Stemgent is an American privately funded biotech company focused on providing reagents and technology developed by some of the world's leading stem cell scientists. Founded in 2008, Stemgent has two fully operational facilities in both San Diego, California and Cambridge, Massachusetts. Stemgent currently has 40 employees.The company is designed to serve researchers who study stem cell biology and regenerative medicine and those who use cells derived from stem cells as tools to advance their understanding of major diseases. Wikipedia.
News Article | May 23, 2017
Joint venture provides quality source of clinically annotated tissue specimens from vast Indian population BELTSVILLE, MD and PHILADELPHIA, PA--(Marketwired - May 23, 2017) - REPROCELL and Fox Chase Cancer Center today announced a joint venture to open a multi-site biosample repository facility in India. Initial operations are underway in Delhi with plans to expand to Hyderabad in the fourth quarter. "The research community's demand for access to clinical-grade bio-specimens from India has gone largely unmet. Through our partnership with Fox Chase, we plan to change that," said Rama Modali, CEO of REPROCELL USA. "We've begun growing our inventory of biosamples in India using the same rigorous quality-assurance standards we employ at our facilities in the US, including the collection and storage of all associated annotated clinical and genetic information and bioinformatics data analytics." The joint venture's India facilities add approximately 3,000 new biosamples monthly. Collected specimens are supported by annotations that include medical history, mutation data and detailed records of treatment protocols as well as outcomes. "This alliance further augments our already impressive and extensive combined biosample inventory, helping us better serve the global cancer research community," said Richard I. Fisher, MD, president & CEO of Fox Chase Cancer Center. As a leading provider of biomaterials, REPROCELL is deeply engaged with cancer and other disease research being conducted by pharma, biotech and leading academic research institutions worldwide. The Hospital of Fox Chase Cancer Center and its affiliates (collectively "Fox Chase Cancer Center"), a member of the Temple University Health System, is one of the leading cancer research and treatment centers in the United States. Founded in 1904 in Philadelphia as one of the nation's first cancer hospitals, Fox Chase was also among the first institutions to be designated a National Cancer Institute Comprehensive Cancer Center in 1974. Fox Chase researchers have won the highest awards in their fields, including two Nobel Prizes. Fox Chase physicians are also routinely recognized in national rankings, and the Center's nursing program has received the Magnet recognition for excellence four consecutive times. Today, Fox Chase conducts a broad array of nationally competitive basic, translational, and clinical research, with special programs in cancer prevention, detection, survivorship and community outreach. For more information, call 1-888-FOX CHASE or (1-888-369-2427). REPROCELL provides services and reagents to support the entire drug discovery pathway. BioServe-brand biorepository and molecular services provide researchers with human tissue samples and services to support a wide variety of research and development, as well as provide a starting point for stem cell research. Stemgent-brand stem cell products and services, along with REPROCELL brand differentiated cells and reagents, enable researchers to bring the power of stem cells to bear on human disease. Alvetex-brand 3D culture products provide a physiologically relevant environment for cells that mimics the in vitro situation. Biopta-brand human tissue assays provide pharmaceutical companies with physiologically relevant information on human tissue prior to clinical trials. REPROCELL, founded in 2002, is based in Yokohama, Japan and has laboratories in Beltsville, MD, USA, and Glasgow, UK to support global research efforts. For more information please visit www.REPROCELLUSA.com or call 301-470-3362.
News Article | March 4, 2016
Mice were housed in the Unit for Laboratory Animal Medicine at the Whitehead Institute for Biomedical Research and Koch Institute for Integrative Cancer Research. The following strains were obtained from the Jackson Laboratory: Lgr5-EGFP-IRES-CreERT2 (strain name: B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, stock number 008875), Rosa26-lacZ (strain name: B6.129S4-Gt(ROSA)26Sortm1Sor/J, stock number 003474), db/db (strain name: B6.BKS(D)-Leprjb/J, stock number 000697), PpardL/L (strain name: B6.129S4-Ppardtm1Rev/J, stock number 005897). Apcloxp exon 14 (ApcL/L) has been previously described41. Villin-CreERT2 was a gift from S. Robine. Long-term HFD was achieved by feeding male and female mice a dietary chow consisting of 60% kcal fat (Research Diets D12492) beginning at the age of 8–12 weeks and extending for a period of 9–14 months. Control mice were sex- and age-matched and fed standard chow ad libitum. GW501516 (Enzo) was reconstituted in DMSO at 4.5 mg ml−1 and diluted 1:10 in a solution of 5% PEG400 (Hampton Research), 5% Tween80 (Sigma), 90% H O for a daily intraperitoneal injection of 4 mg kg−1. Apc exon 14 was excised by tamoxifen suspended in sunflower seed oil (Spectrum S1929) at a concentration of 10 mg ml−1 and 250 μl per 25 g of body weight, and administered by intraperitoneal injection twice over 4 days before collecting tissue. PpardL/L mice were administered 4–5 intraperitoneal injections of tamoxifen on alternate days. Mice were analysed within 2 weeks of the last tamoxifen injection. BrdU was prepared at 10 mg ml−1 in PBS, passed through a 0.22-μm filter and injected at 100 mg kg−1. As previously described1, tissues were fixed in 10% formalin, paraffin embedded and sectioned. Antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical) in a pressurized Decloaking Chamber (Biocare Medical) for 3 min. Antibodies used: rat anti-BrdU (1:2,000 (immunohistochemistry (IHC)), 1:1,000 (immunofluorescence (IF)) Abcam 6326), rabbit chromogranin A (1:4,000 (IHC), 1:250 (IF), Abcam 15160), rabbit monoclonal non-phospho β-catenin (1:800 (IHC), 1:400 (IF), CST 8814S), mouse monoclonal β-catenin (1:200, BD Biosciences 610154), rabbit polyclonal lysozyme (1:250, Thermo RB-372-A1), rabbit polyclonal MUC2 (1:100, Santa Cruz Biotechnology 15334), rabbit monoclonal OLFM4 (1:10,000, gift from CST, clone PP7), Biotin-conjugated secondary donkey anti-rabbit or anti-rat antibodies were used from Jackson ImmunoResearch. The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) followed by Dako Liquid DAB+ Substrate (Dako) was used for visualization. For immunofluorescence, Alexa Fluor 568 secondary antibody (Invitrogen) was used with Prolong Gold (Life Technologies) mounting media. All antibody incubations involving tissue or sorted cells were performed with Common Antibody Diluent (Biogenex). Organoids were fixed with 4% paraformaldehyde, permabilized with 0.5% Triton X-100 in PBS, rinsed with 100 mM glycine in PBS, blocked with 10% donkey serum in PBS, incubated overnight with primary antibody at 4 °C, rinsed and incubated with Alexa Fluor 568 secondary antibody (Invitrogen), and mounted with Prolong Gold (Life Technologies) mounting media. The in situ hybridization probes used in this study correspond to expressed sequence tags or fully sequenced cDNAs obtained from Open Biosystems. The accession numbers (IMAGE mouse cDNA clone in parenthesis) for these probes are as follows: mouse Olfm4 BC141127 (9055739), mouse Crp4 BC134360 (40134597). Both sense and antisense probes were generated to ensure specificity by in vitro transcription using DIG RNA labelling mix (Roche) according to the manufacturer’s instructions and to previously published detailed methods23, 42. Single-molecule in situ hybridization was performed using Advanced Cell Diagnostics RNAscope 2.0 HD Detection Kit. Adult mice were exposed to 15 Gy of ionizing irradiation from a 137-caesium source (GammaCell) and euthanized after 72 h. The number of surviving crypts per length of the intestine was enumerated from haematoxylin-and-eosin-stained sections15. Antibodies: rabbit polyclonal anti-PPAR-δ (1:100, Thermo PA1-823A), rabbit polyclonal anti-CPT1a (1:250, ProteinTech 15184-1-AP), rabbit polyclonal anti-HMGCS2 (1:500, Sigma AV41562), rabbit monoclonal anti-FABP1 (1:1,000, Abcam ab129203), NF-κB Sampler Pathway Kit (CST, 9936S), mouse monoclonal anti-STAT-3 (CST, 9139P), rabbit monoclonal anti-P-STAT3 (Y705) XP (CST, 9145P), mouse monoclonal anti-CREB (CST, 86B10), mouse monoclonal anti-β-catenin (1:200, BD Biosciences 610154), rabbit polyclonal anti-γ-tubulin (1:1,000, Sigma T5192). For immunoprecipitation assays, crypts were collected and nuclear extraction was carried out using Abcam nuclear extraction kit (ab113474) following manufacturer’s instructions. Nuclear extracts were incubated with 5 μg anti-PPAR-δ antibody (Thermo), or anti-rabbit IgG control antibody (Santa Cruz) overnight at 4 °C followed by 2 h of incubation with Dynabeads Protein G for immunoprecipitation. Protein complexes bound to antibody and beads were washed five times and eluted with Laemmli sample buffer. Samples were resolved by SDS–PAGE. Protein interaction was analysed by immunoblotting. Lgr5-GFPhi ISCs or Lgr5-GFPlow progenitors were sorted directly into Laemmli sample buffer and boiled for 5 min. Samples were resolved by SDS–PAGE and analysed by immunoblotting with horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (1:10,000, Santa Cruz Biotechnology sc-2054) and Western Lightning Plus-ECL detection kit (Perkin Elmer NEL104001EA) As previously reported and briefly summarized here, small intestines and colons were removed, washed with cold PBS without magnesium chloride and calcium (PBS−/−) opened longitudinally, and then cut into 3–5-mm fragments. Pieces were washed several times with cold PBS−/− until clean, washed 2–3 with PBS−/− EDTA (10 mM), incubated on ice for 90–120 min, and gently shook at 30-min intervals. Crypts were then mechanically separated from the connective tissue by more rigorous shaking, and then filtered through a 70-μm mesh into a 50-ml conical tube to remove villus material (for small intestine) and tissue fragments. Crypts were removed from this step for crypt culture experiments and embedded in Matrigel with crypt culture media. For ISC isolation, the crypt suspensions were dissociated to individual cells with TrypLE Express (Invitrogen). Cell labelling consisted of an antibody cocktail comprising CD45-PE (eBioscience, 30-F11), CD31-PE (Biolegend, Mec13.3), Ter119-PE (Biolegend, Ter119), CD24-Pacific Blue (Biolegend, M1/69), CD117-APC/Cy7 (Biolegend, 2BS), and EPCAM-APC (eBioscience, G8.8). ISCs were isolated as Lgr5-EGFPhiEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−. EGFPlow progenitors were isolated as EGFPlowEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−, and Paneth cells from small intestine were isolated as CD24hiSidescatterhiLgr5-EGFP−Epcam+CD31−Ter119−CD45−7-AAD− with a BD FACS Aria II SORP cell sorter into supplemented crypt culture medium for culture. Dead cells were excluded from the analysis with the viability dye 7-AAD (Life Technologies). When indicated, populations were cytospun (Thermo Cytospin 4) at 800 r.p.m. for 2 min, or allowed to settle at 37 °C in fully humidified chambers containing 5% CO onto poly-l-lysine-coated slides (Polysciences). The cells were subsequently fixed in 4% paraformaldehyde (pH 7.4, Electron Microscopy Sciences) before staining. Isolated crypts were counted and embedded in Matrigel (Corning 356231 growth factor reduced) at 5–10 crypts per μl and cultured in a modified form of medium as described previously13. Unless otherwise noted, Advanced DMEM (Gibco) was supplemented by EGF 40 ng ml−1 (R&D), Noggin 200 ng ml−1 (Peprotech), R-spondin 500 ng ml−1 (R&D or Sino Biological), N-acetyl-l-cysteine 1 μM (Sigma-Aldrich), N2 1X (Life Technologies), B27 1X (Life Technologies), Chiron 10 μM (Stemgent), Y-27632 dihydrochloride monohydrate 20 ng ml−1 (Sigma-Aldrich). Colonic crypts were cultured in 50% conditioned medium derived from L-WRN cells supplemented with Y-27632 dihydrochloride monohydrate 20 ng ml−1, as described43. Approximately 25–30 μl droplets of Matrigel with crypts were plated onto a flat bottom 48-well plate (Corning 3548) and allowed to solidify for 20–30 min in a 37 °C incubator. Three hundred microlitres of crypt culture medium was then overlaid onto the Matrigel, changed every 3 days, and maintained at 37 °C in fully humidified chambers containing 5% CO . Clonogenicity (colony-forming efficiency) was calculated by plating 50–300 crypts and assessing organoid formation 3–7 days or as specified after initiation of cultures. Palmitic acid (Cayman Chemical Company 10006627 conjugated to BSA), oleic acid (Sigma O1008), lipid mixture (Sigma L0288), or GW501516 (Enzo) were added immediately to cultures at 30 μM (palmitic acid, oleic acid), 2% (lipid mixture), and 1 μM (GW501516). 