An adult myogenic cell line of the Japanese fire-bellied newt Cynops pyrrhogaster

Animals

Sexually mature males of C. pyrrhogaster (total body length: 9–11 cm) were used. They were captured from Miyagi, Niigata, Fukui and Ishikawa prefectures by a supplier (Aqua Grace, Yokohama, Japan), and reared in plastic containers containing shallow water and a resting place (land) at 18–22 °C under natural light conditions until experiments were performed⁴³. All experiments were carried out in accordance with the guidelines and regulations in the University of Tsukuba. All protocols with animals were approved by the Animal Care and Use Committee of the University of Tsukuba (approval number: 220125). Moreover, all methods were performed and reported in compliance with the ARRIVE guidelines 2.0⁴⁴.

Anesthesia

0.1% FA100 (4-allyl-2-methoxyphenol; LF28C054; DS Pharma Animal Health, Osaka, Japan) dissolved in water was used at room temperature (RT: 22 °C). Animals were placed in a sealed bottle containing the anesthetic solution (five or less individuals/300 mL) for 45 min before limb amputation^{11,15,17,18,32}.

Limb amputation

After anesthesia, limb amputation was performed as described previously^{15,18,32,45,46}. To obtain the normal forearm samples for muscle explant culture and biochemistry, the upper arm was amputated in the middle (at the mid-stylopod region) or near the level of the elbow. To induce forearm regeneration, the forearm was amputated in the middle (at the mid-zeugopod region). To obtain samples of regenerating forearms for histology, the upper arm was amputated in the middle, and for biochemistry, the regenerating forearm was amputated again at the level of the amputation plane. After the limb samples had been harvested, amputees were placed on dry paper towels until the bleeding stopped, transferred to moist containers with a lid containing air vents (up to three newts per container of length 200 mm × width 150 mm × height 55 mm) and allowed to recover. The moist container was always kept in a semi-dry condition in which the bottom was covered by a moist paper towel (Elleair Prowipe, Soft High Towel, Unbleached, 4P; Elleair Paper Business Support, Tokyou, Japan) that was tightly wrung. Paper towels were replaced with new ones every other day. The stages of limb regeneration were determined according to previous criteria³² otherwise noted.

Acquisition of the cell line

One male individual (total body length: 9 cm) was used for this purpose. After the right forelimb was amputated in the middle of the upper arm using a surgical blade (No. 19, Futaba, Tokyo, Japan) under a dissecting microscope (SZ61; Olympus/Evident, Tokyo, Japan), the sample was immersed in 70% ethanol (diluted in type1 MilliQ water, Merck Millipore, Merck, Tokyo, Japan) for 5 s, washed twice each with fresh sterile phosphate-buffered saline (PBS; pH 7.5), and then, under the dissecting microscope, immobilized on a silicon base (SILPOT 184 W/C; DuPont Toray Specialty Materials K.K., Tokyo, Japan) of a PBS-containing 35 mm plastic dish (353001; Falcon, Corning, Shizuoka, japan) with fine insect pins (20111; Shiga Konchu Fukyusha, Tokyo, Japan). In this condition, after the skin and bone of the forearm were carefully removed using fine forceps, two major muscles, the flexor digitorum communis (FDC) and extensor digitorum communis (EDC)⁴⁷ without tendons were cut off from the rest. Both muscles were cut into 1 mm blocks along the longitudinal axis, and one block each of FDC and EDC was placed together on a collagen type I-coated glass base dish (27 mm; 4970-011; AGC Techno Glass, Shizuoka, Japan) filled with 1.5 mL of a Leibovitz’s L-15-based medium (72% Leibovitz’s L-15 medium (41300-039; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS (SH30071.03, Hyclone, Cytive, Marlborough, MA, USA), 1% penicillin/streptomycin (15140-122; Gibco, Thermo Fisher Scientific)). A total of six dishes were prepared and incubated at 25 °C in a Parafilm-sealed condition for 3 weeks (Fig. 1a).

