News Article | May 10, 2017
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. Recombinant adenoviruses were constructed with the following inserts. Full-length mouse Dkk1 (ref. 6), and mouse Rspo1-Fc16 with full-length Rspo1 fused to a mouse antibody IgG2α Fc fragment at the C terminus have been described. Human RSPO2 and mouse Rnf43 ECD and Znrf3 ECD similarly contained full-length open reading frames with a C-terminal mouse IgG2α Fc fragment. Mouse Fzd8 CRD (residues 25–173) was cloned with an N-terminal haemagglutinin (HA) epitope tag and C-terminal IgG2α Fc fragment. In addition, a recombinant adenovirus was engineered to express human LGR5 ECD with both C-terminal FLAG and histidine tags. The construction of the adenoviruses encoding the scFv–DKK1c Wnt surrogate agonist and scFv–DKK1c–RSPO2 single-chain polypeptide fusion, each with a C-terminal His tag is described in a companion paper by Janda et al.25 On day 2 after intravenous injection, scFv–DKK1c was found to be expressed in vivo at ~10–20 μg ml−1 (280–560 nM) in mouse sera and the serum potently induced TOPflash activity in vitro. Full-length Wnt3a cDNA (a gift from R. Nusse) was cloned without any epitope tags and detected by western blotting with anti-WNT3A (Cell Signaling 2391) against a recombinant WNT3A protein. No detectable WNT3A protein was found in mouse sera after intravenous injection. All adenoviral constructs contained an N-terminal signal peptide sequence to allow for their secretion. These adenoviruses were cloned by homologous recombination into E1− E3− adenovirus strain 5, purified by double CsCl gradient, and titred as previously described33. Recombinant proteins were expressed in serum-free CD293 medium (Invitrogen) of HEK293 cells infected by adenovirus. Recombinant LGR5-ECD protein was purified by nickel-NTA affinity chromatography (Qiagen) from Ad-LGR5-ECD-infected CD293 medium. Likewise, recombinant RNF43 and ZNRF3 ECD-Fc fusion proteins were purified by protein A affinity chromatography (KPL) from Ad-Rnf43-ECD-infected or Ad-Znrf3-ECD-infected CD293 medium, respectively. Protein purity was verified by Coomassie-stained SDS–PAGE. Adult Lgr5-eGFP-IRES-creER mice7 (Jax) or Axin2-LacZ mice (Jax) between 8 and 12 weeks old were injected intravenously with adenoviruses (doses of 5 × 108 to 1 × 109 pfu per mouse). Lgr5-eGFP-IRES-creER mice were crossed with Rosa26-tdTomato mice to generate Lgr5-eGFP-IRES-creER; Rosa26-tdTomato compound heterozygous mice. Similarly, Villin-creER or Actin-creER mice were crossed to Rosa26-Rainbow mice to generate Villin-creER; Rosa26-Rainbow or Actin-creER; Rosa26-Rainbow compound heterozygous mice. Mice were dosed with adenoviruses as above, and serum expression of all ECDs was confirmed by immunoblotting and histological assessment of intestinal crypt hyperplasia for those treated with Ad-Rspo1 and Ad-RSPO2. Adult mice between 8 and 12 weeks of age were administered tamoxifen (Sigma) dosed at 4 mg per 40 g body weight to genetically label for lineage tracing experiments using the various Rosa26 reporter strains. All in vivo experiments used n = 3–5 mice per group and were repeated at least twice except for the RNA-seq studies. Both male and female mice were used. All animal experiments were conducted in accordance with procedures approved by the IACUC at Stanford University. FACS experiments were performed using fresh small intestine epithelial preparations. A standardized 3 cm segment of proximal jejunum was used for quantitative FACS analysis of ISC populations. Intestinal epithelial cells were extracted from en bloc resected small intestine with 10 mM EDTA and manual shaking, followed by enzymatic dissociation with collagenase/dispase (Roche) to generate a single-cell suspension. Singlet discrimination was sequentially performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H). Dead cells were excluded by scatter characteristics and viability stains. All FACS experiments were performed on an Aria II sorter (BD) or LSRII analyser (BD) at the Stanford University Shared FACS Facility and FACS data were analysed using FlowJo software (TreeStar). Intestinal tissue was collected and fixed in 4% paraformaldehyde. 8-μm OCT frozen sections or 5-μm paraffin-embedded sections were TUNEL-stained using the DeadEnd Fluorometric TUNEL system per manufacturer’s instructions (Promega) or immunostained using the following primary antibodies: anti-Ki67 (ThermoFisher RM-9106), anti-MUC2 (Santa Cruz sc-15334), anti-lysozyme (Dako A0099), anti-chromogranin A (Santa Cruz sc-1488), anti-FABP1 (Novus NBP1-87695), anti-CD44 (BD Pharmingen 550538), anti-cyclin D1 (Abcam ab134175) and anti-CD166 (R&D AF1172). All primary antibodies were used at 1:100 to 1:200 dilutions. Cy3- and Cy5-conjugated secondary antibodies (Santa Cruz and Jackson ImmunoResearch) were used at 1:500 to 1:1,000 dilutions. Alexa Fluor 594-conjugated phalloidin (Invitrogen) was used at 1:500. CD166 immunostained tissue sections34 were analysed and confocal images acquired as 0.5-μm planes using an IX81 Inverted Microscope equipped with Fluoview FV1000-Spinning Disc Confocal scan head and FV10 ASW 1.7 software (Olympus). All other images were captured on a Zeiss Axio-Imager Z1 with ApoTome or Leica SP5 confocal microscope. In situ hybridization for Olfm4 mRNA was performed using the RNAscope kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. In brief, 5 μm formalin-fixed, paraffin-embedded tissue sections or 8 μm OCT frozen sections were pre-treated with heat and protease before hybridization with a target probe to Olfm4 mRNA. A horseradish peroxidase (HRP)-based signal amplification system was then hybridized to the target probes followed by colorimetric development with DAB. Negative control probes for the bacterial gene DapB were also included for each slide. Adult Lgr5-eGFP-IRES-creER mice (Jax) between 10 and 12 weeks old were treated with intravenous adenovirus. After 48 h, these mice were treated by oral gavage for 4 days with twice daily dosing interval with either 50 mg kg−1 of PORCN inhibitor C59 (Cellagen Technology) or vehicle consisting of 0.5% methylcellulose plus 0.1% Tween80, as previously described35. Mice were euthanized 20 h after the last dose of C59 and the intestine was harvested for FACS and histological analysis. Small intestine tissue samples were fixed with 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide in 100 mM phosphate buffer. Tissue was dehydrated, embedded in epoxy resin, and visualized by a JEOL transmission electron microscope at 120 kV (model JEM-1210). L cells stably transfected with TOPflash dual reporter plasmid system (a gift from J. Chen) were used in TOPflash dual luciferase assays (Promega Dual Luciferase kit) with WNT3A conditioned medium from a stably transfected WNT3A-expressing cell line (a gift from R. Nusse) from which activation of the TOPflash reporter has been confirmed; mycoplasma contamination was not tested. Recombinant WNT3A (R&D) was alternatively used. Recombinant mouse RSPO1–RSPO4 proteins (R&D) were used at 5 pM concentration each in these assays. Recombinant LGR5, RNF43 and ZNRF3 ECD proteins were expressed and purified as above and their purity and protein concentrations were determined by Coomassie-stained SDS–PAGE and Bradford assays. Assays were visualized with a Tecan M1000 luminometer. Recombinant scFv–DKK1c was expressed and purified as described in the companion paper25. The kinetics and affinity of interactions between RSPO1–RSPO4 and Flag- and histidine-tagged LGR5 ECD, Fc-tagged RNF43 ECD or Fc-tagged ZNRF3 ECD were determined by surface plasmon resonance. Data were collected on the BIAcore T100 instrument (GE Healthcare). Approximately 1,000 resonance units (RU) of recombinant mouse RSPO1, RSPO2, RSPO3 or RSPO4 (R&D) were immobilized on a CM5 sensor chip (GE Healthcare) using standard amine coupling. Increasing concentrations of LGR5 ECD, RNF43 ECD or ZNRF3 ECD were passed