Induced Pluripotent Stem Cell Elimination in a Cell Sheet by Methionine-Free and 42°C Condition for Tumor Prevention

Katsuhisa Matsuura M.D., Ph.D. 1), 2) Kyoji Ito M.D. 1), 3) Nobuaki Shiraki Ph.D. 4), Shoen Kume Ph.D. 4), Nobuhisa Hagiwara M.D., Ph.D. 2) and Tatsuya Shimizu M.D., Ph.D. 1

Abstract

Pluripotent stem cells including induced pluripotent stem (iPS) cells are promising cell sources for regenerative medicine to replace injured tissues and tissue engineering technologies enable engraftment of functional iPS cell-derived cells in vivo for prolong periods. However, the risk of tumor formation is a concern for the use of iPS cells. Bioengineered tissues provide a suitable environment for cell survival, which require vigorous efforts to eliminate remaining iPS cells and prevent tumor formation. We recently reported three iPS cell elimination strategies including methionine-free medium, TRPV1 activation through 42 °C cultivation, and dinaciclib, a cyclin-dependent kinase 1/9 inhibitor. However, it remains unclear how many iPS cells in bioengineered tissues are eliminated using these strategies alone or in combination and the subsequent tumor prevention. In the present study, we found that 2 days of cultivation at 42 °C sufficiently eliminated 1 × 102 iPS cells in fibroblast sheets and prevented tumor formation. After screening for suitable combinations of these strategies based on Lin28 expression in co-cultures of fibroblasts and 1 × 104 iPS cells, we found that 1 day of cultivation at 42 °C in methioninefree culture medium with or without dinaciclib remarkably decreased Lin28 expression and prevented tumor formation. Furthermore, these culture strategies did not affect spontaneous beating or the cell number of human iPS cell-derived cardiomyocytes. These quantitative findings may contribute to decreasing the tumor formation risk and development of regenerative medicine using iPS cells.

Key words: iPS cell, cell sheet, tumor, methionine, 42 °C culture

Impact statement

An elimination strategy for remaining iPS cells is important to prevent tumor formation in regenerative medicine. Because bioengineered tissues provide a suitable environment for cell survival and the subsequent engraftment, methods are necessary to eliminate remaining iPS cells. Here, we succeeded in eliminating 1 × 104 iPS cells in a fibroblast sheet by cultivation at 42 °C in methionine-free medium and prevented tumor formation. This strategy does not require any additional materials in the culture process and may contribute to decreasing the tumor formation risk and development of regenerative medicine using iPS cells.

Introduction

Regenerative medicine is expected to provide new therapeutic strategies for uncured diseases. Pluripotent stem cells (PSCs) including induced pluripotent stem (iPS) cells (1) generate robust numbers and varieties of cells and are promising cell sources for regenerative medicine to replace injured tissues. Recent advancement of culture technologies, including three-dimensional suspension culture, has enabled generation of robust numbers of iPS cell-derived cells including cardiomyocytes and pancreatic progenitor cells (2, 3). Furthermore, tissue engineering technologies including cell sheet engineering are capable of fabricating human cardiac tissues in vitro (2, 4) and in vivo (5, 6). Pulsation of engrafted cardiac tissues has been reported to generate pulse pressure (6), suggesting that sufficient and long-term engraftment of transplanted cells might directly support the functions of tissues and organs in vivo.
However, the risk of tumor formation is a concern for the use of PSCs and makes it difficult to develop regenerative medicine using PSC-derived cells. There are some risk factors for tumor formation owing to remaining PSCs in regenerative medicine products, including the cell number for transplantation, differentiation efficacy, engraftment efficacy, and efficacy of eliminating any remaining iPS cells. Transplantation of a high cell number increases the risk of remaining PSCs, and >1 × 108 cells are needed in transplants for heart diseases and diabetes. A higher differentiation efficacy is necessary to reduce the risk of tumor formation but does not exclude the need for a purification process. Tissue engineering technology plays an important role in promoting engraftment of cells, while undesired cells such as remaining PSCs might also be retained. In particular, because single cell conditions easily induce cell death of PSCs (7), bioengineered tissues might be a suitable environment for PSC survival. Therefore, strategies to sufficiently eliminate remaining PSCs without affecting the viabilities of desired cells in the tissue fabrication process are important. Recently, we reported iPS cell elimination methods, including methionine-free medium (8), TRPV1 activation through 42 °C cultivation (9), and dinaciclib, a cyclindependent kinase (CDK) 1/9 inhibitor (10), based on the difference of tolerance in culture environments between iPS cells and differentiated cells. In particular, because methioninefree medium and 42 °C cultivation do not require any addition materials, there is no concern for residual substances derived from materials used in the production process of regenerative medicine products. However, it remains unclear how many iPS cells in bioengineered tissues are eliminated using each method or their combinations and the subsequent tumor prevention.
In the present study, we found that 2 days of cultivation at 42 °C sufficiently eliminated 1 × 102 iPS cells in bioengineered tissues and prevented tumor formation. Furthermore, the combination of methionine-free medium at 42 °C eliminated 1 × 104 iPS cells in bioengineered tissues and prevented tumor formation without affecting cardiomyocyte viability. These quantitative findings may contribute to decreasing the tumor formation risk and development of regenerative medicine using iPS cells.

