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The Journal of Reproduction and Development logoLink to The Journal of Reproduction and Development
. 2023 Oct 26;69(6):317–327. doi: 10.1262/jrd.2022-038

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat

Ryoji KANEGI 1,2,*, Shingo HATOYA 1,2,*, Kazuto KIMURA 1,2, Kyohei YODOE 2, Toshiya NISHIMURA 2, Kikuya SUGIURA 1,2, Noritoshi KAWATE 1,2, Toshio INABA 2
PMCID: PMC10721851  PMID: 37880086

Abstract

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Keywords: Basic fibroblast growth factor, Feline, Hematopoietic lineage, Neural stem cell, Transgene silencing

Embryonic stem (ES) cells can self-renew and differentiate into various cell types, including the three germ layers: endoderm, ectoderm, and mesoderm. In 1981, Evans and Kaufman were the first to establish ES cells derived from the inner cell mass (ICM) of mouse blastocysts [1, 2]. Extensive research on mouse ES cells has shown that these cells can be maintained as feeder cells in medium supplemented with leukemia inhibitory factor (LIF), which suppresses differentiation and maintains self-renewal properties. Additionally, mouse ES cells are vital for chimera generation [3] and are therefore highly beneficial for the development of transgenic mice [4]. In 1998, human ES cells were first cultivated in a medium supplemented with basic fibroblast growth factor (bFGF), with the hope of being used to study development and transplantation therapies [5]. However, there are grave ethical considerations and barriers to the use of human embryos in ES research and other applications.

In their groundbreaking work, Takahashi and Yamanaka reprogrammed mouse and human somatic cells, using the transcription factors Oct4, Sox2, Klf4, and c-Myc, to create induced pluripotent stem (iPS) cells, an excellent alternative to ES cells [6, 7]. Similar to ES cells, iPS cells proliferate infinitely and differentiate into various cell types. These cells also express ES cell markers. Furthermore, iPS cells reflect the genetic background of their donor somatic cells, and thus confer a great advantage for modeling diseases in vitro [8].

Advances in mammalian ES and iPS cell technology, as gathered from studies done in rats [9, 10], dogs [11, 12], rabbits [13, 14], cows [15, 16], horses [17, 18], and goats [19, 20], have provided cell resources for transgenic animal development, experimental models for regenerative medicine, and the means to enhance reproductive techniques. Despite these advances in animal iPS research and applications, efforts to generate sustainable domestic cat pluripotent stem cells have been limited. Previous works outline cat ES-like cells generated using in vitro- and in vivo-produced blastocysts [21, 22]. Notably, these cells had a typical ES cell-like morphology, exhibited alkaline phosphatase activity, expressed ES cell markers, and differentiated into various cell types. However, these cells cannot be maintained for prolonged passages.

Regarding iPS cells, a previous study reported that domestic cat iPS-like cells depend on LIF; however, exogenous genes were continuously expressed [23]. These results indicate the necessity of optimizing the culture conditions for cat iPS cells. Additionally, the ability of these cells to differentiate into key cell types such as blood cells and neurons, which are the focus of stem cell research, has not been investigated. The use of domestic cat iPS cells would further enhance the study of spontaneous diseases because domestic cats are highly susceptible to developing spontaneous diseases, such as diabetes mellitus and cardiomyopathy, which closely resemble human cases in etiology and pathology [24]. The domestic cat is larger and lives longer than the mouse, which is a commonly used experimental animal. These differences provide advantages for surgical operations and longer monitoring of side effects. Thus, domestic cats serve as an excellent animal model for spontaneous human diseases [24] and generating sustainable domestic cat iPS cells would greatly progress this field of study. Furthermore, domestic cats are used to develop reproductive techniques to preserve endangered wild felids [25]. The use of efficient cat iPS cells would greatly propel such reproductive techniques because germ cells derived from pluripotent stem cells contribute to the production of mouse offspring [26, 27]. In this study, we aimed to generate domestic cat iPS cells from cat embryonic fibroblasts retrovirally transfected with the mouse reprogramming transcription factors Oct3/4, Klf4, Sox2, and c-Myc.

Materials and Methods

This study was approved by the Institutional Animal Experiment Committee of Osaka Prefecture University and was conducted according to the Animal Experimentation Regulations of the Osaka Prefecture University. In this study, we collected domestic short-haired cat fetuses from a cat that was 30 days pregnant and had undergone ovariohysterectomy at a local veterinary clinic. The cat was privately owned, and the owner’s consent was obtained before fetal collection. No cats were bred or operated specifically for this study.

Cell culture

Six fetuses were washed with phosphate-buffered saline without magnesium and calcium (PBS (–); Nacalai Tesque, Kyoto, Japan), and their heads and livers were removed. The fetuses were then cut into small pieces and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, San Diego, CA, USA) supplemented with 10% (v/v) Fetal Bovine Serum (FBS; PAA Laboratories, Pasching, Australia), 100-IU/ml penicillin and 100-µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 2-mM L-glutamine (Sigma-Aldrich). Cells were maintained at 37°C and 5% CO2. The adherent cells were passaged using 0.25% trypsin (Sigma-Aldrich). After three passages, spindle cells were used as cat embryonic fibroblasts. The cat embryonic fibroblasts used in the present study originated from a single fetus.

