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The Journal of Veterinary Medical Science logoLink to The Journal of Veterinary Medical Science
. 2024 Jan 4;86(3):247–257. doi: 10.1292/jvms.23-0422

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems

Kazuto KIMURA 1, Hiroya NAGAKURA 1, Masaya TSUKAMOTO 1, Takumi YOSHIDA 1, Hiroko SUGISAKI 1, Kohei SHISHIDA 1, Yuta TACHI 1, Shoko SHIMASAKI 1, Kikuya SUGIURA 1, Shingo HATOYA 1,*
PMCID: PMC10963097  PMID: 38171744

Abstract

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Keywords: canine induced pluripotent stem cell, culture system, differentiation propensity, feeder-free, weekend-free

Induced pluripotent stem cells (iPSCs), derived from somatic cells by inducing pluripotency genes, have the ability to self-renew infinitely and differentiate into all three germ layers [43]. These characteristics make them promising resources in regenerative medicine, which is a branch of medicine used to grow, repair, or replace damaged or diseased cells, organs, or tissues. In addition, for ailments that have long been perplexing, such as idiopathic diseases with no known cause, in vitro disease modeling using canine iPSCs (ciPSCs) has emerged as a pivotal tool. By faithfully recapitulating disease conditions in a controlled laboratory environment, researchers gain invaluable insights into the mechanisms underlying these enigmatic diseases and expedites drug discovery efforts.

In contrast, dogs are not only precious family members but may also be excellent models for many diseases in humans, because dogs naturally develop many diseases similar to those in humans and their longer lifespans allow for long-term studies of treatment efficacy, side effects, and safety compared to rodent models [11, 23]. Thus, veterinary clinical trials, such as cutting-edge care using ciPSC-derived cells, may provide useful insights into the clinical efficacy and safety of regenerative medicine for extrapolation in humans [2, 7]. In addition, the differences in genetic backgrounds among dog breeds results in a number of distinctive diseases, which makes them useful for disease modeling and the development of novel therapies for veterinary and human use [42].

However, development of research and clinical applications of ciPSCs requires an efficient method for the generation and culture of ciPSCs in large quantities. Although ciPSCs have been generated by several groups in the past few decades [10, 21, 45, 46, 51], they were previously reported to be maintained on feeder cells such as mouse embryonic fibroblasts in the media, including fetal bovine serum or knockout serum replacement; this has prevented researchers from improving experimental reproducibility because of their undefined components and differences between batches [44]. In contrast, our group showed that a serum- and feeder-free culture system for human pluripotent stem cells (hPSCs) using StemFit AK02N [35] as a medium and laminin-511 E8-fragment (commercial name iMatrix-511) [34] as a coating matrix can be used to culture ciPSCs [22]. However, StemFit AK02N medium is commercially unavailable around the world except for Japan. Additionally, this culture system requires daily medium change. Thus, low accessibility and time-, effort-, and cost-consuming requirements for daily media changes are major restrictions in the advancement of ciPSC research. On the other hand, human iPSC culture conditions influence hepatic differentiation efficiency [30]. Thus, although the differentiation capacity of ciPSCs is highly dependent on the culture conditions, the relationship between ciPSC culture conditions and differentiation capacity is unknown.

In contrast, apart from StemFit and iMatrix-511, many culture systems for hPSCs have been developed. For instance, StemFlex™ Medium (StemFlex; Thermo Fisher Scientific, Waltham, MA, USA) contains proprietary compounds and supports the robust expansion of hPSCs even in a weekend-free schedule. The mTeSR™ Plus medium (STEMCELL Technologies, Vancouver, BC, Canada) was developed based on the TeSR medium, which is the first reported medium for hPSC feeder-free culture [27, 28]. Both media are commercially available for human iPSC maintenance and have been widely used in hPSC research [12, 13, 26]. These media are formulated to contain thermally stabilized basic fibroblast growth factor (bFGF) [8], which results in the stable maintenance of hPSCs. It has been suggested that ciKIC medium can be modified from AKIT medium, which was developed as a growth factor- and albumin-free hPSC medium by combining several small compounds to replace key growth factors, such as bFGF and transforming growth factor (TGF)-β [50]. These media do not require daily change, including on weekends, because of stabilized bFGF or replacement of short-half-life growth factors with small compounds, which results in reduced effort and cost for iPSC culture. Vitronectin, which supports hPSC self-renewal [6], and Geltrex™ (Thermo Fisher Scientific), which is a replacement for Matrigel derived from mouse Engelbreth-Holm-Swarm tumors [19], are widely used as cost-effective coating matrices for hPSC maintenance, and are commercially available and recommended by suppliers when StemFlex or mTeSR Plus are used for human iPSC maintenance in weekend-free culture systems.

