{"id":395019,"date":"2023-12-19T19:00:00","date_gmt":"2023-12-20T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/adult-stem-cell-activity-in-naked-mole-rats-for-long-term-tissue-maintenance-nature-communications\/"},"modified":"2023-12-23T18:49:36","modified_gmt":"2023-12-23T23:49:36","slug":"adult-stem-cell-activity-in-naked-mole-rats-for-long-term-tissue-maintenance-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/adult-stem-cell-activity-in-naked-mole-rats-for-long-term-tissue-maintenance-nature-communications\/","title":{"rendered":"Adult stem cell activity in naked mole rats for long-term tissue maintenance – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

Ethics<\/h3>\n

This study involved undertaking animal procedures in four different countries: U.K, USA, Austria, and the Republic of South Africa. Animal procedures were carried out in accordance with Home Office, UK regulations and the Animals (Scientific Procedures) Act, 1986 of UK, the Institutional Animal Care and Use Committee (IACUC) of USA, Act 7, 1991 of South Africa, and the Directive 2010\/63\/EU of the European Parliament.<\/p>\n

Normal human colonoscopy samples were collected under the research tissue bank ethics 16\/YH\/0247 supported by NIHR Biomedical Research Centre, Oxford, U.K. and under the London Dulwich Research Ethics Committee (reference number 15\/LO\/1998). Written informed consent was obtained from all participants undergoing routine bowel cancer or IBD screening. All samples were anonymized.<\/p>\n

Mouse husbandry<\/h3>\n

Wild-caught mice (F1) were acquired from a founder population trapped in lower Austria and Vienna (2016) and housed at the Konrad Lorenz Institute of Ethology, University of Vienna, Austria. All C57BL\/6\u2009J mice used in this study were purchased from Charles River (Kent, UK) or the Jackson Laboratory (USA) and housed at Biomedical Services Unit in John Radcliffe Hospital, Oxford, UK or at Rutgers University Animal Facility in Newark, New Jersey, USA. Mice were housed in individually ventilated cages under specific pathogen-free conditions and maintained at 19\u201323\u2009\u00b0C temperature with 45-65% relative humidity, in an alternating 12-h light\/12-h dark cycles and fed with food and water ad libitum<\/i>.<\/p>\n

Naked mole rat husbandry<\/h3>\n

Naked mole rats (NMRs) were housed at the Animal Facility of the Department of Zoology and Entomology, University of Pretoria. The NMRs were kept in tunnel systems consisting of several Perspex chambers containing wood shavings as nestling material. The NMR room was maintained at temperatures ranging between 29\u201332\u2009\u00b0C, with relative humidity around 40-60%. NMRs were fed chopped fresh fruits and vegetables (apple, sweet potato, cucumber, and capsicum) daily ad libitum<\/i> along with weekly supplement of ProNutro (Bokomo). Since NMRs obtain all their necessary water from food sources, no drinking water was provided to the animals. All scientific procedures on NMRs were conducted under ethics approval (NAS046-19 and NAS289-2020) by the Animal Ethics Committee, University of Pretoria. In addition, DAFF section 20 approval was granted (SDAH-Epi-20111909592).<\/p>\n

For all analyses, both male and female mice, NMRs, and humans were included in the study.<\/p>\n

In vivo administration of BrdU and EdU<\/h3>\n

15\u2009mg\/mL solution of BrdU (5-bromo-2\u2019-deoxyuridine, Abcam, ab142567) and 12.3\u2009mg\/mL solution of EdU (5-ethynyl-2-deoxyuridine, Merck, 900584) were prepared in sterile 1\u00d7 PBS (Gibco, 10010023) and filtered through a 0.2\u2009\u00b5m strainer. Using a 27-gauge needle and 1\u2009mL syringe, 100\u2009mg per kg bodyweight BrdU and 82.14\u2009mg per kg bodyweight EdU were administered intraperitoneally. Animals were checked regularly for signs of discomfort (hunched back, shivering, low mobility) after the injection.<\/p>\n

For cumulative labelling protocol using BrdU, the first injection in naked mole rats was administered between 14:00 to 15:00. Subsequent BrdU injections were given every 8\u2009h for a duration of 5 days and intestinal tissues were collected every 8\u2009h after the first injection. In C57BL\/6\u2009J mice, the first BrdU injection was also given between 14:00 to 15:00, with further injections given every 6\u2009h for a total of 2.25 days. Mouse intestinal tissues were collected 1\u2009h after each injection. The rationale for the frequency and total number of injections in the two species is discussed in Supplementary Note 1<\/a>.<\/p>\n