SNL 76/7, 293FT, and platinum-E cells were purchased from the Health Protection Agency (London, UK), Life Technologies, and Cell BioLabs (San Diego, CA, USA), respectively. Cells were cultured according to the manufacturer’s protocol. To note, SNL 76/7 cells were inactivated with 10-μg/ml mitomycin C for 2.5 h and used as feeder layers.

Cat iPS-like cells were cultured on 35 mm tissue culture dishes (AGC Techno Glass, Shizuoka, Japan) pretreated with gelatin from bovine skin (Sigma-Aldrich) and 3.4 × 105 inactivated SNL 76/7 cells in primate ES cell medium, which was DMEM Nutrient Mixture F-12 HAM (Sigma-Aldrich) medium supplemented with 100-IU/ml penicillin and 100-µg/ml streptomycin, 2-mM L-glutamine, 0.1-mM non-essential amino acids (Life Technologies), 0.1-mM 2-mercaptoethanol (Sigma-Aldrich), 10-ng/ml human bFGF (Pepro Tech, Rocky Hill, NJ, USA), and 20% (v/v) ES cell FBS (Life Technologies) or KnockOut Serum Replacement (KnockOut SR; Life Technologies). Cells were maintained at 37°C and 5% CO2. Cat iPS-like colonies were mechanically passaged using the sharp edge of a flame-pulled Pasteur pipette every three to four days onto new gelatin-coated dishes and inactivated SNL 76/7 cells.

Lentivirus and retrovirus production

293FT cells, packaging cells for lentivirus production, were plated at a density of 3.65 × 106 cells in 100 mm tissue culture dishes (AGC Techno Glass). On the next day, the cells were transduced with 3 μg of pLenti6/UbC/mSlc7a1 (Addgene plasmid # 17224, Cambridge, MA, USA) and 9 μg of ViraPower Lentiviral Packaging Mix (Life Technologies), including 36 μg of the Lipofectamine 2000 transfection reagent (Life Technologies). The medium was changed 24 h later, and the supernatants were collected 48 h post-transduction. Supernatants were then clarified using sterile Whatman® FP 30/0.45 CA-S filter unit (GE Healthcare UK, Little Chalfont, Buckinghamshire, England), and used immediately without measuring vector biological titer.

Platinum-E cells, packaging cells for retrovirus production, were plated at a density of 1.3 × 106 cells in 60 mm tissue culture dishes (AGC Techno Glass). On the next day, Platinum-E cells were transduced with 3 μg of pMXs-Oct3/4, pMXs-Klf4, pMXs-Sox2, or pMXs-c-Myc (Addgene plasmid # 13366, plasmid #13370, plasmid #13367, and plasmid #13375) along with 9 μl of the Fugene 6 transfection reagent (Roche Diagnostics, Mannheim, Germany). Medium was changed 24 h later, and the supernatants were collected 48 h post-transduction. Supernatants were then clarified using sterile Whatman® FP 30/0.45 CA-S filter unit, and used immediately without measuring vector biological titer.

Generation of domestic cat iPS cells from embryonic fibroblasts

On the day before viral infection, cat embryonic fibroblasts were plated at a density of 2.8 × 105 cells in 60 mm tissue culture dishes. On day 1, cat embryonic fibroblasts were transfected with lentiviruses containing mouse Slc7a1 gene, which encodes an ecotropic retroviral receptor necessary for retroviral transfection. Infected cells were washed with PBS (–) 16 h later, harvested using 0.25% EDTA-trypsin, and reseeded at a density of 1.0 × 105 cells in 35 mm tissue culture dishes. On day 3, lentivirus-infected fibroblasts were transfected with retroviruses containing Oct3/4, Klf4, Sox2, and c-Myc. The medium was then replaced after 16 h. On day 8, the infected cells were harvested using 0.05% EDTA-trypsin and reseeded onto gelatin-coated dishes and inactivated SNL 76/7 cells (3.4 × 105 cells per 35 mm dish) at various densities (5.0 × 103 to 4.0 × 104 cells per 35 mm dish) to explore the optimal density in reseeding cat embryonic fibroblasts. On day 9, the medium was replaced with primate ES cell medium containing FBS or KnockOut SR medium. The culture medium was changed every other day. Primary colonies were mechanically dissociated into small pieces using the sharp edge of a flame-pulled Pasteur pipette, transferred to a new gelatin-coated dish covered with inactivated SNL 76/7 cells, and cultured in the same medium. The protocol for the induction of cat iPS cells is shown in Supplementary Fig. 1. In addition, we calculated the reprogramming efficiency by dividing the number of primary colonies by the number of reseeded cells.