Materials and Methods

Human iPS cell culture

Human iPS cell line Ff-I14 (iPSC Stock for non-clinical use) was provided by the Center for iPS Cell Research and Application, Kyoto University. iPS cells were maintained on iMatrix511 (Nippi, Tokyo, Japan) in StemFit AK03N (Ajinomoto, Tokyo, Japan). Cells were passaged as single cells every 7 days using TrypLE Select (Life Technologies, Carlsbad, CA, USA) as described elsewhere (11).
Human iPS cell line 201B7 was purchased from RIKEN (Tsukuba, Japan). Human iPS cells expressing α-MHC promoter and rex-1 promoter-driven drug-resistance genes were cultured on inactivated mouse embryonic fibroblasts (ReproCELL, Yokohama, Japan) as described previously (6).

Co-culture experiments and cell sheet fabrication

Normal human dermal fibroblasts (NHDFs) were purchased from Lonza Group Ltd (Basel, Switzerland) and maintained on uncoated 10-cm dishes (Corning, Corning, NY, USA) in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. Passage 3–7 cells were used for experiments.
6 On day -2 or -1, 1 × 105 (Figs. 1, 2 and 4) or 1.5 × 105 (Figs. 3 and 5 and Supplemental figure 1) NHDFs were seeded onto 24-well plates (Figs. 1, 3, 4 and Supplemental figure 1) (Corning) or 24-well temperature-responsive culture plates (Figs. 2 and 5) (Upcell; CellSeed, Tokyo, Japan) in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. Prior to cell seeding, the surface of temperature-responsive dishes was coated with FBS for 2 days at 37 °C in a humidified atmosphere with 5% CO2. On day 0, the assigned number of human iPS cells (Ff-I14) were seeded onto NHDFs and cultured for 1 day at 37 °C. The culture medium was then changed from 10% FBS/DMEM to StemFit AK03N with Y27632 (10 M) (Wako, Tokyo, Japan).
Experiment 1 (Figs. 1 and 2): The culture medium was changed from StemFit AK03N with Y27632 to 10% FBS/DMEM on day 1, and cells were cultured at 37 °C or 42 °C for 2 days until day 3 (Fig. 1A). In some experiments (Figs. 1B and 2), cells were cultured at 37 °C for a further 2 days until day 5.
Experiment 2 (Figs. 3 and 5 and Supplemental figure 1): The culture medium was changed from StemFit AK03N with Y27632 to 10% FBS/DMEM or KA01 medium (Ajinomoto) on day 1, and cells were cultured at 37 °C or 42 °C for 1 day until day 2. In some conditions, dinaciclib (6 nM) was added to the culture medium from day 1 to 2 (Figs. 3 and 5 and Supplemental figure 1). The culture medium was changed to 10% FBS/DMEM on day 2, and cells were cultured at 37 °C for a further 2 days until day 4.
Experiment 3 (Fig. 4): The culture medium was changed from StemFit AK03N with Y27632 to 10% FBS/DMEM or KA01 medium or KA01 medium with methionine {KA01 medium [methionine (+)]} on day 1, and cells were cultured at 37 °C for 1 day until day 2. The final concentration of L-methionine in KA01 medium [methionine (+)] was 100 M. The culture medium was changed to 10% FBS/DMEM on day 2, and cells were cultured at 37 °C for a further 2 days until day 4.