4-OH tamoxifen (Calbiochem, 579002, 10 nM) was added to organoid cultures derived from PpardL/L; Villin-CreERT2 (Ppard IKO) crypts to ensure Ppard excision in the ex vivo fatty acid or GW501516 experiments. Isolated ISCs or progenitor cells were centrifuged for 5 min at 250g, re-suspended in the appropriate volume of crypt culture medium (500–1,000 cells μl−1), then seeded onto 25–30 μl Matrigel (Corning 356231 growth factor reduced) containing 1 μM Jagged (Ana-Spec) in a flat bottom 48-well plate (Corning 3548). Alternatively, ISCs and Paneth cells were mixed after sorting in a 1:1 ratio, centrifuged, and then seeded onto Matrigel. The Matrigel and cells were allowed to solidify before adding 300 μl of crypt culture medium. The crypt media was changed every second or third day. Organoids were quantified on days 3, 7 and 10 of culture, unless otherwise specified. For secondary organoid assays, either individual primary organoids or many primary organoids were mechanically dissociated and then replated, or organoids were dissociated for 10 min in TrypLE Express at 32 °C, resuspended with SMEM (Life Technologies), centrifuged (5 min at 250g) and then resuspended in cold SMEM with the viability dye 7-AAD. Live cells were sorted and seeded onto Matrigel as previously described in standard crypt media (not supplemented with lipids or GW501516). Secondary organoids were enumerated on day 4, unless otherwise specified. Human biopsies were obtained from patients with informed consent undergoing intestinal resection at the Massachusetts General Hospital (MGH). The MGH Institutional Review Board committee and Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects approved the study protocols. Crypts were isolated43, embedded in Matrigel and subsequently exposed to lipid mixture, palmitic acid or GW501516 (as described in earlier). Cultures were passaged weekly and maintained for 3–4 weeks. To passage, equal numbers of organoids from each condition were disrupted with trypsin/EDTA. Numbers of organoids were counted 4–7 days after passaging into control media. Counts were normalized to numbers of organoids present in control wells and plotted. Statistical significance was calculated by performing analysis of variance (ANOVA) multiple comparisons of the means for each group. For quantitative RNA expression analysis, organoids were dissociated, cells were selected as a live population by flow cytometry (7-AAD, Life Technologies), and sorted into Tri Reagent (Life Technologies) for RNA isolation. After 5 days of culturing, intestinal organoids were placed into Karnovsky’s KII solution (2.5% glutaraldehyde, 2.0% paraformaldehyde, 0.025% calcium chloride, in a 0.1 M sodium cacodylate buffer, pH 7.4) and fixed overnight. Subsequently, they were post-fixed in 2.0% osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in graded ethanol solutions, infiltrated with propylene oxide/Epon mixtures, flat embedded in pure Epon, and polymerized overnight at 60 °C. Then 1-μm sections were cut, stained with toluidine blue, and examined by light microscopy. Representative areas were chosen for electron microscopic study and the Epon blocks were trimmed accordingly. Thin sections were cut with an LKB 8801 ultramicrotome and diamond knife, stained with Sato’s lead, and examined in a FEI Morgagni transmission electron microscope. Images were captured with an AMT (Advanced Microscopy Techiques) 2K digital CCD camera. For RNA sequencing (RNA-seq), total RNA was extracted from 200,000 sorted Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors by pooling 2–5 71-week-old HFD male or control mice using Tri Reagent (Life Technologies) according to the manufacturer’s instructions, except for an overnight isopropanol precipitation at −20 °C. From the total RNA, poly(A)+ RNA was selected using Oligo(dT) -Dynabeads (Life technologies) according to the manufacturer’s protocol. Strand-specific RNA-seq libraries were prepared using the dUTP-based, Illumina-compatible NEXTflex Directional RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s directions. All libraries were sequenced with an Illumina HiSeq 2000 sequencing machine. For RNA-seq data analysis, raw stranded reads (40 nucleotides) were trimmed to remove adaptor and bases with quality scores below 20, and reads shorter than 35 nucleotides were excluded. High-quality reads were mapped to the mouse genome (mm10) with TopHat version 1.4.1 (ref. 44), using known splice junctions from Ensembl Release 70 and allowing at most two mismatches. Genes were quantified with htseq-count (with the ‘intersect strict’ mode) using Ensembl Release 70 gene models. Gene counts were normalized across all samples using estimateSizeFactors from the DESeq R/Bioconductor package45. Differential expression analysis was also performed between two samples of interest with DESeq. GSEA (http://software.broadinstitute.org/gsea/index.jsp) was performed by using the pre-ranked (according to their ratios) 8,240 differentially expressed genes as the expression data set. Motif Analysis was performed using Haystack motif enrichment tool: http://github.com/lucapinello/Haystack46. In total, 24 single Lgr5-GFPhi ISCs and 72 single Lgr5-GFPlow progenitor cells were sorted from control or HFD-fed mice (n = 2 mice per group) for single-cell gene expression analysis. For one-tube single-cell sequence-specific preamplification, individual primer sets of β-catenin target genes (total of 96, Supplementary Table 2) were pooled to a final concentration of 0.1 mM for each primer. Single cells were directly sorted into 96-well plates containing 5 μl RT–PCR master mix (2.5 μl CellsDirect reaction mix, Invitrogen; 0.5 μl primer pool; 0.1 μl reverse transcriptase/Taq enzyme, Invitrogen; 1.9 μl nuclease-free water) in each well. Immediately after, plates were placed on PCR machine for preamplification. Sequence-specific preamplification PCR protocol was as following: 60 min at 50 °C for cell lysis and sequence-specific reverse transcription; then 3 min at 95 °C for reverse transcriptase inactivation and Taq polymerase activation. cDNA was then amplified by 20 cycles of 15 s at 95 °C for initial denaturation, 15 min at 60 °C for annealing and elongation. After preamplificiation, samples were diluted 1:5 before high-throughput microfluidic real-time PCR analysis using Fluidigm platform. Amplified single-cell cDNA samples were assayed for gene expression using individual qRT–PCR primers and 96.96 dynamic arrays on a BioMark System by following manufacturers protocol (Fluidigm). To confirm PPAR-δ-mediated induction of the most upregulated genes (n = 3 mice, 24 ISCs and 72 progenitors per group), or for single-cell analysis of organoid composition (n = 3 mice, 48 cells per group) and db/db mice (n = 3, 48 cells per group) standard single-cell qRT–PCR was performed using preamplified cDNA with corresponding primers. For Fluidigm analysis, threshold cycle (C ) values were calculated using the BioMark Real-Time PCR Analysis software (Fluidigm). See Supplementary Information for raw gene expression data. Gene expression levels were estimated by subtracting the C values from the background level of 35, which approximately represent the log gene expression levels. The t-Distributed stochastic neighbour embedding (t-SNE) analysis47 was performed using the MATLAB toolbox for dimensionality reduction. Differential expression analysis was conducted using the two-sided Wilcoxon–Mann–Whitney rank sum test implemented in the R coin package (https://www.r-project.org). P values were adjusted for multiple testing48 using the p.adjust function in R with method = ‘fdr’ option. Fold changes were calculated as the difference of median of log expression levels for the two cell populations. Split violin plots were generated using the vioplot package and the vioplot2 function in R (https://gist.github.com/mbjoseph/5852613). The heatmap for β-catenin target genes was generated with the MultiExperiment Viewer (MeV) program (http://www.tm4.org/mev.html) using the correlation-based distance and average linkage method as parameters of the unsupervised hierarchical clustering of genes. The heatmap for organoid composition was generated using MATLAB. The percentages of Jag1/Jag2-upregulated cells were calculated based on the number of single cells whose log expression was above 15. Approximately 25,000 cells were sorted into Tri Reagent (Life Technologies) and total RNA was isolated according to the manufacturer’s instructions with following modification: the aqueous phase containing total RNA was purified using the RNeasy plus kit (Qiagen). RNA was converted to cDNA with the cDNA synthesis kit (Bio-Rad). qRT–PCR was performed with diluted cDNA (1:5) in three wells for each primer and SYBR green master mix (Bio-Rad) on Bio-Rad iCycler RT–PCR detection system. For organoid experiments, 1,000 live cells were sorted and qRT–PCR optimized for low cell numbers (<1,000) was performed after sequence specific pre-amplification (cDNA diluted 1:200 in three wells for each primer) as described in single-cell gene expression analysis. All qRT–PCR experiments were repeated at least three independent times. Primers used are listed on Supplementary Table 1. ApcL/L; Lgr5-EGFP-IRES-CreERT2 mice were treated with vehicle or GW501516 for 1 month, and then injected with two intraperitoneal doses of tamoxifen. Four days later, Apc-null Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors were sorted by flow cytometry, as described earlier. For primary cell transplantations, 10,000 Apc-null Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors were resuspended into 90% crypt culture media (as described) and 10% Matrigel, then transplanted into the colonic lamina propria of C57BL/6 recipient mice by optical colonoscopy using a custom injection needle (Hamilton Inc., 33-gauge, small Hub RN NDL, 16 inches long, point 4, 45 degree bevel, like part number 7803-05), syringe (Hamilton Inc. part number 7656-01), and transfer needle (Hamilton Inc. part number 7770-02). Optical colonoscopy was performed using a Karl Storz Image 1 HD Camera System, Image 1 HUB CCU, 175 Watt Xenon Light Source, and Richard Wolf 1.9mm/9.5 Fr Integrated Telescope (part number 8626.431). Four injections were performed per mouse. Mice then underwent colonoscopy 8 weeks later to assess tumour formation. Colonoscopy videos and images were saved for offline analysis. Following sacrifice, the distal colons were excised and fixed in 10% formalin, then examined by haematoxylin and eosin section to identify adenomas. Histology images were reviewed by gastrointestinal pathologists who were blinded to the treatment groups (S.S., V.D. and Ö.H.Y.). All experiments reported in Figs 1, 2, 3, 4, 5 were repeated at least three independent times, except for Figs 3a, 4c, d, which were repeated twice. All samples represent biological replicates. For mouse organoid assays, 2–4 wells per group with at least 3 different mice were analysed. For human organoid assays, 4 wells per group with 4 different patient samples were analysed and experiments were repeated 4 times. All centre values shown in graphs refer to the mean. For statistical significance of the differences between the means of two groups, we used two-tailed Student’s t-tests. Statistical significance in Fig. 3k was calculated by performing ANOVA multiple comparisons of the means for each group. No samples or animals were excluded from analysis, and sample size estimates were not used. Animals were randomly assigned to groups. Studies were not conducted blinded, with the exception of all histological analyses and Fig. 5c, h. All experiments involving mice were carried out with approval from the Committee for Animal Care at MIT and under supervision of the Department of Comparative Medicine at MIT.
