manufacturer’s protocol. An aliquot of 2 μg of total RNA was reverse-transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, WI) and oligo-dT primers (Promega). Quantitative reverse transcribed–polymerase chain reaction (qRT–PCR) was done using the ABI 7000 machine (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix (Applied Biosystems). Comparative quantization of transcripts was assessed relative to hypoxanthine phosphoribosyl transferase (Hprt) levels and amplified with appropriate primers. Primer sequences used were as follows (mouse genes): Cxcr4 forward 5′- ACGGCTGTAGAGCGAGTGTT-3′; reverse 5′- AGGGTTCCTTGTTGGAGTCA-3′; Fgfr1 forward 5′-CAACCGTGTGACCAAAGTGG-3′; reverse 5′-TCCGACAGGTCCTTCTCCG-3′; Fgfr2 forward 5′-ATCCCCCTGCGGAGACA-3′; reverse 5′-GAGGACAGACGCGTTGTTATCC-3′; Hprt forward 5′-GCAGTACAGCCCCAAAATGG-3′; reverse 5′-GGTCCTTTTCACCAGCAAGCT-3′. All statistical analyses were conducted with Prism 4.0c version or Excel (*P < 0.05, **P < 0.01, ***P < 0.005; NS, not significant). All data are expressed as mean ± standard error (s.e.m) and all n numbers represent biological repeats. Unless indicated otherwise in figure legends, a Student’s two-tailed unpaired t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA was used to compare means among three or more independent groups. Bonferroni post-hoc tests were used to compare all pairs of treatment groups when the overall P value was <0.05. A normal distribution of the data was tested using the Kolmogorov–Smirnov test if the sample size allowed. If normal-distribution or equal-variance assumptions were not valid, statistical significance was evaluated using the Mann–Whitney test and the Wilcoxon signed rank test. Animals were randomly assigned to treatment groups. Tested samples were assayed in a blinded fashion.
News Article | November 7, 2016
This report studies sales (consumption) of ELISA Kits in United States market, focuses on the top players, with sales, price, revenue and market share for each player, covering Split by product types, with sales, revenue, price, market share and growth rate of each type, can be divided into Type I Type II Type III Split by applications, this report focuses on sales, market share and growth rate of ELISA Kits in each application, can be divided into Application 1 Application 2 Application 3 View Full Report With Complete TOC, List Of Figure and Table: http://globalqyresearch.com/united-states-elisa-kits-market-report-2016 United States ELISA Kits Market Report 2016 1 ELISA Kits Overview 1.1 Product Overview and Scope of ELISA Kits 1.2 Classification of ELISA Kits 1.2.1 Type I 1.2.2 Type II 1.2.3 Type III 1.3 Application of ELISA Kits 1.3.1 Application 1 1.3.2 Application 2 1.3.3 Application 3 1.4 United States Market Size Sales (Value) and Revenue (Volume) of ELISA Kits (2011-2021) 1.4.1 United States ELISA Kits Sales and Growth Rate (2011-2021) 1.4.2 United States ELISA Kits Revenue and Growth Rate (2011-2021) 5 United States ELISA Kits Manufacturers Profiles/Analysis 5.1 Thermo Fisher Scientific 5.1.1 Company Basic Information, Manufacturing Base and Competitors 5.1.2 ELISA Kits Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 5.1.3 Thermo Fisher Scientific ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.1.4 Main Business/Business Overview 5.2 Enzo Life Sciences 5.2.2 ELISA Kits Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 5.2.3 Enzo Life Sciences ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.2.4 Main Business/Business Overview 5.3 BioLegend 5.3.2 ELISA Kits Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 5.3.3 BioLegend ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.3.4 Main Business/Business Overview 5.4 Sigma-Aldrich 5.4.2 ELISA Kits Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 5.4.3 Sigma-Aldrich ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.4.4 Main Business/Business Overview 5.5 Aviva Systems Bio 5.5.2 ELISA Kits Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 5.5.3 Aviva Systems Bio ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.5.4 Main Business/Business Overview 5.6 Abnova 5.6.2 ELISA Kits Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 5.6.3 Abnova ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.6.4 Main Business/Business Overview 5.7 Repligen 5.7.2 ELISA Kits Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 5.7.3 Repligen ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.7.4 Main Business/Business Overview 5.8 LSBio 5.8.2 ELISA Kits Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 5.8.3 LSBio ELISA Kits Sales, Revenue, Price and Gross Margin (2011-2016) 5.8.4 Main Business/Business Overview Global QYResearch is the one spot destination for all your research needs. Global QYResearch holds the repository of quality research reports from numerous publishers across the globe. Our inventory of research reports caters to various industry verticals including Healthcare, Information and Communication Technology (ICT), Technology and Media, Chemicals, Materials, Energy, Heavy Industry, etc. With the complete information about the publishers and the industries they cater to for developing market research reports, we help our clients in making purchase decision by understanding their requirements and suggesting best possible collection matching their needs.