Human iPS cell-derived cardiomyocyte preparation

Human iPS cells (201B7; MHC-puro/Rex1-neo) were used for cardiac differentiation. The differentiation protocol using a bioreactor system (ABLE Co., Tokyo, Japan) has been described previously (6). Cell aggregates on day 15 of cardiac differentiation were dissociated to single cells with trypsin and cultured in 10% FBS/DMEM for 7 days on cell culture plates (Corning). During cultivation on cell culture plates, cardiomyocytes were purified by treatment with 1.5 µg/ml puromycin (Sigma-Aldrich) for 1 day. Movies of spontaneous cardiomyocyte beating were obtained under an inverted microscope (Nikon, Tokyo, Japan) with NIS-Elements software (Nikon). In some experiments (Fig. 6C), cardiomyocytes that were not purified with puromycin were used.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde, and then immunocytochemistry was performed as described previously (9). The following antibodies were used: rabbit polyclonal anti-cardiac troponin T antibody (Abcam, Cambridge, UK) and goat polyclonal anti-Oct3/4 antibody (R&D systems). The secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Nuclei were stained with Hoechst 33258 (Life Technologies). Samples were imaged by ImageXpress (Molecular Device, Sunnyvale, CA, USA) with MetaXpress and AcuityXpress software (Molecular Device).

RNA extraction and quantitative RT-PCR

Total RNA extraction and RT-PCR were performed as described previously (9). Quantitative PCR was performed with a 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Relative mRNA expression levels were calculated using a standard curve of GAPDH mRNA levels. All primers were obtained from Applied Biosystems (LIN28: Hs00702808_s1; GAPDH: Hs00266705_g1; ACTB: Hs99999903_m1).

Cell sheet transplantation

Animal experiments were performed according to the Guidelines of Tokyo Women’s Medical University on Animal Use and consistent with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Women’s Medical University. Monolayered dermal fibroblast sheets with or without iPS cells were collected by lowering the culture temperature on 8 day 4 (Fig. 5) or day 5 (Fig. 2) and transplanted onto subcutaneous tissue of male Fischer 344 athymic nude rats (~6 weeks of age; Charles River Japan, Tokyo, Japan) and then covered with a 0.5 mm-thick silicone membrane (Unique Medical, Tokyo, Japan) to prevent adhesion to the skin as described previously (5). Twelve weeks after transplantation, rats were anaesthetized by 2% isoflurane inhalation, and macroscopic images were recorded using a surgical microscope system (M651 Surgical microscope system; Leica, Germany).

Histology

Tumors were resected, fixed with 4% paraformaldehyde, and routinely processed into 7 μm-thick, paraffin-embedded sections. Hematoxylin and eosin (HE) staining was performed using conventional methods. Images were obtained by optical microscopy (Nikon) with NIS-Elements software (Nikon).

Statistical analysis

Data are presented as dot plots or the mean ± standard deviation. Multiple group comparisons were performed using the Kruskal-Wallis test by Steel-Dwass and Steel procedures for comparison of means. In some experiments (Fig. 6B), multiple group comparisons were performed by one-way analysis of variance followed by the TukeyKramer procedure for comparison of means. Time-to-first-occurrence of events was analyzed using the Kaplan-Meier method with the log-rank test. A value of p < 0.05 was considered as statistically significant. EZR software (12) and EXCEL Toukei ver. 7.0 (ESUMI Co., Ltd, Tokyo, Japan) were used for statistical analyses.