In vivo administration of dextran sulphate sodium<\/h3>\n

Dextran sulphate sodium (DSS) salt (Merck, 42867) was dissolved in sterile ddH2<\/sub>O to prepare 0 to 8.75% (w\/V) solution. Using a 2\u2009mL syringe fitted with a plastic feeding tube (Prime Bioscience, FTP-20-38), 50\u2009mL per kg bodyweight of DSS solution in NMRs or 12\u2009mL per kg bodyweight in mice was administered orally at specific intervals for 3 days. Body mass was monitored daily and stool samples collected while animals were also checked for signs of discomfort (e.g. hunched back, shivering, low mobility) every 3\u2009h.<\/p>\n

Intestinal tissue collection and processing<\/h3>\n

After sacrificing the animals by approved procedures, the intestine was immediately isolated from the abdominal cavity and fatty tissue was removed. The small intestine was then divided into three equal sections: SB1 (duodenum), SB2 (jejunum) and SB3 (ileum). All three parts of the small intestine and colon were then flushed with 1\u00d7 PBS (Phosphate Buffered Saline) solution using a P1000 pipette to clean all the faecal material. Each tissue section was then cut open longitudinally using a gut cutting device86<\/a><\/sup> and the edges pinned down onto a 3MM filter paper such that the luminal side was facing upward. The tissue was then fixed in 10% neutral buffered formalin overnight at room temperature. The following day fixed intestinal tissues were rolled using the Swiss-rolling technique87<\/a><\/sup> and stored in 70% ethanol at 4\u2009\u00b0C. Next, formalin-fixed Swiss-rolls were dehydrated through increasing concentrations of ethanol, cleared through xylene, and embedded in paraffin. The paraffin blocks were sectioned at 4\u2009\u00b5m thickness using a microtome (Anglia Scientific).<\/p>\n

Haematoxylin and Eosin staining<\/h3>\n

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were deparaffinized by submerging slides in xylene (2 times, 10\u2009min each) and rehydrated in 100% ethanol (2 times, 5\u2009min each), 95% ethanol (2\u2009min), 70% ethanol (2\u2009min), 50% ethanol (2\u2009min), and distilled water (5\u2009min). Sections were then stained with Harris Haematoxylin (Merck, HHS32) for 2\u2009min 45\u2009s followed by washing in running tap water for 5\u2009min. Next, slides were dipped in 95% ethanol ten times before sections were counter-stained with Eosin solution (Merck, 117081) for 3\u2009min. This was followed by tissue sections being dehydrated in 95% ethanol (15\u2009s) and 100% ethanol (2 times, 15\u2009s each), dipped in xylene (2 times, 5\u2009min each), and finally coverslipped using DPX Mountant (Merck, 06522).<\/p>\n

Alcian blue staining<\/h3>\n

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were first deparaffinized with xylene (2 times, 5\u2009min each). They were rehydrated in 100%, 90%, 70% ethanol (5\u2009min each) and tap water (2\u2009min), dipped in 3% acetic acid solution (3\u2009min) before staining with Alcian blue 8GX (Merck, A5268) solution (pH 2.5) for 30\u2009min. Tissue sections were then washed (5\u2009min) in running tap water and counterstained (5\u2009min) with Nuclear Fast Red (Merck, N3020). After 1\u2009min wash in running tap water again, tissue sections were dehydrated in ethanol, dipped in xylene and finally coverslipped using DPX Mountant (Merck, 06522).<\/p>\n

Measuring the thickness of the mucus layer in the colon<\/h3>\n

To preserve the mucus layer of the colonic epithelium, contact with any aqueous solution was avoided after the excision of the intestinal tissue. Without removing the faecal matter, several segments of the colon were cut using a scalpel and fixed overnight at room temperature in methacran\/Carnoy\u2019s solution which was composed of 60% methanol, 30% chloroform, and 10% glacial acetic acid. On the second day, fixed tissues were processed in 100% methanol (2 times, 30\u2009min each), 100% ethanol (3 times, 60\u2009min each) and xylene (2 times, 60\u2009min each). Processed tissues were embedded in paraffin and 4\u2009\u00b5m thick sections cut and stained with Alcian blue as described above. Stained tissues were photomicrographed at 60\u00d7 magnification on an Olympus BX51 brightfield microscope. For both NMRs and mice, 30 independent measurements of the mucus layer were taken from 3 animals using the \u2018measure\u2019 tool in Fiji package88<\/a><\/sup>.<\/p>\n