Characterization of cat iPS-like cells

The alkaline phosphatase activity of cat iPS-like cells at passage 20 was evaluated using an Alkaline Phosphatase Staining Kit II (Stemgent, Lexington, MA, USA) according to the manufacturer’s protocol. To detect ES cell markers in cat iPS-like cells, immunofluorescence staining was performed at passage 42. Briefly, cat iPS-like cells were washed with PBS (–) and fixed in 4% (w/v) paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) (diluent used: PBS (–)) at room temperature for five minutes. After fixation, the cat iPS-like cells were washed with PBS (–) and treated with 0.05% (v/v) Tween 20 (Nacalai Tesque) (diluent used: PBS (–)) at room temperature for five minutes. Cat iPS-like cells were washed with PBS (–) containing 0.1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) and blocked in 10% (w/v) BSA at room temperature for 30 min. The cat iPS-like cells were incubated with PBS (–) containing primary antibodies against the stage specific embryonic antigen-1 (SSEA-1; 40-µg/ml; MAB4301; Millipore, Darmstadt, Germany), SSEA-4 (40-µg/ml; MAB4304; Millipore), tumor rejection antigen 1-60 (TRA-1-60; 40-µg/ml; MAB4360; Millipore), TRA-1-80 (40-µg/ml; MAB4380; Millipore), NANOG (5-µg/ml; ab77095; Abcam, Cambridge, UK), and OCT3/4 (1-µg/ml; sc-9081; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature overnight. On the next day, iPS-like cells were washed with 0.1% (w/v) BSA and incubated at room temperature for 60 min with PBS (–) containing either of the following secondary antibodies: goat anti-mouse IgM monoclonal antibody conjugated with Cy3 for SSEA-1, TRA-1-60 and TRA-1-81 (1-µg/ml; Millipore), goat anti-mouse IgG monoclonal antibody conjugated with Alexa-488 for SSEA-4 (1-µg/ml; Life Technologies), rabbit anti-goat IgG polyclonal antibody conjugated with Alexa-488 for NANOG (2-µg/ml; Invitrogen, Grand Island, NY, USA), and goat anti-rabbit IgG polyclonal antibody conjugated with Alexa-546 for OCT3/4 (2-µg/ml; Invitrogen). The iPS-like cells were washed with PBS (–) and enclosed with ProLong® Gold Antifade Reagent with 4',6-diamidino-2-phenylindole (DAPI) to stain DNA (Life Technologies). The samples were observed and imaged using a confocal microscope (Nikon CLSI; Nikon, Tokyo, Japan). Cat iPS-like cells incubated only with secondary antibodies and cat embryonic fibroblasts served as negative controls under the same conditions to confirm that they did not show non-specific staining in cats.

Karyotyping

Karyotyping was performed at passage 20 to evaluate the chromosomal makeup of cat iPS-like cells. On the day before karyotyping, colonies of cat iPS-like cells were replated on new gelatin-coated dishes to prevent feeder cell contamination. Colcemid (Life Technologies) was added to the medium at a final concentration of 0.1-μg/ml and incubated for 2 h at 37°C and 5% CO2. Cat iPS-like cells were washed twice with PBS (–) and treated with 0.25% EDTA-trypsin for three minutes. Floating cells were fixed with 0.075 M KCl for five minutes at 37°C and Carnoy’s fluid (1:3 glacial acetic acid and ethanol mixture). The cells were stained with Wright’s stain solution (Nacalai Tesque) and Giemsa’s stain solution (Nacalai Tesque) and observed under a light microscope (BX50; Olympus, Tokyo, Japan).

RT-PCR analysis

Total RNA was extracted from cat embryonic fibroblasts and iPS-like cells using RNeasy Mini and RNeasy Micro kits (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The extracted RNA was used in a 20-µl reverse transcription (RT) reaction containing 0.5-mM dNTPs (TOYOBO, Osaka, Japan), 2.5-µM random 9-mer (TOYOBO), and 5-U/µl ReverTra Ace reverse transcriptase (TOYOBO) in RT buffer. The RT solution was incubated at 30°C for 10 min, at 42°C for 50 min, and finally at 99°C for five minutes.

PCR was carried out in 20-µl reactions containing 0.5 µl cDNA, 0.2-mM dNTPs, 0.025-U/µl Blend Taq polymerase (TOYOBO), and 0.5-µM of each pair of forward and reverse primers in Blend Taq buffer. The thermocycling protocol comprised of preincubation at 94°C for 2 min; 42 cycles of 94°C for 30 sec, 30 sec at the respective annealing temperature for each gene assessed, and 72°C for 90 sec; and finally 72°C for 2 min. The primers, sequences, and annealing temperatures are listed in Supplementary Table 1. Cat embryonic fibroblasts were used as negative controls. PCR products were electrophoresed on 2% (w/v) Tris-Acetate-EDTA agarose gel (Nacalai Tesque) supplemented with 0.5-µg/ml ethidium bromide (Nacalai Tesque). Gels were imaged using a UV transilluminator (DT-20CP; ATTO, Tokyo, Japan).