After culture for 3 weeks, four dishes were selected in which muscles were firmly attached to the bottom and mesenchymal cells appeared surrounding the muscles, the culture medium was replaced with “growth medium” (65% MEM (11090081; Gibco, Thermo Fisher Scientific), 10% FBS, 1% L-glutamine (25030081; Gibco, Thermo Fisher Scientific), 1% penicillin/streptomycin and 10 µg/mL insulin (I6634-100MG, Sigma-Aldrich, Merck, Tokyo, Japan)), and the dishes were incubated at 25 °C under a CO₂ equilibration condition for an additional 1 week (Fig. 1a). During a total of 4 weeks of explant culture, half of the volume of the medium was replaced with fresh medium every 4 days. After 4 weeks of culture, muscle explants were removed from the dishes, and mesenchymal cells on the bottom of the dish were washed with 1 mL PBS three times, and incubated in 1 mL of 1/5x Trypsin-EDTA (0.25% Trypsin/0.53 mM EDTA in Hanks Balanced Salt Solution without calcium or magnesium, 30-2101; ATCC, Gaithersburg, MD, USA) diluted with PBS for 5 min at RT (trypsinization). In the meantime, the cells were separated from the bottom of the dish by tapping the dish lightly with a finger under the dissecting microscope. After 1 mL of growth medium was added to the dish to inactivate trypsin, the cell suspensions from four dishes were combined in one tube and centrifuged at 93 ɡ for 8 min (LC-120; TOMY, Tokyo, Japan). After the supernatant was removed, the cell pellet was resuspended in 1.5 mL of fresh growth medium and cultured in one 35 mm collagen type I-coated plastic dish (4000-010, IWAKI, Shizuoka, Japan) until the cells had grown to cover 80–90% of the bottom of the dish, i.e., 80–90% confluence (passage 1). The cells in the dish were collected in one tube as before, divided equally into two dishes, and cultured until each was 80–90% confluent (passage 2). The cells in the two dishes were collected in one tube, divided equally into four dishes, and cultured until each was 80–90% confluent (passage 3). The cells in the four dishes were collected in one tube, and a half volume of the cell suspension collected was divided equally into four dishes, and cultured until each was 80–90% confluent (passage 4). This step was repeated six more times until passage 10 to obtain CpM01 (Fig. 1a). The incubation period per passage was 6–10 days.

CpM01 cells in four dishes at passage 10 were collected by trypsinization, mixed in growth medium containing 8% dimethyl sulfoxide (DMSO; 4-X; ATCC, Manassas, VA, USA), transferred equally into two cryotubes (~ 2.0 × 10⁵ cells/tube; 377267, Nunc™ Biobanking and Cell Culture Cryogenic Tubes; Thermo Fisher Scientific), and stored in LN2 (Fig. 1a). The cells in these two tubes were recovered in one dish (recovery rate was about 47%) and amplified through the following three passages (Supplementary Fig. S6a). Note that passages were performed so that cells from one dish were equally seeded into two dishes and cultured until each was 70–80% confluent. The cells after the culture of passage 3 (8 dishes) were mixed, transferred into three cryotubes (~ 2.0 × 10⁵ cells/tube), and stored in LN2 (master stock). Remaining cells were used to check the proliferative activity of the cells for the master stock (Fig. 2a,b; see below). The cells in one tube of the master stock were amplified as before and stored in LN2 (working stock), and were further amplified to either replenish the working stock or use for experiments (Fig. 1a and Supplementary Fig. S6b).

Evaluation of proliferative capacity

To evaluate the proliferative capacity of CpM01, ~ 6.0 × 10⁴ cells for the master stock (before freezing) were seeded into a collagen type I-coated glass base dish, cultured for 6 days until 80% confluence, and then examined for their PCNA immunoreactivity (Fig. 2a). This test was not repeated because of sample limitations. For the proliferative capacity of the working stock, cells amplified through 3–4 passages from the working stock were seeded at ~ 2.0 × 10⁴ cells in a collagen type I-coated glass base dish and cultured for 2 days. The cells attached on the bottom were further cultured in the presence of 10 µM BrdU (B5002-100MG; Sigma-Aldrich, Merck) for 24 h, and then examined for their BrdU immunoreactivity (Fig. 2b).