over the chip in HBS supplemented with 0.005% surfactant P20 (HBS+P). Binding phases for the LGR5-ECD were performed at 50 μl min−1 for 240 s and dissociation phases were performed at 50 μl min−1 for 1,850 s. The chip was regenerated after each injection with 240-s washes with 0.5 M magnesium chloride. Binding and dissociation phases for RNF43 ECD and ZNRF3 ECD were each performed at 50 μl min−1 for 120 s. The chip was regenerated after each injection with 120-s washes with 1 M magnesium chloride. All curves were reference-subtracted from a flow cell containing 1,000 RU of a negative control protein (hen egg white lysozyme or BSA). Curves were fitted using the BIAcore T100 evaluation software to a 1:1 model to determine the association rate (k ), dissociation rate (k ) and dissociation constant (K ). The kinetics and affinity of anti-RSPO antibody interactions with RSPO1–RSPO4 were determined as described for RNF43 and ZNRF3, except that the regeneration buffer was 25% ethylene glycol and 2.25 M magnesium chloride. The kinetics and affinity of Fc-tagged RNF43 and ZNRF3 ECDs are enhanced by avidity effects due to Fc-dimerization. The furin 1 and 2 repeats of human RSPO2 were cloned into the pCT302 vector as a C-terminal fusion to a c-Myc epitope and the cell-wall protein AGA2. RSPO2 was displayed on the EBY100 strain of Saccharomyces cerevisiae as previously described36. Competent yeast cells were electroporated with the RSPO2 expression plasmid and recovered in SDCAA selection media. The cultures were harvested in log phase, and yeast cells were then pelleted and resuspended in SGCAA induction media. Surface expression of RSPO2 was detected by staining yeast with a 488-labelled antibody to the c-Myc epitope (Cell Signaling 279), and then analysed by flow cytometry. Binding of LGR5, RNF43 and ZNRF3 ECDs was tested by incubating yeast with 200 nM recombinant Flag-tagged LGR5 ECD or with Fc-tagged RNF43 ECD or ZNRF3 ECD in PBS and 0.1% BSA for 2 h, washing twice with PBS and 0.1% BSA and then incubating for 30 min with an Alexa Fluor 647-labelled antibody to the Flag epitope (Cell Signaling 3916S) (for LGR5 binding) or a PE-labelled anti-IgG antibody (eBioscience 12-4998-82). Cells were washed twice with PBS and 0.1% BSA and then analysed by flow cytometry. Sequential staining of yeast was performed by incubating samples with 200 nM LGR5-ECD, 200 nM RNF43 ECD, or 200 nM ZNRF3 ECD alone, washing and then incubating with a mixture of (200 nM LGR5-ECD and 200 nM RNF43-ECD) or (200 nM LGR5-ECD and 200 nM ZNRF3 ECD). Cells double-stained with both LGR5-ECD and either RNF43 or ZNRF3 ECD were then washed and incubated with a mixture of PE-anti-IgG and 647-anti-Flag before a final wash and analysis by flow cytometry. Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n = 2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. A 1.8× volume of AMPure beads (Beckman Coulter) was added to the thawed cell lysates. After a 30-min incubation at room temperature, the samples were washed twice with 70% ethanol and eluted in 22 μl water. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB buffer 1 (NEB) at 50 °C for 20 min, followed by a heat-inactivation step at 65 °C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37 °C for 30 min. The samples were cleaned with a 1.8× volume of AMPure XP beads (Beckman Coulter). 1 ng of purified total RNA, as determined by Agilent Bioanalyzer (Agilent Technologies), was processed with the mRNA direct micro kit (Life Technologies) to select for poly A RNA. Each entire sample was input into the Ambion WT Expression Kit (Life Technologies) to perform double-stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA. The cRNA was purified following the manufacturer’s instructions and the concentration was determined with a NanoDrop instrument (ThermoFisher). 1 μg of cRNA was fragmented in 1× fragmentation buffer (mRNA-Seq Sample Prep Kit, Illumina) at 94 °C for 5 min, then placed on ice and the reaction was stopped by the addition of 20 mM EDTA. The fragments were precipitated with 70 mM sodium acetate (Life Technologies), 40 μg glycogen (Life Technologies) and 70% ethanol at −80 °C for 1 h followed by centrifugation and washing with 70% ethanol. 3 μg of random hexamer (Life Technologies) was added to the fragmented, purified cRNA and incubated at 70 °C for 10 min to anneal the primer. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25 °C for 10 min, then 42 °C for 50 min, then 75 °C for 15 min and cooled to 4 °C. In second-strand synthesis, 1× second strand buffer (Illumina) and 0.3 mM dNTPs (Illumina) were added and the samples were incubated at 4 °C for 5 min before adding 50 U of DNA Polymerase (NEB) and 5 U of Rnase H (NEB). The samples were mixed well and incubated at 16 °C for 2.5 h, followed by purification with the MinElute Kit (Qiagen). To perform library prep, the samples were end repaired using a Quick Blunting Kit (NEB) and incubated at 20 °C for 1 h, then 75 °C for 30 min to inactivate the enzyme. To produce overhangs aimed to improve subsequent ligation efficiency, a single A base was added to the 3′ ends of each fragment with 2 mM dATP and 5 units of Klenow fragment 3′-5′ exo- DNA Polymerase (NEB) at 37 °C for 45 min, followed by 75 °C for 30 min to inactivate the enzyme. Using a quick ligase kit (NEB), 0.5 μM of adaptors containing single T base overhangs were ligated to the cDNA fragments at 12 °C for 75 min, then 80 °C for 20 min and cooled to 4 °C. These adaptors contain barcodes to facilitate sample multiplexing during sequencing. The adaptor sequence is preceded by four random nucleotides to add diversity to the pooled library. The samples were pooled by combining 5 μl of each library. After AMPure XP cleanup, one-half of the pooled library was run on the Pippin Size Selection Instrument (Sage Sciences) to select for 200 bp fragments. Library amplification was performed on one-half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 U Phusion Hot Start II Polymerase (Thermo Fisher Scientific). The reaction was run with the following program: 98 °C for 30 s, then 15 cycles of 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s, then 72 °C for 4 min and cooled to 4 °C. The amplified library was cleaned using a 1× volume of AMPure XP beads and QC was run with the Agilent Bioanalyzer DNA 1000 kit, followed by concentration determination by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). To perform sequencing, the library was diluted to 4 nM and denatured with 0.1 N NaOH. Following denaturation, the library was further diluted to 4 pM and run on the Illumina HiSeq 2500 in paired-end, 100 × 100 bp format. Sequenced reads were aligned to the mouse reference genome mm9 (UCSC) using TopHat37 with the transcript annotation supplied. The mapped reads was assigned to gene using the tool htseq-count of the Python package HTseq38, with the default union-counting mode. The output of htseq-count was used as input for DESeq2 (ref. 39) to perform differential expression analysis, with a false discovery rate (FDR) of 10% as the cutoff. In addition, a filtering criterion of mean fragments per kilobase of transcript per million mapped reads (FPKM) of 1 in at least one condition was used to define expressed transcripts in each differential expression analysis. Cufflinks40 was used to calculate gene count and perform FPKM normalization. Gene Ontology term analysis was performed using DAVID functional annotation tool41. A FDR of 10% was applied to evaluate the significance. Lgr5-eGFP-IRES-creER mice were treated with adenovirus in vivo, and then 26 h after treatment the proximal jejunum was harvested to generate a single-cell suspension and FACS isolated using the endogenous GFP signal, as above. The sorted cellular suspensions were loaded on a GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads in emulsion (GEMs). Approximately 1,200–2,800 cells were loaded per channel. Two technical replicates were generated per sorted cell suspension. Single-cell RNA-seq libraries were prepared using GemCode Single Cell 3′ Gel Bead and Library Kit (now sold as P/N 120230, 120231, 120232, 10x Genomics) as described previously29. Sequencing libraries were loaded at 2.1 pM on an Illumina Next-Seq500 with 2 × 75 paired-end kits using the following read length: 98 bp read1, 14 bp I7 index, 8 bp I5 index and 5 bp read2. Note that these libraries were generated before the official launch of GemCode Single Cell 3′ Gel Bead and Library Kit. Thus, 5 bp UMI was used (the official GemCode Single Cell 3′ Gel Bead contains 10 bp UMI). The Cell Ranger Single Cell Software Suite was used to perform sample de-multiplexing, barcode processing, and single-cell 3′ gene counting (http://software.10xgenomics.com/single-cell/overview/welcome). 