Alkaline phosphatase staining<\/h3>\n

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were deparaffinized in xylene (2 times, 5\u2009min each) and rehydrated in 100%, 90%, 70% ethanol (5\u2009min each) and distilled water (5\u2009min). A hydrophobic barrier was drawn around the tissue sections using a PAP pen (Vector Lab, H-4000) before incubating in the AB solution (AP Staining kit, SystemBio, AP100B-1) for 20\u2009min at room temperature in the dark. All sections were then washed in 1\u00d7 PBS (5\u2009min, on a shaker), counterstained with Nuclear Fast Red (5\u2009min), washed in running tap water (1\u2009min), dehydrated in ethanol, dipped in xylene and finally coverslipped with DPX Mountant (Merck, 06522).<\/p>\n

Immunohistochemistry<\/h3>\n

4\u2009\u00b5m thick formalin-fixed paraffin-embedded (FFPE) sections were cut using a microtome and dried overnight on SuperFrost Plus slides (VWR, 6310108). Tissue sections were baked at 60\u2009\u00b0C for 1\u2009h the next day, deparaffinized in 3 rounds of xylene (5\u2009min each) and rehydrated in 100%, 90%, 70% ethanol and distilled H2<\/sub>O (5\u2009min each). Endogenous peroxidase activity was quenched by incubating sections in 3% H2<\/sub>O2<\/sub> (Merck, 8222871000) for 20\u2009min. A heat mediated antigen retrieval was performed by boiling sections in 10\u2009mM sodium citrate buffer (pH 6.0) for 10\u2009min which was followed by 20\u2009min of cooling down in the same solution. This was followed by incubating the tissue sections in 1\u00d7 PBSTX (0.1% Triton X) for 10\u2009min. All sections were then blocked for 1\u2009h at room temperature using 5% serum which matched the species of the secondary antibody. Next, primary antibodies were diluted in antibody diluent (1% BSA dissolved in 1\u00d7 PBS) which was applied to the tissue sections and incubated overnight at 4\u2009\u00b0C. The primary antibodies used in this study were Chromogranin A (Abcam, ab15160) at 1:2000 and BrdU (Abcam, ab6326) at 1:500. It is noteworthy that in our BrdU staining, we did not use HCl-mediated DNA denaturation and only performed heat-mediated antigen retrieval (98-100\u2009\u00b0C) which has been shown to produce a brighter signal than acid hydrolysis89<\/a><\/sup>. After 3 rounds of washes (5\u2009min each) with 1\u00d7 PBST (0.1% Tween20 in 1\u00d7 PBS), tissue sections were then incubated for 1\u2009h at room temperature with biotinylated secondary antibodies diluted at 1:300. For our study specifically, we used goat anti-rabbit IgG (Vector Laboratories, BA-1000) and goat anti-rat IgG (Abcam, ab207997). To detect the biotinylated target, we used the Avidin\/Biotinylated enzyme Complex (ABC) kit (Vector Laboratories, PK-6101) and developed the signal using the DAB (3,3\u2019-diaminobenzidine) solution (R&D systems, 4800-30-07). The tissue sections were then counterstained with Harris Haematoxylin (Merck, HHS32) for 5\u2009s, dehydrated in 70%, 90% and 100% ethanol for 15\u2009s each, dipped in xylene and coverslipped using DPX Mountant (Merck, 06522).<\/p>\n

mRNA ISH<\/h3>\n

Species-specific RNAscope probes from ACD Bio-techne were used to detect Lgr5<\/i> mRNA expression in NMR (584631), mouse (312171) and human (311021) intestinal tissues. We used the RNAscope Multiplex Fluorescent Detection Kit v2 (ACD Bio-techne, 323110) and followed the instructions of the manufacturer (document number 323100-USM, ACD Bio-techne) to detect Lgr5<\/i> mRNA targets at a single cell level in FFPE tissue sections mounted on SuperFrost Plus slides (VWR, 6310108).<\/p>\n

