Animal experiments
Teratoma formation experiments were performed at Iwate University. All surgical procedures and animal husbandry were performed in accordance with the international guidelines of the Animal Experiments of Iwate University and were approved by the university’s Animal Research Committee (approval number A201734).
Chicken embryonic fibroblasts (Rhode Island Red) were obtained from a primary culture of chicken embryonic tissue provided by Prof. Atsushi Tajima, Tsukuba University. Chicken culture cells were obtained from chicken embryos, and the acquisition of these cells did not require approval. Mouse embryonic fibroblasts (CF-1 strain) were purchased from a manufacturer (CMPMEFCFL; DS Pharma Biomedical, Osaka, Japan). Approval was not required to obtain these cells.
Somatic cells were obtained from wild animals (ex., Okinawa rail). The sampling details described below do not include the exact location of sampling to protect against poaching.
Fibroblast cells from Okinawa rail and Japanese ptarmigan were obtained from dead animals, such as those killed by vehicles (Fig. 1A and Supplementary Fig. 1). Approval was not required to obtain these samples.
Dead Okinawa rail were found on May 21, 2008, by the Okinawa Wildlife Federation, a nonprofit organization that focuses on the conservation of wild animals in the Okinawa area in the southwest region of Japan. The organization has permission from the Japanese Ministry of the Environment (MOE) to handle and perform first aid activities on endangered animals. The dead birds were transferred the following day to the National Institute for Environmental Studies (NIES). Primary cell culture was carried out from muscle tissue and skin of the dead birds (NIES ID: 715A).
On July 8, 2004, tissues recovered from dead Japanese ptarmigan (e.g., skin and retina tissues) were also transferred to NIES from Gifu University Department of Veterinary Medicine. Primary cell culture from this tissue was performed (NIES ID: 22A).
Somatic cells from Blakiston’s fish owl and Japanese golden eagle were obtained from emerging pinfeathers. Concerning the Blakiston’s fish owl, the MOE carries out bird banding, of wild birds with identification tags. The emerging pinfeathers we used had been accidentally release during banding. The banding had been performed by a veterinarian at the Institute for Raptor Biomedicine Japan (IRBJ) in the Hokkaido area on June 2, 2006. IRBJ is a private organization that primarily focuses on emergency medicine first aid and care for wild avians in Hokkaido region of Japan. IRBJ is contracted to MOE to handle and administer first aid for endangered animals. The MOE banding ring was 14C0242. Since banding was carried out with the permission of MOE for capturing wildlife, we did not require the approval to obtain these avian somatic cells. On July 8, 2006, Blakiston’s fish owl pinfeathers were transferred to from IRBJ to NIES, where primary cell culture was performed (NIES ID: 215A).
Concerning the Japanese golden eagle, an emerging pinfeather accidentally fell off a bird during blood collection at the Yagiyama Zoo in Sendai, Japan on July 11, 2018. Dr. Yukiko Watanabe, an IRBJ veterinarian, collected the emerging pinfeather. The sample was shipped the following day to NIES where primary cell culture was performed (NIES ID: 5228).
In addition to these birds, we obtained somatic cells emerging avian pinfeathers of Steller’s sea eagle, white-tail eagle, mountain hawk-eagle, northern goshawk, Taiga bean goose, and Latham’s snipe. These samples were provided by IRBJ.
Concerning the Steller’s sea eagle, an injured individual was found in Hokkaido on July 11, 2006 (ID: 06-NE-SSE-1). The eagle was transferred to IRBJ. On December 4, 2006, IRBJ veterinarian Dr. Keisuke Saito collected fallen pinfeathers. Primary cell culture was performed at NIES on December 8, 2006 (NIES ID: 369A).
Concerning the white-tailed eagle, an injured individual was found in Hokkaido, Japan, on July 12, 2007 (ID: 07-NE-WTE-4). The bird was transferred to IRBJ the same day for emergency treatment. On January 15, 2008, Dr. Saito collected fallen pinfeathers. Primary cell culture was performed on January 18, 2008 at NIES (NIES ID: 492A).