News Article | January 20, 2016
Timed natural matings were used for all experiments, where noon of the next day after the vaginal plugs of mated females were identified was scored as E0.5. Animal studies were authorized by a UK Home Office Project License and carried out in a Home Office-designated facility. No statistical methods were used to predetermine sample size. ∆PE-Oct3/4-GFP (GOF-GFP)31, 32, Prdm1-GFP and Prdm1−/− ES cell lines were established previously14, 19, 22, 23. Inducible Sox2-knockout (2CG2) ES cell line was a gift from H. Niwa27. All ES cell lines were maintained in naive ‘ground state’33 condition; that is, in N2B27 medium (R&D) with 2i (PD0325901, 1 μM; CHIR99021, 3 μM; Stemgent), LIF and 1% KnockOut Serum Replacement (KSR; Life Technologies) on fibronectin-coated dishes (16.7 μg ml−1; Millipore). Medium was changed daily. ES cell colonies were passaged by dissociating with TrypLE (Life Technologies). Oct3/4, Sox2, Nanog, Prdm1, Prdm14 and Brachyury complementary DNAs were cloned from mouse cDNA pool. cDNAs were inserted into PiggyBac-based doxycycline (Dox)-inducible vectors (a gift from H. Niwa). These vectors were transfected using Lipofectamine 2000 (Life Technologies) into ES cells together with a pPyCAG-PBase vector and a pPBCAG-rtTAIRESNeor vector, which harbours a neomycin resistance gene. After 1 week of neomycin (80 μg ml−1; Life Technologies) selection, pooled or single clones were used for experiments. To induce transgene expression, various concentrations of Dox (Sigma-Aldrich) were added to the media. EpiLCs and PGCLCs were induced as described previously5. Transgenes were induced by addition of Dox at day 0 of PGCLC induction. 100 ng ml−1 Dox was used in experiments shown in Figs 3f, 4e, g and Extended Data Figs 3b, c, 7g–i, 8a–c. 200 ng ml−1 Dox was used in Fig. 4b–d, f. 700 ng ml−1 of Dox was used in all other experiments. PGCLCs were induced as described in the manuscript. For inhibition of the BMP–SMAD pathway, noggin (200 ng ml−1; R&D) was added to the media at day 0 of PGCLC induction. For inhibition of WNT signalling, XAV939 (1 μM; Sigma-Aldrich) was added to the media. Day-1 or day-2 EpiLCs were transferred into GMEM 15%KSR 2i/LIF with or without Dox in monolayer culture. In addition, day-1 or day-2 EpiLCs were aggregated in low-cell-binding U-bottom-shaped 96-well plates (Thermo Scientific) (1,000–2,000 cells per well) in PGCLC induction media (GMEM with l-glutamine (Life Technologies), 15% KSR (Life Technologies), 1× MEM NEAA (Life Technologies), 1× sodium pyruvate (Life Technologies), 1× 2-mercaptoethanol (Life Technologies), 1× penicillin/streptomycin (Life Technologies)) and Dox. The medium was replaced daily. After 3 days, the GFP reporter signal was analysed with a fluorescence microscope and via FACS analysis. RNA was collected from pooled cells for qRT–PCR. Day-4 aggregates were dissociated with TrypLE and plated on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells with PGC selection medium (DMEM with l-glutamine (Life Technologies), 15% fetal bovine serum (FBS; Sigma-Aldrich)), LIF, 15 ng ml−1 bFGF, 30 ng ml−1 SCF (R&D) and 2 μM all trans-retinoic acid (Sigma-Aldrich). Retinoic acid promotes germ cell self-renewal while promoting differentiation of ES cells20, 21. The media was replaced daily. After 5 days, proliferating GFP+ cells were dissociated with TrypLE and plated on fibronectin-coated dishes with ESC medium (N2B27 with 2i/LIF). PGCLCs were dissociated with TrypLE, washed with DMEM containing 10% FBS and resuspended with 1×PBS containing 0.1% BSA. Large clumps of cells were removed using a cell strainer (BD Biosciences). The cells were analysed and sorted on flow cytometers (FACS Calibur, BD Biosciences; MoFlo high speed cell sorter, Beckman Coulter; S3 cell sorter, Biorad). Total RNAs from ES cells, EpiLCs and FACS-sorted cells were extracted using the RNeasy Mini Kit (Qiagen) or Picopure RNA Isolation Kit (Life Technologies). The total RNAs were reverse transcribed by the Quantitect Reverse Transcription Kit (QIAGEN). The first-strand cDNAs were used for RT–qPCR analysis with SYBR Green PCR reagent (Sigma-Aldrich). The primer sequences used for the qRT–PCR are listed in Supplementary Table 1. Student’s t-test was used to test for significance. ES cells and day-4 PGCLCs were dissociated and sorted with a MoFlo high-speed cell sorter (Beckman Coulter). Total RNAs were extracted using the RNeasy Mini Kit (QIAGEN). Complementary RNA (cRNA) generation, quality control, hybridization and data analysis were performed by Cambridge Genomic Services at the University of Cambridge. Raw intensity values from Illumina MouseWG-6 v.2.0 expression beadchip microarrays were pre-processed with the Bioconductor lumi and preprocessCore packages (http://www.bioconductor.org): Probes that were not detected in at least one sample were removed, Variance stabilization transformation (VST) was applied, and samples were quantile-normalized. Differential expression was evaluated with the Bioconductor limma package. Comparison with published microarray data (Extended Data Fig. 3j). Our data set was assayed on an Illumina MouseWG-6 v.2.0 expression beadchip, the data set from ref. 5 was assayed on the Affymetrix Mouse Genome 430 2.0 Array platform. We therefore quantile-normalized the data sets to ensure that the data sets span comparable ranges of expression values. PCA was performed on the centre-scaled expression values, where systematic differences between platforms are mainly captured by the first principal component. Day-3, day-4 and day-6 aggregates were fixed with 2% or 4% paraformaldehyde for 20 min at room temperature or for 2 h at 4 °C. Fixed aggregates were washed several times in PBS and transferred into 10% sucrose/PBS (2 h), 20% sucrose/PBS (2 h) and finally into OCT embedding matrix (overnight; CellPath). Next day, cell aggregates were embedded in OCT in tissue moulds and stored at −80 °C. A Leica Cryostat CM3050S was used to cut the OCT blocks in 6–8-μm-thick sections, which were collected on SuperFrost Plus slides (VWR). For immunofluorescence staining, the slides were washed with PBS, permeabilized with PBS/0.1–1% Triton X-100 and then incubated with primary antibodies in permeabilization buffer including 5% donkey serum (Sigma-Aldrich) overnight at 4 °C. Next day, slides were washed with PBS and incubated with secondary antibodies in permeabilization buffer for 2 h at room temperature, washed with PBS, incubated with 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 15–30 min, and mounted using Vectashield Mounting Medium (VECTOR Labs). Images were acquired using a Leica SP5 or SP8 confocal microscope. For 5hmC stainings, it was required to perform an additional antigen retrieval step before incubation with primary antibodies: slides with sections were transferred into TE buffer, pH 8, at ~95 °C and microwaved at very low power for 45 min. The following primary antibodies were used: mouse anti-OCT3/4 (1:100, BD Biosciences, O50808), rat anti-BLIMP1 (1:50, eBioscience, clone 6D3, 14-5963), rabbit anti-AP-2γ (1:250, SantaCruz, sc-8977), rabbit anti-PRDM14 (1:250, a gift from D. Reinberg), rabbit anti-DAZL (1:500, Abcam, ab34139), mouse anti-H3K9me2 (1:250, Abcam, ab1220 and 1:500, Millipore, 07-441), rabbit anti-H3K27me3 (1:500, Millipore, 07-449), rabbit anti-TET1 (1:500, Millipore, 09-872), rabbit anti-5hmC (1:500, Active Motif, 39791), goat anti-KLF4 (1:100, R&D, AF3158), rabbit anti-H3S10ph (1:500, Millipore, 06-5770), mouse anti-γH2A.X (1:250, Millipore, 05-636), rat anti-GFP (1:500, Nakalai Tesque, GF090R). Alexa Fluor488 and 568 were used as secondary antibodies (1:500, Life Technologies). All quantifications were preformed using Fiji34. The DAPI, H3S10ph and γH2A.X channels were processed by applying a Gaussian Blur (H3S10ph staining: DAPI/H3S10ph: σ 0.5/1.1; DAPI/ γH2A.X: σ 1.0/1.5) to reduce noise. The images were then binarized using the Otsu thresholding algorithm and holes were filled before the total signal area was measured. In day-6 Prdm1−/− plus Dox aggregates, many cells underwent cell death. Therefore, nuclei with bright discrete spots of DAPI signal, which indicates chromatin condensation, were excluded from the analysis. The diameter of ~10 cells was measured and used to calculate the average area of one cell to estimate the number of cells in the field of view (DAPI+ area/area of one cell). For all other quantifications on a single-cell level, we developed ‘Object Scan’, which is an object mapping and analysis plugin for Fiji that combines advanced functions with a user-friendly interface. Images are processed with a choice of feature enhancement algorithms, objects are identified by patch sampling to detect intensity edges based on the local energy gradient, and the generated two-dimensional masks are clustered in three dimensions to define the final object map for analysis. We used Object Scan to carry out DoG processing and contained signal analysis using the DAPI channel for object mapping, watershed segmentation, a scan radius of one and the following channel specific settings: edge gradient = 10, estimated object radius = 9 μm. The results were scale normalized (X − X /X − X ) to the range 0 to 1 for comparison. Student’s t-test was used to test for significance. The Object Scan plugin is available from this link: http://www.gurdon.cam.ac.uk/stafflinks/downloadspublic/imaging-plugins. Low cell number ChIP-qPCR was performed as previously described35. 3 × 105 cells per ChIP were fixed in 1% formaldehyde (room temperature, 10 min), quenched with 1 vol. of 250 mM glycine (room temperature, 5 min), and rinsed with chilled TBSE buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA) twice before storage at −80 °C. After thawing the cells on ice, fixed cells were lysed with 100 μl 1% SDS lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, Roche protease inhibitor cocktail; on ice, 5 min) and then centrifuged (2,000 r.p.m., 10 min). Pellet was resuspended in 100 μl of dilution buffer (16.7 mM Tris-HCl, pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS, Roche protease inhibitor cocktail). Samples were sonicated nine times (30-s pulses with 30-s break interval) using the Bioruptor water bath sonicator (Diagenode). Chromatin extracts were then precleared with Dynal Magnetic Beads (Invitrogen) (4 °C, 1 h) followed by centrifugation (2,000 r.p.m., 30 min). Supernatant (precleared chromatin) was immunoprecipitated overnight with Dynal Magnetic Beads coupled with anti-NANOG antibody (1 μg per ChIP, Cosmo Bio Co., RCAB0001P) or normal rabbit serum (1 μg per ChIP). On the next day, beads were washed (nutate in wash buffer for 5 min at 4 °C) in low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl) and LiCl buffer (0.25 M LiCl, 1% NP400, 1% Na deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), for a total of three washes. Following an additional wash in TE, elution was performed in a PCR machine (68 °C, 10 min). After digesting and reverse crosslinking (with Proteinase K at 42 °C for 2 h and 68 °C for 6 h) DNA was purified (phenol-chloroform extraction) and used for qPCR analysis. For the negative control region, we used the Snai3 locus as described previously36. Student’s t-test was used to test for significance. The same protocol was used for the SOX2 ChIP with some deviations. Day-2 EpiLCs were aggregated in low-binding plates for 6 h in the presence of 200 ng ml−1 of Dox before collection. 5 × 106 ES cells and EpiLCs, respectively, were fixed and processed as described earlier. Samples were sonicated 20 times (30-s pulses with 30-s break interval) using a Bioruptor water bath sonicator (Diagenode). Samples were divided for immunoprecipitations with SOX2 antibody (10 μg per ChIP, Santa Cruz, sc-17320 X) or normal rabbit IgG (10 μg per ChIP, Santa Cruz, sc-2027 X) as a negative control. Beads were washed with low-salt buffer, and twice with high-salt buffer for 10 min each. The beads were rinsed in TE, resuspended in Proteinase K digestion buffer (20 mM HEPES, 1 mM EDTA, 0.5% SDS) with 2 μl of 10 mg ml−1 Proteinase K and incubated for 15 min at 50 °C. In parallel, 2 μl of 10 mg ml−1 Proteinase K was added to the saved input samples. Three microlitres 5 M NaCl was added to the supernatants and the input samples. To reverse the crosslinks, samples were incubated at 42 °C for 2 h and 68 °C overnight. Next day, the DNA was purified using Agencourt Ampure XP beads (Beckman Coulter) according to the manufacturer’s instructions. The purified DNA was used for qPCR analysis. For the negative control region, we used the Snai3 locus. Student’s t-test was used to test for significance. The primer sequences used for RT–qPCRs are listed in Supplementary Table 1. The NANOG ChIP for subsequent sequencing was performed as described earlier with some deviations. Day-1 or day-2 EpiLCs were aggregated in low-binding plates for 3 h in the presence of 200 ng ml−1 of Dox. ES cells and EpiLCs were fixed and processed as described earlier. 