Multiplex mRNA ISH with immunofluorescence<\/h3>\n

To enable multiplexing of mRNA and proteins, we adapted the manufacturer\u2019s instructions (document number 323100-USM, ACD Bio-techne) for RNAscope Multiplex Fluorescent Detection Kit v2 (ACD Bio-techne, 323110) to exclude the step involving protease treatment. Once the mRNA signal was developed, we proceeded to detect proteins by first washing tissue sections (2 times, 2\u2009min each) in 1\u00d7 TBST (0.1% Tween20 in 1\u00d7 Tris-buffered saline). This was followed by blocking for 1\u2009h at room temperature with 10% serum which matched the species of the secondary antibodies. Multiple primary antibodies (diluted in 1% BSA in 1\u00d7 TBS) were then applied to the tissue sections and incubated overnight at 4\u2009\u00b0C. The dilutions of various primary antibodies used in our study were 1:500 for EpCAM (Abcam, ab71916), 1:500 for Ki67 (Cell Signaling, 12202), 1:200 for p27Kip1<\/sup> (Cell Signaling, 3686 and 2552), 1:500 for BrdU (Abcam, ab6326) and 1:2000 for PHH3-S28 (Abcam, ab32388). Following primary antibody incubation, the next day we washed the sections thrice in 1\u00d7 TBST (5\u2009min each) before incubating them with fluorophore-linked secondary antibodies (at 1:500 dilution) for 1\u2009h at room temperature. Fluorescent secondary antibodies used in our study included goat anti-rabbit Alexa 488 (Invitrogen, A11008), goat anti-rat Alexa 488 (Invitrogen, A11006), goat anti-rabbit Alexa 555 (Invitrogen, A21428) and goat anti-rabbit Alexa 633 (Invitrogen, A21070). Following the secondary antibody incubation, tissue sections were washed three times in 1\u00d7 TBST (5\u2009min each) and counterstained with DAPI (Invitrogen, D1306) for 15\u2009min at room temperature before mounting with coverslips (VWR, 631-0138) using Diamond Antifade Mountant (Invitrogen, P36961).<\/p>\n

TUNEL assay<\/h3>\n

Click-iT\u2122 Plus TUNEL Assay Kit (Invitrogen, C10617) was used following the manufacturer\u2019s instructions to detect apoptotic cells FFPE tissue sections.<\/p>\n

EdU detection<\/h3>\n

EdU-Click 488 kit (Base Click, BCK-EdU488-1) was used according to the instructions provided by the manufacturer to detect EdU-positive cells in FFPE tissue sections.<\/p>\n

Measuring plasma BrdU concentration<\/h3>\n

Plasma BrdU concentration was determined following the protocol described by Barker et al.90<\/a><\/sup>. In brief, 100\u2009\u00b5L naked mole rat blood was collected by a tail vein puncture after 8\u2009hand 16\u2009h of BrdU injection. The blood was mixed with heparin to stop clotting and centrifuged at 13,000g<\/i> for 15\u2009min to separate all blood cells. Plasma was collected from the top layer and stored at \u221280\u2009\u00b0C.<\/p>\n

HEK293T cells (ATCC, CRL-3216) were cultured in high-glucose DMEM (Merck, D6546) containing 10% FBS (Gibco, 10270), 1\u00d7 Penicillin-Streptomycin (Merck, P4333-100ML), and 2\u2009mM l<\/span>-glutamine (Gibco, 25030-024) at 37\u2009\u00b0C with 5% CO2<\/sub>. Cells were plated on a 13\u2009mm sterile glass coverslip precoated with poly l<\/span>-lysine (VWR, 631-0149) in a 24-well plate (Starlab, CC7682-7524) and cultured overnight. The media was replaced with 500\u2009\u00b5L fresh culture media containing 10\u2009\u00b5L plasma or standard BrdU solution (3, 10, 20, 30, 40, 50\u2009\u00b5g\/ml) and incubated at 37\u2009\u00b0C for 4\u2009h. Cells were then washed with 1\u00d7 PBS and fixed in 4% paraformaldehyde for 20\u2009min at room temperature. Fixed cells were kept in 1\u00d7 PBS at 4\u2009\u00b0C before proceeding to immunocytochemical detection of BrdU.<\/p>\n