Concerning the mountain hawk-eagle, an injured individual was found in the Hokkaido area on August 10, 2008 (ID: 08-Tokachi-HHE-2). The bird was transferred to IRBJ the same day. The bird was treated by an IRBJ veterinarian, but died on September 8, 2008. Emerging pinfeathers were collected from the dead bird by Dr. Saito. Primary cell culture was performed on September 11, 2008 at NIES (NIES ID: 847A).
Concerning the Northern Goshawk, IRBJ accepted an injured bird for treatment on June 12, 2006. Following treatment and recovery, the bird was released into the wild in the Hokkaido area on August 1, 2006. During the treatment (July 4, 2006), Dr. Saito collected fallen pinfeathers. The primary cell culture was performed at NIES on July 6, 2006 (NIES ID: 222A).
Concerning the Taiga bean geese, an injured individual was found in Hokkaido on September 15, 2016 (ID: 13B8005). The injured bird was transferred to IRBJ the same day for emergency treatment. On September 16, 2016, IRBJ veterinarian Dr. Yukiko Watanabe collected fallen emerging pinfeathers. Primary cell culture was performed on September 20, 2016 (NIES ID: 4420A).
Finally, concerning the Latham’s snipe, fallen pinfeathers were collected during MOE approved bird banding performed on September 17, 2006, by Dr. Saito. Dr. Saito also collected fallen emerging pinfeathers (ID: 6A22598). The samples were transferred to NIES on September 20, 2006, for primary cell culture (NIES ID: 338A).
All records are available at NIES.
Cell culture and preservation
Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl-derived fibroblasts were preserved in liquid nitrogen for 8–12 years (Fig. 1f). The preservation solution contained 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide. Cells were preserved at a cell density of 1 × 106–4 × 106 cell/mL. During the freezing period, the cells were maintained at minus The cells were frozen at a temperature of −135 °C. Japanese golden eagle fibroblasts were used without freezing.
Avian-derived fibroblasts were cultured with Kuwana’s modified avian culture medium-1 (KAv-1), which is based on alpha-MEM containing 5% FBS and 5% chicken serum23. Mouse embryonic fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic–antimycotic mixed stock solution (161–23181; Wako Pure Chemical Industries, Osaka, Japan). All avian and mouse cells were cultured at 37 °C under 5% CO2.
Reprogramming vector
We chemically synthesized an expression cassette that included seven reprogramming factors (MyoD-derived transactivation domain-linked Oct3/4, Sox2, Klf4, c-Myc, Klf2, Lin28, and Nanog; all genes derived from mice). The self-cleaving 2A peptide was inserted at the junction of the coding region (Fig. 1g). We transferred the complementary DNA (cDNA) insert from the shuttle vector to the PiggyBac transposon vector containing green fluorescent protein (PB-CAG-GFP). Although the original transposon vector drive the expression of cDNA with the elongation factor-1 (EF1) promoter (PJ547-17; DNA 2.0, Menlo Park, CA, USA), we replaced the EF1 promoter to CAG promoter in our previous study22,24. The reprogramming vector was designated PB-TAD-7F (Fig. 1g).
In addition to the PB-TAD-7F reprogramming vector, we used the PB-DDR-8F reprogramming vector to establish Japanese golden eagle iPSCs. The complete coding sequence of DDR-8F (DDR-Oct3/4, Sox2, Klf4, c-Myc, Klf2, Nanog, Lin28, and Yap) was chemically synthesized. The expression cassettes containing the eight reprogramming factors were excised from the shuttle vector using restriction enzymes. The cDNA fragments were transferred to the PB-CAG-GFP PiggyBac transposon-based vector22,24. Detailed information regarding the PB-DDR-8F reprogramming vectors is shown in Fig. 10a.
