Human fibroblast isolation and culture

Isolation was performed at Coriell Institute on healthy patients and from Alzheimer patient samples at Stanford Hospital, in accordance to the methods and protocols approved by the Institutional Review Board of Stanford University, biopsied for skin mesial aspect of mid-upper arm or abdomen using 2-mm punch biopsies from both male and female patients 60–70 years old (n = 8) and 25–35 years (n = 3). Cells were cultured out from these explants and maintained in Eagle’s Minimum Essential Medium with Earl’s salts supplemented with nonessential amino acids, 10% fetal bovine serum, and 1% Penicillin/Streptomycin. Cells were cultured at 37 °C with 5% CO 2 .

Human endothelial cell isolation and culture

Isolation was performed at Coriell Institute from iliac arteries and veins, and muscle biopsies from Stanford Hospital, in accordance to the methods and protocols approved by the Institutional Review Board of Stanford University, from otherwise healthy 45–60 years old (n = 7). Tissue was digested with collagenase and cells released from the lumen were used to initiate cultures. Plates for seeding were coated with 2% gelatin, then washed with PBS before use. Cells were maintained in Medium 199 supplemented with 2 mM l-glutamine, 15% fetal bovine serum, 0.02 mg/ml Endothelial Growth Supplement, 0.05 mg/ml Heparin, and 1% Penicillin/Streptomycin. Cells were cultured at 37 °C with 5% CO 2 .

Human articular chondrocyte isolation and culture

In accordance to the methods and protocols approved by Institutional Review Board of Stanford University, the human OA chondrocytes were derived from discarded tissues of OA patients (50–72 years of age, n = 6) undergoing total knee arthroplasty. The samples were surgical waste and were fully deidentified prior to procurement, hence no prior patient consent was required. Cartilage pieces were shaved off bone by scalpel, taking care to avoid any fat, then digested with collagenase in DMEM/F12 media (supplemented with 25 mg/ml ascorbate, 2 mM l-glutamine, 1% penicillin/streptomycin antibiotics, and 10% fetal bovine serum) for 1–2 days until shavings were substantially dissolved. Supernatant from cultures was strained, filtered, and centrifuged, and the cells were then resuspended in fresh media. The chondrocytes were cultured in high-density monolayer at 37 °C with 5% CO 2 .

Mice

C57BL/6 male and NSG mice were obtained from Jackson Laboratory. NOD/MrkBomTac-Prkdcscid mice were obtained from Taconic Biosciences. Mice were housed and maintained in the Veterinary Medical Unit at the Veterans Affairs Palo Alto Health Care Systems. The Administrative Panel on Laboratory Animal Care of Stanford University approved animal protocols.

Human skeletal muscle specimens

The human muscle biopsy specimens were taken after patients (10–30 years, n = 2; 30–55 years, n = 2; 60–80 years, n = 3) gave informed consent as part of a human studies research protocol that was approved by the Stanford University Institutional Review Board. Sample processing for cell analysis began within 1–12 h of specimen isolation. In all studies, standard deviation reflects variability in data derived from studies using true biological replicates (i.e., unique donors). Data were not correlated with donor identity.

MuSC isolation and purification

Muscles were harvested from mouse hind limbs (n = 4) and mechanically dissociated to yield a fragmented muscle suspension. This was followed by a 45–50-min digestion in a Collagenase II-Ham’s F10 solution (500 U ml−1, Invitrogen). After washing, a second digestion was performed for 30 min with Collagenase II (100 U ml−1) and Dispase (2 U ml−1, ThermoFisher). The resulting cell suspension was washed, filtered, and stained with VCAM-biotin, CD31-FITC, CD45-APC, and Sca-1-Pacific-Blue antibodies, all at dilutions of 1:100. Human MuSCs were purified from fresh operative samples. Operative samples were carefully dissected from adipose and fibrotic tissue and a dissociated muscle suspension prepared as described for mouse tissue. The resulting cell suspension was then washed, filtered, and stained with anti-CD31-Alexa Fluor 488, anti-CD45-Alexa Fluor 488, anti-CD34-FITC, anti-CD29-APC, and anti-NCAM-Biotin antibodies. Unconjugated primary antibodies were then washed and the cells were incubated for 15 min at 4 °C in streptavidin-PE/Cy7 to detect NCAM-biotin. Cell sorting was performed on calibrated BD-FACSAria II or BD FACSAria III flow cytometers equipped with 488-, 633-, and 405-nm lasers to obtain the MuSC population. A small fraction of sorted cells was plated and stained for Pax7 and MyoD to assess the purity of the sorted population.