Fixed cells on coverslips in 24 well plates were incubated with 3% H2<\/sub>O2<\/sub> for 10\u2009min at room temperature. After washing with 1\u00d7 PBS, cells were incubated in 2\u2009N HCl for 1\u2009h at room temperature to denature DNA strands. Fixed cells were then incubated in 0.1\u2009M Borate buffer (pH 8.5) for 30\u2009min at room temperature and in 1\u00d7 PBSTX (0.1% Triton X) for 10\u2009min. Cells were blocked with 5% goat serum for 1\u2009h at room temperature and incubated with rat anti-BrdU primary antibody (Abcam, ab6326, 1:2000) overnight at 4\u2009\u00b0C. The next day, cells were washed three times in 1\u00d7 PBST and incubated with goat anti-rat-biotin-linked secondary antibody (Abcam, ab207997, 1:400) for 1\u2009h at room temperature. The biotinylated signal was developed using the ABC Kit (Vectastain, PK-6101) following the manufacturer\u2019s instructions and detected with DAB solution (R&D systems, 4800-30-07). Gill\u2019s No. 3 Haematoxylin (Merck, GHS316-500ML) was used for counterstaining and cells on the coverslips were mounted on glass slides using Aquatex mounting agent (Merck, 108562).<\/p>\n

Crypt-villous isolation and qRT-PCR<\/h3>\n

Intestinal tissue was washed with PBS, cut open longitudinally and laid flat on a glass slide with the luminal side facing upward. The small intestinal villi were scrapped off the flat tissue by a glass slide and collected in cold 1\u00d7 PBS. The remaining tissue containing crypts was chopped into <2\u2009mm pieces using a scalpel, washed three times with ice-cold 1\u00d7 PBS and incubated in chelation medium (2\u2009mM EDTA in 1\u00d7 PBS without Ca2+<\/sup> and Mg2+<\/sup>, Gibco 10010023) for 40\u2009min with agitation at 4\u2009\u00b0C. The digested tissue was shaken vigorously for 30\u2009s in 1\u00d7 PBS to release crypts and villi. To separate out crypts and villi of the small intestine, the solution was passed through a 100\u2009\u00b5m cell strainer. The isolated crypts in the flow through were pelleted and transferred to RLT Buffer (Qiagen, 79216). RNeasy microkit (Qiagen, 74004) was used for RNA extraction. Extracted RNAs were incubated with DNase1 (ThermoFisher, EN0521) at 37\u2009\u00b0C for 30\u2009min, followed by a 10\u2009min incubation with EDTA at 65\u2009\u00b0C. High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) was used to generate complementary DNA from total RNA. Quantitative real-time-PCR (qRT-PCR) was performed on LightCycler96 (Roche) with mouse and naked mole rat Gapdh used as an endogenous control. The IDs of Taqman Gene expression assays (Applied Biosystems) used in this study are Gapdh<\/i> (Mm99999915_g1, Hg05064520_gH), Muc2<\/i> (Mm01276681_m1, Hg05250665_g1), Synaptophysin<\/i> (Mm00436850_m1, Hg05249763_m1), and Aldolase B<\/i> (Mm00523293_m1, Hg05103981_m1). The 2-\u0394\u0394Ct<\/sup> method was used to calculate the relative gene expression levels.<\/p>\n

Brightfield microscopy<\/h3>\n

Brightfield images of tissue sections were captured using an Olympus BX51 microscope coupled with an Olympus DP70 camera system using DP controller software. Villi were imaged using 10\u00d7 objective while crypts were imaged with 20\u00d7 (for colon) or 60\u00d7 (for small intestine) objective lens. Histopathological scoring in this study was performed based on the digital images obtained on Hamamatsu (Nanozoomer HT) scanner at 40\u00d7 magnification.<\/p>\n

Histological quantification (brightfield)<\/h3>\n

To quantify cell numbers in crypt-villous structures from brightfield images, \u2018cell counter\u2019 plugin of Fiji software was used. The dimensions of crypt-villous structure were calculated using the \u2018measure\u2019 tool in Fiji.<\/p>\n

Fluorescent microscopy<\/h3>\n

Fluorescent images of intestinal crypts were acquired from 4\u2009\u00b5m thick tissue sections with a Plan Apochromat 63\u00d7 or 100\u00d7 1.4 oil objective on a Zeiss LSM 780 upright or inverted confocal microscope. Images were acquired in Zen SP7 FP3 (black) software using 405\u2009nm, 488\u2009nm, 561\u2009nm, and 633\u2009nm laser lines in sequential tracks. Z<\/i>-stacks of 6-12 optical sections with 50% overlap between subsequent planes were captured within the span of a single cell at 0.3\u2009\u00b5m z<\/i>-distance, 0.087\u2009\u00b5m pixel dimension, and 12-bit depth.<\/p>\n