Results

Quantitative evaluation of iPS cell elimination in bioengineered tissues by cultivation at 42 °C

We previously reported that cultivation at 42 °C induces apoptosis of human iPS cells in bioengineered tissues with the time dependent manner and 2 days of cultivation at 42 °C did not affect cardiomyocyte viability (9). However, it remains unclear how cultivation at 42 °C eliminates iPS cells in bioengineered tissues. To address this issue, we co-cultured human iPS cells in the range of 10 to 1 × 104 cells with NHDFs for 1 day in AK03N medium containing Y27632, which is suitable medium for iPS cells, and then cultured these cells for 2 days in 10% FBS/DMEM, suitable medium for fibroblasts, at 37 °C or 42 °C until day 3 (Fig. 1A). Lin28 mRNA expression levels were significantly increased compared with those in fibroblasts without iPS cells when co-cultured with >1 × 102 iPS cells at 37 °C (Fig. 1A, median values: 0; 0, 10; 2.4 × 10-5, 1 × 102; 5.1 × 10-4, 1 × 103; 5.0 × 10-3, 1 × 104; 9.6 × 10-2 ). Conversely, the expression levels of Lin28 mRNA were remarkably increased when cocultured with >1 × 103 iPS cells at 42 °C (Fig. 1A, median values: 0; 5.6 × 10-6, 10; 0, 1 × 102; 1.1 × 10-4, 1 × 103; 1.7 × 10-3, 1 × 104; 2.3 × 10-2), suggesting that 42 °C cultivation might not be able to sufficiently eliminate >1 × 103 iPS cells in bioengineered tissues. Based on these findings, we hypothesized that 42 °C cultivation for 2 days might be sufficient to eliminate 1 × 102 iPS cells in bioengineered tissues and prevent tumor formation upon transplantation. Next, we examined Lin28 expression levels in co-cultures of NHDFs and 1 × 102 iPS cells on day 5, which were cultivated at 37 °C or 42 °C for 2 days (Day 1–3) (Fig. 1B). Lin28 expression levels in co-cultures after cultivation at 37 °C (Day 1–5) were still significantly higher compared with those in fibroblasts without iPS cells, whereas 2 days of cultivation at 42 °C (Day 1–3) maintained low Lin28 expression even after cultivation at 37 °C for 2 days (Day–5) [Fig. 1B, median values: 0; 7.4 × 10-5, 1 × 102 (37 °C to 37 °C); 1.5 × 10-2, 1 × 102 (42 °C to 37 °C); 1.2 × 10-4]. In addition, Lin28 expression levels were not different between co-cultures (42 °C to 37 °C) and fibroblasts without iPS cells. These findings suggest that almost all 1 × 102 iPS cells might be eliminated by 2 days of cultivation at 42 °C, and Lin28 expression levels might not be increased even after cultivation at 37 °C for 2 days.

Cultivation at 42 °C for tumor prevention

Next, we elucidated the effects of 42 °C cultivation on tumor prevention. First, we fabricated monolayered cell sheets using NHDFs after cultivation at 37°C or 42°C for 2 days, and transplanted them onto subcutaneous tissues of nude rats (n = 5). As shown in Fig. 2A and C, no tumor formation was observed in all rats at 3 months after transplantation, suggesting that fibroblast sheets did not have tumorigenicity and 42 °C cultivation might not affect tumorigenicity of fully differentiated cells. When fibroblast 10 sheets co-cultured with 1 × 102 iPS cells at 37 °C were transplanted, tumor formation was observed in one out of 10 rats (Fig. 2B–D, n = 10). However, when fibroblast sheets cocultured with 1 × 102 iPS cells at 42 °C for 2 days were transplanted, tumor formation was not observed in all rats (Fig. 2B and C, n = 10). These findings suggest that 42 °C cultivation for 2 days might sufficiently eliminate 1 × 102 iPS cells in bioengineered tissues and prevent tumor formation upon transplantation.