mRNA transfection

Cells were transfected using either mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes, to reduce cell toxicity, or Lipofectamine MessengerMax (ThermoFisher) for endothelial cells and MuSCs, which were more difficult to transfect, using the manufacturer’s protocol. For fibroblast and endothelial cells, serum free Pluriton medium with bFGF was used for transfection, while muscle stem cells and chondrocytes were kept in their original media—the former lacking serum and the later requiring serum to prevent the natural dedifferentiation of chondrocytes in culture. Culture medium was changed for fibroblasts and endothelial cells 4 h after transfection, but not for chondrocytes or MuSCs as overnight incubation was needed to produce a significant uptake of mRNA. Efficiency of delivery was confirmed by both GFP mRNA and immunostaining for individual factors in OSKMLN cocktail, the former also being used as a transfection control with the same protocol.

Immunocytochemistry

Cells were washed with HBSS/CA/MG and then fixed with 15% paraformaldehyde in PBS for 15 min. Cells were then blocked for 30 min to 1 h with a blocking solution of 1% BSA and 0.3% Triton X-100 in PBS for fibroblasts, endothelial cells, and 20% donkey serum/0.3% Triton in PBS for MuSCs. Primary antibodies were then applied in blocking solution and allowed to incubate overnight at 4 °C. The following day, the cells were washed with HBSS/CA/MG or PBST for MuSCs before switching to the corresponding Alexa Fluor-labeled secondary antibodies and incubated for 2 h. The cells were then washed again and stained with DAPI for 30 min and switched to HBSS/CA/MG for imaging or Fluoview for MuSCs.

Autophagosome formation staining

Cells were washed with HBSS/Ca/Mg and switched to a staining solution containing a proprietary fluorescent autophagosome marker (Sigma). The cells were then incubated at 37 °C in 5% CO 2 for 20 min, washed two times using HBSS/Ca/Mg, and stained for 15 min using CellTracker Deep Red cell labeling dye. Cells were then switched to HBSS/Ca/Mg for single-cell imaging using the Operetta High-Content Imaging System (Perkin Elmer).

Proteasome activity measurement

Wells were first stained with PrestoBlue Cell Viability dye (Life Technologies) for 10 min. Well signals were read using a TECAN fluorescent plate reader as a measure of cell count. Then cells were washed with HBSS/Ca/Mg before switching to original media containing the chymotrypsin-like fluorogenic substrate LLVY-R110 (Sigma), which is cleaved by proteasome 20S core particle. Cells were then incubated at 37 °C in 5% CO 2 for 2 h before signals were again read on the TECAN fluorescent plate reader. Readings were then normalized by PrestoBlue cell count.

Mitochondrial membrane potential staining

Tetramethylrhodamine Methyl Ester Perchlorate (Thermo) was added to cell culture media. This dye is sequestered by active mitochondria based on their membrane potential. Cells were incubated for 30 min at 37 °C in 5% CO 2 and washed two times with HBSS/Ca/Mg before staining for 15 min using CellTracker Deep Red. Finally, cells were imaged in fresh HBSS/Ca/Mg using the Operetta High-Content Imaging System (Perkin Elmer).