For generating the RGB images used in the figures (Figs. 1<\/a>a, b, 2<\/a>a\u2013d, 3<\/a>d, 4<\/a>d, 7<\/a>a, d, Supplementary Figs. 1<\/a>\u20134<\/a>, 5d<\/a>, e, 11b<\/a>), the original.czi raw files were imported into Fiji software package and a maximum intensity z<\/i>-projection was created from the stacks. Using the \u201csplit channel\u201d option of Fiji, the multicolour fluorescent images were separated into individual channels (DAPI, Alexa 488, Cy3, Alexa 633). The maximum and minimum displayed pixel values of individual channels were adjusted across the entire image set including in negative controls (i.e. linear adjustment) to correct for autofluorescence that had been introduced in the image stacks during acquisition. Then, using \u201cmerge channel\u201d option in Fiji, two\/more channels were combined to create a composite image (Lgr5<\/i>\/Ki67 or LGR5<\/i>\/KI67, Lgr5<\/i>\/EpCAM or LGR5<\/i>\/EPCAM, Lgr5<\/i>\/p27 or LGR5<\/i>\/P27, Lgr5<\/i>\/BrdU, Lgr5<\/i>\/pHH3 or LGR5<\/i>\/PHH3) while keeping the individual channels intact. Finally, all the individual and composite images were converted into \u2018RGB color type\u2019 and saved in TIFF format. These images (TIFF) were compiled in Adobe Illustrator 2020 software to produce the panels presented in the figures.<\/p>\n

Histological quantification (fluorescent)<\/h3>\n

Z<\/i>-stack images were processed in batch mode of Fiji package. Firstly, a maximum intensity projection was created to generate a 2D image from the stacks. Next, each channel of the image was separated, and maximum and minimum displayed pixel values were adjusted across the entire image set including negative controls. To quantify the number of rodent Lgr5<\/i> or human LGR5<\/i> mRNA expressed in a single cell, all the ISH dots were manually counted within the cell periphery demarcated by EpCAM staining. As the Lgr5<\/i> or LGR5<\/i> signal was captured using confocal microscopy at a resolution of 237\u2009nm, overlapping\/merged Lgr5<\/i> or LGR5<\/i> mRNA signal dots were rarely observed. To calculate the distribution of Lgr5<\/i>+<\/i><\/sup> or LGR5<\/i>+<\/i><\/sup> cells relative to other cells along the crypt axis, the cell present at the crypt apex was assigned position 0 and we counted cells on each side of this cell to acquire datapoints in our quantifications. Any cell containing more than three Lgr5<\/i> or LGR5<\/i> mRNA puncta was considered positive for Lgr5<\/i> or LGR5<\/i> expression (Lgr5<\/i>+<\/i><\/sup> or LGR5<\/i>+<\/i><\/sup>).<\/p>\n

We observed significant variation in autofluorescence levels between mouse, human and NMR intestinal tissues, with mouse tissue emitting the most and naked mole rats the least. This variation necessitated adjusting the laser powers of the confocal microscope during image acquisition so that maximal image contrast was achieved while also reducing the autofluorescence signals. The maximum and minimum displayed pixel values of individual channels were adjusted across the entire image set (i.e. linear adjustment), including in negative controls, to correct for autofluorescence. These adjustments resulted in varying intensities for specific signals in the three species and, therefore, we took a binary approach for the quantification of the antibody-based signals. The presence of any specific signal in the target compartment inside a cell was considered positive regardless of the staining intensity.<\/p>\n

Estimating the length of the total cell cycle (TT<\/sub>) and S-phase (Ts<\/sub>) by cumulative labelling with BrdU<\/h3>\n