Real-time PCR analysis

To assess the expression of endogenous pluripotency markers, Real-time PCR was performed three times with three different clones using the Taq Pro Universal SYBR qPCR Master Mix (NIPPON Genetics, Tokyo, Japan) according to the manufacturer’s instructions. The average value of the two runs was used. The mRNA expression levels of pluripotency markers were compared with those of cat embryonic fibroblasts and three different passages of cat iPS-like cells. The primers used are listed in Supplementary Table 1.

Genomic DNA PCR and real-time PCR analysis

Genomic DNA was extracted from iPS-like cells with the NucleoSpin® Tissue XS (Takara, Shiga, Japan) and Proteinase K (Takara) according to the manufacturer’s protocol. PCR and real-time PCR were carried out as described above; however, the extracted DNA was used instead of cDNA. The copy numbers of the transgenes were calculated and compared with that of endogenous Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). The primers used are listed in Supplementary Table 1.

In vitro differentiation and teratoma formation of cat iPS-like cells

To test the properties of differentiation in vitro, cat iPS-like colonies were mechanically dissociated into small clumps at passage 20 using the sharp edge of a flame-pulled Pasteur pipette. Dissociated colonies were cultured in Petri dishes in primate ES cell medium without bFGF or feeder cells for eight days at 37°C and 5% CO2. The formed embryonic bodies (EBs) were re-plated in gelatin-coated cover glass chambers and cultured for an additional eight days, and the expanded cells were then used for immunofluorescence staining. Primary antibodies against tubulin-β-III-isoform (1:1000; MAB1637; Millipore), SOX17 (1-µg/ml; ab84990; Abcam), and desmin (1-µg/ml; ab82506; Abcam) were used. The secondary antibodies used were goat anti-mouse IgG monoclonal antibody conjugated with Alexa-488 for tubulin-β-III-isoform and SOX17 (1-µg/ml) and goat anti-rabbit IgG monoclonal antibody conjugated with Alexa-546 for desmin (1-µg/ml; Life Technologies). The expanded cells were washed with PBS (–) and enclosed ProLong® Gold Antifade Reagent with DAPI. The samples were observed and imaged using a confocal microscope (Nikon CLSI), and expanded cells incubated only with secondary antibodies, embryonic fibroblasts, and undifferentiated iPS-like cells served as negative controls under the same conditions to confirm that they did not show nonspecific staining in the cat.

To differentiate the formed EBs into blood lineage cells, we followed the protocol described in a previous study, with a slight modification [28]. Formed EBs were cultured in hematopoietic differentiation medium consisting of Stem-Pro-34 (Life Technologies) supplemented with 100-IU/ml penicillin and 100-µg/ml streptomycin, 2-mM L-glutamine, 50-mg/ml ascorbic acid, 50-mM monothioglycerol, and 10% (v/v) FBS in floating culture at 37°C and 5% CO2. The hematopoietic differentiation protocol consisted of supplementing the medium with a changing set of cytokines at different stages to enhance hematopoietic differentiation. On day 1, EBs were cultured in medium supplemented with 10-ng/ml human bone morphogenetic protein 4 (BMP4; R&D systems, Minneapolis, MN, USA). Then, EBs were harvested and cultured in a medium supplemented with 10-ng/ml BMP4 and 5-ng/ml human bFGF for three days. On day 4, EBs were harvested and cultured in a medium supplemented with 1-ng/ml bFGF, 10-ng/ml canine vascular endothelial growth factor (VEGF; R&D Systems), 100-ng/ml canine stem cell factor (SCF; R&D Systems), 5-ng/ml human interleukin 11 (IL-11; R&D Systems), 10-ng/ml feline IL-6 (R&D Systems), and 40-ng/ml canine IL-3 (MyBioSource, San Diego, CA, USA). On day 8, the medium was changed to a medium supplemented with 10-ng/ml VEGF, 100-ng/ml SCF, 5-ng/ml IL-11, 10-ng/ml IL-6, 40-ng/ml IL-3, 20-ng/ml canine erythropoietin (R&D Systems), and 50-ng/ml human thrombopoietin (R&D Systems). On day 18, EBs were harvested, dissociated by pipetting, and re-plated on methylcellulose-based medium (STEMCELL technologies, Vancouver, Canada) in 35 mm tissue culture dishes for an additional 10 days. Colonies were washed with PBS (–), dissociated by pipetting, and spread onto slides using Shandon Cytospin 4 (Thermo Fisher Scientific, Waltham, MA, USA). The samples were stained using Wright’s staining solution and Giemsa staining solution and observed under a light microscope (OLYMPUS BX50).