Mitochondrial ROS measurement

Cells were washed with HBSS/Ca/Mg and then switched to HBSS/Ca/Mg containing MitoSOX (Thermo), a live-cell-permeant fluorogenic dye that selectively targeted to mitochondria and fluoresces when oxidized by superoxide. Cells were incubated for 10 min at 37 °C in 5% CO 2 . Cells were then washed twice with HBSS/Ca/Mg, and stained for 15 min using CellTracker Deep Red. Finally, cells were imaged in fresh HBSS/Ca/Mg using the Operetta High-Content Imaging System (Perkin Elmer).

SAβGal histochemistry

Cells were washed twice with PBS then fixed with 15% Paraformaldehyde in PBS for 6 min. Cells were rinsed three times with PBS before staining with X-gal chromogenic substrate, which is cleaved by endogenous Beta galactosidase. Plates were kept in the staining solution, Parafilmed, to prevent from drying out, and incubated overnight at 37 °C with ambient CO 2 . The next day, cells were washed again with PBS before switching to a 70% glycerol solution for imaging under a Leica bright-field microscope.

Fixed and live-cell imaging

Samples were imaged using fluorescent microscopes—the Operetta High-Content Imaging System (Perkin Elmer) or the BZ-X700 (Keyence)—and either a 10× or 20× air objective. Harmony (Operetta) or Volocity (BZ-X700) imaging software was used to adjust excitation and emission filters and came with preprogrammed Alexa Fluor filter settings which were used whenever possible. All exposure times were optimized during the first round of imaging, and then kept constant through all subsequent imaging.

Image analysis

Columbus (Operetta) or Image J (BZ-X700) was used for image analysis. Columbus software was to identify single cells utilizing DAPI of CellTracker Re d to delineate nuclear and cell boundaries and calculate the signal statistics for each cell. Image J was used for muscle fibers to calculate the percentage of area composed of collagen by using the color threshold plug-in to create a mask of only the area positive for collagen. That area was then divided over the total area of the sample, which was found using the free draw tool. All other fiber analyses were performed using Volocity software and manually counting fibers using the free draw tool.

Statistics

Statistical analysis for physiological hallmarks of aging was done as described previously in Miller et al.2. Briefly, 100 cells were randomly selected from each experimental group (data depicted in Supplementary Figs. 2–5), and they were then pooled in a unique population of 800 cells for aged fibroblasts (100 cells × 8 individuals for both aged and aged treated); 300 cells for young fibroblasts (100 cells × 3 individuals); 700 cells for aged endothelial cells (100 cells × 7 individuals for both aged and aged treated); 300 cells for young endothelial cells (100 cells × 3 individuals). Box distribution plots display the fluorescence intensity quantification of 100 cells from each patient. Distributions were compared by statistical analysis by using multiple-comparison ANOVA. Arbitrary units for frequency distributions of different cell types should not be compared because staining was performed at different times. Matlab 2017 (MathWorks) was used for data presentation and analysis.

Cytokine profiling

This work was performed together with the Human Immune Monitoring Center at Stanford University. Cell media was harvested and spun at 400 rcf for 10 min at room temperature. The supernatant was then snap frozen with liquid nitrogen until analysis. Analysis was done using the human 63-plex kit (eBiosciences/Affymetrix). Beads were added to a 96-well plate and washed in a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 h followed by overnight incubation at 4 °C with shaking. Cold and room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation, plates were washed in a Biotek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. Plates were washed as above and streptavidin-PE was added. After incubation for 30 min at room temperature, wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay Control beads by Radix Biosolutions were added to all wells.

Antibodies

The following antibodies were used in this study. The source of each antibody is indicated. Rabbit::H3K9me3 (Abcam #ab8898 1:4000), LAP2α (Abcam #ab5162 1:500), SIRT1 (Abcam #ab7343 1:200); Rabbit: Mouse: HP1γ (Millipore Sigma #05-690 1:200), Lamin A/C (Abcam #ab40567 1:200), GFP (Invitrogen, #A11122, 1:250); Luciferase (Sigma-Aldrich, #L0159, 1:200); Collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200), Laminin (Abcam, #AB11576, 1:1000), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75) and anti-NCAM-Biotin (clone HCD56; BioLegend; #318319, 1:75), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56; BioLegend; #318319, 1:75).