We determined the length of the cell cycle (TT<\/sub>) and S-phase (TS<\/sub>) in CBC cells (Lgr5<\/i>+CBC<\/i><\/sup>) of naked mole rats by counting the fraction of BrdU-labelled Lgr5<\/i>+CBC<\/i><\/sup> cells after successive pulsing over 5 days in NMRs and 2.25 days in mice. As the CBC cells (Lgr5<\/i>+CBC<\/i><\/sup>) cells are on average asynchronously and asymmetrically dividing45<\/a><\/sup>, the labelling index (LI) which provides the ratio of labelled cells to the total population (LI\u2009=\u2009Lgr5<\/i>+<\/i>CBC<\/sup>BrdU+<\/sup>\/Lgr5<\/i>+<\/i>CBC<\/sup>) at any given time (t<\/i>) can be modelled by Eq. 1<\/a> below where TT<\/sub> is the total cell division time33<\/a><\/sup>.<\/p>\n

\n

$${{{{{rm{LI}}}}}}=\t(1\/{{{{{rm{T}}}}}}_{{{{{rm{T}}}}}}){{{{{rm{X}}}}}}t+({{{{{rm{T}}}}}}_{{{{{rm{S}}}}}}\/{{{{{rm{T}}}}}}_{{{{{rm{T}}}}}}),{{{{{rm{for}}}}}},{t}{{{{{rm{le }}}}}}{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}-{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}} {{{{{rm{LI}}}}}}=\t1,{{{{{rm{for}}}}}},t > {{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}-{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}$$<\/span><\/p>\n

\n (1)\n <\/p>\n<\/div>\n

Equation 1<\/a> assumes that there are no or only very few stem cells (based on p27 negativity in NMR and mouse Lgr5<\/i>+<\/i>CBC<\/sup> cells) that remain quiescent for the duration of the BrdU experiment. The lfit<\/i> tool in STATA was used to calculate the least square fit of the data by considering the time points before LI reached saturation. We derived TT<\/sub> from the slope of the regression (TT<\/sub>\u2009=\u20091\/slope). When t<\/i>\u2009=\u20090, LI0<\/sub>\u2009=\u2009TS<\/sub>\/TT<\/sub> which is the y-intercept of the graph. Thus, the duration of S-phase (TS<\/sub>) was estimated from the y-intercept of the regression line.<\/p>\n

Estimating the duration of the specific cell cycle phases<\/h3>\n

For human LGR5<\/i>+CBC<\/i><\/sup> cells, we assumed KI67 is undetectable at G1\/S transition and detected in the S to M phases of the cell cycle46<\/a><\/sup>. We determined the fraction of LGR5<\/i>+CBC<\/i><\/sup> cells that expressed KI67 and calculated the length of S, G2 and M-phase (T(S, G2, M)<\/sub>) using Eq. 2<\/a>:<\/p>\n

\n

$${{{{{{rm{T}}}}}}_{{({{{{{rm{S}}}}}},{{{{{rm{G}}}}}}2,{{{{{rm{M}}}}}})}}}{{{{{rm{KI}}}}}}67^{+}={{{{{{{rm{T}}}}}}}_{{{{{rm{T}}}}}}^{({{{{{{rm{Ref}}}}}}},31)}}{{{{{rm{X}}}}}},{{{{{{rm{LGR}}}}}}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{KI}}}}}}67^{+}\/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$<\/span><\/p>\n

\n (2)\n <\/p>\n<\/div>\n

The time in mitosis (TM<\/sub>) was calculated after quantifying the fraction of rodent (mouse or NMR) Lgr5<\/i>+<\/i>CBC<\/sup> or human LGR5<\/i>+<\/i>CBC<\/sup> cells positive for phospho-histone H3 using Eq. 3<\/a>:<\/p>\n

\n

$${{{{{{rm{T}}}}}}{{{{{rm{M}}}}}}}^{{{{{{rm{Ki}}}}}}67+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}^{({{{{{rm{linear}}}}}},{{{{{rm{regression}}}}}})}{{{{{rm{X}}}}}},{{Lgr}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{pHH}}}}}}3+({{{{{rm{Ser}}}}}}28)\/{{Lgr}5}^{+{{{{{rm{CBC}}}}}}}$$<\/span><\/p>\n

\n (3)\n <\/p>\n<\/div>\n

or<\/p>\n

\n

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{LGR}5}^{+{{{{{rm{CBC}}}}}}}{{{{{{rm{PHH}}}}}}3}^{+}({{{{{rm{Ser}}}}}}28)\/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$<\/span><\/p>\n<\/div>\n

Using TS<\/sub> estimated by Ishikawa et al.31<\/a><\/sup> previously, the length of G2-phase (TG2<\/sub>) was calculated using Eq. 4<\/a>:<\/p>\n