Establishment of iPSCs
We transfected PB-R6F or PB-TAD-7F reprogramming vectors into mouse, chicken, Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl-derived fibroblasts using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). After hygromycin selection (Wako Pure Chemical Industries), the cells were reseeded onto a mouse embryonic fibroblast (MEF) feeder layer. On days 14–32, we picked primary iPSC-like colonies and seeded them on new MEF feeder cell plates. The detailed protocol is shown in Fig. 1h.
To establish Japanese golden eagle-derived iPSCs, we transduced PB-TAD-7F or PB-DDR-8F reprogramming vectors into Japanese golden eagle pinfeather-derived somatic cells. Transfection was performed using Lipofectamine 2000 transduction reagent (11668019; Thermo Fisher Scientific) according to the manufacturer’s instructions. After hygromycin selection (Wako Pure Chemical Industries), cells were seeded onto feeder culture plates. The golden eagle iPSCs were cultured in KAv-1-based medium5.
The medium used to establish avian iPSCs was supplemented with 1000 × human Leukemia Inhibitory Factor (LIF) (125–05603; Wako Pure Chemical Industries), 4.0 ng/ml basic FGF (064–04541; Wako Pure Chemical Industries), 0.75 μM CHIR99021 glycogen synthase kinase-3 inhibitor (034–23103; Wako Pure Chemical Industries), 0.25 μM PD0325901 mitogen-activated protein kinase inhibitor (163–24001; Wako Pure Chemical Industries). In addition to those supplements, 0.25 μM thiazovivin (202–18011; Wako Pure Chemical Industries) was added in the media used to generate Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, and chicken iPSCs. In the medium used to generate mouse iPSCs, we added 1000 × LIF, 0.75 μM CHIR99021, and 0.25 μM PD0325901.
iPSC culture conditions
Two types of cell culture media were used: KAv-1 for avian iPSCs and DMEM for mouse iPSCs. The composition of KAv-1 for avian iPSCs was as follows: alpha-MEM containing 5% FBS and 5% chicken serum 1% antibiotic–antimycotic mixed solution, 1% nonessential amino acids (Wako Pure Chemical Industries), and 2 mM glutamic acid was added (Nacalai Tesque, Kyoto, Japan). The composition of DMEM for mouse was follows: DMEM supplemented with 15% SSR, 0.22 mM 2-mercaptoethanol (21438–82, Nacalai Tesque), 1% antibiotic–antimycotic mixed solution, 1% nonessential amino acids5,22. As a supplement to the iPSC medium, we used 1000 × human LIF (125–05603; Wako Pure Chemical Industries), 4.0 ng/ml basic FGF (064–04541; Wako Pure Chemical Industries), 0.75 μM CHIR99021 (034–23103; Wako Pure Chemical Industries), 0.25 μM PD0325901 (163–24001; Wako Pure Chemical Industries) for the media used to culture Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, Japanese golden eagle, and chicken-derived iPSCs. The supplements for media used to culture Okinawa rail and Japanese ptarmigan-derived iPSCs included 2.5 μM Gö6983 (074–06443, Wako Pure Chemical Industries). To analyze the cellular characteristics, we focused on the Janus kinase (JAK), FGF, ROCK, and glycolytic pathways, since the dependency of these pathways can indicate differences in cellular characteristics. We used 1–10 μM JAK inhibitor I (4200099; MERCK, Darmstadt, Germany), 0.5–4 μM of PD173074, which inhibits FGF receptor (FGFR) inhibitor (160–26831; Wako Pure Chemical Industries), 10 μM of Y27632, which inhibits ROCK (036–24023; Wako Pure Chemical Industries), and 2 or 4 mM 2-deoxyglucose (2DG, D0051; Tokyo Chemical Industry, Tokyo, Japan).