RNA sequencing and data analysis

Cells were washed and digested by TRIzol (Thermo). Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek Corp) and RNA quality was assessed by the RNA analysis screentape (R6K screentape, Agilent); RNA with RIN > 9 was reverse transcribed to cDNA. cDNA libraries were prepared using 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA quality was assessed by an Agilent Bioanalyzer 2100; RNA with RIN > 9 was reverse transcribed to cDNA. cDNA libraries were prepared using 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) with the added benefit of molecular indexing. Prior to any PCR amplification steps, all cDNA fragment ends were ligated at random to a pair of adapters containing a 8-bp unique molecular index. The molecular indexed cDNA libraries were than PCR amplified (15 cycles) and then QC’ed using a Bioanalzyer and Qubit. Upon successful QC, they were sequenced on an Illumina Nextseq platform to obtain 80-bp single-end reads. The reads were trimmed by 2 nt on each end to remove low-quality parts and improve mapping to the genome. The 78-nt reads that resulted were compressed by removing duplicates, while keeping track of how many times each sequence occurred in each sample in a database. The unique reads were then mapped to the human genome using exact matches. This misses reads that cross exon–exon boundaries, as well as reads with errors and SNPs/mutations, but it does not have substantial impact on estimating the levels of expression of each gene. Each mapped read was then assigned annotations from the underlying genome. In case of multiple annotations (e.g., a miRNA occurring in the intron of a gene), a hierarchy based on heuristics was used to give a unique identity to each read. This was then used to identify the reads belonging to each transcript and coverage over each position on the transcript was established. This coverage is nonuniform and spiky. Therefore, we used the median of this coverage as an estimate of the expression value of each gene. In order to compare the expression levels in different samples, quantile normalization was used. Further data analysis was done in Matlab. Ratios of expression levels were then calculated to estimate the log (base 2) of the fold change. Student’s t test was used to determine significance with a p < 0.05 cutoff. Molecular Signatures Database categorization was done using Broad Institute online tools https://software.broadinstitute.org/gsea/msigdb/.

Gene expression analysis

Total RNA was purified using the RNeasy Plus Mini kit (Qiagen), and cDNA was prepared with the First-strand cDNA synthesis kit (Applied Biosystems). The quantitative polymerase chain reaction was performed using VeriQuest Mastermix (ThermoFisher Scientific) for SYBR Green and Taqman primer sets, respectively. The relative gene expression was analyzed by the ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Taqman probes for human GAPDH (Hs02758991); COL2A1 (Hs00264051); SOX9 (Hs00165814); MMP3 (Hs00233962) and MMP13 (Hs00233992) were purchased from Applied Biosystems. The SYBR green primer sequences used are: Human SOD2 (F)-5′GGC CTA CGT GAA CAA CCT GA3′; Human SOD2 (R)-5′TGG GCT GTA ACA TCT CCC TTG3′; Human iNOS (F)-5′GTC CCG AAG TTC TCA AGG CA3′; Human iNOS (R)-5′GTT CTT CAC TGT GGG GCT TG3′; Human RANKL (F)-5′CAG GTT GTC TGC AGC GT3′ and Human RANKL (R)-5′GAT CCA TCT GCG CTC TGA AAT A3′; Human GAPDH (F)- 5′TGT CCC CAC TGC CAA CGT GTC3′; Human GAPDH (R)-5′AGC GTC AAA GGT GGA GGA GTG GGT3′.

ATP assay

ATP in the chondrocytes was measured using colorimetric assay and the ATP assay kit (ab83355; Abcam, Cambridge, MA) following the manufacturer’s instructions. Cells were washed in cold phosphate buffered saline and homogenized and centrifuged to collect the supernatant. The samples were loaded with assay buffer in triplicate. ATP reaction mix and background control (50 µL) was added to the wells and incubated for 30 min in dark. The plate was read at OD 570 nm using SpectraMax M2e (Molecular Devices, Sunnyvale, CA). The mean optical density was used to estimate of the intracellular ATP concentration relative to the standard curve.