Therefore, we investigated the applicability of some common hPSC culture systems for ciPSC culture and the influence on the differentiation capacity of ciPSCs by changing the culture conditions. In this study, we first verified whether hPSC culture media without the requirement of daily media changes (StemFlex, mTeSR Plus, and ciKIC) could be used to culture ciPSCs. Next, we investigated the availability of vitronectin and Geltrex for ciPSC maintenance. Finally, the differentiation propensities of the ciPSCs maintained in these culture systems were studied using two ciPSC lines.

MATERIALS AND METHODS

Cell culture

We used peripheral blood mononuclear cells-derived ciPSC lines generated at our laboratory, which have teratoma formation ability (OPUiD04-B and OPUiD05-A) [22]. The ciPSCs were maintained on iMatrix-511 silk (0.25 µg/cm2, Nippi, Inc., Tokyo, Japan)-coated dishes in StemFit AK02N medium (Ajinomoto, Tokyo, Japan) at 5% CO2 and 37°C. They were passaged mechanically using a glass Pasteur pipet with a split ratio of 1:5 to 1:20 every 3–5 days or enzymatically using TrypLE Select (Thermo Fisher Scientific) at a density of 1–3 × 103/cm2 every 3–5 days. The ciPSCs at passages 15–40 were used for further experiments.

Feasibility of using hPSC media to culture ciPSCs

The OPUiD04-B cell line was used for this experiment. Three ciPSC colonies of nearly identical sizes were picked and seeded onto iMatrix-511-coated 4-well plates in ciKIC iPS medium (ciKIC; Kanto Chemical, Tokyo, Japan), StemFlex, or mTeSR Plus supplemented with 10 µM Y-27632 (Nacalai Tesque, Kyoto, Japan). The medium was changed every two days in case ciKIC, StemFlex, and mTeSR Plus were used, and the medium was changed every day in case StemFit was used. The ciPSCs were then passaged mechanically. This experiment was repeated three times for each culture condition.

Examination of multiple weekend-free culture conditions for ciPSCs

The ciPSC cell lines OPUiD04-B and OPUiD05-A were used for this experiment. The ciPSCs were dissociated into single cells using TrypLE Select and were seeded onto a 24-well cell culture plate coated with 0.5 µg/cm2 human recombinant vitronectin-N truncated (VTN-N; Thermo Fisher Scientific) or Geltrex™ (Geltrex; Thermo Fisher Scientific) at 1:100 dilution in StemFlex or mTeSR Plus at a density of 5 × 104/cm2 by adding 10 µM Y-27632. Medium changes and passages were performed on a weekend-free schedule as recommended by the suppliers. After changing the media and coating matrix, ciPSCs were passaged using 0.5 mM ethylenediaminetetraacetic acid (EDTA)/phosphate-buffered saline (PBS)-based buffer (Nacalai Tesque) in Dulbecco’s phosphate-buffered saline without calcium and magnesium (D-PBS (−); Nacalai Tesque) with a split ratio of 1:3 to 1:20 every 2–4 days. Briefly, the cells were washed once with D-PBS (−), an appropriate volume of EDTA was added to the plates, and the cells were incubated for 4 min at 37°C. Then, the EDTA solution was aspirated, and fresh medium was added. Digested cells were gently dissociated by pipetting and seeded onto a cell culture plate coated with VTN-N or Geltrex at the desired density.

Karyotyping

The ciPSCs were incubated in a medium containing 0.04 µg/mL colcemid (Thermo Fisher Scientific) for 1 hr, followed by trypsinization and incubation in 0.05 M potassium chloride (KCl) at 37°C for 20 min. The cells were fixed in a mixture of acetic acid and methanol (1:3), assessed by Q-banding, and analyzed under a confocal laser microscope (LSM980; Carl Zeiss, Oberkochen, Germany).