To differentiate cat iPS-like cells into neural stem cells, colonies were mechanically dissociated into small clumps at passage 20. Dissociated colonies were cultured in a non-treated 60 mm dish using nerve-cell culture medium (Sumitomo Bakelite, Tokyo, Japan) for 10 days at 37°C and 5% CO2. Half of the medium was replaced every other day and supplemented daily with bFGF at a final concentration of 20-ng/ml. Then, formed spheres were replated on 35 mm L-lysine coated dishes (AGC Techno Glass) covered with laminin (AGC Techno Glass) and cultured for an additional seven days in Neurobasal Medium (Life Technologies) supplemented with 100-IU/ml penicillin and 100-µg/ml streptomycin, 2-mM L-glutamine, and 1X B-27®supplement (Thermo Fisher Scientific). Cells were maintained at 37°C and 5% CO2. Half of the medium was replaced every other day and supplemented with human bFGF and human epithelial growth factor (R&D systems) daily at final concentrations of 20-ng/ml respectively. The expanded cells were used for immunofluorescence staining. The primary antibody used was rabbit anti-nestin polyclonal antibody (1-μg/ml; MAB353; Millipore) and the secondary antibody was goat anti-rabbit IgG monoclonal antibody conjugated with Alexa-546 (1-µg/ml). The expanded cells were washed with PBS (–) and enclosed with ProLong® Gold Antifade reagent with DAPI. The samples were observed and imaged using a confocal microscope (Nikon CLSI), and the expanded cells incubated with only the secondary antibody served as a negative control.

To test the properties of teratoma formation, 1 × 106 cat iPS-like cells were suspended in Matrigel diluted 1:1 in DMEM/Nutrient Mixture F-12 Ham and administered into the testis capsules of male severe combined immunodeficiency (SCID) mice. Tumor growth was periodically monitored for 12 weeks. After 12 weeks, the mice were euthanized to evaluate teratoma formation.

Growth Factor Assay of Cat iPS-like Cells

To confirm the growth factor dependency of cat iPS-like cells, we cultured in KSR medium, or in FBS medium supplemented with no bFGF, 10-ng/ml human bFGF, 10-ng/ml bFGF and dimethyl sulfoxide (DMSO), or 10-ng/ml bFGF and 1-μM JAK Inhibitor I (Cayman, Ann Arbor, MI, USA). Except for the growth factor conditions, the cat iPS-like cell culture method was the same as mentioned above.

Results

iPS-like cells can be derived from domestic cat embryonic fibroblasts

We assessed the impact of different cultivation methods (primate ES cell medium containing either 20% (v/v) ES cell Fetal FBS or KnockOut SR) and the number of seeded cat embryonic fibroblasts on iPS cell production. In the present study, we observed primary colonies of domestic cat iPS-like cells derived from embryonic fibroblasts beginning on day 18 in both media at all cell concentrations. However, cat embryonic fibroblasts seeded at higher densities (more than 1.0 × 104 cells in 35 mm tissue culture dishes) in medium containing FBS overgrew and inhibited primary colony growth, which was not observed in medium supplemented with KnockOut SR (Figs. 1A–B). Therefore, we reseeded 5.0 × 103 fibroblasts in FBS medium and 4.0 × 104 fibroblasts in KnockOut SR medium. More primary colonies appeared in the medium containing KnockOut SR than in the medium containing FBS (approximately 0.03% and 0.01%, respectively). Some of the primary colonies in both media grew sufficiently large to be passaged between days 25 and 32. We mechanically picked iPS-like colonies from both culture conditions, transferred them to a new gelatin-coated dish covered with inactivated SNL 76/7 cells, and subsequently cultured them in the same medium. Most of these colonies were not fully reprogrammed or differentiated, or stopped growing beyond passage 6 (Table 1). However, we were able to maintain cat iPS-like cells with high nucleus-to-cytoplasm ratios, clear edges, and compact shapes over passage 45 in medium with FBS (Figs. 1C–D), which was further analyzed in later experiments.

Fig. 1.

Fig. 1.

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Cat induced pluripotent stem (iPS)-like colonies. Cat embryonic fibroblasts in the medium including Knockout serum replacement (KnockOut SR) (A), or fetal bovine serum (FBS) (B). Cat iPS-like colonies maintained in the medium including FBS at passage 20 (C) and cat iPS-like cells composing the colony (D). Scale bars = 100 µm.

Table 1. Number of cell lines surviving to passage.

Number of cell lines surviving to passage
1 2 3 4 5 6 7
FBS 9 5 3 2 2 1 1
KnockOut SR 47 26 7 2 1 1 0
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In total, we cultured 1.2 × 105 and 8.8 × 105 cat embryonic fibroblasts in the medium supplemented with fetal bovine serum (FBS) and KnockOut serum replacement (KnockOut SR), respectively. Primary colonies were selected and cultured in the same medium.

Domestic cat iPS-like cells possess alkaline phosphatase activity and key pluripotent cell markers

We next, we characterized cat iPS-like cells. In particular, we assessed alkaline phosphatase activity and performed immunostaining to determine whether the cells expressed the hallmark iPS markers. Most cat iPS-like cells were positive for alkaline phosphatase staining at passage 20 (Fig. 2A). Additionally, immunostaining revealed that cat iPS-like cells were positive for SSEA-4, TRA-1-60, TRA-1-81, NANOG, and OCT3/4 proteins and partially positive for SSEA-1 at passage 42 (Figs. 2B–G).

Fig. 2.

Fig. 2.

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Characteristics of cat induced pluripotent stem (iPS)-like cells. Alkaline phosphatase staining of cat iPS-like cells at passage 20 (A; scale bar = 100 µm). Immunostaining of cat iPS-like cells combined with DAPI (nucleus; blue) for stage specific embryo antigen (SSEA)-1 (B), SSEA-4 (C), tumor rejection antigen (TRA)-1-60 (D), TRA-1-81 (E), NANOG (F), OCT3/4 (G) at passage 42. Scale bars = 20 µm.