5 bp UMI tags were extracted from read2. We analysed a total of 13,247 single cells, consisting of 11,268 FACS-sorted Lgr5–eGFP+and 1,979 Ad-Fc-treated Lgr5–eGFP− cells. Two technical replicates (the number of cells recovered per channel ranges from around 400 to 1,400 cells) were generated from each treatment condition. The mean raw reads per cell varied from ~45 k to 86 k. Each sample was downsampled to 28,439 confidently mapped reads per cell. Then the gene-cell barcode matrix from each sample was concatenated. The gene-cell barcode matrix was filtered based on number of genes detected per cell (any cells with less than 400 or more than 4,400 genes per cell were filtered) and percentage of mitochondrial UMI counts (any cells with more than 10% of mitochondrial UMI counts were filtered). Altogether, 13,176 cells, and 15,865 genes were kept for analysis by the Seurat R package30. Among these 13,176 cells, 74 did not show any epithelial cell markers so they were removed leaving a final total of 13,102 cells, consisting of 1,925 Ad-Fc-treated Lgr5–eGFP− cells and 11,177 Lgr5–eGFP+ cells across six conditions. 2,289 variable genes were selected based on their expression and dispersion (expression cutoff = 0.0125, and dispersion cutoff = 0.5). The first 11 principal components were used for the t-SNE projection and clustering analysis (resolution = 0.3, k.seed = 100). We applied sSeq from ref. 42 to identify genes that are enriched in a specific cluster (the specific cluster is assigned as group a, and the rest of clusters is assigned as group b). There are a few differences between our implementation and ref. 42. First, we used the ratio of total UMI counts and median of total UMI counts across all cells as the size factors. Second, the quantile rule of thumb was used to estimate the shrinkage target. Third, for genes with large counts, an asymptotic approximation from the edgeR package43 was used instead of the negative binomial exact test to speed up the computation. For the heatmap in Extended Data Fig. 9h, the gene list was furthered filtered requiring minimum UMI counts of 5 in each group, with a positive log fold change of mean expression between the two groups, and an adjusted P < 0.01. The top 10 genes specific to each cluster were picked, and their mean expression was centre scaled before used for the heatmap. Classification of cells was inferred from the annotation of cluster-specific genes. The stem cell clusters (clusters 0 and 1) were marked by enrichment of Lgr5, Olfm4 and Ascl2. Non-cycling and cycling stem cells were distinguished by the enrichment of cell cycle markers such as Mki67 and Tuba1b. Transit amplifying cells (cluster 2) were classified based on the enrichment of cell cycle markers and lack of Lgr5+ stem-cell marker expression. Enterocytes (clusters 3 and 4) were annotated based on the enrichment of markers such as Alpi and Reg1 and prior studies31. Goblet cells (cluster 5) were annotated based on the enrichment of markers such as Muc2 and Guca2a. Paneth cells (cluster 6) were annotated based on the enrichment of Defa genes. Tuft cells (cluster 7) were annotated based on the enrichment of markers such as Dclk1. EE cells (cluster 8) were annotated based on the enrichment of markers such as Chga and Chgb. To compare the global expression difference between samples and the Fc control, we first normalized gene expression by the sum of their UMI counts across all cells in the sample (adding 1 to the numerator and denominator to avoid dividing by 0 for genes that were not detected at all). Then we compared the normalized gene expression between the samples and the Fc control. To generate the heatmap, we furthered filtered the gene list: (1) Only genes with UMI counts >2 in each sample and a log fold change of >1 were considered. (2) The top 15 up- or downregulated genes were picked per sample–Fc comparison, and the union of all genes was used for the heatmap. Data generated during this study are available in the Gene Expression Omnibus (GEO) repository under accession numbers GSE92377 and GSE92865. All other data are available from the corresponding author upon reasonable request.
News Article | May 10, 2017
No statistical methods were used to predetermine sample size. All cells were tested for mycoplasma and included for analysis only upon testing negative. The identity of all cell lines was confirmed by whole-exome sequencing and SNP array analysis. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. As a source of hES cells for this study, we focused on those that had been voluntarily listed by research institutions on the registry of hES cell lines maintained by the US National Institutes of Health (NIH) (http://grants.nih.gov/stem_cells/registry/current.htm). As of 8 July 2015, a total of 307 hES cell lines were listed on this registry. Of these, we requested viable frozen stocks of the 182 lines annotated to be available for distribution and to lack known karyotypic abnormalities or disease-causing mutations. During our effort to obtain these cell lines, we found that 45 were subject to overly restrictive material transfer agreements that precluded their use in our studies and 11 could not be readily obtained as frozen stocks owing to differences in human subjects research regulations between the US and the UK. Nine cell lines were unavailable upon request or were overly difficult to import, and three could not be cultured despite repeated attempts. Further details on the availability of cell lines can be found in Supplementary Table 1. The generation of hES cells used in this study was previously approved by the institutional review boards (IRBs) of all providing institutions. Use of the hES cells for sequencing at Harvard was further approved and determined not to constitute Human Subjects Research by the Committee on the Use of Human Subjects in Research at Harvard University. A protocol for the adaptation of hES cell lines from diverse culture conditions can be found at Protocol Exchange30. In brief, we considered that different laboratories employ different methods to culture hES cells, raising the question of how best to thaw and culture the cell lines we obtained from multiple sources. Traditionally, hES cells are maintained on gelatinized plates and co-cultured with replication-incompetent mouse embryonic fibroblast (MEF) feeder cells in tissue culture medium containing knockout serum replacement (KOSR). More recently, hES cells have been cultured on a substrate of cell-line-derived basal membrane proteins known by the trade names of Matrigel (BD Biosciences) or Geltrex (Life Technologies), in mTeSR1 (ref. 31), E8 (ref. 32) or similar in the absence of feeder cells. In previous work, we found that a medium containing an equal volume of KOSR-based hES cell medium (KSR) and mTeSR1 (STEMCELL Technologies) (KSR–mTeSR1) robustly supports the pluripotency of hES cells undergoing antibiotic selection during the course of gene-targeting experiments under feeder-free conditions33. To minimize stress to hES cells previously cultured and frozen under diverse conditions, cell