In the growth test (Fig. 2c), cells of the working stock were allowed to recover for 1–3 days and then passaged twice. When the culture of the second passage reached 70–80% confluence, the cells were collected and subcultured at an initial density of either 2.0, 4.0, or 6.0 × 10⁴ cells/35 mm collagen type I-coated plastic dish for 10 days. The total number of cells per dish was counted every other day. Note that it always took 6–10 days for the cells to reach 70–80% confluence, when culturing cells from the working stock was started from a density of 6.0 × 10⁴/35 mm dish.

In the immortality test (Fig. 2d), cells of the working stock were allowed to recover for 1–3 days and then passaged at an initial density of 6.0 × 10⁴ cells/35 mm dish. Passages in the same manner were repeated six more times every 8–10 days.

Induction of differentiation

To induce the differentiation of CpM01 into myotubes, cells of the working stock were allowed to recover for 1–3 days and then passaged three times. As the cells of the third passage reached 80–90% confluence, the culture medium was changed from growth medium to “differentiation medium” (65% MEM, 1% horse serum (H1138-100ML; Sigma-Aldrich, Merck), 1% L-gultamine, 1% penicillin/streptomycin and 10 µg/mL insulin). The cells were cultured in this condition for 6 days. During this period, the medium was refreshed every other day.

Molecular cloning of C. pyrrhogaster MRFs

Contigs for the orthologs of Myf5, MRF4, and myogenin were found in a C. pyrrhogaster comprehensive transcriptome database, TOTAL (http://antler.is.utsunomiya-u.ac.jp/imori/)³² Supplementary Figs. S7–S11). Of note, however, contigs for MyoD were not found. The existence of these three transcripts in the limb muscle and blastema was validated by standard PCR-based molecular cloning (Supplementary Fig. S1b–e) and subsequent nucleotide sequencing by MinION (MIN-101B, Flow Cell (R10.4.1) FLO-MIN114, SQK-LSK114 sequencing kit, MinKNOW Stand-alone NC Windows version 23.11.5; Oxford Nanopore Technologies plc., Oxford, UK), using cDNAs which were constructed from normal forearm muscles and stage III forearm blastemas with Nucleospin RNA (Mini kit for RNA purification; Takara Bio Inc., Shiga, Japan) and the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific)^17,32. The nucleotide sequences determined here were deposited at GenBank (accession numbers: Myf5, PP934189; MRF4, PP934188; myogenin, PP934190). MEGA11 (version 11.0.013; https://www.megasoftware.net/) was used for molecular phylogenetic tree analysis with sequence data of 15 vertebrate species, and 500 bootstrap replicates (a maximum likelihood method based on the JTT matrix-based model) were performed. Amino acid (AA) sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/) except for those of urodele amphibians, which were obtained from databases of Ambystoma mexicanum (AmexT_v47 – v.47; https://www.axolotl-omics.org/), P. waltl (iNewt; https://www.nibb.ac.jp/imori/main/), C. pyrrhogaster (TOTAL) and N. viridescence (RSNRP; http://sandberg.cmb.ki.se/redspottednewt/).

Antibodies

Antibodies used in this study are listed in Supplementary Fig. S12. Based on the nucleotide sequences, rabbit polyclonal antibodies against Myf5, MRF4, and myogenin of C. pyrrhogaster were generated (Eurofin genomics, Tokyo, Japan), and their specificity was confirmed by Western blotting (Supplementary Figs. S1g, S2).