Immunocytochemistry

The ciPSCs were dissociated into clumps, seeded onto Geltrex-coated 8-well chamber slides (AGC Techno Glass, Shizuoka, Japan), and cultured using StemFit, StemFlex, or mTeSR Plus. For differentiation, after ciPSCs were seeded as described above, the medium was changed to bovine serum albumin polyvinylalchohol essential lipid (BPEL) medium [36] with slight modification supplemented with/without 100 ng/mL Activin A (Nacalai Tesque) and cultured for approximately 10 days. The components of BPEL media are listed in Supplementary Table 1. The ciPSCs or differentiated cells were washed with D-PBS (−), fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, and permeabilized with 0.1% Tween 20 in PBS (−) for 15 min at approximately 25°C. The cells were incubated with 10% bovine serum albumin (Nacalai Tesque) for 1 hr, followed by overnight incubation at 4°C in the presence of primary antibodies against OCT3/4 (clone: C-10), SRY-box transcription factor 2 (SOX2) (clone: E-4), Nanog homeobox (NANOG), Tubulin Beta 3 (TUBB3) (clone: TU-20), alpha-smooth muscle actin (α-SMA) (clone: 1A4), and Forkhead box A2 (FOXA2). Negative control cells were incubated with D-PBS (−) without the primary antibodies. The next day, cells were washed with D-PBS (−) and incubated for 1 hr at approximately 25°C with the following secondary antibodies. All antibodies used are listed in Supplementary Table 2. The cells were washed with D-PBS (−), labeled with DNA using ProLong Gold Antifade Reagent 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific), and observed using confocal laser microscopy (FV3000; Olympus, Tokyo, Japan).

Quantitative reverse transcription PCR (RT-qPCR)

Total RNA was isolated using a FastGene RNA Premium Kit (Nippon Genetics, Tokyo, Japan) and reverse-transcribed into complementary DNA using random primers and ReverTra Ace (Toyobo, Osaka, Japan). RT-qPCR was performed in triplicate using Taq Pro Universal SYBR qPCR Master Mix (Nanjing Vazyme Biotech, Nanjing, China) and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), according to the manufacturer’s instructions. All primers used are listed in Supplementary Table 3. PCR data were analyzed using the ∆/∆CT method and normalized to β-ACTIN expression.

Quantitative analysis of differentiation markers for three germ layers

The ciPSCs were dissociated into single cells, seeded onto Nunclon™ Sphera™ 96-well U-shaped-bottom microplates (Thermo Fisher Scientific), and cultured for 10 days in BPEL medium with slight modifications to form multiple embryoid bodies (EBs). Total RNA isolation, reverse transcription, and qPCR analyses were performed as described above.

Statistical analysis

Data are expressed as the mean ± standard deviation. Statistical significance was determined using the Tukey–Kramer multiple comparison procedure and Statcel software (OMS Ltd., Tokyo, Japan). A significant difference was determined in P<0.05.

RESULTS

ciPSCs can be maintained in StemFlex and mTeSR Plus media with media change every two days

After one passage, ciPSCs maintained in StemFit on iMatrix-511 survived and proliferated in StemFlex and mTeSR, and had similar colony morphologies compared to those before the passage; in contrast, ciPSCs died immediately in ciKIC (Fig. 1A). The ciPSCs were maintained in StemFlex and mTeSR Plus media even with media change every two days, and these cultured ciPSCs formed similar colonies four passages after transfer from culture media (Fig. 1A). The colony numbers after one passage were 29.8 ± 5.01 and 30.2 ± 4.01 in StemFlex and mTeSR Plus, respectively, compared to 37.5 ± 4.77 in StemFit, with no significant difference. Meanwhile, barely any colony was formed in ciKIC and the number of colonies were significantly lower than those in other experimental groups (Fig. 1B). After five passages, the colony numbers were 63.0 ± 7.37 and 54.8 ± 4.01 in StemFit and StemFlex, respectively, which were significantly higher than those in mTeSR Plus (30.3 ± 1.61) (Fig. 1B). To confirm whether ciPSCs cultured in StemFlex and mTeSR Plus maintained pluripotency, the pluripotency marker OCT3/4 and their expression levels in ciPSCs were verified. The ciPSCs cultured in both media expressed OCT3/4 at levels similar to those of ciPSCs cultured in StemFit (Fig. 1C). These data suggest that StemFlex and mTeSR Plus can be used as ciPSC culture media.

