Hampton A.L.,Unit for Laboratory Animal Medicine |
Hish G.A.,Unit for Laboratory Animal Medicine |
Aslam M.N.,Medical School |
Rothman E.D.,University of Michigan |
And 6 more authors.
Journal of the American Association for Laboratory Animal Science
Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD. Copyright © 2012 by the American Association for Laboratory Animal Science. Source
Hampel J.A.,Unit for Laboratory Animal Medicine |
Rinkinen J.,University of Michigan |
Peterson J.R.,University of Michigan |
Agarwal S.,University of Michigan |
And 6 more authors.
Journal of Craniofacial Surgery
Introduction: Analytical morphomics focuses on extracting objective and quantifiable data from clinical computed tomography (CT) scans to measure patients' frailty. Studies are currently retrospective in nature; therefore, it would be beneficial to develop animal models for well-controlled, prospective studies. The aim of this study is to develop an in vivo microCT protocol for the longitudinal acquisition of whole-body images suit able for morphomic analyses of bone. Methods: The authors performed phantom studies on 2 microCT systems (Inveon and CT120) to study tissue radiodensity and further characterize system performance for collecting animal data. The authors also describe their design of a phantomimmobilization device using phantoms and an ovariectomized (OVX) mouse. Results: The authors discovered increased consistency along the zaxis for scans acquired on the Inveon compared with CT120, and calibration by individual slice reduces variability. Objects in the field of view had more impact on measurement acquired using the CT120 compared with the Inveon. The authors also found that using the middle 80% of slices for data analysis further decreased variability, on both systems. Moreover, bone-mineral-density calibration using the QCT Pro Mini phantom improved bonemineral-density estimates across energy spectra, which helped confirm our technique. Comparison of weekly body weights and terminal uterine mass between sham and OVX groups validated our model. Discussion: The authors present a refined microCT protocol to collect reliable and objective data. This data will be used to establish a platform for research animal morphomics that can be used to test hypotheses developed from clinical human morphomics. Copyright © 2016 by Mutaz B. Habal, MD. Source
Bell J.D.,University of Michigan |
Bergin I.L.,Unit for Laboratory Animal Medicine |
Harris L.H.,University of Michigan |
Chai D.,Institute of Primate Research |
And 8 more authors.
Journal of Infectious Diseases
Background. The baboon (Papio hamadryas anubis) can be transcervically instrumented, facilitating studies of intrauterine contraception and reproductive tract infection. We sought to determine if the baboon could become infected with a single cervical inoculation of Chlamydia trachomatis.Methods.Ten female baboons were randomized and inoculated cervically with C. trachomatis serovar E (or buffer alone). Animals underwent weekly clinical and laparoscopic evaluations for four weeks and at post-inoculation week 8, to monitor upper tract infection. Cervical culture and nucleic acid amplification testing (NAAT) were completed weekly throughout the study. Animals were euthanized at week 16 and the reproductive tracts were examined histologically.Results.All inoculated animals developed cervical infection. The average duration of positive NAAT results was 6.8 weeks (range 2-16). Two of eight (25%) animals tested positive from fallopian tube samples. Infected animals showed histological findings consistent with chlamydial infection, such as germinal centers. Five of ten animals seroconverted to C. trachomatis.Conclusions.Baboons cervically inoculated once with C. trachomatis develop infection similar to humans, with a low incidence of upper tract infection. This novel model of Chlamydia infection closely resembles human disease and opens new avenues for studying the pathogenesis of sexually transmitted infections and contraceptive safety. © 2011 The Author. Source
Makidon P.E.,Unit for Laboratory Animal Medicine |
Makidon P.E.,University of Michigan |
Smith D.M.,University of Michigan |
Groom J.V.,University of Michigan |
And 3 more authors.
Uremia due to chronic kidney disease (CKD) in humans is associated with immune dysfunction, increased susceptibility to infections, immune-activation-associated inflammation, and poor responses to vaccines. The pathophysiologic basis of these immune defects is hypothesized to be associated with a wide range of immunologic abnormalities, including an inability to sufficiently express the B7 family (B7-1, CD80; B7-2, CD86) of T-cell costimulatory molecules. However, testing the hypothesis that a state of chronic uremia contributes to attenuated expression of CD80 or CD86 has been difficult because few animal models faithfully recapitulate the immune defects observed in human CKD patients. We used a humanized mouse in a model of surgically induced renal failure and secondary chronic uremia to evaluate the effect of uremia on the expression of these markers. In a manner that resembles the changes observed in CKD patients, surgically induced CKD in mice resulted in decreased costimulatory CD86 expression compared with that in sham-operated controls. Immunodeficiency was functionally demonstrated in this mouse model by documenting an attenuated immune response to a cholera-toxin-based hepatitis B vaccine. This model will be useful for investigating the mechanisms involved in chronic uremia-associated immunodeficiency, poor response to vaccination, and problems associated with immunization of CKD patients. © 2015. Source
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.