lines were thawed in the presence of 10 μM Y-27632 (DNSK International) into two wells of a 6-well plate, one of which contained KSR–mTeSR1 on a substrate of Matrigel, and the other containing KOSR-based hES cell medium on a monolayer of irradiated MEFs. After 24 h, Y-27632 was removed and cells were fed daily with the aforementioned media in the absence of any antibiotics. All cultures were tested for the presence of mycoplasma and cultured in a humidified 37 °C tissue culture incubator in the presence of 5% CO and 20% O . Colonies of cells with hES cell morphology and with a diameter of approximately 400 μm were transferred into KSR–mTeSR1 medium containing 10 μM Y-27632 on a substrate of Matrigel by manual picking under a dissecting microscope. Cells with differentiated morphology were removed from plates by aspiration during feeding. Once cultures consisting of cells with homogeneous pluripotent stem cell morphology had been established, they were passaged by brief (2–10 min) incubation in 0.5 mM EDTA in PBS followed by gentle trituration in KSR–mTeSR1 medium containing 10 μM Y-27632 and re-plating. Once cultures had reached approximately 90% confluence in one well of a six-well plate, they were passaged with ETDA onto a Matrigel-coated 10 cm plate. Upon reaching approximately 90% confluence, cell lines were dissociated with EDTA as described above and banked for later use in cryoprotective medium containing 50% KSR–mTeSR1, 10 μM Y-27632, 10% DMSO, and 40% fetal bovine serum (HyClone). A subset of hES cell lines (Supplementary Table 1) were passaged enzymatically with TrypLE Express (Life Technologies), expanded onto two 15 cm plates, and frozen down in 25 cryovials. Cell pellets of approximately 1–5 million cells were generated from banked cryovials of research-grade hES cell lines, or were obtained directly from institutions providing GMP-grade hES cell lines. Cell pellets were digested overnight at 50 °C in 500 μl lysis buffer containing 100 μg ml−1 proteinase K (Roche), 10 mM Tris (pH 8.0), 200 mM NaCl, 5% w/v SDS, 10 mM EDTA, followed by phenol:chloroform precipitation, ethanol washes, and resuspension in 10 mM Tris buffer (pH 8.0). Genomic DNA was then transferred to the Genomics Platform at the Broad Institute of MIT and Harvard for Illumina Nextera library preparation, quality control, and sequencing on the Illumina HiSeq X10 platform. Sequencing reads (150 bp, paired-end) were aligned to the hg19 reference genome using the BWA alignment program. Genotypes from WES data for the cell lines were computed using best practices from GATK software34 compiled on 31 July 2015. Sequencing quality and coverage were analysed using Picard tool metrics. Cross sample contamination was estimated using VerifyBamID (v1.1.2)35, and none was detected. Data from each cell line were independently processed with the HaplotypeCaller walker and further aggregated with the CombineGVCFs and GenotypeGVCFs walkers to generate a combined variant call format (VCF) file. Genotyped sites were finally filtered using the ApplyRecalibration walker. To determine whether lines with or without acquired TP53 mutations showed other chromosomal aberrations or smaller regional changes in copy number, additional genotyping of the 140 hES cell lines was performed using a custom high density SNP array (‘Human Psych array’) that contains more than half a million SNPs across the genome. CNVs larger than 500 kb were identified using the PennCNV (v1.0.0)36 tool (http://penncnv.openbioinformatics.org). All CNVs were manually reviewed and are shown in Supplementary Table 6. To identify candidate mosaic variants, a table of heterozygous variants was generated from the VCF (Supplementary Table 2). To limit the frequency of false positive calls due to sequencing artefacts and PCR errors, variants were included if they had a variant read depth of at least 10, if they were either flagged as a ‘PASS’ site or were not reported in the Exome Aggregation Consortium (ExAC) database11, and if they were not located in regions of the genome with low sequence complexity, common large insertions and segmental duplications, as described by Genovese and colleagues5. Multiallelic sites were split, left-aligned, and normalized. The resulting list of 2.1 million ‘high-quality heterozygous variants’ was further refined to include sites that were covered by at least 60 unique reads and had a high confidence variant score (‘PASS’) as ascertained by GATK’s Variant Quality Score Recalibration software (840,222 variants). To exclude common inherited variants, we selected variants present in less than 0.01% of the (ExAC) control population and restricted our analysis to only singleton or doubleton variants (9,490 variants present in 1–2 of the 140 samples). Coverage was calculated by summing reference and alternate allele counts for each variant. Allelic fraction was calculated by dividing the alternate allele count by the total read coverage (both alleles) of the site. Although the allelic fraction of inherited heterozygous variants is expected to be 50%, reference capture bias (a tendency of hybrid selection to capture the reference allele more efficiently than alternative alleles) causes the actual expected allele fraction for SNPs and indels to be closer to 45% and 35%, respectively5. To account for these technical biases, we used a binomial test with a null model centred at 45% allelic fraction for inherited SNPs and 35% for inherited indels. Variants for which this binomial test was nominally significant (P < 0.01) were deemed to be candidate mosaic variants. The nominal P-value threshold of 0.01 was chosen as an inclusive threshold in order to screen sensitively for potentially mosaic variants, at the expense of also capturing false positives for which low allelic fractions represented statistical sampling fluctuations. For this reason, we considered it important to further evaluate putative mosaic variants by independent molecular methods that deeply sample alleles at the nominated sites (Fig. 3). A more stringent computational screen based on a P-value threshold of 1 × 10−7 identified three of the six TP53 variants, and TP53 was also the only gene with multiple putatively mosaic variants in this screen. We also identified all high quality heterozygous variants that passed the inclusive statistical threshold of (P < 0.01) in our binomial test and could potentially be mosaic (n = 36,396). These data are included in Supplementary Table 2. Variant annotation was performed using SnpEff with GRCh37.75 Ensembl gene models. Variants with moderate effect were classified as damaging by a consensus model based on seven in silico prediction algorithms37. We turned to the ExAC database11 that compiles the whole-exome sequences of over 60,000 individuals to assess the frequency at which the amino acid residues we observed to be mutated in some hES cells were affected in the general population. We then consulted the COSMIC12 (http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=TP53), ICGC13 (https://dcc.icgc.org/), and IARC P53 (ref. 14) (http://p53.iarc.fr/TP53SomaticMutations.aspx) databases and plotted the percentage of tumours carrying a mutation in each codon (Fig. 2d, Extended Data Fig. 2b). To visualize the spatial location of the amino acid residues affected by TP53 mutations observed in hES cells by WES on the P53 protein, we downloaded the 1.85 Angstrom X-ray diffraction-based structure file from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (file 2AHI) and built the model protein/DNA system (chain IDs D, G, and H) to visualize the secondary structure of a P53 monomer complexed to DNA as a ribbon diagram. DNA was illustrated as a space-filling model. Water molecules were discarded when building the wild-type model and minimized in two steps using the AMBER 16 package38. Affected residues were indicated as space-filling model superimposed on the ribbon diagram of P53 