AP and immunological staining of fibroblasts and iPSCs
A red-color AP staining kit (AP100 R-1; System Bioscience, Palo Alto, CA, USA) was used to detect AP activity of iPSCs. iPSCs were stained for SSEA-1, SSEA-3, and SSEA-4 antibodies (Supplementary Table 2). To stain the iPSCs with the SSEA antibodies, the cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 3 min. Cells were permeabilized by 0.5% Triton X-100 (35501-15; Nacalai Tesque, Kyoto, Japan) for 60 min. After three washes with PBS, the iPSCs were blocked with 1% bovine serum albumin (BSA, 01863-06; Nacalai Tesque) for 45 min. iPSCs were incubated with a primary antibody overnight and then exposed to the corresponding fluorescent-labeled secondary antibodies for 60 min. Counterstaining was performed with a 4′,6-diamidino-2-phenylindole (DAPI) solution (Cellstain-DAPI solution, DOJINDO, Kumamoto, Japan).
Japanese golden eagle and chicken-derived fibroblasts were seeded in 12-well cell culture plates for immunological staining. After 48 h of incubation, F-actin staining was performed using Alexa Fluor 568 phalloidin (A12380; Thermo Fisher Scientific) according to the manufacturer’s protocol. Double staining was performed with an anti-vimentin antibody (MA5-11883; Thermo Fisher Scientific) and Alexa Fluor 488-labeled secondary antibody (A-11001; Thermo Fisher Scientific) (Supplementary Table 2). The samples were counterstained with Cellstain-DAPI solution (DOJINDO) as described above.
Detection of reprogramming vectors and internal control genes from iPSCs
DNA was isolated using the EZ1 DNA Tissue Kit (953034; QIAGEN, Hilden, Germany). PCR was performed with 100 ng of template DNA. Primer sequences are listed in Supplementary Tables 3 and 4. We performed PCR assays using KOD FX Neo (KFX-201; TOYOBO, Osaka, Japan). PCR was conducted by predenaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, and extension at 68 °C for 30 s, with 40 cycles of denaturation and extension. PCR products were analyzed by electrophoresis on 2.0% agarose/Tris-acetate–ethylenediaminetetraacetic acid (EDTA) gels.
Sequential passaging
Mouse, Okinawa rail, and Japanese ptarmigan-derived primary cells and iPSCs were seeded in six-well plates with feeder cells for analysis. When cell growth became confluent, all cells and the number of cells per dish was enumerated using a Countess cell counter (Thermo Fisher Scientific). The harvested and seeded cell numbers were used to calculate the PD time as an indicator of the speed of cell growth, using the formula PD = log2 (A/B), where A is the number of harvested cells at the end of each passage, and B is the number of seeded cells at the start25.
Detection of mRNA expression
Total RNA was isolated from iPSCs using an EZ1 RNA Tissue Mini Kit (959034; QIAGEN). cDNA was synthesized from total RNA using the PrimeScript reverse transcription (RT) reagent kit (Perfect Real Time, RR047A; TaKaRa Bio, Ohtsu, Japan). Real-time PCR was performed in a 12.5 μl volume containing 2 × KOD SYBR qPCR Mix (QKD-201; Toyobo), 10 ng of cDNA solution, and 0.3 μM of each primer. The primer sequences are listed in Supplementary Tables 5–10. The reaction was performed in duplicate. The cycling program was as follows: 98 °C for 120 s (initial denaturation), 98 °C for 10 s (denaturation), 58 °C for 10 s (annealing), and 68 °C for 32 s (extension) for 40 cycles. We normalized the expression levels of the target genes to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Mitochondria staining
Mitochondria were stained by incubation with 50 nM MitoTracker Orange (M7510; Thermo Fisher Scientific) or 20 nM tetramethyl rhodamine ethyl ester perchlorate (TMRE, T669; Thermo Fisher Scientific) for 10 min. After staining, the solution was removed, and fresh medium was added for observation.
EB formation and in vitro differentiation
In vitro differentiation of Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, and Japanese golden eagle iPSCs was performed. To generate EBs, iPSCs were seeded in low-binding dishes in KAv-1 medium. After 7–14 days, floating EBs were selected and seeded in 0.1% gelatin-coated 6-well plates with KAv-1 medium. To induce differentiation into neural cells, the floating EBs were cultured in 0.1% gelatin-coated plates containing KAv-1 supplemented with 10 μM ATRA and 4.0 ng/ml FGF for 7 days.