News Article | November 21, 2016
This report studies Monensin in Global market, especially in North America, Europe, China, Japan, Southeast Asia and India, focuses on top manufacturers in global market, with Production, price, revenue and market share for each manufacturer, covering CAYMAN CHEMICAL Elanco Bio Agri Mix BioLegend Ranch-Way Feed’s R&D Systems Enzo Biochem, Inc. Santa Cruz Biotechnology Cayman Chemical CEVA Hubbard Feeds SRL Hi-Pro Feeds Market Segment by Regions, this report splits Global into several key Regions, with production, consumption, revenue, market share and growth rate of Monensin in these regions, from 2011 to 2021 (forecast), like North America Europe China Japan Southeast Asia India Split by product type, with production, revenue, price, market share and growth rate of each type, can be divided into Type I Type II Type III Split by application, this report focuses on consumption, market share and growth rate of Monensin in each application, can be divided into Application 1 Application 2 Application 3 1 Monensin Market Overview 1.1 Product Overview and Scope of Monensin 1.2 Monensin Segment by Type 1.2.1 Global Production Market Share of Monensin by Type in 2015 1.2.2 Type I 1.2.3 Type II 1.2.4 Type III 1.3 Monensin Segment by Application 1.3.1 Monensin Consumption Market Share by Application in 2015 1.3.2 Application 1 1.3.3 Application 2 1.3.4 Application 3 1.4 Monensin Market by Region 1.4.1 North America Status and Prospect (2011-2021) 1.4.2 Europe Status and Prospect (2011-2021) 1.4.3 China Status and Prospect (2011-2021) 1.4.4 Japan Status and Prospect (2011-2021) 1.4.5 Southeast Asia Status and Prospect (2011-2021) 1.4.6 India Status and Prospect (2011-2021) 1.5 Global Market Size (Value) of Monensin (2011-2021) 2 Global Monensin Market Competition by Manufacturers 2.1 Global Monensin Production and Share by Manufacturers (2015 and 2016) 2.2 Global Monensin Revenue and Share by Manufacturers (2015 and 2016) 2.3 Global Monensin Average Price by Manufacturers (2015 and 2016) 2.4 Manufacturers Monensin Manufacturing Base Distribution, Sales Area and Product Type 2.5 Monensin Market Competitive Situation and Trends 2.5.1 Monensin Market Concentration Rate 2.5.2 Monensin Market Share of Top 3 and Top 5 Manufacturers 2.5.3 Mergers & Acquisitions, Expansion 3 Global Monensin Production, Revenue (Value) by Region (2011-2016) 3.1 Global Monensin Production and Market Share by Region (2011-2016) 3.2 Global Monensin Revenue (Value) and Market Share by Region (2011-2016) 3.3 Global Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.4 North America Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.5 Europe Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.6 China Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.7 Japan Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.8 Southeast Asia Monensin Production, Revenue, Price and Gross Margin (2011-2016) 3.9 India Monensin Production, Revenue, Price and Gross Margin (2011-2016) 4 Global Monensin Supply (Production), Consumption, Export, Import by Regions (2011-2016) 4.1 Global Monensin Consumption by Regions (2011-2016) 4.2 North America Monensin Production, Consumption, Export, Import by Regions (2011-2016) 4.3 Europe Monensin Production, Consumption, Export, Import by Regions (2011-2016) 4.4 China Monensin Production, Consumption, Export, Import by Regions (2011-2016) 4.5 Japan Monensin Production, Consumption, Export, Import by Regions (2011-2016) 4.6 Southeast Asia Monensin Production, Consumption, Export, Import by Regions (2011-2016) 4.7 India Monensin Production, Consumption, Export, Import by Regions (2011-2016) 5 Global Monensin Production, Revenue (Value), Price Trend by Type 5.1 Global Monensin Production and Market Share by Type (2011-2016) 5.2 Global Monensin Revenue and Market Share by Type (2011-2016) 5.3 Global Monensin Price by Type (2011-2016) 5.4 Global Monensin Production Growth by Type (2011-2016) 6 Global Monensin Market Analysis by Application 6.1 Global Monensin Consumption and Market