3 × 106 fixed cells were lysed with 1 ml 1% SDS lysis buffer and then centrifuged (2,000 r.p.m., 15 min). Nuclear fraction was resuspended in 0.9 ml of dilution buffer. Samples were sonicated ten times (30-s pulses with 30-s break interval) using a Bioruptor water bath sonicator (Diagenode). Immunoprecipitations were performed with anti-NANOG antibody (2 μg per ChIP, Cosmo Bio Co., RCAB0001P). After elution, samples were digested with Proteinase K and reverse crosslinked for 6 h at 68 °C. Twelve nanograms of purified DNA was used for library preparation using Ovation Ultralow DR Multiplex System (Nugen). Once prepared, library was size selected and sequenced using HiSeq2000 with single-end 50 nucleotides read length. ChIP-seq reads were aligned with the bwa aligner (http://bio-bwa.sourceforge.net) to the mouse reference genome (GRCm38/mm10). Peaks were called with MACS (version 2.1.0; https://github.com/taoliu/MACS) and visualized using the Integrative Genomics Viewer (https://www.broadinstitute.org/igv/). Peak regions from two biological replicates were intersected using bedops (http://bedops.readthedocs.org). Overlapping peak regions with peak summits within <50 nucleotides distance in both replicates were retained. Peak regions from the three cell types were merged. Differences in ChIP-seq read intensities on peak regions were evaluated by using diffReps (http://code.google.com/p/diffreps) and MACS (macs2 bdgdiff). High-confidence sets of differentially bound regions that were detected by both methods were selected for further analysis by applying the following thresholds for diffReps: pValue <0.001 and abs(log2FC) > 1. Previously published H3K27ac ChIP-seq data sets9, 30 were aligned to the mouse reference genome in a similar manner as described earlier, and H3K27ac enrichment (log(ChIP/input) values were determined on NANOG peak regions. High-confidence MACS peaks, for which the distance of the peak summits in both replicates was <50 nucleotides, were selected. De novo motifs were determined with HOMER (http://homer.salk.edu/homer) in the 2,000 top-enriched peaks in ES cells, day-1 and day-2 EpiLCs for both repeat-masked and repeat-unmasked regions within ± 50 nucleotides of the peak summit. Genomic regions containing putative enhancers of Prdm1 and Prdm14, as well as a negative control region depleted of enhancer signatures, were amplified from mouse E14 ES-cell genomic DNA. These regions were cloned into a PiggyBAC-based firefly luciferase reporter plasmid upstream of a minimal TK promoter. Stable luciferase reporter GOF-GFP ES cell lines, which can overexpress Nanog, Nanog/Sox2 or Brachyury upon Dox addition, were established. Cell pellets were collected from ES cells cultured in N2B27 2i/LIF, day-2 EpiLCs and EpiLCs after PGCLC induction ±Dox at 12/24 h. Luciferase assays were performed with the ONE-Glo Luciferase Assay System (Promega). Protein concentration in each lysate was quantified by Pierce 660 nm Protein Assay (Thermo Scientific). Relative luciferase activities were obtained by dividing luciferase activity by protein concentration in each sample. ES-cell clones carrying both the Nanog transgene and a CAG monomeric Kusabira-orange (mKO) fluorescence reporter were selected by neomycin (Sigma-Aldrich) and zeocine (Life Technologies). Day-4 PGCLCs were induced from day-2 EpiLCs with Nanog and used for derivation of EGCLCs. For ES cells or day-4 PGCLC injections, GOF-GFP ES cells were co-transfected with a vector, which enabled inducible expression of Nanog and constitutive expression of Venus, a variant of eGFP. For day-4 PGCLCs, after induction of PGCLCs with Nanog, cells were stained with PE-conjugated-CD61 antibody (1:10, Biolegend, 104308) and Alexa660-conjugated-SSEA-1 antibody (2.5 μl per 105 cells, eBioscience, clone eBioMC-480, 50-8813) according to the manufacturer’s instructions. Double-positive PGCLC cells were collected by using a S3 cell sorter (Biorad). Embryos for chimaera experiments were obtained from CBA/C57BL/6 F1 crossed with C57BL/6 mice. Blind tests or randomization methods were not used. The sex of embryos was not determined. Manipulations of embryos were performed as described previously37. Briefly, five cells were injected into a morula, which were subsequently cultured in KSOM (Millipore). On the following day, the embryos were transferred into the uteri of pseudopregnant mice. All embryos were analysed 1 week after embryo transfer, which corresponded to E9.5. The CRISPR–Cas9 system was used to generate Nanog-knockout ES cells. Guide RNAs (gRNAs) targeting exon 1 of the Nanog gene were cloned into pX330 (Addgene)38. One microgram of this plasmid was transfected with a pPyCAG-monomeric Kusabira Orange-IRES-Pac plasmid. Transfected cells were selected by puromycin (1 μg ml−1) for 2 days. Clonal Nanog-knockout ES cell lines were established and mutations of Nanog alleles were confirmed by qPCR, western blotting and DNA sequencing. Subsequently, pPBhCMV*1-Nanog-pA plasmid was transfected into those lines with pPyCAG-PBase and pPBhCMV*1-rtTA-IRESNeor to generate Nanog-knockout ES cell lines carrying a Dox-inducible Nanog transgene. Loss of Nanog affected the growth of ES cells. Thus, these cell lines were maintained in N2B27 2i/LIF with a low dose of Dox (100 ng ml−1). gRNA sequences are listed in Supplementary Table 1. 5 × 104 cells were lysed in lysis buffer (50 mM Tris-HCl (pH 8.0), 1% SDS, 10 mM EDTA). Protein concentration was measured by Bicinchoninic Acid Kit (Sigma-Aldrich). The protein amount was adjusted among samples, then 4 × Laemmli buffer was added. Samples were boiled at 95 °C for 5 min. Proteins were separated on 10% acrylamide gels, blotted on Immobilon-P transfer membrane (Millipore). The membrane was blocked with 5% skimmed milk and incubated with primary antibodies: anti-NANOG (1:500, mouse IgG, eBioscience, clone eBioMLC-51, 14-5761), anti-SOX2 (1:500, rabbit IgG, Cell Signaling, 2748), anti-α-tubulin (1:1,000, mouse IgG, Sigma-Aldrich, clone DM1A, T9026). Primary antibodies were detected on X-ray film with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (Dako) followed by detection using Western Detection System (GE Healthcare). For gel source data, see Supplementary Fig. 1. pPBhCMV*1-Nanog-pA, pPBCAG-rtTA-IRESNeor and pPyCAG-PBase were transfected into the Sox2-conditional-knockout ES cell line (2CG2)27. After 1 week of neomycin selection (80 μg ml−1), pooled cells were used for the subsequent experiments. Dexamethasone-inducible Sox2-knockout and Dox-inducible Nanog expression were confirmed by qPCR and western blotting.
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 | October 27, 2016
Dublin, Oct. 27, 2016 (GLOBE NEWSWIRE) -- Research and Markets has announced the addition of the "Genome Editing Global Market-Forecast to 2022" report to their offering. The genome editing global market over the forecast period 2015 to 2022 and the market is expected to grow at a CAGR of 31.1%. The Genome editing global market is segmented based on technology, applications, products, end users and geography. Technologies segment consists of Zinc Finger Nucleases (ZFN), Transcriptor-activator-like effector nuclease (TALEN); Clustered regularly interspaced short palindromic repeats (CRISPR) and other gene modification techniques such as Recombinant adeno-associated virus (rAAV), piggyBac transposon, megatales etc. Applications identified includes, basic research, agriculture biotechnology, animal research, drug discovery and development. Products are classified into reagents, enzymes and consumables, instruments, cell lines & animal models and software. Depending on the end users, the genome editing global market is sectioned into academic & government institutions, pharmaceutical & biotechnology companies, plant biotechnology and others. Increased R&D expenditure and growth of biotechnology and pharmaceutical industries, increasing private and public sector funding, rapid advancements in sequencing and gene editing technologies, non-labeling of gene-edited products as Genetically Modified Organisms (GMOs), applications in various drug discovery processes are some of the factors driving the genome editing global market growth. Factors such as stringent regulatory framework, ethical issues concerning editing human embryo and adverse public perception, unavailability of gene-editing based therapeutics in the market, off-target effects of CRISPR and patent disputes associated with CRISPR technology are hampering the market growth. The genome editing global market is a consolidated market with key players such as Applied Stemcell (U.S.), Cellectis S.A. (France), Genscript (U.S.), Horizon Discovery Group (U.K.), Merck KGaA (Germany), Origene Technologies (U.S.), Sangamo Biosciences (U.S.), System Biosciences (U.S.), Thermo Fisher Scientific (U,S,), Transposagen (U.S.). Key Topics Covered: 1 Executive Summary 2 Introduction 2.1 Key Takeaways 2.2 Report Description 2.3 Markets Covered 2.4 Stakeholders 2.5 Research Methodology 3 Market Overview 3.1 Introduction 3.2 Market Segmentation 3.3 Factors Influencing Market 3.4 Market Dynamics 3.4.1 Drivers And Opportunities 18.104.22.168 Increased R&D Expenditure And GROWth Of Biotechnology And Pharmaceutical Industries. 22.214.171.124 Investments From Both Public And Private Sectors For Gene-Editing Technology 126.96.36.199 Technological Advancements In Sequencing And Gene Editing Technologies 188.8.131.52 Non-Transgenic Breeding Technologies And Gene-Edited Plants Not Labelled As Gmo In Many Countries 184.108.40.206 Improvement In Drug Discovery Process 220.127.116.11 Varied Applications In Drug Discovery And Development, Plant Engineering, Improvement In Animal Traits, Therapeutics 18.104.22.168 Precision Medicine And New Therapeutics For Genetic And Other Disorders 3.4.2 Restraints And Threats 22.214.171.124 Stringent Regulatory Frameworks 126.96.36.199 Ethical Issues Concerning Editing Human Embryo And Adverse Public Perception 188.8.131.52 Unavailability Of Gene-Editing Based Therapeutic Product In The Market And Less Geographic Penetration Due To Uncertain Regulations 184.108.40.206 Patent Dispute Associated With Crispr/Cas 220.127.116.11 Off-Target Effects Of Crispr/Cas9 Genome Editing Technology 3.5 Porter's Five Force Analysis 3.6 Regulatory Affairs 3.7 Patent Analysis 3.8 Funding Scenario 3.9 Collaboration, Joint Venture, Partnership, Agreement 3.1 Market Share Analysis 4 Genome Editing Global Market, By Technology 4.1 Introduction 4.2 Zinc Finger Nuclease (Zfn) 4.3 Transcription Activator-Like Effector Nucleases (Talen) 4.4 Clustered Regularly Interspaced Short Palindromic Repeats (Crispr/Cas9) 4.4.1 Wt Crispr/Cas9 4.4.2 Crispr/Cas9 Nickase 4.4.3 Crispr Dcas9 4.5 Others 4.5.1 Recombinant Adeno-Associated Virus (R Aav) 4.5.2 Piggybac Transposase And Sleeping Beauty Transposon 4.5.3 Arcus (Homing Endonuclease) And Megatal 4.5.4 Targatt And Rapid Trait Development System (Rtds) 5 Genome Editing Global Market, By Application 5.1 Introduction 5.2 Basic Research 5.2.1 Transcription Activation/Repression 5.2.2 Genomic Screening 5.2.3 Genomic Visualization 5.3 Plant Biotechnology/Agriculture 5.4 Animal Biotechnology 5.4.1 Animal Health 5.4.2 Livestock 5.4.3 Other 5.5 Drug Discovery And Development 5.5.1 Pre-Clinical 5.5.2 Clinical 6 Genome Editing Global Market, By Products And Services 6.1 Introduction 6.2 Reagents, Enzymes And Consumables 6.2.1 Genome Editing Tools And Kits 6.2.2 Delivery Tools 6.2.3 Other Reagents, Enzymes And Consumables 6.3 Cell Lines And Animal Models 6.4 Genome Editing Services 6.5 Instruments 6.6 Software 7 Genome Editing Global Market, By End Users 7.1 Introduction 7.2 Academic And Government Research Institutes 7.3 Plant Biotechnology Companies 7.4 Pharmaceutical And Biotechnology Companies 7.5 Others 7.5.1 Animal Biotechnology 7.5.2 Contract Research Organizations (Cro) 8 Genome Editing Global Market, By Region 9 Competitive Landscape 9.1 Introduction 9.1.1 Licensing Agreement And Others As A Major GROWth Strategy Of Market Players 9.2 Licensing Agreement 9.3 Other Developments 9.4 Agreement/Collaboration/Partnership/Joint Venture 9.5 Approval 9.6 New Product Launch 9.7 Acquisitions 10 Major Companies - Abcam - Addgene - Agilent Technologies - Applied Biological Materials (Abm) - Axol Bioscience Ltd - Bio-Rad Laboratories - Caribou Biosciences - Charles River Laboratories - Cibus - Crispr Therapeutics - Desktop Genetics - Discovery Genomics - Editas Medicine - Eisai - Ers Genomics - GE Healthcare - Genecopoeia - Genus Inc - Homology Medicines - Illumina - Integrated Dna Technologies (Idt) - Intellia Therapeutics - Lonza - New England Biolabs (Neb) - Pacific Bioscience - Precision Biosciences - Proteonic B.V. - Recombinetics - Shire Plc - Stemgent, Inc - Syngenta - Takara Bio - Targetgene Biotechnologies - Tide - Toolgen - Transgenic - Viravecs Labs For more information about this report visit http://www.researchandmarkets.com/research/c2g57b/genome_editing