Karyotyping of iPS-like cells

To assess the chromosomal landscape of cat iPS-like cells, we performed karyotyping (Table 2). Karyotyping revealed that these cells had a normal chromosomal complement (2N) of 38, with 18 pairs of autosomes and XY sex chromosomes, (at passage 20, the number of cat iPS-like cells analyzed = 42) (Fig. 3).

Table 2. Karyotyping.

Number of chromosomes < 38 38 > 38
Number of cells 0 42 0
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To assess the chromosomal landscape of the cat iPS-like cells, we performed karyotyping. Karyotyping revealed that these cells had a normal chromosomal complement (2N) of 38, with 18 pairs of autosomes and the XY sex chromosomes, (at passage 20, the number of cat iPS-like cells analyzed = 42).

Fig. 3.

Fig. 3.

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Karyotype of Cat induced pluripotent stem (iPS)-like cells at passage 20 (the number of cat iPS-like cells analyzed = 42).

RT-PCR kinetics of iPS-like cells

RT-PCR analysis of iPS-like cells was performed to assess the transcriptional abundance of the transfected mouse transcription factors Oct3/4, Klf4, Sox2, and c-Myc. Three exogenous genes, Oct3/4, Klf4, and Sox2, were gradually silenced during prolonged passages, with no expression observed at passages 21 or 38 (Fig. 4A). However, c-Myc was expressed continuously.

Fig. 4.

Fig. 4.

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Gene expression of cat induced pluripotent stem (iPS)-like cells. (A) RT-PCR of exogenous genes (mouse Oct3/4, Klf4, Sox2 and c-Myc). Cat embryonic fibroblasts (CEF) and no reverse transcription mRNA (RT-) were used as negative control, and genomic DNA was used as positive control for transgenes. GAPDH was selected as endogenous control. (B) Real-time PCR of endogenous genes (NANOG, OCT3/4, and SOX2). Expression of endogenous genes were relative to CEF. P, Passage.

Real-time PCR analysis

We detected the stable expression of endogenous NANOG and SOX2. Endogenous OCT3/4 was also expressed; however, its expression was not as high as that in cat embryonic fibroblasts (Fig. 4B).

Copy number analysis of transgenes

Integration of transgenes into the host genome was confirmed by PCR against genomic DNA. We detected two exogenous genes, OCT3/4 and SOX2 and five exogenous c-MYC genes (Supplementary Fig. 2). We attempted to examine the copy number of exogenous KLF4; however, we could not design an appropriate primer pair for exogenous KLF4.

In vitro differentiation and teratoma formation

The ability of cat iPS-like cells to differentiate was assessed based on EB formation at passage 20. The cat iPS-like cells could differentiate into various cell types, such as neuron-like and epithelial-like cells (Figs. 5A–B). Moreover, some of these cells differentiated into either of the three germ layers in vitro, ectoderm, endoderm, and mesoderm, as determined by expression of tubulin-β-III-isoform (Fig. 5C), SOX17 (Fig. 5D), or desmin proteins (Fig. 5E), respectively. Additionally, we observed blood lineage differentiation of cat iPS-like cells, which formed small red colonies on the methylcellulose medium at passage 20 (Fig. 5F). These colonies consisted of two cell types: many monocyte-like cells and a few erythroid progenitor-like cells (Figs. 5G–H). Upon differentiation of these iPS-like cells to neural stem cells, we obtained spindle cells expanding from the EBs (Figs. 5I–J), which were positive for nestin (Fig. 5K). To analyze teratoma formation, cat iPS-like cells were injected into four male SCID mice. However, these cells did not contribute to teratoma formation and instead formed hematomas (Fig. 5L).

Fig. 5.

Fig. 5.

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Differentiation property of cat induced pluripotent stem (iPS)-like cells. Several types of cells expanding from embryo bodies at passage 20 (EBs); neuron-like cells (A) and epithelial-like cells (B). Immunostaining of cells expanding from EBs combined with DAPI for Tublin-β-III-isoform (ectoderm; C), SOX17 (endoderm; D) or Desmin (mesoderm; E). A little red colony on methylcellulose medium after blood lineage differentiation (F). The components of a little red colonies were monocyte-like cells (G) and erythroid progenitor-like cells (H). After differentiation to neural stem cells, spindle cells expanding from EBs (I, J), and its immunostaining for Nestin with DAPI (K). Teratoma formation assay, in which cat iPS-like cells were administered into the testis capsule of male mice with severe combined immunodeficiency (SCID) mice (L). Scale bar = 20 µm.