Cell proliferation assay

Cell viability was assayed using the PrestoBlue Cell Viability (Life Technologies) reagent consecutively for 3 days post transfection in accordance with the manufacturer’s instructions. PrestoBlue reagent was added to the cell culture medium, and the cells were incubated at 30 °C for 30 min. Absorbance of the PrestoBlue was measured daily using SpectraMax M2e (Molecular Devices, Sunnyvale, CA).

EDU staining

Staining was done according to the manufacturer’s protocol using the Click-iT EdU kit. Cells were labeled with Edu after switching to growth media. Cells were allowed to grow 1 or 2 days before fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100 in PBST. Cells were the incubated in Click-It reaction cocktail for 30 min before washing in PBS and imaging.

MitoTracker staining and flow cytometry analysis

Cells were washed twice with Ham’s F10 (no serum or pen/strep). Subsequently, MuSCs were stained with MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 min at 37 °C, washed three times with Ham’s F10, and analyzed using a BD FACSAria III flow cytometer.

Myogenic colony-forming cell assay for MuSCs

Single treated and control MuSCs were deposited into wells of collagen- and laminin-coated plates at one cell per well by BD FACSAria III flow cytometer. Collagen/laminin coating was accomplished by overnight incubation of the plates rocking at 4 °C with a 1:1 mixture of laminin (10 μg/ml ThermoFisher 23017-015) and collagen (10 μg/ml Sigma C8919) in PBS. Coated wells were washed three times with PBS before use. The cells were cultured in grow media, F10 medium supplemented with 20% horse serum, and 5 ng/ml basic fibroblast growth factor (bFGF; PeproTech 100-18B). After 6 days of culture, plates were fixed with 4% paraformaldehyde (Electron Microscopy Services 15710), stained with DAPI (Invitrogen D1306), and scored by microscopy to determine the number of myogenic colony-forming cells, defined by wells that contained at least eight cells.

Myogenic/fusion index

Myogenic analysis was completed as previously described36. After MuSCs underwent reprogramming or control treatment, cells were cultured in grow media. To induce differentiation, myoblast cultures were maintained in DMEM supplemented with 2% horse serum. The myogenic/fusion index was determined as the percentage of myonuclei in myotubes (defined as cells with three or more nuclei) compared with the total number of nuclei in the field.

Lentiviral transduction

Luciferase and GFP protein reporters were subcloned into a third-generation HIV-1 lentiviral vector (CD51X DPS, SystemBio). To transduce freshly isolated MuSCs, cells were plated on a Poly-D-Lysine (Millipore Sigma, A-003-E) and ECM coated eight-well chamber slide (Millipore Sigma, PEZGS0896) and were incubated with 5 μl of concentrated virus per well and 8 μg/mL polybrene (Santa Cruz Biotechnology, sc-134220). Plates were spun for 5 min at 3200 g, and for 1 h at 2500 g at 25 °C. Cells were then washed with fresh media two times, scraped from plates, and resuspended in the final volume according to the experimental conditions.

Bioluminescence imaging

Bioluminescent imaging was performed using the Xenogen IVIS-Spectrum System (Caliper Life Sciences). Mice were anesthetized using 2% isoflurane at a flow rate of 2.5 l/min Intraperitoneal injection of d-Luciferin (50 mg/ml, Biosynth International Inc.) dissolved in sterile PBS was administered. Immediately following the injection, mice were imaged for 30 s at maximum sensitivity (f-stop 1) at the highest resolution (small binning). Every minute, a 30-s exposure was taken until the peak intensity of the bioluminescent signal began to diminish. Each image was saved for subsequent analysis.

Bioluminescence image analysis

Analysis of each image was performed using Living Image Software, version 4.0 (Caliper Life Sciences). A manually generated circle was placed on top of the region of interest and resized to completely surround the limb or the specified region on the recipient mouse. Similarly, a background region of interest was placed on a region of a mouse outside the transplanted leg.