Western blotting

Proteins were extracted from normal forearm muscles and stage III forearm blastemas, each of which were collected from four individuals (two males and two females) in a 1.5 mL protein low-binding tube (0030 108.116; Eppendolf, Hamburg, Germany) filled with 0.5 mL of chilled PBS. After centrifugation (93 ɡ, 1 min), PBS was carefully removed from the tube, and the weight of the sample was measured. The sample was added with lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA-2Na, 1% Igepal CA-630, 1% sodium deoxycholate, 0.1% SDS) containing 1% proteinase inhibitor cocktail (P8340; Sigma-Aldrich, Merck) in the volume equivalent to the same weight of water, and mashed (Biomasher, 893062; Nippi, Tokyo, Japan). To make the lysate smooth, it was frozen with LN2 and sonicated (46 kH, VS-70U; Iuchi/AS ONE, Osaka, Japan) in chilled water for 5 min. This step was repeated five times. After spinning down the content (15,000 ɡ, 10 min, 4 °C), the supernatant was transferred to a fresh 1.5 mL protein low-binding tube. It was mixed with the same volume of 2x Laemmli Sample Buffer (161–0737; Bio-Rad, Hercules, CA, USA), heat-denatured by placing it in boiling water for 5 min, and stored at − 80 °C until use. Western blotting was performed as described previously^11,32,46. Briefly, protein samples and the molecular weight marker proteins (All Blue Prestained Protein Standards, 1610373; Bio-Rad, Tokyo, Japan) were separated on a 4–15% gradient gel (4561083, Mini-PROTEAN TGX Precast Gels; Bio-Rad) by SDS-PAGE, and transferred to an activated Immun-Blot^® PVDF membrane (1620–174; Bio-Rad).

Tissue preparation

Tissue sections for immunostaining were prepared as described previously^{11,15,17,18,32}. Normal and regenerating limb samples were fixed in a modified Zambony’s fixative (2% paraformaldehyde and 0.2% picric acid dissolved in PBS) for 5 h at 4 °C. Exceptionally, for normal forearm muscle, the skin was removed from the limb samples before fixation. Fixed samples were thoroughly washed with chilled PBS and immersed in 30% sucrose-PBS solution overnight (15 h) at 4 °C. They were embedded in Tissue-Tek O.C.T. Compound (4583; Sakura Finetek USA, Inc., Torrance, CA, USA), frozen at − 20 °C in a cryostat (CM1860; Leica Biosystems, Tokyo, Japan), and then stored at − 80 °C until use. The samples were sectioned at between − 30 and − 20 °C by the cryostat: for normal muscle, transverse sections of limbs were made at a thickness of 12 μm (30–40 slices/sample); for regenerating limbs, sections along the dorsoventral axis were made at a thickness of 20 μm (20–30 slices/sample). Tissue sections were placed on gelatin-coated coverslips, air-dried, and then stored at − 20 °C until use.

Culture preparation

Cultures for immunostaining were fixed in 2% paraformaldehyde in PBS solution (pH 7.5) for 20 min at 4 °C and rinsed gently (PBS, 0.2% TritonX-100 in PBS (PBST), PBS; each for 15 min). In the case of BrdU staining, they were incubated in 2 N HCl (080-01066; Wako; Fujifilm, Osaka, Japan) diluted in type1 MilliQ water for 30 min at RT, and then rinsed in PBS before immunostaining.

Immunostaining

For each immunoblotting, a Western-blotted membrane from a gel electrophoresed with the same amount of protein in adjacent lanes was cut into two pieces prior to immunostaining for the test and control antibodies (Supplementary Figs. S1g, S2). Protein bands on the membranes were immunostained as described previously^11,32,46. Signals were visualized with an ABC (Vectastain ABC Elite kit, PK-6100; Vector Laboratories, Newark, CA 94560)–DAB (SK-4100; Vector Laboratories) system.

Fixed tissue sections were immunostained as described previously^{11,15,17,18,32}. Signals were visualized with fluorescent secondary antibodies.