Fig. 1.

Fig. 1.

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Analysis of various chemically-defined culture media for canine iPSC (ciPSC) culture. (A) Morphology of ciPSCs in each culture medium at passage 1 (upper columns) and at passage 5 (lower columns) after changing the media type. Scale bar=100 µm. (B) Number of colonies at passage 1 (left) and passage 5 (right) after changing the media type. **P<0.01; n=3. (C) OCT3/4 mRNA expression levels relative to those in ciPSCs maintained in StemFit. mRNA expression levels were normalized using β-ACTIN as the housekeeping gene. n=3.

ciPSCs can be maintained in weekend-free culture systems

To verify if ciPSCs can be maintained in weekend-free culture systems, we investigated whether the hPSC weekend-free culture systems using StemFlex or mTeSR Plus with VTN-N or Geltrex, which are widely used in hPSC research, are applicable to ciPSC culture. Both ciPSC lines could be maintained in StemFlex or mTeSR Plus with VTN-N or Geltrex and had typical ciPSC morphology, such as clear borders of ciPSC colonies and a flat and compact colony (Fig. 2A). Moreover, under culture conditions using VTN-N or Geltrex, a non-enzymatic dissociation solution (EDTA) that dissociates ciPSC colonies into small cell clumps can be used for passaging ciPSCs without Y-27632 (a Rho-associated protein kinase (ROCK) inhibitor), which inhibits cell apoptosis induced by single-cell dissociation [47]. All tested culture conditions and the EDTA passaging method enabled stable growth of ciPSCs under weekend-free conditions (Fig. 2B, 2C). Furthermore, ciPSCs maintained in these culture systems had normal karyotypes with 38 matched pairs of autosomes and XX gonosomes (Fig. 2D). The percentage of cells with normal karyotypes was 75–91% (OPUiD04-B: StemFlex, VTN-N: 11/13, StemFlex, Geltrex: 12/14, mTeSR Plus, VTN-N: 10/12, mTeSR Plus, Geltrex: 9/12, OPUiD05-A; StemFlex, VTN-N: 9/11, StemFlex, Geltrex: 10/11, mTeSR Plus, VTN-N: 9/10, mTeSR Plus, Geltrex: 12/14).

Fig. 2.

Fig. 2.

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Examination of various weekend-free culture conditions for ciPSC culture. (A) Morphology of ciPSCs in each culture system at passage 5 after changing the culture conditions. The upper and lower columns show the morphologies of the OPUiD04-B and OPUiD05-A ciPSC lines, respectively. Scale bar=100 µm. (B) Schema for ciPSC culture in a weekend-free manner. Mon, Monday; Tue, Tuesday; Wed, Wednesday; Thu, Thursday; Fri, Friday; Sat, Saturday; Sun, Sunday; P, Passage; F, Media change; 2F, Media change using 2-fold amount of media. (C) Fold expansion of ciPSCs maintained under each culture condition relative to cell number of day 0. Total cell number at passage N=Cell number counted at passage N × passage ratio at passage N-1. n=3. (D) Karyotype analysis of ciPSCs at passage 10–15 after culture condition change. Normal: 78, XX with 38 matched pairs of autosomes in cells.

To evaluate the pluripotency of the ciPSCs maintained in these culture systems, immunocytochemistry and qRT-PCR were performed. Immunocytochemistry showed that both ciPSC lines maintained under the four conditions as well as those maintained in StemFit and iMatrix-511 were positive for pluripotency markers, such as OCT3/4, NANOG, and SOX2 (Fig. 3A). RT-qPCR analysis indicated that culture using mTeSR Plus with VTN-N or Geltrex resulted in lower OCT3/4 expression level in iPSCs compared to StemFit and iMatrix-511 in the OPUiD04-B line (Fig. 3B). In contrast, in the OPUiD05-A line, ciPSCs in StemFlex and VTN-N showed higher expression of NANOG than those in StemFit and iMatrix-511 (Fig. 3B). The expression of other genes was not significantly different among the culture conditions (Fig. 3B). These data indicate that these culture conditions can be used to maintain expression levels of most pluripotency markers, although the media and matrices may affect ciPSC characteristics.