News Article | March 4, 2016
Human ES cell line H9 (WA-09) and derivatives (SOX10::GFP; SYN::ChR2-EYFP; SYN::EYFP;PHOX2B:GFP;EF1::RFP Ednrb−/−) as well as two independent human iPS cell lines (healthy and familial dysautonomia, Sendai-based, OMSK (Cytotune)) were maintained on mouse embryonic fibroblasts (Global Stem) in knockout serum replacement (KSR; Life Technologies, 10828-028) containing human ES cell medium as described previously7. Cells were subjected to mycoplasma testing at monthly intervals and short tandem repeats (STR) profiled to confirm cell identity at the initiation of the study. Human ES cells were plated on matrigel (BD Biosciences, 354234)-coated dishes (105 cells cm−2) in ES cell medium containing 10 nM FGF2 (R&D Systems, 233-FB-001MG/CF). Differentiation was initiated in KSR medium (knockout DMEM plus 15% KSR (Life Technologies, 10828-028), l-glutamine (Life Technologies, 25030-081), NEAA (Life Technologies, 11140-050)) containing LDN193189 (100 nM, Stemgent) and SB431542 (10 μM, Tocris). The KSR medium was gradually replaced with increasing amounts of N2 medium from day 4 to day 10 as described previously7. For CNC induction, cells were treated with 3 μM CHIR99021 (Tocris Bioscience, 4423) in addition to LDN193189 and SB431542 from day 2 to day 11. ENC differentiation involves additional treatment with retinoic acid (1 μM) from day 6 to day 11. For deriving MNCs, LDN193189 is replaced with BMP4 (10 nM, R&D, 314-BP) and EDN3 (10 nM, American Peptide company, 88-5-10B) from day 6 to day 11 (ref. 3). The differentiated cells are sorted for CD49D at day 11. CNS precursor control cells were generated by treatment with LDN193189 and SB431542 from day 0 to day 11 as previously described7. Throughout the manuscript, day 0 is the day the medium is switched from human ES cell medium to LDN193189 and SB431542 containing medium. Days of differentiation in text and figures refer to the number of days since the pluripotent stage (day 0). For immunofluorescence, the cells were fixed with 4% paraformaldehyde (Affymetrix-USB, 19943) for 20 min, then blocked and permeabilized using 1% bovine serum albumin (BSA) (Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). The cells were then incubated in primary antibody solutions overnight at 4 °C and stained with fluorophore-conjugated secondary antibodies at room temperature for 1 h. The stained cells were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, the cells are dissociated with Accutase (Innovative Cell Technologies, AT104) and fixed and permeabilized using BD Cytofix/Cytoperm (BD Bioscience, 554722) solution, then washed, blocked and permeabilized using BD Perm/Wash buffer (BD Bioscience, 554723) according to manufacturer’s instructions. The cells are then stained with primary (overnight at 4 °C) and secondary (30 min at room temperature) antibodies and analysed using a flow Cytometer (Flowjo software). A list of primary antibodies and working dilutions is provided in Supplementary Table 4. The PHOX2A antibody was provided by J.-F. Brunet (rabbit, 1:800 dilution). Fertilized eggs (from Charles River Farms) were incubated at 37 °C for 50 h before injections. A total of 2 × 105 CD49D-sorted, RFP-labelled NC cells were injected into the intersomitic space of the vagal region of the embryos targeting a region between somite 2 and 6 (HH 14 embryo, 20–25 somite stage). The embryos were collected 36 h later for whole-mount epifluorescence and histological analyses. For RNA sequencing, total RNA was extracted using RNeasy RNA purification kit (Qiagen, 74106). For qRT–PCR assay, total RNA samples were reverse transcribed to cDNA using Superscript II Reverse Transcriptase (Life Technologies, 18064-014). qRT–PCR reactions were set up using QuantiTect SYBR Green PCR mix (Qiagen, 204148). Each data point represents three independent biological replicates. ENC cells from the 11-day induction protocol were aggregated into 3D spheroids (5 million cells per well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF). After 4 days of suspension culture, the spheroids are plated on poly-ornithine/laminin/fibronectin (PO/LM/FN)-coated dishes (prepared as described previously26) in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1–2 weeks. The cells were fixed for immunostaining or collected for gene expression analysis at days 25, 40 and 60 of differentiation. Mesoderm specification is carried out in STEMPRO-34 (Gibco, 10639-011) medium. The ES cells are subjected to activin A treatment (100 ng ml−1, R&D, 338-AC-010) for 24 h followed by BMP4 treatment (10 ng ml−1, R&D, 314-BP) for 4 days9. The cells are then differentiated into SMC progenitors by treatment with PDGF-BB (5 ng ml−1, Peprotech, 100-14B), TGFb3 (5 ng ml−1, R&D systems, 243-B3-200) and 10% FBS. The SMC progenitors are expandable in DMEM supplemented with 10% FBS. The SMC progenitors were plated on PO/LM/FN-coated culture dishes (prepared as described previously26) 3 days before addition of ENC-derived neurons. The neurons were dissociated (using accutase, Innovative Cell Technologies, AT104) at day 30 of differentiation and plated onto the SMC monolayer cultures. The culture is maintained in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). Functional connectivity was assessed at 8–16 weeks of co-culture. SMC-only and SMC-ENC-derived neuron co-cultures were subjected to acetylcholine chloride (50 μM, Sigma, A6625), carbamoylcholine chloride (10 μM, Sigma,C4382) and KCl (55 mM, Fisher Scientific, BP366–500) treatment, 3 months after initiating the co-culture. Optogenetic stimulations were performed using a 450-nm pigtailed diode pumped solid state laser (OEM Laser, PSU-III LED, OEM Laser Systems, Inc.) achieving an illumination between 2 and 4 mW mm−2. The pulse width was 4 ms and stimulation frequencies ranged from 2 to 10 Hz. For the quantification of movement, images were assembled into a stack using Metamorph software and regions with high contrast were identified (labelled yellow in Supplementary Fig. 5). The movement of five representative high-contrast regions per field was automatically traced (Metamorph software). Data are presented in kinetograms as movement in pixels in x and y direction (distance) with respect to the previous frame. We used the previously described method for generation of tissue-engineered colon11. In brief, the donor colon tissue was collected and digested into organoid units using dispase (Life Technologies, 17105-041) and collagenase type 1 (Worthington, CLS-1). The organoid units were then mixed immediately (without any in vitro culture) with CD49D-purified human ES-cell-derived ENC precursors (day 15 of differentiation) and seeded onto biodegradable polyglycolic acid scaffolds (2-mm sheet thickness, 60 mg cm−3 bulk density; porosity >95%, Concordia Fibres) shaped into 2 mm long tubes with poly-l lactide (PLLA) (Durect Corporation). The seeded scaffolds were then placed onto and wrapped in the greater omentum of the adult (>2 months old) NSG mice. Just before the implantation, these mice were irradiated with 350 cGy. The seeded scaffolds were differentiated into colon-like structures inside the omentum for 4 additional weeks before they were surgically removed for tissue analysis. All mouse procedures were performed following NIH guidelines, and were approved by the local Institutional Animal Care and Use Committee (IACUC), the Institutional Biosafety Committee (IBC) as well as the Embryonic Stem Cell Research Committee (ESCRO). We used 3–6-week-old male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice or 2–3-week-old Ednrbs-l/s-l (SSL/LeJ) mice27 (n = 12, 6 male, 6 female) for these studies. Animal numbers were based on availability of homozygous hosts and on sufficient statistical power to detect large effects between treatment versus control (Ednrbs-l/s-l) as well as for demonstrating robustness of migration behaviour (NSG). Animals were randomly selected for the various treatment models (NSG and Ednrbs-l/s-l) but assuring for equal distribution of male/female ratio in each group (Ednrbs-l/s-l). All in vivo experiments were performed in a blinded manner. Animals were anaesthetized with isoflurane (1%) throughout the procedure, a small abdominal incision was made, abdominal wall musculature lifted and the caecum is exposed and exteriorized. Warm saline is used to keep the caecum moist. Then 20 μl of cell suspension (2–4 million RFP+ CD49D-purified human ES-cell-derived ENC precursors) in 70% Matrigel (BD Biosciences, 354234) in PBS or 20 μl of 70% Matrigel in PBS only (control-grafted animals) were slowly injected into the caecum (targeting the muscle layer) using a 27-gauge needle. Use of 70% matrigel as carrier for cell injection assured that the cells stayed in place after the injection and prevented backflow into the peritoneum. After injection that needle was withdrawn, and a Q-tip was placed over the injection site for 30 s to prevent bleeding. The caecum was returned to the abdominal cavity and the abdominal wall was closed using 4-0 vicryl and a taper needle in an interrupted suture pattern and the skin was closed using sterile wound clips. After wound closure animals were put on paper on top of their bedding and attended until conscious and preferably eating and drinking. The tissue was collected at different time points (ranging from two weeks to four months) after transplantation for histological analysis. Ednrbs-l/s-l mice were immunosuppressed by daily injections of cyclosporine (10 mg kg−1 i.p, Sigma, 30024). The collected colon samples were fixed in 4% paraformaldehyde at 4 °C overnight before imaging. Imaging is performed using Maestro fluorescence imaging system (Cambridge Research and Instrumentation). The tissue samples were incubated in 30% sucrose (Fisher Scientific, BP220-1) solutions at 4 °C for 2 days, and then embedded in OCT (Fisher Scientific, NC9638938) and cryosectioned. The sections were then blocked with 1% BSA (Thermo Scientific, 23209) and permeabilized with 0.3% Triton X-100 (Sigma, T8787). The sections are then stained with primary antibody solution at 4 °C overnight and fluorophore-conjugated secondary antibody solutions at room temperature for 30 min. The stained sectioned were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before they were mounted with Vectashield Mounting Medium (vector, H1200) and imaged using fluorescent (Olympus IX70) or confocal microscopes (Zeiss SP5). Mice are gavaged with 0.3 ml of dye solution containing 6% carmine (Sigma, C1022-5G), 0.5% methylcellulose (Sigma, 274429-5G) and 0.9 NaCl, using a #24 round-tip feeding needle. The needle was held inside the mouse oesophagus for a few seconds after gavage to prevent regurgitation. After 1 h, the stool