Fixed cultures were immunostained as follows: they were incubated with blocking solution (PBST containing 5% serum of the same species as that in which secondary antibody was produced) at RT for 30 min, and then incubated with primary antibody diluted in the blocking solution at 4 °C for 15 h. After washing, they were incubated in secondary antibody diluted in the blocking solution at RT for 30 min. After washing, cell nuclei were counter stained with 4,6-diaminodino-2-phenylindole (DAPI, 1: 50,000; D1306; Thermo Fisher Scientific). In these experiments, normal donkey serum (017-000-001; Jackson ImmunoResearch, West Grove, PA, USA) or normal goat serum (S-1000; Vector Laboratories) was used for the blocking solution.

Image acquisition

Images of living cultures were captured by a single-lens reflex camera system (EOS Kiss X7; EOS Utility, Version 2.13.30.0; Cannon, Tokyo, Japan) mounted on an inverted phase contrast microscope (IMT-2, Olympus/Evident). Images of immunostained cultures were acquired by an all-in-one fluorescence inverted microscope system (BZ-X800; KEYENCE, Osaka, Japan) with filter sets for EGFP (OP-87763; exciter: 470/40 nm; emitter: 525/50 nm), TRITC (OP-87764; exciter: 545/25 nm; emitter: 605/70 nm), and DAPI (OP-87762; exciter: 360/40 nm; emitter: 460/50 nm). Images of immunostained tissue sections were acquired through a confocal microscope system (LSM700; ZEN 2009, ver. 6.0.0.303; Carl Zeiss, Oberkochen, Germany) with filter sets for rhodamine (Diode 555 Laser; emitter: BP 575–640 nm), Alexa Fluor 488 (Diode 488 Laser; emitter: BP 515–565 nm) and DAPI (Diode 405-5 Laser; emitter: BP 445/50 nm).

Single cell mRNA sequencing

CpM01 cells were harvested from three dishes at the third passage from a master stock, as usual (Supplementary Fig. S6b), except for the time of culture (8 days), cell density (80–90% confluence), and trypsinization conditions (1x Trypsin-EDTA, 28 °C for 1 h). The cells were stored in LN2 as for the master stock and transported to the Life Science Data Research Center (LiSDaC) in Tokyo University (Kashiwa, Japan), the site for the cDNA library construction and sequencing (see below), in a frozen condition.

For erythroid cells, blood/mesenchymal cells were collected from regenerating parts of the right forelimbs of 30 adult males (mid bud stage (or stage III³¹), 20; late bud stage, 1; palette stage, 6; early digit stage, 3)⁴⁸ as follows: the regenerate samples were harvested in 100 µL drops (4–5 samples/drop) of 30 mM tetrasodium 1,2-bis(2-aminophenoxy)ethane-N, N,N’,N’-tetraacetate (BAPTA-Na₄) dissolved in PBS (T2844; Tokyo Chemical Industry, Tokyo, Japan), which were placed on the bottom of a 90 mm plastic dish (01–013; Sansei Medical, Kyoto, Japan) on ice. The regenerates were minced by a blade (FA-10; FEATHER Safety Razor, Osaka, Japan), and released cells were carefully transferred into a 1.5 mL protein low-binding tube with cell saver micropipettes (200 µL, PT-004; 1000 µL, PT-004; INA OPTIKA, Osaka, Japan). The cells were washed twice in PBS by centrifuging at 1800 ɡ for 10–20 s, placed on ice, and brought to LiSDaC within 1 h.