and highlighted in blue (wild-type) or red (mutated) without consideration of how the mutations might affect the secondary or tertiary structure of the protein. We assayed the allelic fraction of the four distinct TP53 mutations identified by WES (Supplementary Table 3) in the 140 hES cell lines by droplet digital PCR (ddPCR). Each ddPCR analysis incorporated a custom TaqMan assay (IDT). Assays were designed with Primer3Plus and consisted of a primer pair and a 5′ fluorescently labelled probe (HEX or FAM) with 3′ quencher (Iowa Black with Zen) for either the control (reference) or mutant (alternative) base for each identified P53 variant (Supplementary Table 4). Genomic DNA from each hES cell line was analysed by ddPCR according to the manufacturer’s protocol (BioRad). The frequency of each allele for a given sample was estimated first by Poisson correction of the endpoint fluorescence reads21. These corrected counts were then converted to fractional abundance estimates of the mutant allele and multiplied by two to determine the fraction of cells carrying the variant allele. To assess how the allelic fraction of TP53 mutations might change over time in culture, hES cell lines CHB11 (passage 22 or 25), WA26 (passage 13 or 15), and ESI035 (passage 36 in two separate experiments) were serially passaged in mTeSR1 media (STEMCELL Technologies) at a density of approximately 30,000 cells cm−2 in the presence of 10 μM Y-27632 on the day of passaging. Cells were fed daily with mTeSR1 and passaged with Accutase (Innovative Cell Technologies Inc.) at approximately 90% confluence. To monitor changes in allelic fractions, genomic DNA from cells at the indicated passages were analysed by ddPCR. To calculate the relative expansion rate of mutant relative to wild-type cells, we applied the following formula: where R is defined as the ratio of (variant positive cells)/(variant negative cells) after some number of starting passages and R and R represent the aforementioned ratios measured on the same sample at T and T > T passages respectively. From this equation, the estimation of variant positive cells after T passages from starting ratio R can be defined as R egT. Note that this equation estimates the relative growth rate of cells carrying the variant allele with a round of passaging as unit of time, with both relative survival and growth being incorporated. These data are included in Supplementary Table 5. For the subsequent calculation of the earliest passage at which these mutations might have become detectable, the detection thresholds (R ) for WES and ddPCR was assumed to be 0.1 (10 / 100 reads) and 0.001 (1 per 1,000 droplets), respectively. In order to identify TP53 mutations in hPS cells, we analysed 256 publicly available high-throughput RNA sequencing samples of hPS cells from the SRA database39 (http://www.ncbi.nlm.nih.gov/sra). Data accession numbers for SRA (and GEO, where applicable) are provided in Supplementary Table 7. 5 of these 256 samples were not considered further as they were from single cells rather than cell lines. Following sequence alignment to the hg19 human reference genome with Tophat2 (ref. 40), single nucleotides divergent from the reference genome were identified using GATK HaplotypeCaller34. As sufficient sequencing depth is required to deduce sequence mutation, a threshold of 25 reads per nucleotide was set. Under this criterion, 43 samples (40 hES cell lines and 3 hiPS cell lines) had a missense mutation in TP53. 10 of the 40 hES cell samples (WA09) carried two separate mutations (Supplementary Table 7). Upon the identification of cell lines carrying mutant reads, RNA sequencing data from studies containing differentiated samples were included for analysis. In order to evaluate TP53 alleles, we assessed the level of polymorphism by calculating the ratio between the minor and major alleles across chromosome 17. So as to minimize sequencing noise and errors, we included SNPs covered by more than 10 reads and that are located in the dbSNP build 142 database41. The resulted wig files were then plotted using Integrative Genomics Viewer (IGV)42 (Extended Data Fig. 4). In order to quantify the difference in polymorphism between samples, we converted the wig files to BigWig using UCSC Genome Browser utilities43 and summed the allelic ratios between the distal part of the short arm of chromosome 17 (17p), the proximal side of this arm and the long arm of chromosome 17 (17q). The allelic ratio sum was then divided by the region’s length (bp), which resulted in the proportion of SNPs, followed by one-sided Z-score test for two population proportion to compare between the chromosome 17 areas within each sample. Whereas most samples with mutations in TP53 showed a comparable, non-significant rate of polymorphic sites along the chromosome, WIBR3 samples with H193R mutations and WA09 samples with both P151S and R248Q mutations had a significantly different proportion (P < 0.001) of polymorphic sites, in the distal part of the short arm of the chromosome (first 16 × 106 base pairs), including the TP53 site. Unlike the three mutant WIBR3 samples, the wild-type WIBR3 sample had a normal distribution of polymorphic sites with no significant difference between the short and long arms. Sequence data from cell lines listed on the NIH hES cell registry have been deposited in the NCBI database of Genotypes and Phenotypes (dbGaP) under accession number phys001343.v1.p1 (at https://www.ncbi.nlm.nih.gov/gap/?term=phys001343.v1.p1). Sequence data from the remaining cell lines reported in our study have been deposited at the European Genome-phenome Archive (EGA), which is hosted by the EBI and the CRG, under accession number EGAS00001002400 (at https://www.ebi.ac.uk/ega/search/site/EGAS00001002400).
News Article | May 17, 2017
The Cdh5(PAC)-CreERT2 transgenic mice (iECre) were a gift from R. H. Adams39. Krit1fl/fl and Ccm2fl/fl animals have been previously described40, 41. Tlr4fl/fl, Cd14−/−, Ai14 (R26-LSL-RFP), and R26-CreERT2 animals42, 43, 44, 45 were obtained from the Jackson Laboratories. The Slco1c1(BAC)-CreERT2 transgenic mice have been previously described18. All experimental animals were maintained on a mixed 129/SvJ, C57BL/6J, DBA/2J genetic background unless specifically described. C57BL/6J and timed pregnant Swiss Webster mice were purchased from Charles River Laboratories. Germ-free Swiss Webster mice were purchased from Taconic. Breeding pairs between two and ten months of age were used to generate the neonatal CCM mouse model pups. Mice were housed in a specific pathogen-free facility where cages were changed on a weekly basis; ventilated cages, bedding, food, and acidified water (pH 2.5–3.0) were autoclaved before use, ambient temperature maintained at 23 °C, and 5% Clidox-S was used as a disinfectant. Experimental breeding cages were randomly housed on three different racks in the vivarium, and all cages were kept on automatic 12-h light/dark cycles. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. A group of the resistant Ccm2ECKO colony was exported to the Centenary Institute, Sydney, Australia, where the mice were permanently maintained as an inbred colony in a quarantine facility. After several generations, this colony uniformly converted to lesion susceptibility. Cages were changed on a weekly basis; ventilated cages, bedding, food and acidified water (pH 2.5–3.0) were autoclaved before use. Ambient temperature was maintained between 22–26 °C, and 80% ethanol and F10SC (1:125 dilution of the concentrate, a quaternary ammonium compound) were used as disinfectants. Experimental breeding cages were randomly distributed throughout the vivarium, and all cages were kept on 12-h light/dark cycles. The Sydney Local Health District Animal Welfare Committee approved all animal ethics and protocols. All experiments were conducted under the guidelines/regulations of Centenary Institute and the University of Sydney. Germ-free Swiss Webster mice were purchased from Taconic and directly transferred into sterile isolators (Class Biologically Clean Ltd) under the care of the Penn Gnotobiotic Mouse Facility. Food, bedding and water (non-acidified) were autoclaved before transfer into the sterile isolators. Ventilated cages were changed weekly, and all cages in the vivarium were kept under 12-h light/dark cycles. Microbiology testing (aerobic and anaerobic culture, 16S qPCR) was performed every ten days and faecal samples were sent to Charles Rivers Laboratories for pathology testing on a quarterly basis. Further details regarding the sterile C-section fostering can be found below. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. For all neonatal CCM mouse model experiments, at one day post-birth (P1), pups were intragastrically injected by 30-gauge needle with 40 μg of 4-hydroxytamoxifen (4OHT, Sigma Aldrich, H7904) dissolved in a 9% ethanol/corn oil (volume/volume) vehicle (50 μl total volume per injection). This solution was freshly prepared from pre-measured, 4OHT powder for every injection. Before injection, the pup skin was sanitized using ethanol wipes. The P1 time point was defined by checking experimental breeding pairs every evening for new litters. The following morning (P1), pups were injected with 4OHT. All experimental pups were subjected to this induction regimen. For the Tlr4 rescue experiment (Fig. 2), and all lineage-tracing experiments, an additional dose of 40 μg 4OHT was intragastrically delivered at P2 (P1+2, two total doses). Pups were then harvested as previously described9 at the specified time points. Tissue samples were fixed in 4% formaldehyde overnight, dehydrated in 100% ethanol, and embedded in paraffin. 5-μm-thick sections were used for haematoxylin and eosin (H&E) and immunohistochemistry staining. The following antibodies were used for immunostaining: rat anti-PECAM (1:20, Histo Bio Tech DIA-310), rabbit anti-pMLC2 (1:200, Cell Signaling 3674S), goat anti-KLF4 (1:100, R&D AF3158), and rabbit anti-RFP (1:50, Rockland 600-401-379). Littermate control and experimental animal sections were placed on the same slide and immunostained at the same time under identical conditions. Images were taken at the same time using the same exposure times and colour channels, and were subsequently overlaid using ImageJ. Intra-abdominal abscesses were dissected and triturated in 500 μl of SOC medium. Drops of the mixture were placed on a microscope slide, briefly exposed to heat, and Gram staining was performed using a kit from Sigma Aldrich (77730) following the manufacturer’s protocol. Eyes from euthanized P17 mice were removed and fixed overnight in cold 4% PFA/PBS solution. The following day, retinas were dissected, cut into petals, and stained with isolectin-B4 conjugated to Alexa488 fluorophore (Thermo Fisher I21411) as previously described46. The retinas were then whole-mounted on microscopy slides in a flat, four-petal shape for fluorescence imaging. B. fragilis was purchased directly from the ATCC (strain 25285) and grown in chopped meat glucose (CMG) broth (Anaerobe Systems AS-813) under anaerobic conditions at 37 °C. Autoclaved, degassed caecal contents (ACC) were generated by collecting caecal contents from the colons of euthanized adult mice between 2–8 months of age. Caecal contents were then autoclaved and pulverized in an equal volume of CMG broth. This slurry was filtered through a 70-μm nylon strainer and degassed overnight in the anaerobic chamber. 1 ml of CMG broth was inoculated with B. fragilis and grown overnight to an optical density of between 0.8 and 1.0. An equal volume of ACC was mixed with the overnight bacterial culture. 100 μl of this B. fragilis–ACC mixture was injected intraperitoneally into five-day-old pups with a 31-gauge needle. Control littermates were simultaneously injected intraperitoneally with 100 μl of ACC alone. Pups were harvested at P17. Spleen weight was measured immediately after dissection, and all tissue was subsequently processed as described above. LPS from E. coli O127:B8 was purchased from Sigma (L3129) and administered to the low-lesion-penetrance, resistant Ccm2ECKO neonatal CCM disease model. At P5, a 3 μg dose of LPS dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, a 5 μg dose of LPS was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Peptidoglycan from Bacillus subtilis (a Gram-positive gut commensal) was purchased from Invivogen (tlrl-pgnb3) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Poly(I:C) was purchased from Invivogen (tlrl-picw) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Mouse IL-1β was purchased from Genscript (Z02988) and administered to the resistant Ccm2ECKO neonatal CCM disease model. At P5, a 5 ng dose of IL-1β dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, an 8-ng dose of IL-1β was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Mouse TNFα was purchased from Genscript (Z02918) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the IL-1β experiments. For all experiments using microCT quantification of CCM lesion volume, brains were harvested and immediately placed in 4% PFA/PBS fixative. Brains remained in fixative until staining with non-destructive, iodine contrast and subsequent microCT imaging performed as previously described47. All tissue processing, imaging and volume quantification were performed in a blinded manner by investigators at the University of Chicago without any knowledge of experimental details. We blinded samples at three distinct points in the analysis. First, neonatal CCM model pups were injected with 4OHT without knowledge of genotypes. Second, hindbrains from genotyped animals were given randomized, de-identified labels to provide for blinded microCT scanning by an independent operator. Third, randomized microCT image stacks were analysed in a blinded manner by individuals not involved in any prior experimental steps. Mice were anaesthetized with Avertin and underwent intra-cardic perfusion with 10 ml of cold PBS. The brain was separated from the brainstem, and the cerebellum was separated from the remaining brain and processed in parallel. The tissue was minced with scissors, placed in digestion buffer (RPMI, 20 mM HEPES, 10% FCS, 1 mM CaCl , 1 mM MgCl , 0.05 mg ml−1 Liberase (Sigma), 0.02 mg ml−1 DNase I (Sigma)), and incubated for 40 min at 37 °C with shaking at 200 r.p.m. The mixture was passed through a 100-μm strainer and washed with FACS buffer (PBS, 1% FBS). Cells were resuspended in 4 ml of 40% Percoll (GE Healthcare) and overlaid on 4 ml of 67% Percoll. Gradients were centrifuged at 400g for 20 min at 4 °C and cells at the interface were collected, washed with 10 ml of FACS buffer, and stained for flow cytometric analysis. Neonatal P10 mice were anaesthetized with Avertin and underwent intracardiac puncture/blood draw using a 27-gauge needle/syringe coated with 0.5 M EDTA, pH 8.0 immediately before use. Cells were pelleted by centrifugation at 300g for 5 min at 4 °C. Serum was removed and red blood cells were lysed using ACK lysis buffer. Spleens were dissected in parallel, hand-homogenized using a mini-pestle and red blood cells were lysed using ACK lysis buffer. Cells from both sets of tissues were passed through a 70-μm cell-strainer, pelleted and resuspended in FACS buffer (PBS, 2% FBS, 0.1% NaN ) for immunostaining and subsequent FACS analysis. Cells were isolated from the indicated tissues. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Multi-laser, flow cytometry analysis procedures were performed at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analysers running FACSDiva software (BD Biosciences). Two-laser, flow cytometry analyses were performed at the University of Pennsylvania iPS Cell Core using BD Accuri C6 instruments. FlowJo software (v.10 TreeStar) was used for data analysis and graphics rendering. All fluorochrome-conjugated antibodies used are listed as follows (Clone, Company, Catalog Number): CD11b (M1/70, Biolegend, 101255); CD11c (N418, Biolegend, 117318); CD16/32 (93, Biolegend, 101319); CD16/32 (93, eBiosciences, 56D0161D80); CD19 (6D5, Biolegend, 115510); CD3ε (145D2C11, Biolegend, 100304); CD4 (GK1.5, Biolegend, 100406); CD45 (30-F11, Biolegend, 103121 or 103151), CD8a (53D6.7, Biolegend, 100725); Foxp3 (FJK-16 s, eBiosciences, 50-5773-82); Ly-6G (1A8, Biolegend, 127624); Live/Dead (N/A, Thermofisher, LD34966); NK1.1 (PK136, Biolegend, 108745); RORγt (B2D, eBiosciences, 12-6981-82); Siglec-F (E50D2440, BD, 562757); TCRγδ (UC7-13D5, Biolegend, 107504) At the specified time points, cerebellar endothelial cells were isolated through enzymatic digestion followed by separation using magnetic-activated cell sorting by anti-CD31-conjugated magnetic beads (MACS MS system, Miltenyl Biotec), as previously described9. Lung endothelial cells were isolated through enzymatic digestion, as previously described, followed by separation using anti-CD31-conjugated magnetic beads and the MACS MS system48. Isolated endothelial cells were pelleted and total RNA was extracted using the RNeasy Micro kit (Qiagen 74004). For qPCR analysis, cDNA was synthesized from 300 ng to 500 ng total RNA using the SuperScript VILO cDNA Synthesis Kit and Master Mix (Thermo Fisher 11755050). Real-time PCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher 4368577) using the primers listed (all mouse): Gapdh forward: 5′-AAATGGTGAAGGTCGGTGTGAACG-3′; Gapdh reverse: 5′-ATCTCCACTTTGCCACTGC-3′; Klf2 forward: 5′-CGCCTCGGGTTCATTTC-3′; Klf2 reverse: 5′-AGCCTATCTTGCCGTCCTTT-3′; Klf4 forward: 5′-GTGCCCCGACTAACCGTTG-3′; Klf4 reverse: 5′-GTCGTTGAACTCCTCGGTCT-3′; Krit1 forward: 5′-CCGACCTTCTCCCCTTGAAC-3′; Krit1 reverse: 5′-TCTTCCACAACGCTGCTCAT-3′; Il1b forward: 5′-GCAACTGTTCCTGAACTCAACT-3′; Il1b reverse: 5′-ATCTTTTGGGGTCCGTCAACT-3′; Sele forward: 5′-ATGCCTCGCGCTTTCTCTC-3′; Sele reverse: 5′-GTAGTCCCGCTGACAGTATGC-3′; Tlr4 forward: 5′-ACTGGGGACAATTCACTAGAGC-3′; Tlr4 reverse: 5′-GAGGCCAATTTTGTCTCCACA-3′. As part of the Brain Vascular Malformation Consortium (BVMC) CCM study (Project 1), a large cohort of familial CCM individuals with identical KRIT1(Q455X) mutations were enrolled between 2009–2014 at the University of New Mexico. All study protocols were approved by the Institutional Review Boards at the University of New Mexico and University of California San Francisco (UCSF) and all procedures were performed in accordance with these protocols. Prior to participation in the study, written informed consent was obtained from every patient and properly documented by UNM investigators. At study enrollment, participants received a neurological examination and 3T MRI imaging using a volume T1 acquisition (MPRAGE, 1-mm slice reconstruction) and axial TSE T2, T2 gradient recall, susceptibility-weighted, and FLAIR sequences. Lesion counting by the neuroradiologist was based on concurrent evaluation of axial susceptibility-weighted imaging with 1.5-mm reconstructed images and axial T2 gradient echo 3-mm images. Participants also provided blood or saliva samples for genetic studies. Genomic DNA was extracted using standard protocols. De-identified samples were normalized, plated on 96-well plates, and genotyped at the UCSF Genomics Core Facility using the Affymetrix Axiom Genome-wide LAT1 Human Array. Affymetrix Genotyping Console (GTC) 4.1 Software package was used to generate quality control metrics and genotype calls. All samples had genotyping call rates of ≥ 97% and were further checked for sample mix-ups (sex check, Mendelian errors and cryptic relatedness), resulting in 188 samples for genetic analysis. 21 candidate genes were further examined in the TLR4 and MEKK3–KLF2/4 signalling pathways (TLR4, CD14, MD-2, LBP, MYD88, TICAM1, TIRAP, TRAF1-6, MAP3K3, MEK5, ERK5, MEF2C, KLF2, KLF4, ADAMTS4, ADAMTS5) including 467 SNPs within 20 kb upstream or downstream of each gene locus using UCSC Genome Browser coordinates (GRCh37/hg19). Because total lesion counts are highly right-skewed, raw counts were log-transformed and analysis was performed on residuals (adjusted for age at enrollment and sex). To identify genotypes associated with log-transformed residual counts, linear regression analysis was implemented using QFAM family-based association tests for quantitative traits (PLINK v1.07 software), with stringent multiple testing correction (Bonferroni correction for the number of SNPs tested within each gene) given that some SNPs on the Affymetrix array were in linkage disequilibrium with each other, that is, statistically correlated with R2 > 0.8. The Fehrmann dataset used for eQTL lookups consisted of peripheral blood samples from the UK and the Netherlands49, 50. Samples were genotyped with Illumina HumanHap300, HumanHap370 or 610 Quad platforms. Genotypes were input by Impute v2 (ref. 51) using the GIANT 1000G p1v3 integrated call set for all ancestries as a reference52. Gene expression levels were measured by Illumina HT12v3 arrays. Gene expression pre-processing involved quantile normalization, log transformation, probe centring and scaling, population stratification correction (first four genetic multi-dimensional scaling components were removed from gene expression data) and correction for unknown confounders (first 20 gene expression principal components not associated with genetic variants were removed from gene expression data). Identification of potential sample mix-ups was conducted by MixupMapper21 and finally 1,227 samples remained. All pre-processing steps were performed with the QTL mapping pipeline v1.2.4D (https://github.com/molgenis/systemsgenetics/tree/master/eqtl-mapping-pipeline - downloading-the-software). These results are corroborated by an independently conducted GTEX Consortium study (http://www.gtexportal.org/home/snp/rs10759930 and http://www.gtexportal.org/home/snp/rs778587). TAK-242 was purchased from EMD Millipore (614316) and administered to the neonatal CCM disease model. Five, seven and nine days after birth, a 60-μg dose of TAK-242 was dissolved in DMSO/sterile intralipid (Sigma, I141) vehicle and administered retro-orbitally in a total volume of 30 μl. Control animals were identically injected with sterile DMSO/intralipid vehicle alone. Pups were euthanized and brains dissected 10 days after birth. LPS-RS ultrapure was purchased from Invivogen (tlrl-prslps) and administered to the neonatal CCM disease model. Starting at P5, a 20 μg dose dissolved in sterile PBS was administered retro-orbitally in a total volume of 30 μl every 24 h. Control animals were identically injected with sterile PBS alone. Pups were euthanized and brains dissected 10 days after birth. Experimental breeding pairs of mice, yielding susceptible neonatal CCM pups, were identified by induction of a neonatal CCM litter and evaluation of lesion burden. These breeding pairs then underwent timed matings and at E14.5, both male and female adult mice received antibiotic-laced drinking water mixed with 40 g l−1 of sucralose and red food colouring. Antibiotic water was replaced daily. The following antibiotics were mixed with 0.22-μm-filtered water: penicillin (500 mg l−1), neomycin (500 mg l−1), streptomycin (500 mg l−1), metronidazole (1 g l–1) and vancomycin (1 g l−1). Antibiotics were purchased from the Hospital of the University of Pennsylvania pharmacy. The neonatal CCM model was induced as described above. At P10, pups were euthanized and antibiotic water switched to normal drinking water. Experimental breeding pairs were then mated to obtain third generation, post-antibiotic pups. Co-housed, susceptible Krit1fl/fl females underwent evening–morning timed matings with a single susceptible Krit1ECKO male. Upon detection of a plug in the morning, the females were subsequently separated into singly-housed cages. At E14.5, female mice were received either vancomycin (1 g l−1)-laced or untreated (vehicle) sterile-filtered