Development of a suitable combined culture strategy to eliminate 1 × 104 iPS cells in bioengineered tissues

To apply fabricated bioengineered tissues to regenerative medicine, a strategy to eliminate remaining iPS cells is necessary. In the case of fabricating cell sheets using culture dishes of 10-cm in diameter, ~1 × 107 cells are cultured. Because the detection limit of Tra-1 60expressing cells by flow cytometric analysis is 0.1%, there might be 1 × 104 remaining iPS cells among 1 × 107 iPS cells. Therefore, a strategy to eliminate 1 × 104 iPS cells in bioengineered tissues will improve the safety of regenerative medicine. Recently, we reported iPS cell elimination strategies including methionine-free medium and dinaciclib, a CDK1/9 inhibitor and also observed that 2 days of cultivation in methionine free medium and 1 day of cultivation with dinaciclib did not affect cardiomyocyte viability (8, 10). Therefore, we next elucidated which method alone or in combination would sufficiently eliminate 1 × 104 iPS cells in bioengineered tissues. A total of 1 × 104 iPS cells were cocultured with fibroblasts for 1 day in AK03N medium containing Y27632 and then cultured for 1 day (Day 1–2) under the following conditions: 1) 10%FBS/DMEM without dinaciclib at 37 °C [Dina(-) 37°C DMEM], 2) 10% FBS/DMEM with dinaciclib (6 nM) at 37 °C [Dina(+) 37°C DMEM], 3) 10% FBS/DMEM without dinaciclib at 42 °C [Dina(-) 42°C DMEM], 4) 10% FBS/DMEM with dinaciclib (6 nM) at 42 °C [Dina(+) 42°C DMEM], 5) KA01 medium without dinaciclib at 37 °C [Dina(-) 37°C KA01], 6) KA01 medium with dinaciclib (6 nM) at 37 °C [Dina(+) 37°C KA01], 7) KA01 medium without dinaciclib at 42 °C [Dina(-) 42°C KA01], and 8) KA01 medium with dinaciclib (6 nM) at 42 °C [Dina(+) 42°C KA01]. KA01 medium is methionine-free AK03N medium. Cells were cultured for a further 2 days (Day 2–4) in 10% FBS/DMEM at 37 °C, and then Lin28 expression was evaluated (Day 4). As shown in Fig. 3A, the mono-culture strategy including dinaciclib, methionine-free medium, or 42°C cultivation alone failed to prevent the significant increase of Lin28 expression compared with fibroblasts without iPS cells (Fig. 3A, median values: 0; 0, 1 × 104 [Dina(-) 37°C DMEM]; 3.3 × 10-2, 1 × 104 [Dina(+) 37°C DMEM]; 6.3 × 10-3, 1 × 104 [Dina(-) 42°C DMEM]; 4.2 × 10-3, 1 × 104 [Dina(-) 37°C KA01]; 3.1 × 10-2, n = 5–6). However, there were no significant differences in Lin28 expression levels among fibroblasts without iPS cells and cells after combined strategies in the limited number of experiments (Fig. 3A, median values: 1 × 104 [Dina(+) 42°C DMEM]; 1.1 × 10-3, 1 × 104 [Dina(+) 37°C KA01]; 8.2 × 10-4, 1 × 104 [Dina(-) 42°C KA01]; 1.8 × 10-4, 1 × 104 [Dina(+) 42°C KA01]; 8.9 × 10-5, n = 5–6).
Because the median values of Lin28 expression levels under the conditions of Dina(+) 42°C KA01 and Dina(-) 42°C KA01 were the lowest and second lowest respectively, among the examined conditions, we further evaluated Lin28 expression levels under these condition using another 10 samples (Fig. 3B). Cultivation in Dina(+) 42°C KA01 and Dina(-) 42°C KA01 also maintained Lin28 expression at quite low levels compared with that in conventional culture [Dina(-) 37°C DMEM]. Although Lin28 expression under the conditions of Dina(+) 42°C KA01 and Dina(-) 42°C KA01 was quite low and cultivation under the Dina(+) 42°C KA01 condition further decreased Lin28 expression levels compared with Dina(-) 42°C KA01, a significant increase of Lin28 expression levels was still observed under these conditions compared with fibroblasts without iPS cells (Fig. 3B, n=10, median values: 0; 0, 1 × 104 [Dina(+) 42°C KA01]; 3.8 × 10-4, 1 × 104 [Dina(-) 42°C KA01]; 1.3 × 10-3, 1 × 104 [Dina(-) 37°C DMEM]; 5.7 × 10-1). Consistent with the results of Lin28 expression levels, immunocytochemical analysis revealed that almost all of iPS cells were eliminated after the cultivation in Dina(+) 42°C KA01 and Dina(-) 42°C KA01 conditions, but small number of Oct3/4 positive cells (not more than around 10 cells) was remained in some samples (Supplemental figure 1). When 1 × 104 iPS cells were co-cultured with fibroblasts in KA01 medium with methionine at 37 °C, Lin28 expression levels were significantly higher compared with co-cultures in 10% FBS/DMEM or KA01 medium (Fig. 4, n=10, median values: 10% FBS/DMEM; 3.9 × 10-2, KA01 (methionine(+)); 7.6 × 10-2, KA01; 3.1 × 10-3), suggesting that the decrease in Lin28 expression by cultivation in KA01 medium was caused by methionine deficiency.