Share by Application (2011-2016) 6.2 Global Monensin Consumption Growth Rate by Application (2011-2016) 6.3 Market Drivers and Opportunities 6.3.1 Potential Applications 6.3.2 Emerging Markets/Countries 7 Global Monensin Manufacturers Profiles/Analysis 7.1 CAYMAN CHEMICAL 7.1.1 Company Basic Information, Manufacturing Base and Its Competitors 7.1.2 Monensin Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 7.1.3 CAYMAN CHEMICAL Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.1.4 Main Business/Business Overview 7.2 Elanco 7.2.1 Company Basic Information, Manufacturing Base and Its Competitors 7.2.2 Monensin Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 7.2.3 Elanco Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.2.4 Main Business/Business Overview 7.3 Bio Agri Mix 7.3.1 Company Basic Information, Manufacturing Base and Its Competitors 7.3.2 Monensin Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 7.3.3 Bio Agri Mix Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.3.4 Main Business/Business Overview 7.4 BioLegend 7.4.1 Company Basic Information, Manufacturing Base and Its Competitors 7.4.2 Monensin Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 7.4.3 BioLegend Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.4.4 Main Business/Business Overview 7.5 Ranch-Way Feed’s 7.5.1 Company Basic Information, Manufacturing Base and Its Competitors 7.5.2 Monensin Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 7.5.3 Ranch-Way Feed’s Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.5.4 Main Business/Business Overview 7.6 R&D Systems 7.6.1 Company Basic Information, Manufacturing Base and Its Competitors 7.6.2 Monensin Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 7.6.3 R&D Systems Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.6.4 Main Business/Business Overview 7.7 Enzo Biochem, Inc. 7.7.1 Company Basic Information, Manufacturing Base and Its Competitors 7.7.2 Monensin Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 7.7.3 Enzo Biochem, Inc. Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.7.4 Main Business/Business Overview 7.8 Santa Cruz Biotechnology 7.8.1 Company Basic Information, Manufacturing Base and Its Competitors 7.8.2 Monensin Product Type, Application and Specification 22.214.171.124 Type I 126.96.36.199 Type II 7.8.3 Santa Cruz Biotechnology Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.8.4 Main Business/Business Overview 7.9 Cayman Chemical 7.9.1 Company Basic Information, Manufacturing Base and Its Competitors 7.9.2 Monensin Product Type, Application and Specification 188.8.131.52 Type I 184.108.40.206 Type II 7.9.3 Cayman Chemical Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.9.4 Main Business/Business Overview 7.10 CEVA 7.10.1 Company Basic Information, Manufacturing Base and Its Competitors 7.10.2 Monensin Product Type, Application and Specification 220.127.116.11 Type I 18.104.22.168 Type II 7.10.3 CEVA Monensin Production, Revenue, Price and Gross Margin (2015 and 2016) 7.10.4 Main Business/Business Overview 7.11 Hubbard Feeds 7.12 SRL 7.13 Hi-Pro Feeds 8 Monensin Manufacturing Cost Analysis 8.1 Monensin Key Raw Materials Analysis 8.1.1 Key Raw Materials 8.1.2 Price Trend of Key Raw Materials 8.1.3 Key Suppliers of Raw Materials 8.1.4 Market Concentration Rate of Raw Materials 8.2 Proportion of Manufacturing Cost Structure 8.2.1 Raw Materials 8.2.2 Labor Cost 8.2.3 Manufacturing Expenses 8.3 Manufacturing Process Analysis of Monensin 9 Industrial Chain, Sourcing Strategy and Downstream Buyers 9.1 Monensin Industrial Chain Analysis 9.2 Upstream Raw Materials Sourcing 9.3 Raw Materials Sources of Monensin Major Manufacturers in 2015 9.4 Downstream Buyers 12 Global Monensin Market Forecast (2016-2021) 12.1 Global Monensin Production, Revenue Forecast (2016-2021) 12.2 Global Monensin Production, Consumption Forecast by Regions (2016-2021) 12.3 Global Monensin Production Forecast by Type (2016-2021) 12.4 Global Monensin Consumption Forecast by Application (2016-2021) 12.5 Monensin Price Forecast (2016-2021)