colour was monitored for gavaged mice every 10 min. For each mouse, total gastrointestinal transit time is between the time of gavage and the time when red stool is observed. The double nickase CRISPR/Cas9 system28 was used to target the EDNRB locus in EF1–RFP H9 human ES cells. Two guide RNAs were designed (using the CRISPR design tool; http://crispr.mit.edu/) to target the coding sequence with PAM targets ~20 base pairs apart (qRNA #1 target specific sequence: 5′-AAGTCTGTGCGGACGCGCCCTGG-3′, RNA #2 target specific sequence: 5′-CCAGATCCGCGACAGGCCGCAGG-3′). The cells were transfected with guide RNA constructs and GFP-fused Cas9-D10A nickase. The GFP-expressing cells were FACS purified 24 h later and plated in low density (150 cells cm−2) on mouse embryonic fibroblasts. The colonies were picked 7 days later and passaged twice before genomic DNA isolation and screening. The targeted region of EDNRB gene was PCR amplified (forward primer: 5′-ACGCCTTCTGGAGCAGGTAG-3′, reverse primer: 5′-GTCAGGCGGGAAGCCTCTCT-3′) and cloned into Zero Blunt TOPO vector (Invitrogen, 450245). To ensure that both alleles (from each ES cell colony) are represented and sequenced, we picked 10 bacterial clones (for each ES cell clone) for plasmid purification and subsequent sequencing. The clones with bi-allelic nonsense mutations were expanded and differentiated for follow-up assays. The ENC cells are plated on PO/LM/FN coated (prepared as described previously26) 96-well or 48-well culture plates (30,000 cm−2). After 24 h, the culture lawn is scratched manually using a pipette tip. The cells are given an additional 24–48 h to migrate into the scratch area and fixed for imaging and quantification. The quantification is based on the percentage of the nuclei that are located in the scratch area after the migration period. The scratch area is defined using a reference well that was fixed immediately after scratching. Migration of cells was quantified using the open source data analysis software KNIME29 (http://knime.org) with the ‘quantification in ROI’ plug-in as described in detail elsewhere30. To quantify proliferation, FACS-purified ENC cells were assayed using CyQUANT NF cell proliferation Assay Kit (Life Technologies, C35006) according to manufacturer’s instructions. In brief, to generate a standard, cells were plated at various densities and stained using the fluorescent DNA binding dye reagent. Total fluorescence intensity was then measured using a plate reader (excitation at 485 nm and emission detection at 530 nm). After determining the linear range, the CD49D+ wild-type and Ednrb−/− ENC precursors were plated (6,000 cell cm−2) and assayed at 0, 24, 48 and 72 h. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To monitor the viability of wild-type and Ednrb−/− ENC precursors, cells were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 cytotoxicity assay kit (Promega, G1780). In brief, the cells are plated in 96-well plates at 30,000 cm−2. The supernatant and the cell lysate is collected 24 h later and assayed for LDH activity using a plate reader (490 nm absorbance). Viability is calculated by dividing the LDH signal of the lysate by total LDH signal (from lysate plus supernatant). The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. The chemical compound screening was performed using the Prestwick Chemical Library. The ENC cells were plated in 96-well plates (30,000 cm−2) and scratched manually 24 h before addition of the compounds. The cells were treated with two concentrations of the compounds (10 μM and 1 μM). The plates were fixed 24 h later for total plate imaging. The compounds were scored based on their ability to promote filling of the scratch in 24 h. The compounds that showed toxic effects (based on marked reduction in cell numbers assessed by DAPI staining) were scored 0, compounds with no effects were scored 1, compounds with moderate effects were scored 2, and compounds with strong effects (that resulted in complete filling of the scratch area) were scored 3 and identified as hit compounds. The hits were further validated to ensure reproducibility. The cells were treated with various concentrations of the selected hit compound (pepstatin A) for dose response analysis. The optimal dose (10 μM based on optimal response and viability) was used for follow-up experiments. For the pre-treatment experiments, cells were CD49D purified at day 11 and treated with pepstatin A from day 12 to day 15 followed by transplantation into the colon wall of NSG mice. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To inhibit BACE2, the ENC precursors were treated with 1 μM β-secretase inhibitor IV (CAS 797035-11-1; Calbiochem). To knockdown BACE2, cells were dissociated using accutase (Innovative Cell Technologies, AT104) and reverse-transfected (using Lipofectamine RNAiMAX-Life Technologies, 13778-150) with an siRNA pool (SMARTpool: ON-TARGETplus BACE2 siRNA, Dharmacon, L-003802-00-0005) or four different individual siRNAs (Dharmacon, LQ-003802-00-0002, 2 nmol). The knockdown was confirmed by qRT–PCR measurement of BACE2 mRNA levels in cells transfected with the BACE2 siRNAs versus the control siRNA pool (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05). The transfected cells were scratched 24 h after plating and fixed 48 h later for migration quantification. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. Data are presented as mean ± s.e.m. and were derived from at least three independent experiments. Data on replicates (n) is given in figure legends. Statistical analysis was performed using the Student’s t-test (comparing two groups) or ANOVA with Dunnett test (comparing multiple groups against control). Distribution of the raw data approximated normal distribution (Kolmogorov–Smirnov normality test) for data with sufficient number of replicates to test for normality. Survival analysis was performed using a log-rank (Mantel–Cox) test. Z-scores for primary hits were calculated as Z = (x − μ)/σ, in which x is the migration score value and is 3 for all hit compounds; μ is the mean migration score value, and σ is the standard deviation for all compounds and DMSO controls (n = 224).
News Article | November 21, 2016
Notes: Sales, means the sales volume of Alkaline Phosphatase Kit Revenue, means the sales value of Alkaline Phosphatase Kit This report studies sales (consumption) of Alkaline Phosphatase Kit in Global market, especially in United States, China, Europe, Japan, focuses on top players in these regions/countries, with sales, price, revenue and market share for each player in these regions, covering Interlab Srl GeneTex Stemgent EMD Millipore Corporation Formosa Biomedical Technology BioVision Abcam Eurogentec AnaSpec Arrayit Corporation ScienCell Research Laboratories Beyotime BioAssay Systems Market Segment by Regions, this report splits Global into several key Regions, with sales (consumption), revenue, market share and growth rate of Alkaline Phosphatase Kit in these regions, from 2011 to 2021 (forecast), like United States China Europe Japan Split by product Types, with sales, revenue, price and gross margin, 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 Alkaline Phosphatase Kit in each application, can be divided into Application 1 Application 2 Application 3 Global Alkaline Phosphatase Kit Sales Market Report 2016 1 Alkaline Phosphatase Kit Overview 1.1 Product Overview and Scope of Alkaline Phosphatase Kit 1.2 Classification of Alkaline Phosphatase Kit 1.2.1 Type I 1.2.2 Type II 1.2.3 Type III 1.3 Application of Alkaline Phosphatase Kit 1.3.1 Application 1 1.3.2 Application 2 1.3.3 Application 3 1.4 Alkaline Phosphatase Kit Market by Regions 1.4.1 United States Status and Prospect (2011-2021) 1.4.2 China Status and Prospect (2011-2021) 1.4.3 Europe Status and Prospect (2011-2021) 1.4.4 Japan Status and Prospect (2011-2021) 1.5 Global Market Size (Value and Volume) of Alkaline Phosphatase Kit (2011-2021) 1.5.1 Global Alkaline Phosphatase Kit Sales and Growth Rate (2011-2021) 1.5.2 Global Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) 2 Global Alkaline Phosphatase Kit Competition by Manufacturers, Type and Application 2.1 Global Alkaline Phosphatase Kit Market Competition by Manufacturers 2.1.1 Global Alkaline Phosphatase Kit Sales and Market Share of Key Manufacturers (2011-2016) 2.1.2 Global Alkaline Phosphatase Kit Revenue and Share by Manufacturers (2011-2016) 2.2 Global Alkaline Phosphatase Kit (Volume and Value) by Type 2.2.1 Global Alkaline Phosphatase Kit Sales and Market Share by Type (2011-2016) 2.2.2 Global Alkaline Phosphatase Kit Revenue and Market Share by Type (2011-2016) 2.3 Global Alkaline Phosphatase Kit (Volume and Value) by Regions 2.3.1 Global Alkaline Phosphatase Kit Sales and Market Share by Regions (2011-2016) 2.3.2 Global Alkaline Phosphatase Kit Revenue and Market Share by Regions (2011-2016) 2.4 Global Alkaline Phosphatase Kit (Volume) by Application Figure Picture of Alkaline Phosphatase Kit Table Classification of Alkaline Phosphatase Kit Figure Global Sales Market Share of Alkaline Phosphatase Kit by Type in 2015 Figure Type I Picture Figure Type II Picture Table Applications of Alkaline Phosphatase Kit Figure Global Sales Market Share of Alkaline Phosphatase Kit by Application in 2015 Figure Application 1 Examples Figure Application 2 Examples Figure United States Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) Figure China Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) Figure Europe Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) Figure Japan Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) Figure Global Alkaline Phosphatase Kit Sales and Growth Rate (2011-2021) Figure Global Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) Table Global Alkaline Phosphatase Kit Sales of Key Manufacturers (2011-2016) Table Global Alkaline Phosphatase Kit Sales Share by Manufacturers (2011-2016) Figure 2015 Alkaline Phosphatase Kit Sales Share by Manufacturers Figure 2016 Alkaline Phosphatase Kit Sales Share by Manufacturers Table Global Alkaline Phosphatase Kit Revenue by Manufacturers (2011-2016) Table Global Alkaline Phosphatase Kit Revenue Share by Manufacturers (2011-2016) Table 2015 Global Alkaline Phosphatase Kit Revenue Share by Manufacturers Table 2016 Global Alkaline Phosphatase Kit Revenue Share by Manufacturers Table Global Alkaline Phosphatase Kit Sales and Market Share by Type (2011-2016) Table Global Alkaline Phosphatase Kit Sales Share by Type (2011-2016) Figure Sales Market Share of Alkaline Phosphatase Kit by Type (2011-2016) Figure Global Alkaline Phosphatase Kit Sales Growth Rate by Type (2011-2016) Table Global Alkaline Phosphatase Kit Revenue and Market Share by Type (2011-2016) Table Global Alkaline Phosphatase Kit Revenue Share by Type (2011-2016) Figure Revenue Market Share of Alkaline Phosphatase Kit by Type (2011-2016) Figure Global Alkaline Phosphatase Kit Revenue Growth Rate by Type (2011-2016) Table Global Alkaline Phosphatase Kit Sales and Market Share by Regions (2011-2016) Table Global Alkaline Phosphatase Kit Sales Share by Regions (2011-2016) Figure Sales Market Share of Alkaline Phosphatase Kit by Regions (2011-2016) Figure Global Alkaline Phosphatase Kit Sales Growth Rate by Regions (2011-2016) Table Global Alkaline Phosphatase Kit Revenue and Market Share by Regions (2011-2016) …. 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News Article | December 5, 2016