Cells were immunochemically stained after in vitro differentiation using antibody to TUJ1, alpha-smooth muscle, or Gata4 (Supplementary Table 2). Differentiated cells were stained based on the immunological staining procedure of iPSCs described above.
Teratoma formation and tissue sectioning
The Animal Committee of Iwate University approved the experimental protocol for teratoma formation (approval numbers A201734, A201737). For teratoma formation, 1 × 106 iPSCs were injected into the testes of SCID mice (C.B-17/Icr-scid/scidJcl; CLEA Japan, Tokyo, Japan). After 4–34 weeks post-injection, tumor tissues were excised from the mice. Each tumor tissue was fixed with 10% formaldehyde in PBS. Fixed tissue sections were stained with hematoxylin-eosin (HE) and observed by microscopy.
Immunological staining was performed in addition to HE staining. For immunological staining, antibody to TUJ1, alpha-smooth muscle, or Gata4 was used (Supplementary Table 2). The paraffin block of each teratoma was sliced to produce a section 5 μm thick. After deparaffinization, the antigen was activated with citric acid buffer (SignalStain Citrate Unmasking Solution (10×), 14746; Cell Signaling Technology, Beverly, MA, USA) by microwaving for 10 min. To block endogenous peroxidase, tissue sections were incubated with 3% hydrogen peroxide (081–04215; Wako Pure Chemical). After washing with purified water, the tissue sections were incubated with 5% goat serum (555–76251; Wako Pure Chemical) in PBS. Next, the section were incubated in a solution containing a 1:100 dilution of primary antibody overnight at 4 °C. After washing with PBS, the tissue sections were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (anti-IgG (H+L chain), mouse, pAb-HRP, code no. 330; MBL Co., Ltd., Nagoya, Japan) or anti-IgG (H+L chain, rabbit, pAb-HRP, code no. 458; MBL) for 1 h (Supplementary Table 2). After washing with PBS, the tissue sections were incubated with 3,3′-diaminobenzidine substrate solution (Histostar, code no. 8469; MBL) for 5–20 min. After washing with purified water, tissue sections were counterstained with hematoxylin for 1–2 min.
DNA component analysis
Cultured cells fixed with 70% ethanol at least 4 h under −20 °C condition. The fixed cells stained with the Muse Cell Cycle Assay Kit (Merck Millipore Corporation, Darmstadt, Germany). The stained cells analyzed with Muse Cell Analyzer (Merck Millipore Corporation) were used for DNA content analysis.
Karyotype analysis
Our iPSCs were treated with 0.02 mg/ml colcemid. Those iPSCs exposed to a hypotonic solution and fixed with Carnoy’s fluid. We counted the chromosomal number in 50 cells and performed a G-banding analysis in 20 cells22.
Production of interspecific chimeras and their immunological staining
To evaluate whether iPSCs derived from Japanese ptarmigan could contribute to the generation of interspecific chimeras in chick embryos, iPSCs were stained with 10 μM CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C7025; Thermo Fisher Scientific) for 30 min. Eggs of white leghorn chicken were purchased from a local farm (Goto-furanjyo, Gifu, Japan). We injected the labeled Japanese ptarmigan iPSCs into stage X chick blastoderms and cultured the embryos26. To confirm the contribution of chimera, fluorescence was observed after 72 h. To analyze the tissue-level contribution of chimera, embryos on day 5. The embryos were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan), frozen in liquid nitrogen, and stored at −80 °C until use. Cryosections 20 μm in thickness were prepared using a cryostat, air-dried for 30 min at room temperature, and fixed with 4% paraformaldehyde for 2 min at room temperature. After washing three times with PBS, sections were incubated with PBS containing 5% FBS for 1 h. After blocking with FBS, the sections were incubated with an anti-hygromicin resistance gene antibody (anti-HPT2; Supplementary Table 2) overnight. After washing three times with PBS, the sections were incubated with secondary antibody (goat anti-mouse IgG, Alexa Fluor 568; Supplementary Table 2) and Cellstain- DAPI solution (DOJINDO) for 1 h.