Tissue harvesting

TiA muscles were carefully dissected away from the bone, weighed, and placed into a 0.5% PFA solution for fixation overnight. The muscles were then moved to a 20% sucrose solution for 3 h or until muscles reached their saturation point and began to sink. The tissues were then embedded and frozen in Optimal Cutting Temperature (OCT) medium and stored at −80 °C until sectioning. Sectioning was performed on a Leica CM3050S cryostat that was set to generate 10-μm sections. Sections were mounted on Fisherbrand Colorfrost slides. These slides were stored at −20 °C until immunohistochemistry could be performed.

Flow cytometry

For mouse MuSC sorting scheme, we followed the same gating strategy previously published37. For human MuSC sorting scheme, we followed the same strategy previously published38.

Histology

TiA muscles were fixed for 5 h using 0.5% electron-microscopy-grade paraformaldehyde and subsequently transferred to 20% sucrose overnight. Muscles were then frozen in OCT, cryosectioned at a thickness of 10 μm, and stained. For colorimetric staining with Hematoxylin and Eosin (Sigma) or Gomori Trichrome (Richard-Allan Scientific), samples were processed according to the manufacturer’s recommended protocols.

Ex vivo force measurement

To measure the force, we isolated the TiA in a bath of oxygenated Ringer’s solution and stimulated it with plate electrodes. Immediately after euthanasia, the distal tendon of the TiA, the TiA, and the knee (proximal tibia, distal femur, patella, and associated soft tissues) were dissected out and placed in Ringer’s solution (Sigma) maintained at 25 °C with bubbling oxygen with 5% carbon dioxide. The proximal tibia was sutured to a rigid wire attached to the force transducer, and the distal tendon was sutured to a rigid fixture. No suture loops or slack was present in the system. The contralateral limb was immediately dissected and kept under low passive tension in oxygenated Ringer’s solution bath until measurement. Supramaximal stimulation voltage was found, and the active force-length curve was measured in a manner similar to the in vivo condition. After measurement, the muscle was dissected free and the mass measured. An Aurora Scientific 1300-A Whole Mouse Test System was used to gather force production data.

DNA methylation data

The human Illumina Infinium EPIC 850K chip was applied to n = 16 DNA samples (corresponding to two treatment levels (before/after treatment) of four fibroblasts and four endothelial cells). The raw image data were normalized using the “preprocessQuantile” normalization method implemented in the “minfi” R package39,40.

Epigenetic clock analysis

Several DNAm-based biomarkers have been proposed in the literature, which differ in terms of their applicability (most were developed from blood), and in terms of their biological interpretation (reviewed in ref. 11). We focused on two epigenetic clocks that apply to fibroblasts and endothelial cells. In our primarily analysis, we used the pan-tissue epigenetic clock3 because it applies to all sources of DNA (with the exception of sperm). A previously defined mathematical algorithm is used to combine the methylation levels of 353 CpG into an age estimate (in units of years), which is referred to as epigenetic age or DNAm age3. In our secondary analysis, we used the skin-and-blood epigenetic clock (based on 391 CpGs) because it is known to lead to more accurate DNAm age estimates in fibroblasts, keratinocytes, buccal cells, blood cells, saliva, and endothelial cells13.

We used the online version of the epigenetic clock software to arrive at DNA methylation age estimates from n = 16 samples collected from n = 8 individuals3. Although the chronological age range was relatively narrow (ranging from 47 to 69 years, median age = 55), the two DNAm age estimates exhibited moderately high correlations with chronological age (r = 0.42 and r = 0.63, P = 0.0089 for the pan-tissue- and the skin-and-blood clock, respectively).

Two samples (before and after rejuvenation treatment) were generated from each of n = 8 individuals. To properly account for the dependence structure in the data, we used linear mixed effects models to regress DNAm age (dependent variable) on treatment status, chronological age, and individual identifier (coded as random effect). Toward this end, we used the “lmer” function in the “lmerTest” R package41.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.