Growth factor assay of cat iPS-like cells

iPS-like cells cultured with 10-ng/ml bFGF, or 10-ng/ml bFGF and DMSO possessed high nucleus-to-cytoplasm ratios, clear edges, and compact shapes (Figs. 6A–B). In contrast, iPS-like cells cultured in KSR medium or FBS medium without-FGF gradually changed into spindle shapes, and the density of their colonies decreased (Figs. 6C–D). Furthermore, colonies of iPS-like cells cultured with 10-ng/ml bFGF and 1-μM JAK Inhibitor I collapsed in a few days (Figs. 6E–F).

Fig. 6.

Fig. 6.

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Growth Factor Assay of Cat induced pluripotent stem (iPS)-like cells. Cat iPS-like cells cultured in fetal bovine serum (FBS) medium with 10-ng/ml basic fibroblast growth factor (bFGF) (A), in FBS medium with 10-ng/ml bFGF and dimethyl sulfoxide (B), in Knockout serum replacement medium with 10-ng/ml bFGF (C), or in FBS medium without bFGF (D) during 3 passages. Day 1 (E) and day 2 (F) of cat iPS-like-cells cultured in FBS medium with 10-ng/ml bFGF and 1-μM JAK Inhibitor I. Scale bars = 200 µm.

Discussion

In this study, we investigated the viability of domestic cat iPS cells via retroviral transfection of cat embryonic fibroblasts with mouse Oct3/4, Klf4, Sox2, and c-Myc genes. We selected cat embryonic fibroblasts as parent cells because embryonic fibroblasts are often used for iPS cell generation in other mammals [6, 7]. However, we observed that these embryonic fibroblasts grew too quickly and primary colony expansion was inhibited in cell medium containing FBS. Our observations corroborated those of a previous study conducted in rabbits [14]. Therefore, we reseeded the cat embryonic fibroblasts at a lower density in medium with FBS than we did with embryonic fibroblasts maintained in medium with KnockOut SR. While iPS-like colonies appeared under both culture conditions, the reprogramming efficiency was higher in medium supplemented with KnockOut SR medium. Interestingly, the proliferation of cat iPS-like cells was higher in medium supplemented with FBS. Similar findings have been reported for cat ES cell generation [21]. It is better to evaluate the appearance of colonies based on not only the morphology but also the alkaline phosphatase staining of those cells. However, we were unable to perform additional experiments due to a shortage of parent cells. The observed reprogramming efficiency and cell proliferation dynamics may be due to differences in the media in which either FBS or KnockOut SR was supplemented. Many medium components such as albumin, insulin, and transforming growth factor-beta affect reprogramming efficiency and cell proliferation [29]. While a rigorous comparison of the composition of FBS and KnockOut SR will help in the optimization of cultivation methods, such a task may be difficult because the composition of FBS is complex and varies among lots. Additionally, the composition of the KnockOut SR is proprietary information, and hence is not openly accessible [30].

Most cat iPS-like cells stained positive for alkaline phosphatase. This observation corroborated a previous study conducted on cat pluripotent stem cells and suggested that cat iPS-like cells were pluripotent [21,22,23]. Immunostaining analyses demonstrated that cat iPS-like cells were positive for SSEA-4, TRA-1-60, TRA-1-81, NANOG, and OCT3/4 and partially positive for SSEA-1. This expression pattern was similar to that observed in human pluripotent stem cells, but not in mouse pluripotent stem cells [6, 7]. However, there are conflicting reports regarding SSEA-1 and SSEA-4 expression in cat pluripotent stem cells [21,22,23]. Commercial monoclonal antibodies against human SSEA-1 and SSEA-4 were used in our study; therefore, it is possible that these antibodies did not react appropriately with cat iPS-like cells. These antibodies have been used in previous studies [21, 22]. In addition, OCT3/4 expression was detected not only in the inner cell mass but also in the trophoblast in the domestic cat [22]. Therefore, we assessed NANOG, TRA-1-60, and TRA-1-81, whose expression has been reported in some mammals, such as pigs and dogs [31, 32], to support the pluripotency of our cells. Future studies should simultaneously compare cat ES and iPS cells to better understand feline ES cell marker expression.

Previous reports revealed that chromosomal abnormalities sometimes occur during iPS cell establishment [6]. In the present study, cat iPS-like cells proliferated without any anomalies. Therefore, we concluded that a medium containing FBS and bFGF could maintain cat iPS-like cells. However, to deny the possibility of subtle chromosomal abnormalities such as translocations, transversions, and deletions, a more detailed analysis is needed.