Tumor prevention of cell sheets with 1 × 104 iPS cells

We next elucidated whether iPS elimination strategies using Dina(+) 42°C KA01 and Dina(-) 42°C KA01 prevented tumor formation upon transplantation of bioengineered tissues containing 1 × 104 iPS cells onto subcutaneous tissues of nude rats (n=5 for each condition). When fibroblast sheets with 1 × 104 iPS cells cultivated in 10% FBS/DMEM without dinaciclib at 37 °C were transplanted, one rat died of unknown causes at 18 days after transplantation and tumor formation was observed in four rats within 2 months (Fig. 5A–C). However, no tumors were observed in all rats that were transplanted with cell sheets containing 1 × 104 iPS cells after cultivation in Dina(+) 42°C KA01 and Dina(-) 42°C KA01 at 3 months (Fig. 5A and B). These findings suggest that the combined strategy by cultivation in methionine-free medium at 42 °C might sufficiently eliminate remaining robust iPS cells in bioengineered tissues to prevent tumor formation, and that additional treatment with dinaciclib might be applicable to further improve safety.

Influence of combined iPS elimination strategies on human iPS cell-derived cardiomyocytes

Finally, we evaluated the influence of the combined iPS elimination strategies on human iPS cell-derived cardiomyocytes. When iPS cell-derived cardiomyocytes that were purified with puromycin were cultivated under Dina(+) 42°C KA01 and Dina(-) 42°C KA01 conditions for 1 day, spontaneous beating was clearly observed (Supplementary video 1–3) and the number of cardiac troponin T-positive cells was not different compared with that under the conventional culture condition [Dina(-) 37°C DMEM] (Fig. 6A and B). When iPS cellderived cardiomyocytes that were not purified with puromycin were cultured in 10% FBS DMEM without dinaciclib at 37°C, Lin28 expression levels were variable among samples and were classified to high expression samples (Lin28 expression levels > 0.004) and low expression samples (Lin28 expression levels < 0.002). However, after the cultivation in Dina(+) 42°C KA01 and Dina(-) 42°C KA01 conditions, high Lin28 expression samples were not observed (Fig. 6C, n=5, median values: [Dina(-) 37°C DMEM]; 1.9 × 10-3, [Dina(-) 42°C KA01]; 1.1 × 10-3, [Dina(+) 42°C KA01]; 8.0 × 10-4). These results suggested that the combined culture strategies with methionine-free medium with or without dinaciclib at 42 °C might be useful to eliminate remaining iPS cells in the process of cardiac tissue production.