Detection of contribution of chimera from genome
We injected Japanese ptarmigan iPSCs (without CellTracker Green CMFDA label) into a stage X chicken blastoderms. On day 5, the entire chicken embryos were collected. The genome of each embryo was collected using NucleoSpin Tissue (U0952S; MACHEREY-NAGEL, Düren, Germany). After collecting the chimeric genome, we detected the reprogramming vector cassette using genomic PCR analysis using 50 ng of template genome. To extend the target sequence, we used the KOD FX Neo (KFX-201; TOYOBO). Primer information is provided in Supplementary Table 11. This analysis was performed according to the manufacturer’s protocol. The cycling program comprised 45 cycles of 94 °C for 120 s (initial denaturation), 98 °C for 10 s (denaturation), and 68 °C for 50 s (annealing and extension). After PCR, 2% agarose gel electrophoresis was performed. Gels were stained with GelGreen (517–53333; Biotium, Inc., Fremont, CA, USA).
Real-time PCR was also performed to detect the contribution of chimera. The fluorescence probe and primers designed to detect chimeric contributions are summarized in Supplementary Table 12. The template was a 30 ng genome. The analysis was performed using 1 × THUNDERBIRD Probe qPCR Mix (QPS-101; TOYOBO), 0.3 μM of each primer, 0.2 μM of probe, and 1 × Rox. Fifty cycle of 95 °C for 60 s (initial denaturation), 95 °C for 15 s (denaturation), and 60 °C for 60 s (annealing and extension) were used. The expression levels of the target genes were normalized to that of chicken Tsc-2.
RNA preparation and sequencing for RNA-seq analysis
Total RNA from iPSCs, fibroblasts, and chicken embryo stage X was collected using NucleoSpin Tissue (740952.50; MACHEREY-NAGEL). Triplicate samples of all iPSCs, fibroblasts, and chicken embryo stage X were prepared. To prepare the library, we used the TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-2101; Illumina, San Diego, CA, USA). The quality of the library was evaluated using the Qubit DNA Assay (Thermo Fisher Scientific) on a TapeStation with a D1000 screen tape (Agilent Technologies, Santa Clara, CA, USA). The cDNA samples were used for the sequencing reaction on an Illumina HiSeq X sequencing machine, resulting in more than 40 M reads with 150 bp ends for each sample, except chicken fibroblast No. 3, which displayed more than 40 M reads with 75 bp ends. To analyze the RNA-seq data, we used the CLC Genomic Workbench (CLC Bio, Aarhus, Denmark). In the trim read step, low-quality sequence with the quality score of the CLC workbench, 5′ end, 3′ end, and short sequences (shorter than 15 sequences) were removed. The trimmed sequence data were mapped onto the chicken reference genome. Gene expression data were obtained in this step. PCA was performed and a heat map created with CLC Genomic Workbench using gene expression data. In this step, normalization was automatically performed using TMM methods. To compare chicken cells, RNA-seq data from SRA (SRP115012 (GEO: GSE102353) and SRP087639 (GSE86592) were used. The RNA-seq data has been submitted to the DNA DataBank of Japan under accession number DRA013522 (Submission), PRJDB13093(BioProject), SAMD00444261–SAMD00444287 (BioSample).
Statistics and reproducibility
Nonparametric multiple comparison analysis used the Steal–Dwass test (Figs. 2e, 3 [Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl], 4d, 4f, 5b, 5d, 5f, 5h, 10i). For nonparametric independent two-group analysis, we used the Mann–Whitney U test (Fig. 3, for mouse and chicken, and 4b). Statistically significant differences are indicated by *(p < 0.05) and **(p < 0.01). Sample sizes and replicates are described in legends of figures.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Source: Ecology - nature.com