\n

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}2}}^{{{{{{rm{KI}}}}}}67+}={{{{{{rm{T}}}}}}}_{({{{{{rm{S}}}}}},{{{{{rm{G}}}}}}2,{{{{{rm{M}}}}}})}{{{{{{rm{KI}}}}}}67}^{+}-left({{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}right)$$<\/span><\/p>\n

\n (4)\n <\/p>\n<\/div>\n

After quantifying the fraction of LGR5<\/i>+<\/i>CBC<\/sup> cells expressing P27, we calculated the time spent in G0 and G1 (T(G1, G0)<\/sub>P27+<\/sup>) using Eq. 5<\/a>:<\/p>\n

\n

$${{{{{{{rm{T}}}}}}}_{({{{{{rm{G}}}}}}1,{{{{{rm{G}}}}}}0)}}^{{{{{{rm{P}}}}}}27+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{LGR}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{P}}}}}}27+\/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$<\/span><\/p>\n

\n (5)\n <\/p>\n<\/div>\n

We took the fraction of LGR5+<\/i><\/sup>P27+<\/sup> cells in G0 phase (QF) from Ishikawa et al. 31<\/a><\/sup> to calculate the length of G0 in human LGR5<\/i>+CBC<\/sup> cells using Eq. 6<\/a>:<\/p>\n

\n

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}0}}^{{{{{{rm{P}}}}}}27+}={{{{{{rm{QF}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{{{{{{rm{T}}}}}}}_{(G1,G0)}}^{{{{{{rm{P}}}}}}27+}$$<\/span><\/p>\n

\n (6)\n <\/p>\n<\/div>\n

Finally, using Eq. 7<\/a>, we quantified the time human colonic LGR5<\/i>+CBC<\/i><\/sup> cells spend in G1 (TG1<\/sub>):<\/p>\n

\n

$${{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}={{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}0}}^{{{{{{rm{P}}}}}}27+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}1}}^{{{{{{rm{P}}}}}}27+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}2}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}$$<\/span><\/p>\n

\n (7)\n <\/p>\n<\/div>\n

In NMR and mouse, Lgr5<\/i>+CBC<\/i><\/sup> cells are negative for p27 such that TG0<\/sub>\u2009=\u20090. For these species, we derived the combined length of time spent in G1 and G2 (TG1+<\/sub>TG2<\/sub>) from Eq. 7<\/a>.<\/p>\n

Estimating cell division time of Lgr5<\/i>
\n +above crypt base<\/sup> cells from single time point analysis of BrdU labelling<\/h3>\n

Using the length of TS<\/sub> from cumulative BrdU labelling in Lgr5<\/i>+CBC<\/i><\/sup> cells and assuming no change in TS<\/sub> in Lgr5<\/i>+<\/i><\/sup> cells located at different positions within the crypt31<\/a><\/sup>, we measured the total cell division time (TT<\/sub>) of Lgr5<\/i>+<\/i>above crypt base<\/sup> cells using Eq. 1<\/a> by measuring the labelling index (LI) at a single time point (t<\/i>) after pulsing animals with BrdU in vivo. More specifically, in C57BL\/6 mice (n<\/i>\u2009=\u20093 animals, 4 months old), we administered BrdU once and analysed intestinal tissue at t<\/i>\u2009=\u20090.5\u2009h. In NMRs (n<\/i>\u2009=\u20093 animals, 6-24 months-old), we pulsed the animals with BrdU every 8\u2009h and analysed the intestine after t<\/i>\u2009=\u20091 day.<\/p>\n

Statistical analysis<\/h3>\n

We used Microsoft Excel (v16.77.1) for inputting raw data after collection. All statistical tests and graphs displayed in this paper were generated using StataMP 14.1. Details of statistical tests performed are described in figure legends. P<\/i>-values are generated by conducting two-tailed t<\/i>-tests, F<\/i>-test and Wilcoxon rank sum test as indicated in each figure legend. No blinding and randomization were performed during the analysis.<\/p>\n

Illustration<\/h3>\n

All the figures presented in this manuscript were prepared using Adobe Illustrator 2020 (version 24.1). Vector line arts shown in Figs. 1c, d<\/a>, 3<\/a>a, h, 4<\/a>a, h, 6a<\/a>, Supplementary Figs. 5d, e<\/a>, and 9a, b<\/a> were created using the curvature tool of Adobe Illustrator.<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n