Regarding the dynamics of the exogenous transgenes, Oct3/4, Klf4, and Sox2 were silenced. However, c-Myc was continuously expressed, which may have been caused by higher copy numbers than those of the other transgenes. The LTR promoter, utilized in pMXs vectors, is generally easily silenced. Therefore, we hypothesized that c-Myc expression continuously supports the prolonged passage of cat iPS-like cells [33]. After the three transgenes were silenced, cat iPS-like cells were maintained in FBS medium, which was conducive to the endogenous expression of genes critical for pluripotency, such as NANOG and OCT3/4. We detected NANOG, OCT3/4, and SOX2 expression in all passages. This result indicated that only four mouse transgenes could reprogram cat embryonic fibroblasts, and that FBS medium containing bFGF maintained cat iPS-like cells. In a previous study, cat iPS-like cells derived from adipose tissue fibroblasts were generated using five human transgenes driven by the LTR promoter and cultured in FBS medium containing LIF [23]. However, the transgenes of cat iPS-like cells treated with LIF alone were continuously expressed. However, we must consider the impact of cytokines and growth factors secreted by SNL 76/7 cells, that are genetically modified to synthesize LIF proteins [34]. In ES and iPS cell generation in mammals, such as mice, dogs, rabbits, cows, horses, goats, and cats, LIF is often added to the medium with or without bFGF to maintain pluripotency and proliferation abilities [3, 35, 37]. However, the necessity of LIF and bFGF during pluripotent stem cell culture has not yet been confirmed in most mammals, including cats [35, 36]. Therefore, we performed a growth factor assay to assess the utility of LIF in cat iPS-like cells. To completely exclude the effects of LIF, we added JAK inhibitor I, as described previously [38]. As a result, colonies of iPS-like cells cultured with 10-ng/ml bFGF and 1-μM JAK Inhibitor I collapsed in a few days. This suggested that cat iPS-like cells depend on both bFGF and LIF, which will improve the culture conditions for cat iPS cells in future studies.

In a previous study, the expression pattern of endogenous pluripotent factors in cat embryonic stem-like cells was found to differ from that in human and mouse pluripotent stem cells [22]. OCT3/4 expression was observed not only in the inner cell mass but also in trophoblasts and was not stable in ES-like cells. SOX2 was not expressed in the inner cell mass but was expressed in differentiated cells. Additionally, KLF4 and c-MYC were expressed in embryonic fibroblasts, which are the origin of iPS-like cells. Conversely, Filliers reported that OCT3/4, SOX2, and NANOG were expressed in cat blastocysts, and their expression patterns in cats resembled those of mice [37]. However, whole blastocysts were used for RT-PCR and were not separated into ICM or trophoblasts. Therefore, we should carefully consider whether these genes can be regarded as pluripotency markers in domestic cats.

In the present study, cat iPS-like cells differentiated into three germ layers in vitro, suggesting that these cells were pluripotent. Furthermore, these cells also had the capacity to differentiate into blood cells. There is a shortage of blood for domestic cats, making blood transfusion for domestic cats very problematic owing to the lack of blood banks and the capacity to screen for blood-borne viruses [39, 40]. The use of cat iPS-like cells on a large scale to generate a stable blood supply would greatly advance domestic cat medicine.

In this study, we showed that cat iPS-like cells could also differentiate into neural stem cells. Neurons differentiated from iPS cells reflect the genetic background of their donor cells and hence may serve as excellent models of disease [8]. Several genetic neural degenerative diseases have been reported in domestic cats [24], and the ability of cat iPS-like cells to differentiate into neural cell lineages is advantageous in the study of these diseases. Collectively, we showed that domestic cat iPS-like cells can differentiate into neural stem cells, monocyte-like cells, and erythroid progenitor-like cells, highlighting the potential utility of these cells in a diverse array of research and clinical applications.

However, in our study, we could not detect teratoma formation, which is a key criterion for pluripotency, after cat iPS-like cells were injected into SCID mice, as in a previous study [23]. In a previous study, we confirmed the procedure for teratoma formation in mouse ES cells. Thus, we considered that the failure of teratoma formation depended on the cell characteristics. Quantitative RT-PCR revealed that cat iPS-like cells expressed OCT3/4 at a weak level, which plays a key role in the reprogramming and maintenance of pluripotency. This may be one of the reasons why our cells were not fully reprogrammed, and hence were iPS-like. Therefore, we focused on new vector systems in which transgene expression can be controlled with doxycycline [31, 41, 42] or suppressed when iPS cells begin to differentiate [43]. Furthermore, the domestic cat had some differences or gaps in the sequences of OCT3/4, KLF4, SOX2, and c-MYC between human and mouse genes (Supplementary Figs. 3–6). Thus, the combination and species of transgenes should be considered to generate cat iPS cells that can form teratomas.

In conclusion, we generated cat iPS-like cells from cat embryonic fibroblasts that were retrovirally transfected with mouse reprogramming factors. These cells expressed the hallmark ES/iPS cell markers, had a normal set of chromosomes, and could differentiate into several cell types. However, these cells did not exhibit all the characteristics of iPS cells, as they could not form teratomas. Additionally, the present study was limited in that we could only establish and evaluate a single cat iPS-like cell line. Thus, we should consider whether the results of the present study reflect clonal variation. The groundwork we have laid in this study will drive further optimization and development of true cat iPS cells. A successful method for generating cat iPS cells will enhance transgenic animal research, provide models for a plethora of diseases and regenerative medicine, and promote reproductive techniques.

Conflict of interests

The authors have nothing to declare.

Supplementary

Supplementary Materials
jrd-69-317-s001.pdf (1.4MB, pdf)

Acknowledgments

This study was supported by JSPS KAKENHI Grant Numbers 24380172, JP15K07747, JP18K19273, JP18H02349, and 21K14974.

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Supplementary Materials

Supplementary Materials
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