drinking water, changed daily. The drinking water was further mixed with 40 g l−1 sucralose and red food colouring. Pups were harvested at P11. The entire neonatal gut was dissected, snap-frozen on dry ice, and stored at −80 °C. The QIAamp DNA Stool Mini Kit (Qiagen 51504 or 51604) was used to extract bacterial DNA from the neonatal gut. Before commencing the standard Qiagen protocol, the frozen gut was mixed in the included stool lysis buffer and homogenized with a 5 mm stainless steel bead in a TissueLyser LT (Qiagen 69980) at 50 Hz for 10 min at 4 °C. Concentration of the extracted DNA was equalized and 16 ng of DNA was used per qPCR reaction with universal bacterial 16S rRNA gene primers53, two different sets of previously characterized Bacteroidetes s24-7 primers54, 55, and Firmicutes primers56. Universal 16S rRNA forward: 5′-ACTGAGAYACGGYCCA-3′; universal 16S rRNA reverse: 5′-TTACCGCGGCTGCTGGC-3′; Bacteroidetes s24-7 rRNA set 1 forward: 5′-GGAGAGTACCCGGAGAAAAAGC-3′; Bacteroidetes s24-7 rRNA set 1 reverse: 5′-TTCCGCATACTTCTCGCCCA-3′; Bacteroidetes s24-7 rRNA set 2 forward: 5′-CCAGCAGCCGCGGTAATA-3′; Bacteroidetes s24-7 rRNA set 2 reverse: 5′-CGCATTCCGCATACTTCTC-3′; Firmicutes rRNA forward: 5′-TGAAACTYAAAGGAATTGACG-3′; Firmicutes rRNA reverse: 5′-ACCATGCACCACCTGTC-3′. Evening–morning timed matings to generate donor susceptible or resistant females yielding Krit1ECKO or Ccm2ECKO pups were performed and timed pregnant Swiss Webster females (Charles River 024) served as foster mothers. To prevent delivery of the pups, at E16.5, donor females were injected subcutaneously with 100 μl of a 15 μg ml−1 solution of medroxyprogesterone (Sigma Aldrich, M1629) dissolved in DMSO. The morning of E19.5, the donor mother was euthanized by cervical dislocation and submerged in a warm sterile solution of 1% VirkonS/PBS (weight/volume) for one minute. The uterus was then dissected in a sterile laminar flow hood, submerged in a warm sterile solution of 1% VirkonS/PBS for one minute and quickly rinsed with warm sterile PBS. Pups were then removed from the uterus and fostered to the Swiss Webster recipient female. The following morning, induction of the neonatal CCM model was performed as described above. Timed matings were performed using germ-free Swiss Webster mice housed in sterile isolators under care of the University of Pennsylvania Gnotobiotic Mouse Facility. Simultaneous evening–morning timed matings were also performed using co-housed, susceptible Krit1fl/fl females and Krit1ECKO males previously characterized to yield CCM-susceptible pups. Medroxyprogesterone was administered to donor females and the sterile C-section was performed at E19.5 as described in the previous section. The intact uterus was passed through a J-tube filled with warm 1% VirkonS/PBS that was hermetically sealed to the sterile isolator. Pups were dissected from the uterus inside the sterile isolator and fostered to the recipient germ-free Swiss Webster mother. Approximately one week later, faecal samples were collected for microbiology testing. Germ-free status was further confirmed by 16S qPCR of bacterial DNA isolated from maternal faeces and neonatal guts. Fresh faecal pellets were collected from experimental females yielding susceptible or resistant pups one day after harvesting the pups to determine phenotypic severity. Collection was performed between 16:00 and 18:00, pellets were immediately snap-frozen on dry ice, and stored at −80 °C. DNA was extracted from stool samples using the Power Soil htp kit (Mo Bio Laboratories) following the manufacturer’s protocol. Library preparation was performed by using previously described barcoded primers targeting the V1/V2 region of the 16S rRNA gene57. PCR reactions were performed in quadruplicate using AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen). Each PCR reaction consisted of 0.4 μM primers, 1× AccuPrime Buffer II, 1 U Taq, and 25 ng DNA. PCRs were run using the following parameters: 95 °C for 5 min; 20 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s; and 72 °C for 8 min. Quadruplicate PCR reactions were pooled and products were purified using AMPureXP beads (Beckman-Coulter). Equimolar amounts from each sample were pooled to produce the final library. Positive and negative controls were carried through the amplification, purification and pooling procedures. Negative controls were used to assess reagent contamination and consisted of extraction blanks and DNA-free water. Positive controls were used to assess amplification and sequencing quality and consisted of gBlock DNA (Integrated DNA Technologies) containing non-bacterial 16S rRNA gene sequences flanked by bacterial V1 and V2 primer binding sites. Paired-end 2 × 250 bp sequence reads were obtained from the MiSeq (Illumina) using the 500 cycle v2 kit (Illumina). Sequence data were processed using QIIME version 1.9.1 (ref. 58). Read pairs were joined to form a complete V1/V2 amplicon sequence. Resulting sequences were quality filtered and demultiplexed. Operational taxonomic units (OTUs) were selected by clustering reads at 97% sequence similarity59. Taxonomy was assigned to each OTU with a 90% sequence similarity threshold using the Greengenes reference database60. A phylogenetic tree was inferred from the OTU data using FastTree61. The phylogenetic tree was then used to calculate weighted and unweighted UniFrac distances between each pair of samples in the study62, 63. Microbiome compositional differences were visualized using principle coordinates analysis (PCoA). Community-level differences between mice genetic background as well as disease susceptibility groups were assessed using a PERMANOVA test64 of weighted and unweighted UniFrac distances. To assess significance in the PERMANOVA test, each cage was randomly re-assigned to groups 9,999 times. Differential abundance was assessed for taxa present in at least 80% of the samples, using generalized linear mixed-effects models. For tests of taxon abundance, the cage was modelled as a random effect, as previous research has established that the faecal microbiota of mice are correlated within cages65. The P values were corrected for multiple testing using Benjamini–Hochberg method. Sample sizes were estimated on the basis of our previous experience with the neonatal CCM model and lesion volume quantification by microCT9. Using 40 historically collected, susceptible Krit1ECKO and Ccm2ECKO P10 brains, we calculated a sample standard deviation of 0.250 mm3. Between Krit1ECKO and Ccm2ECKO genotypes, an F-test to compare variances confirmed no significant difference (P = 0.340). Thus, for a two-group comparison of lesion volumes, each sample group requires seven animals for a desired statistical power of 95% (β = 0.05), and a conventional significance threshold of 5% (α = 0.05) assuming an effect size of 50% (0.5) and equal standard deviations between sample groups. These predictive calculations were corroborated by our recent publication in which larger effect sizes (>90%) were found to be statistically significant with four to five samples per group9. All experimental and control animals were littermates and none were excluded from analysis at the time of harvest. Experimental animals were lost or excluded at two pre-defined points: (i) failure to properly inject 4OHT and observation of significant leakage; (ii) death before P10 because of injection or chaos. Given the early time points, no attempt was made to distinguish or segregate results based on neonatal genders. P values were calculated as indicated in figure legends using an unpaired, two-tailed Student’s t-test; one-way ANOVA with multiple comparison corrections (Holm–Sidak or Bonferroni); PERMANOVA; or linear mixed effects modelling. As indicated in the figure legends, the standard error of the mean (s.e.m.), 95% confidence interval, or boxplot is shown. All relevant data are available from the authors upon reasonable request.