For retinal cells, retinal pigment epithelium cells were collected from 20 normal retina-less eye-cups (a posterior half of eyeball from which the neural retina was removed) prepared from adult females and males (5 each) as described previously⁴⁹. Briefly, the animals were anesthetized as for limb amputation, except that they were placed in the anesthetic solution for 2 h in dark⁵⁰. After decapitation, normal eyeballs were enucleated from the head under the dissecting microscope, and placed cornea side up on a membrane filter (0.45 μm MF-Millipore MCE Membrane, HAWP01300; Merck) in a 35 mm Petri dish (Nunc™ Cell Culture/Petri Dishes 171099; Thermo Fisher Scientific) (one eyeball/dish). PBS was poured into the dish so that the eyeball was immersed. A portion of the eyeball anterior to the cornea-sclera boundary that includes the ciliary marginal zone was excised, and the posterior portion was used as an eye-cup (this process was repeated for all eyeballs). Then, the neural retina was carefully removed from the eye-cup in PBS, making a retina-less eye-cup (this process was repeated for all eye-cups). The retina-less eye-cups on the membrane filter were transferred into 180 µL drops of newt normal saline (in mM: NaCl, 115; KCl, 3.7; CaCl₂, 3; MgCl₂, 1; D-glucose, 18; HEPES, 5; pH 7.5 adjusted with 0.3 N NaOH) (one sample/drop) placed on the bottom of a 90 mm plastic dish (01–013; Sansei Medical), and incubated at RT for 1 min. These samples were transferred into 180 µL drops of 0.2 mM collagenase I (SCR103; Sigma-Aldrich, Merck) in the newt normal saline (one sample/drop) placed in another 90 mm plastic dish, and incubated at 28 °C for 90 min. The collagenase I solution on the sample was replaced with PBS, the volume of PBS was carefully reduced as much as possible, and then the samples were immersed in 180 µL of 1x Trypsin-EDTA solution and further incubated at 28 °C for 45 min. The sample was transferred into PBS in a 40 μm cell strainer (pluriStrainer, 45-50040-03; pluriSelect Life Science, Deutscher Platz 5c, 04103 Leipzig, Germany) placed in a well of a 6-well plate (Nunc™ Non-Treated Multidishes 6-well, 150239; Thermo Fisher Scientific), the membrane filter was removed, and then cells on the surface of the sample, including RPE cells, were dissociated by pipetting with cell saver micropipettes under a dissecting microscope. This process was repeated for all the samples on the same strainer in the same well. The cells collected in the well were transferred into PBS in a 5 μm cell strainer (pluriStrainer, 43-50005-13; pluriSelect Life Science) placed in a plastic Tupperware (length 200 mm × width 150 mm × height 55 mm) to remove small debris and pigment granules. Cells remaining on the mesh of the strainer were transferred into several 1.5 mL protein low-binding tubes with cell saver micropipettes, and integrated into one tube after centrifugation at 80 ɡ for 1–2 min. After the supernatant was removed, cells were mixed with growth medium containing 8% DMSO, as was done for CpM01, and stored at − 80 °C. The cell-containing tube was transported to LiSDaC in a frozen condition.

At LiSDaC, 3’ scRNA-seq was carried out with Chromium Next GEM Single Cell 3ʹ Kit v3.1 (16 rxns, PN-1000268; 10x Genomics, Pleasanton, CA, USA) on Chromium iX (10x Genomics) and NovaSeq 6000 (Illumina, San Diego, CA, USA) according to manufacturers’ instructions. Read data are available in NCBI (BioProject accession number: PRJNA1138950; SRA accession number: for CpM01, SRR29978956; for erythroid cells, SRR29978954; for retinal cells, SRR29978918).

Data analysis

The total number of cells in a dish was estimated per passage by counting cells in cell suspension for the next passage using C-Chip (DHC-N01-M5; AR BROWN, Tokyo, Japan). Images were analyzed with software for image acquisition systems and by Adobe Photoshop 2024 (Adobe Systems, San Jose, CA, USA). In investigations of the expression of MRFs in cultures, the mean luminance of fluorescence in the nucleus was measured in Adobe Photoshop 2024.