This report studies sales (consumption) of Alkaline Phosphatase Kit in Europe market, especially in Germany, UK, France, Russia, Italy, Benelux and Spain, focuses on top players in these countries, with sales, price, revenue and market share for each player in these Countries, covering Interlab Srl GeneTex Stemgent EMD Millipore Corporation Formosa Biomedical Technology BioVision Abcam Eurogentec AnaSpec Arrayit Corporation ScienCell Research Laboratories Beyotime BioAssay Systems View Full Report With Complete TOC, List Of Figure and Table: http://globalqyresearch.com/europe-alkaline-phosphatase-kit-market-report-2016 Market Segment by Countries, this report splits Europe into several key Countries, with sales (consumption), revenue, market share and growth rate of Alkaline Phosphatase Kit in these countries, from 2011 to 2021 (forecast), like Germany France UK Russia Italy Spain Benelux Split by product type, with sales, 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 sales, market share and growth rate of Alkaline Phosphatase Kit in each application, can be divided into Application 1 Application 2 Application 3 Europe Alkaline Phosphatase Kit Market Report 2016 1 Alkaline Phosphatase Kit Overview 1.1 Product Overview and Scope of Alkaline Phosphatase Kit 1.2 Classification of Alkaline Phosphatase Kit 1.2.1 Type I 1.2.2 Type II 1.2.3 Type III 1.3 Application of Alkaline Phosphatase Kit 1.3.1 Application 1 1.3.2 Application 2 1.3.3 Application 3 1.4 Alkaline Phosphatase Kit Market by Countries 1.4.1 Germany Status and Prospect (2011-2021) 1.4.2 France Status and Prospect (2011-2021) 1.4.3 UK Status and Prospect (2011-2021) 1.4.4 Russia Status and Prospect (2011-2021) 1.4.5 Italy Status and Prospect (2011-2021) 1.4.6 Spain Status and Prospect (2011-2021) 1.4.7 Benelux Status and Prospect (2011-2021) 1.5 Europe Market Size (Value and Volume) of Alkaline Phosphatase Kit (2011-2021) 1.5.1 Europe Alkaline Phosphatase Kit Sales and Growth Rate (2011-2021) 1.5.2 Europe Alkaline Phosphatase Kit Revenue and Growth Rate (2011-2021) 10 Europe Alkaline Phosphatase Kit Manufacturers Analysis 10.1 Interlab Srl 10.1.1 Company Basic Information, Manufacturing Base and Competitors 10.1.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.1.2.1 Type I 10.1.2.2 Type II 10.1.3 Interlab Srl Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.1.4 Main Business/Business Overview 10.2 GeneTex 10.2.1 Company Basic Information, Manufacturing Base and Competitors 10.2.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.2.2.1 Type I 10.2.2.2 Type II 10.2.3 GeneTex Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.2.4 Main Business/Business Overview 10.3 Stemgent 10.3.1 Company Basic Information, Manufacturing Base and Competitors 10.3.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.3.2.1 Type I 10.3.2.2 Type II 10.3.3 Stemgent Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.3.4 Main Business/Business Overview 10.4 EMD Millipore Corporation 10.4.1 Company Basic Information, Manufacturing Base and Competitors 10.4.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.4.2.1 Type I 10.4.2.2 Type II 10.4.3 EMD Millipore Corporation Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.4.4 Main Business/Business Overview 10.5 Formosa Biomedical Technology 10.5.1 Company Basic Information, Manufacturing Base and Competitors 10.5.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.5.2.1 Type I 10.5.2.2 Type II 10.5.3 Formosa Biomedical Technology Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.5.4 Main Business/Business Overview 10.6 BioVision 10.6.1 Company Basic Information, Manufacturing Base and Competitors 10.6.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.6.2.1 Type I 10.6.2.2 Type II 10.6.3 BioVision Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.6.4 Main Business/Business Overview 10.7 Abcam 10.7.1 Company Basic Information, Manufacturing Base and Competitors 10.7.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.7.2.1 Type I 10.7.2.2 Type II 10.7.3 Abcam Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.7.4 Main Business/Business Overview 10.8 Eurogentec AnaSpec 10.8.1 Company Basic Information, Manufacturing Base and Competitors 10.8.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.8.2.1 Type I 10.8.2.2 Type II 10.8.3 Eurogentec AnaSpec Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.8.4 Main Business/Business Overview 10.9 Arrayit Corporation 10.9.1 Company Basic Information, Manufacturing Base and Competitors 10.9.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.9.2.1 Type I 10.9.2.2 Type II 10.9.3 Arrayit Corporation Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.9.4 Main Business/Business Overview 10.10 ScienCell Research Laboratories 10.10.1 Company Basic Information, Manufacturing Base and Competitors 10.10.2 Alkaline Phosphatase Kit Product Type, Application and Specification 10.10.2.1 Type I 10.10.2.2 Type II 10.10.3 ScienCell Research Laboratories Alkaline Phosphatase Kit Sales, Revenue, Price and Gross Margin (2011-2016) 10.10.4 Main Business/Business Overview 10.11 Beyotime 10.12 BioAssay Systems Global QYResearch ( http://globalqyresearch.com/ ) is the one spot destination for all your research needs. 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News Article | November 23, 2016
The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. No statistical methods were used to determine sample size. Intestinal crypts were extracted from 5–10-week-old heterozygous Lgr5–eGFP-IRES-CreERT2 mice (Jackson Laboratory), following animal experimentation protocols prescribed by EPFL and FELASA. Murine intestinal crypts were isolated following procedures described previously. In brief, the proximal part of the intestine was collected, opened longitudinally and washed with ice-cold PBS. The luminal side of the intestine was scraped using a glass slide to remove luminal content and villous structures. After washing with ice-cold PBS again, the intestine was cut into 2–4 mm pieces with scissors. The pieces were transferred to a 50 ml Falcon tube and further washed with cold PBS (5–10 times) with gentle vortexing. Intestinal fragments were then incubated in 20 mM EDTA/PBS for 20 min on ice. EDTA was removed, 10 ml of cold PBS was added and crypts were released by manual shaking of the suspension for 5 min. The supernatant was collected and passed through a 70-μm strainer (BD Biosciences). The remaining tissue fragments were resuspended in 10 ml cold PBS, triturated 5–10 times and the supernatant was passed through a 70-μm strainer. The previous step was repeated a second time. The three crypt-containing fractions were combined and centrifuged at 110g for 5 min. The pellet was resuspended in 10 ml cold Advanced DMEM/F12 (Invitrogen) and centrifuged at 84g to remove single cells and tissue debris. The resulting pellet was enriched in crypts, which were subsequently dissociated or directly embedded in PEG gels or in Matrigel (BD Biosciences; growth factor reduced, phenol red-free formulation). When needed, crypts or ISC colonies were dissociated enzymatically by incubating for 8 min at 37 °C in 1 ml TrypLE Express (Life Technologies), supplemented with DNase I (2000 U ml−1; Roche), 0.5 mM N-acetylcysteine (Sigma) and 10 μM Y27632 (Stemgent). Undigested clusters were removed by passing the suspension through a 40 μm strainer. Freshly isolated mouse crypts or single cells from dissociated mouse ISC colonies were embedded in Matrigel or PEG gels, which were cast into 20-μl droplets at the bottom of wells in 24-well plate. Following polymerization (15 min, 37 °C), the gels were overlaid with 500 μl of ISC expansion medium (Advanced DMEM/F12 containing Glutamax, HEPES, penicillin-streptomycin, B27, N2 (Invitrogen) and 1 μM N-acetylcysteine (Sigma)), supplemented with growth factors, including EGF (50 ng ml−1; R&D), Noggin (100 ng ml−1; produced in-house) and R-spondin (500 ng ml−1; produced in-house), and small molecules, including CHIR99021 (3 μM; Millipore) and valproic acid (1 mM; Sigma). For single-cell culture, thiazovivin (2.5 μM; Stemgent) was included in the medium during the first two days. To induce stem cell differentiation and organoid formation, the medium was removed, the gels were washed with PBS and fresh medium containing EGF, Noggin and R-spondin was added. Human small intestinal and colorectal cancer organoids were generated as described previously31, 32 and grown in 20-μl droplets of Matrigel or PEG gels overlaid with Advanced DMEM/F12 containing Glutamax, HEPES, penicillin-streptomycin, B27 (Life Technologies), Wnt3a (50% conditioned medium; produced in-house; only for small intestinal organoids), R-spondin 1 (20% conditioned medium; produced in-house), Noggin (10% conditioned medium; produced in-house), N-acetylcysteine (2 μM; Sigma), Nicotinamide (10 mM; Sigma), human EGF (50 ng ml−1; Peprotech), A83-01 (500 nM; Tocris), SB202190 (10 μM; Sigma), Prostaglandin E2 (10 nM; Tocris); Gastrin (10 nM; Tocris), and Y-27632 (10 μM; Abmole). In general, growth factors were replenished every two days, with full medium change taking place every four days. Where indicated, the following compounds were used at the specified concentrations: blebbistatin (Sigma, 12.5 μM), ML7 (Calbiochem, 10 μM), cytochalasin D (Merck-Millipore, 0.1 μg ml−1), echistatin (Sigma, 500 nM). Vinylsulfone-functionalized 8-arm PEG (8-arm PEG-VS or sPEG) was purchased from NOF, and acrylate-functionalized 8-arm PEG (8-arm PEG-Acr or dPEG) was purchased from Creative PEGWorks. The transglutaminase (TG) factor XIII (FXIIIa) substrate peptides Ac-FKGGGPQGIWGQ-ERCG-NH2 (TG-DG-Lys), Ac-FKGG-GDQGIAGF-ERCG-NH2 (TG-NDG-Lys) and H-NQEQVSPL-ERCGNH2 (TG-Gln) and the RGD-presenting adhesion peptide H-NQEQVSPL-RGDSPG-NH2 (TG-Gln-RGD) were purchased from GL Biochem. To couple the FXIIIa substrate peptides to the 8-arm PEG-VS or 8-arm PEG-Acr, they were mixed with the PEG powder in a 1.2 stoichiometric excess (peptide-to-VS group); the combined solids were dissolved in triethanolamine (0.3 M, pH 8.0), and allowed to react for 2 h at 37 °C. The reaction solution was dialysed (Snake Skin, MWCO 10K, PIERCE) against ultrapure water for 3 days at 4 °C, after which the five products ((PEG-VS)-DG-Lys, (PEG-VS)-NDG-Lys, (PEG-VS)-Gln, (PEG-Acr)-NDG-Lys, (PEG-Acr)-Gln) were lyophilized. The resulting solid precursors were dissolved in ultra-pure water to make 13.33% w/v stock solutions. Appropriate volumes of 13.33% w/v PEG precursor solutions were mixed in stoichiometrically balanced ratios to generate hydrogel networks of a desired final PEG content. Hydrogel formation was triggered by the addition of thrombin-activated FXIIIa (10 U ml−1; Galexis) in the presence of Tris-buffered saline (TBS; 50 mM, pH 7.6) and 50 mM CaCl . The spare reaction volume was used for the incorporation of dissociated ISCs, fragments of human small intestinal or colorectal cancer organoids, and ECM components: TG-RGD-Gln, fibronectin (0.5 mg ml−1; Invitrogen), laminin-111 (0.1 mg ml−1; Invitrogen), collagen-IV (0.25 mg ml−1; BD Bioscience), hyaluronic acid (0.5 mg ml−1; gift from D. Ossipov, Uppsala University), perlecan (0.05 mg ml−1; Sigma). Gels were allowed to crosslink by incubating at 37 °C for 15 min. Dissociation and release of colonies grown in PEG for downstream cell processing or re-embedding was accomplished by enzymatic digestion of the gels. Gels were carefully detached from the bottom of the plate using the tip of a metal spatula and transferred to a 15-ml Falcon tube containing 1 ml of TrypLE Express (Life Technologies), supplemented with DNase I (2,000 U ml−1; Roche), 0.5 mM N-acetylcysteine (Sigma) and 10 μM Y27632 (Stemgent). Following digestion (10 min, 37 °C), the cell suspension was washed with 10 ml of cold medium, passed through a 40-μm strainer (BD Biosciences) and centrifuged at 1,200 r.p.m. for 5 min. To form mechanically dynamic PEG hydrogels, which underwent varying extents of spontaneous softening, hybrid hydrogels were formed from both PEG-VS and PEG-Acr hydrogel precursors. Specifically, to form a fast-softening 100% Acr gel, stoichiometric quantities of (PEG-Acr)-NDG-Lys and (PEG-Acr)-Gln precursors were allowed to crosslink. A slow-softening 50% Acr gel was formed by crosslinking stoichiometric amounts of (PEG-VS)-NDG-Lys and (PEG-Acr)-Gln precursors. A 75% Acr gel with intermediate kinetics of softening was formed by crosslinking the (PEG-Acr)-Gln precursor with half of the stoichiometric equivalent of (PEG-VS)-NDG-Lys and half of the stoichiometric equivalent of (PEG-Acr)-NDG-Lys. Regardless of the relative proportions of (PEG-VS) and (PEG-Acr) precursors within the hydrogel, its overall PEG content was varied to tune its initial mechanical properties. It should be noted that, by providing an initially stiff and later a soft environment, the mechanically dynamic matrices support both ISC expansion and organoid formation. Hence, ISCs can be expanded and organoids can be formed in the same hydrogel. Hybrid PEG–alginate (PEG–alg) gels