Fig. 3.

Fig. 3.

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Expression of pluripotency markers in ciPSCs was maintained under each culture condition. (A) Immunocytochemistry for pluripotency markers OCT3/4, Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2). The left blocks show results in the OPUiD04-B ciPSC line. The right blocks show results in the OPUiD05-A ciPSC line. Scale bar=100 µm. (B) Relative mRNA expression levels of pluripotency markers OCT3/4, NANOG, and SOX2 relative to those in ciPSCs maintained in StemFit on iMatrix-511. mRNA expression levels were normalized using β-ACTIN as the housekeeping gene. *P<0.05; StemFit-iMatrix, n=3; other culture conditions, n=5.

We then examined whether ciPSCs cultured in StemFlex or mTeSR Plus with VTN-N or Geltrex could differentiate into cells of the three germ layers. With both cell lines, differentiated cells were positive for TUBB3, α-SMA, and FOXA2 under all culture conditions (Fig. 4A, 4B).

Fig. 4.

Fig. 4.

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Analysis of differentiation marker expression. (A) Immunocytochemistry for the differentiation markers of the three germ layers, Tubulin Beta 3 (TUBB3), alpha-smooth muscle actin (α-SMA), and Forkhead box A2 (FOXA2), in the OPUiD04-B ciPSC line. Scale bar=100 µm. (B) Immunocytochemistry for the differentiation markers of the three germ layers, TUBB3, α-SMA, and FOXA2, in the OPUiD05-A ciPSC line. Scale bar=100 µm.

Quantitative analysis of differentiation markers of the three germ layers

EBs were formed from ciPSCs under all culture conditions and were similar in shape and size (Fig. 5A). When ciPSCs were maintained in mTeSR Plus with VTN-N, the expression levels of the ectodermal marker TUBB3 in EBs were significantly higher than those in StemFlex with VTN-N and mTeSR Plus with Geltrex in the OPUiD04-B line (Fig. 5B). In addition, the expression levels of Paired Box 6 (PAX6) in EBs formed from ciPSCs maintained in StemFlex with Geltrex were significantly higher than those in other culture conditions in the OPUiD04-B line (Fig. 5B). In contrast, in the OPUiD05-A line, the mesodermal marker, kinase insert domain receptor (KDR) in EBs were expressed although the level was less than 10-fold, and another representative mesodermal marker, platelet derived growth factor receptor alpha (PDGFRα) was hardly expressed in the EBs formed from ciPSCs maintained under all culture conditions (Fig. 5B). In the OPUiD04-B line, KDR was expressed in EBs formed from ciPSCs cultured in StemFlex and VTN-N or Geltrex as well as mTeSR Plus and Geltrex; however, PDGFRα was not expressed (Fig. 5B). The expression levels of the endodermal marker, C-X-C motif chemokine receptor 4 (CXCR4) were approximately 10-fold higher than those in ciPSCs under all culture conditions (Fig. 5B). However, another endodermal marker SOX17 was barely expressed under all culture conditions in either cell line (Fig. 5B). These data indicate that culture conditions are unlikely to significantly influence the differentiation propensity of ciPSCs under the experimental conditions of this study.

Fig. 5.

Fig. 5.

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Quantitative analysis of differentiation markers of the three germ layers. (A) Morphologies of EBs formed from ciPSCs maintained under each culture condition on day 10. The upper and lower columns show the morphologies of the OPUiD04-B and OPUiD05-A ciPSC lines, respectively. Scale bar=100 µm. (B) Relative mRNA expression levels of differentiation markers of the three germ layers: TUBB3 and paired box 6 (PAX6) (ectoderm), kinase insert domain receptor (KDR) and platelet derived growth factor receptor alpha (PDGFRa) (mesoderm), and C-X-C motif chemokine receptor 4 (CXCR4) and SOX17 (endoderm) relative to the undifferentiated state under each culture condition. mRNA expression levels were normalized using β-ACTIN as the housekeeping gene. The upper and lower columns show the results for OPUiD04-B and OPUiD05-A, respectively. *P<0.05, **P<0.01; n=3.