For cell clustering with the scRNA-seq data, a SuperTranscript database (918,436 nucleotide sequences) was used as a reference⁵¹. It was constructed by Trinity_gene_splice_modeler.py in Trinity v.2.15.1⁵², using 1,336,820 contigs (mean length: 714.7 base) constructed by Trinity v.2.15.1 itself with all the read data for TOTAL, the comprehensive transcriptome database of C. pyrrogaster³². Using this reference database, the scRNA-seq data were analyzed by CellRanger (pipeline version: cellranger-7.1.0; 10x Genomics) and Loupe Browser 8.0.0 (10x Genomics). In the CpM01 sample, estimated number of cells: 6,413; mean reads per cell: 71,686; median genes per cell: 323. In the sample for erythroid cells, estimated number of cells: 6,426; mean reads per cell: 74,596; median genes per cell: 676. In the sample for retinal cells, estimated number of cells: 7,490; mean reads per cell: 67,213; median genes per cell: 499. In preliminary examinations of gene expression profiling of cell clusters on the Loupe Browser, cross contamination was suspected in some transcripts that are abundantly expressed in certain types of cells, such as hemoglobin of erythrocytes. Therefore, in this study, raw_feature_bc_matrix.h5 files deduced by CellRanger were further filtered by CellBender⁵³ (version 0.3.0 for CpM01 and erythroid cells; version 0.2.2 for retinal cells), and using the filtered files re-clustering was made by Seurat⁵⁴ (for CpM01 and erythroid cells, version 5.0.1; for retinal cells, version 4.4.0) as follows.

The filtered file was loaded on the ‘CreateSeuratObject’ function of Seurat (for CpM01 and erythroid cells, min.cells = 1, min.feature = 200; for retinal cells, min.cells = 3, min.feature = 200), the count data was normalized by the ‘NormalizeData’ function, 2,000 differentially expressed genes were selected by the ‘FindVariableFeature’ function (nfeature = 2000), scaling of the gene expression levels was made by the ‘ScaleData’ function, the dimension reduction was made by the functions ‘RunPCA’, and then clustering was made by the functions ‘FindNeighbors’ and ‘FindClusters’. Exceptionally, for CpM01, after the filtered data was loaded on the ‘CreateSeuratObject’ function, the number of cells that had a Unique Molecular Identifier (UMI) between 13,246 and 164,705 and the number of expressed genes between 690 and 18,202 were selected by the ‘subset’ function (nCount_RNA > 13245 & nCount_RNA < 164706 & nFeature_RNA > 689 & nFeature_RNA < 18203). Clustering was made by the ‘FindCluster’ function with a low resolution parameter (= 0.1) so that all the cells belonged to the same cluster. Finally, for CpM01, erythroid cells and retinal cells, a total of 1 (#0), 22 (#0–#21) and 13 (#0–#12) clusters, respectively were deduced.

The results of clustering (Seurat objects) for CpM01, erythroid cells and retinal cells were merged by the ‘merge’ function, cluster #0 of CpM01, #13 of erythroid cells, and #6 of retinal cells were isolated and integrated into one object by the ‘JoinLayer’ function, 2,000 differentially expressed genes were selected by the ‘FindVariableFeature’ function (nfeature = 2000), scaling of the gene expression levels of the three clusters was made by the ‘ScaleData’ function, and then the dimension reduction was made by the functions ‘RunPCA’ for principal component analysis (PCA), ‘RunUMAP’ for Uniform Manifold Approximation and Projection (UMAP), and ‘RunTSNE’ for t-SNE. The result of t-SNE is shown in Fig. 6b.

To find differentially expressed genes among the three clusters, after the gene expression levels of the three clusters were scaled, the FindAllMarker function was applied to select those genes whose expression levels were significantly higher in a given cluster. For CpM01, the genes were functionally annotated via Blast2GO 6.0.3⁵⁵ and the results were exported to a csv file and analyzed by Microsoft Excel (Supplementary Data). The top 20 genes for each cluster are shown as a heatmap by the ‘DoHeatmap’ function (Fig. 6c).

Loupe Browser was applied to visualize and analyze Seurat data, producing UMAP and violin plots that show the expression patterns of featured genes.

Figures were prepared using Adobe Photoshop 2024. Image brightness, contrast and sharpness were adjusted according to the journal’s guidelines. Statistical analysis was made using BellCurve for Excel (version 4.07, Social Survey Research Information, Tokyo, Japan). Data in the text are presented as the mean ± SE.

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• Source: https://www.nature.com/articles/s41598-024-81899-6