were employed to induce a controlled drop in stiffness at a desired time. PEG–alg gels were formed by the simultaneous presence of activated FXIII enzyme—to drive the crosslinking of the PEG macromers—and Ca2+ ions, which induce the crosslinking of the alginate polysaccharides. Hybrid gels were formed by casting a solution, containing 2% (w/v) of stoichiometrically balanced (PEG-VS)-NDG-Lys and (PEG-VS)-Gln precursors, 10 mM TG-RGD-Gln, 10 U ml−1 FXIIIa, 0.8% (w/v) alginate (Sigma) and dissociated ISCs, within a 1% agarose/2% gelatin mould, containing 20 mM CaCl . The solution was incubated at 37 °C for 15 min, carefully de-molded from the agarose substratum and transferred to a 12-well plate containing 1 ml of complete ISC expansion medium. Matrix softening was induced at the desired time by adding 1 U ml−1 alginate lyase (Sigma), and incubating for 1 h at 37 °C. The digested gels were washed and transferred to freshly prepared ISC expansion medium. The shear modulus of the PEG gels was determined by performing small-strain oscillatory shear measurements on a Bohlin CVO 120 rheometer. In brief, preformed hydrogel discs 1–1.4 mm in thickness were allowed to swell in complete cell culture medium for at least 3 h, and were subsequently sandwiched between the parallel plates of the rheometer. The mechanical response of the gels was recorded by performing frequency sweep (0.1–10 Hz) measurements in a constant strain (0.05) mode, at 37 °C. The shear modulus (G') is reported as a measure of gel mechanical properties. To quantify the colony formation efficiency of single embedded ISCs, phase contrast z-stacks spanning the entire thickness of the cell-laden Matrigel or PEG gels were collected (Zeiss Axio Observer Z1) at 5 different locations within the gels. The Cell Counter plugin in ImageJ (NIH) was used to quantify the fraction of cells which had formed colonies at day 4 after seeding. To quantify colony circularity, phase contrast images of ISC colonies grown in the condition of interest (between 5 and 38 colonies per condition) were taken, and their contours traced manually in ImageJ. The circularity of the contours was measured using the Measure algorithm in ImageJ. To characterize the cell morphology within ISC colonies grown in different conditions, phase contrast images of at least 50 colonies were taken and the numbers of colonies containing packed columnar cells versus spread cells were counted. To quantify Lgr5 expression within ISC colonies grown within different matrices, fluorescence images of at least 50 colonies per condition were recorded and the number of colonies expressing Lgr5–eGFP was counted. To identify a short sequence that supports intestinal organoid culture, we created a library of soft (G' = 200 Pa) hydrogels in which binding sequences from the laminin α1 subunit previously shown to be biofunctional33, 34 (Extended Data Table 4) were tethered to the PEG backbone. Embedding fragments of pre-formed organoids and screening the library for organoid survival and growth revealed that two laminin-derived peptides—AG73 and A55—significantly enhanced organoid viability and supported further growth (Extended Data Fig. 5a). Presenting these two sequences (A55 and AG73) alongside in the same gel did not appear to have an additive effect, likely owing to a redundant adhesion mechanism. Hence, we focused on the sequence with a stronger individual effect, that is, AG73 and the corresponding PEG gels (referred to as TG PEG-AG73). Varying the amount of AG73 peptide tethered to the PEG gel backbone revealed a dose-dependent effect on intestinal organoid viability and growth (Extended Data Fig. 5b, c). Despite the improved rate of survival and morphogenesis in TG PEG-AG73 matrices compared with plain PEG or PEG RGD, the process was significantly less efficient compared with Matrigel, and morphological differences were apparent. Keeping in mind that the effect of AG73 was concentration-dependent, we attributed these differences to a potentially sub-optimal AG73 ligand density within the synthetic system. By design, there is an upper limit to the concentration of tethered factors that can be incorporated into the PEG system used thus far in the study: exceeding this limit disrupts the structural integrity of the gels. To overcome this limitation and enhance the biofunctionality of the synthetic matrix by increasing the concentration of AG73 ligands, we turned to chemically crosslinked PEG gels. Here, vinyl sulfone (VS)-conjugated 4-arm PEG precursors are covalently linked into solid hydrogels through Michael-type addition between VS groups and the thiols of a short crosslinker containing two cysteine residues. To incorporate the AG73 ligand at a high density, we designed a crosslinker in which the AG73 sequence was flanked by two short cysteine-containing sequences. The resulting gels (hereafter referred to as MT PEG-AG73) presented the AG73 ligand at a concentration of 3.1 mM, thus significantly surpassing the highest concentration achieved in the enzymatically crosslinked matrices. Embedding intestinal organoid fragments into MT PEG-AG73 revealed that the percentage of tissues that remained viable and continued to undergo morphogenesis approached that observed in Matrigel (Extended Data Fig. 5e). To verify the maintenance of ISCs within the organoids grown in MT PEG-AG73, we embedded tissues extracted from the Lgr5-eGFP reporter mouse and monitored eGFP expression. We observed that Lgr5–eGFP was expressed in the expected pattern: localized to the crypt-like buds of the organoids (Extended Data Fig. 5f). The fraction of organoids expressing the marker was significantly higher than in those cultured in TG PEG AG73, and at least as high as in organoids cultured in Matrigel (Extended Data Fig. 5g). We also confirmed that the organoids cultured in MT PEG-AG73 were polarized and contained differentiated cells (Extended Data Fig. 5h). ISC colonies or organoids embedded in Matrigel or PEG gels were fixed with 4% paraformaldehyde (PFA) in PBS (30 min, room temperature). The fixation process typically led to complete degradation of the Matrigel. Suspended tissues were collected and centrifuged (800 r.p.m., 5 min) to remove the PFA, washed with ultra-pure water and pelleted. Following resuspension in water, the organoids were spread on glass slides and allowed to attach by drying. Attached organoids were rehydrated with PBS. Following fixation, organoids embedded in PEG or spread on glass were permeabilized with 0.2% Triton X-100 in PBS (1 h, room temperature) and blocked (10% goat serum in PBS containing 0.01% Triton X-100) for at least 3 h. Samples were subsequently incubated overnight at 4 °C with phalloidin-Alexa 546 (Invitrogen) and primary antibodies against lysozyme (1:50; Thermo Scientific PA1-29680), mucin-2 (1:50; Santa Cruz sc-15334), chromogranin-A (1:50; Santa Cruz sc-13090), L-FABP (1:50; Santa Cruz sc-50380) and YAP1 (1:50; Santa Cruz sc-101199) diluted in blocking buffer. After washing with PBS for at least 3 h, samples were incubated overnight at 4 °C with secondary antibody Alexa 647 goat-α-rabbit (1:1000 in blocking solution; Invitrogen). Following extensive washing, stained organoids were imaged in epifluorescence (Zeiss Axio Observer Z1) or confocal (Zeiss LSM 710) mode. Alternatively, ISC colonies or organoids cultured in PEG were released from the hydrogel before PFA fixation, by incubating the gels with 1 mg ml−1 Dispase (Gibco) for 7 min at 37 °C. The released colonies or organoids were fixed with PFA in suspension, and attached to glass coverslips, as described above. Human organoids were fixed in 10% neutral buffered formalin, washed with PBS, dehydrated, and embedded in paraffin. Sections were stained with H&E or Ki67 antibody (1:250; Monosan). Lentiviral particles encoding for shRNA recognizing YAP (two sequences validated for knockdown, purchased from Sigma) or the pLKO.1-puro Non-Target control shRNA (Sigma) were generated in HEK 293T cells, using third generation lentivirus packaging vectors. Transfection was carried out using the X-tremeGENE HP Transfection kit (Roche). After 48 h, the supernatant was collected, filtered and ultracentrifuged at 50,000g for 2 h at 20 °C. The resulting pellet was resuspended in PBS and stored at −80 °C. Lentiviral infection of ISCs was performed by dissociating the ISC colonies (described above), resuspending the resulting single cells in ice-cold liquid Matrigel, containing 10 μM Y27632 and the concentrated lentiviral particles at a dilution of 1:10. ISCs were incubated with viral particles in a liquid suspension for 45 min on ice. The suspension was subsequently cast into droplets and allowed to form gels, which were overlaid with ISC expansion medium. The embedded ISCs proceeded to form colonies, which carried the transgene encoded by the virus. The cells were allowed to recover and form colonies for 36 h, after which they were dissociated and encapsulated within PEG hydrogels or used for quantification of knockdown efficiency by qPCR. ISC colonies or organoids grown in Matrigel or PEG gels were dissociated as described above, and RNA was extracted using an RNeasy Micro Kit (Qiagen). cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). qPCR was carried out using the Power SYBR Green PCR Master Mix (Applied Biosystems) and the primers listed Extended Data Table 1. RNA-seq libraries were prepared using 500 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina; San Diego, California, USA) on a Sciclone liquid handling robot (PerkinElmer; Waltham, Massachusetts, USA) using a PerkinElmer-developed automated script. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were processed using the Illumina Pipeline Software version 1.84. Purity-filtered reads were adapters and quality trimmed with Cutadapt and filtered for low complexity with seq_crumbs (v. 0.1.8). Reads were aligned against the Mus musculus.GRCm38.82 genome using STAR35. The number of read counts per gene locus was summarized with htseq-count36 using the Mus musculus.GRCm38.82 gene annotation. Quality of the RNA-seq data alignment was assessed using RSeQC37. Reads were also aligned to the Mus musculus.GRCm38.82 transcriptome using STAR and the estimation of the isoforms abundance was computed using RSEM38. Statistical analysis was performed for genes in the R software package. Genes with low counts were filtered out according to the rule of 1 count per million in at least 1 sample. Library sizes were scaled using TMM normalization in the EdgeR package39 and log-transformed with the limma voom function40. Differential expression was computed with limma41 by fitting paired samples data into a linear model and performing all pairwise comparisons. To control for false discovery and multiple testing, we computed an adjusted P value, using the Benjamini–Hochberg method. Gene set expression analysis was performed with the freely available GSEA software42 (Broad Institute), using differential expression values and pre-defined gene signatures as inputs. In particular, to test for upregulation of stress-related genes, the MSigDB gene set ‘BIOCARTA_STRESS_PATHWAY’ was used. We checked for upregulation of colon cancer-related genes by using the inflammatory colon cancer signature, as identified by Sadanandam et al.43. Functional annotation and gene ontology analysis of significantly enriched gene sets was conducted using the MetaCore software. The accession numbers for the gene expression profiles described here are GEO: GSE85391. Statistically significant differences between the means of two groups were assessed by using a Student’s t-test, whereas data containing more than two experimental groups were analysed with a one-way ANOVA followed by a Bonferroni’s multiple comparison test. All statistical analyses were performed in the GraphPad Prism 6.0 software. RNA sequencing data that support the findings of this paper have been deposited to the Gene Expression Omnibus (GEO) public repository (GSE85391; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE85391). Source Data for Figs. 1, 2, 3, 4 and Extended Data Figs 1, 2, 3, 4, 5 are provided with the paper. All additional relevant data are available upon request from the corresponding author.