DISCUSSION

Although our previous study enabled easy and reproducible maintenance of ciPSCs by application of culture conditions to StemFit and iMatrix-511 in a feeder-free and chemically defined manner [22], low accessibility around the world and high costs, effort, and long duration of daily ciPSC maintenance persist as complications that can hamper the expansion and progression of research using ciPSCs. Therefore, we investigated whether widely available and weekend-free culture systems for hPSC culture could be applied for ciPSC maintenance. StemFlex and mTeSR Plus can be used to maintain ciPSCs under feeder-free conditions and enable weekend-free culture, resulting in wide access to medium appliable for ciPSC culture and a reduced cost, effort, and time for ciPSC maintenance.

We showed that StemFlex and mTeSR Plus can be used to maintain ciPSCs with typical morphology, colony formation, and expression of the pluripotency marker OCT3/4 under feeder-free conditions, such as StemFit. These data suggest that ciPSCs can be maintained at conditions similar to those for hPSCs. Ludwig et al. revealed that high concentration of bFGF plays an important role in the culture of hPSCs under feeder-free condition and developed the TeSR medium [28]. Chen et al. optimized the TeSR medium and revealed the eight essential components required for hPSC culture, including bFGF, TGFβ, insulin, transferrin, sodium selenite, and L-ascorbic acid, and developed the Essential 8 (E8) medium [9]. Although the complete compositions of StemFlex and mTeSR Plus are unavailable, at least mTeSR Plus has been developed based on the TeSR medium. In addition, we showed that StemFit, which contains components similar to E8, except for albumin [35], could maintain ciPSCs [22]. Therefore, identical elements as those in the TeSR or E8 media may possibly contribute to positive effects on ciPSC culture. In contrast, our data indicated that ciKIC cannot be used to culture ciPSCs. ciKIC can be used to culture hPSCs by replacing TGFβ and bFGF, which play important roles in hPSC growth in their undifferentiated states [1, 15, 17, 49] and with glycogen synthase kinase-3β (GSK-3β) inhibitors, dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), and calcineurin/nuclear factor of activated T cells (NFAT) [50]. This suggests that although most receptors of key growth factors, such as bFGF and TGFβ, may be common in canine and human iPSCs, the downstream signaling pathways modulated by key growth factors for iPSCs may be different. Additionally, StemFit, StemFlex and mTeSR Plus contain bovine or human serum albumin but not ciKIC. Albumin has several important physiological and pharmacological functions, including antioxidant properties [40], which may positively affect ciPSC maintenance. Further studies using completely defined media are required to understand the key factors and intracellular signaling pathways that contribute to ciPSC maintenance, and may lead to the development of an optimal medium for ciPSC maintenance and realization of a homemade, inexpensive medium, as has been done for hPSCs [25].

Moreover, combining StemFlex or mTeSR Plus with VTN-N or Geltrex allowed weekend-free culture of ciPSCs with typical morphology, stable growth rate, normal karyotypes, pluripotency marker expression, and differentiation capacity. These data reveal that ciPSCs and hPSCs can be successfully maintained in weekend-free culture systems. This positive result may be because of the effectiveness of thermally stabilized bFGF in StemFlex and mTeSR Plus for ciPSC culture, similar to that reported in humans [8]. Although the necessity of bFGF and/or leukemia inhibitory factor has been discussed for ciPSC research [29, 32, 37, 48], our data supports the hypothesis that bFGF is essential for ciPSC culture and that the pluripotency maintenance in ciPSCs is similar to that in hPSCs.

VTN-N and Geltrex were used as coating matrices for ciPSC culture. Vitronectin supports sustained self-renewal and pluripotency in hPSCs by binding with the αVβ5 integrins expressed on the surface of PSCs [24]. Matrigel is similar to Geltrex and laminin-511 mainly bind α6β1 integrins [31, 41]. Our data showed that the difference in cell adhesion receptors between VTN-N and Geltrex or iMatrix-511 was not critical for ciPSC maintenance, suggesting that both αVβ5 and α6β1 integrins can be expressed on ciPSCs and contribute to their attachment to plates, cell growth, and maintenance of pluripotency similar to that in hPSCs [6, 33].

In addition, weekend-free culture systems enabled the passage of ciPSCs with EDTA, whereas enzymatic reagents, TrypLE Select, and ROCK inhibitor Y-27632 were used to passage ciPSCs in the culture system using StemFit and iMatrix-511. Enzyme-free passaging without ROCK inhibitor has the great advantage of karyotype stability in routine ciPSC maintenance because enzyme reagents dissociate ciPSCs into single cells, which leads to poor cell survival and increases the risk of acquiring genomic mutations or chromosomal abnormalities [3, 4, 14]. In hPSC research, EDTA-based passaging procedure is recommended for routine hPSC culture because of the risk of abnormal karyotypes [5].

Interestingly, the expression levels of NANOG in the OPUiD05-A line maintained using StemFlex and VTN-N were significantly higher than those in the ciPSCs maintained using StemFit and iMatrx-511. This data may be consistent with hPSC research data, which indicated that NANOG levels were elevated with increased FGF stability [8]. In contrast, the OPUiD04-B line maintained in mTeSR Plus showed significantly reduced OCT3/4 expression compared to that maintained in StemFit. In humans, it is reported that the expression levels of pluripotency markers including OCT3/4 varied among hPSC culture conditions [16]. The expression levels of pluripotency markers such as OCT3/4 in ciPSCs may also be affected by culture conditions.

Finally, we investigated the qualitative differentiation capacity and quantitative differentiation propensity of two ciPSC lines maintained in weekend-free culture systems. There were few differences in differentiation propensities between the tested culture conditions for ciPSCs except for some markers in one cell line. Our data showed that all tested culture conditions could maintain the differentiation ability of ciPSCs, suggesting that these weekend-free culture systems can be used for ciPSC culture. In contrast, quantitative analysis of differentiation markers revealed that both cell lines preferentially differentiate into ectoderm lineages under spontaneous differentiation conditions without growth factors that induce specific lineage differentiation. These observations suggest that the ciPSC lines used in this study have biased potential to differentiate into ectodermal lineage. In hPSCs, variability in the differentiation capacity of hPSC lines related to the cell types or donors of iPSC origin, genetic diversity, and epigenetic variance determine the preference for differentiation propensity towards specific germ layers or cell types [18, 20, 24, 38, 39]. The ciPSC lines used in this study were generated from separate donors [22]; both donors were experimental beagle dogs, which likely have similar genetic background. Hence, genetic background may influence the differentiation capacity of the tested ciPSC lines. In contrast, both tested iPSC lines were established and acclimated under similar culture conditions using StemFit and iMatrix-511 [22]. It has been reported that hPSCs maintained in StemFit on iMatrix-511 are unlikely to express endodermal markers such as GATA4 and GATA6, but are likely to express SOX2, a pluripotency and ectodermal marker, compared to those maintained in E8 or TeSR with vitronectin [30]. Thus, culture conditions, especially during PSC establishment, may influence the differentiation propensity of ciPSCs. To confirm the importance of culture conditions, more studies comparing ciPSCs established under different culture conditions are required.

In conclusion, we showed that ciPSCs can be cultured under weekend-free culture conditions using StemFlex or mTeSR Plus with VTN-N or Geltrex and can be passaged with EDTA in an enzyme- and ROCK inhibitor-free manner. Since there were few significant differences in differentiation propensities among the various ciPSC culture conditions analyzed, further studies are needed to identify the factors that determine ciPSC differentiation propensities. This study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems commercially available in the world, which may lead to further development in research using ciPSCs.

CONFLICT OF INTEREST

The authors declare no conflicts of interest directly relevant to the content of this article.

Supplementary

Supplement Tables
jvms-86-247-s001.pdf (246.4KB, pdf)

Acknowledgments

This work was supported by JSPS KAKENHI (Grant numbers JP18H02349, and 22J14623, 22H02525) and the 2022 Osaka Metropolitan University (OMU) Strategic Research Promotion Project (Priority Research).

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