Discussion

In the present study, we showed that 42 °C cultivation sufficiently eliminated 1 × 102 iPS cells in bioengineered tissues and prevented tumor formation upon transplantation. Furthermore, the combined culture strategies of methionine-free culture medium at 42 °C eliminated 1 × 104 iPS cells in bioengineered tissues and prevented tumor formation without affecting cardiomyocyte viability.
Various types of methods have been reported to eliminate iPS cells from differentiated cells (13-16). However, the strategy in the present study might have some advantages compared with those in previous reports. Many studies have used small molecules or antibodies to eliminate iPS cells. Because residual substances derived from materials used in the production process of regenerative medicine products are one of the concerns for their applications in clinical settings, a strategy that does not add materials to the production process is desirable. Methionine depletion from standard medium for iPS cell culture and 42 °C cultivation do not increase concerns of residual substances in final products, and the combination of these strategies might be useful to manufacture regenerative medicine products. We used bioengineered tissues for transplantation, while almost all previous studies have transplanted cells as a single cell injection. Tissue engineering technologies are widely used for regenerative medicine to better engraft desired cells compared with single cell injection (17). Human PSCs are well known to die easily as single cells (7). Furthermore, we previously reported that cultivation at 42 °C or with dinaciclib eliminates almost all human iPS cells cultured under a feeder-less condition (9, 10), while some iPS cells remained when iPS cells were co-cultured with fibroblasts at 42 °C or with dinaciclib (9, 10). These findings suggest that co-cultivation with differentiated cells in bioengineered tissues might promote iPS cell survival. Therefore, transplantation of cells including iPS cells as a single cell suspension might underestimate the risk of tumorigenicity, and the combined culture method with methionine-free culture medium at 42 °C might be more useful for iPS cell elimination and tumor prevention.
Notwithstanding cell sheet-based cardiac tissues (2, 18), several types of cardiac microtissues including cardiac organoids and bioengineered human myocardium have been reported (19-22). As these cardiac microtissues are also composed of several types of cells including cardiomyocytes and mesenchymal cells, and the contamination of undifferentiated cells is the common issue for the risk of tumorigenicity, the culture strategies shown in the present study might be applicable for many types of cardiac microtissues.
To estimate remaining PSCs in the regenerative medicine final products is quite important to evaluate the risk of tumor formation. There are several methods to estimate remaining PSCs including flow cytometric analysis and Lin28 expression analysis (23, 24). However in the case of flow cytometric analysis for bioengineered tissues, it is necessary to dissociate tissues to single cells, which might affect cell viability and lead to underestimate the remaining PSCs. Therefore, Lin28 expression analysis might be useful to estimate remaining PSCs in bioengineered tissues. As shown in figure 6C, Lin28 expression levels in iPS cell-derived cardiomyocytes were low in principle, but relatively higher Lin28 expression levels were also observed in some samples. These findings indicate that Lin28 expression analysis might be useful to screen the samples containing some amount of remaining iPS cells. On the other hand, since the combined culture conditions attenuated the occasional increase of Lin28 expression levels in iPS cell-derived cardiomyocytes, these strategies might further reduce the risk of tumorigenicity.
The influence of an iPS cell elimination strategy on differentiated cell viability and tissue fabrication is a concern. We previously reported that methionine-free medium, 42 °C cultivation, and dinaciclib do not affect cell sheet fabrication or cardiomyocyte viability (810). In accordance with the results from each strategy, the combination of iPS cell elimination strategies including methionine-free medium at 42 °C cultivation with or without dinaciclib did not affect cell sheet fabrication or cardiomyocyte viability. The mechanisms of iPS cell apoptosis are different among these strategies. Methionine-free medium has been reported to activate the p53-p38 signaling pathway through decreased levels of s-adenosyl methionine (25). Cultivation at 42 °C upregulates TRPV-1 expression, leading to iPS cell apoptosis and higher expression levels TRPV-1 in iPS cells compared with cardiomyocytes, which might account for their high sensitivity to 42 °C (9). A low concentration of dinaciclib induces iPS cell apoptosis through CDK-1-mediated MCL-1 degradation, whereas a high concentration of dinaciclib suppresses MCL-1 transcription through CDK-9 inhibition (10). On the other hand, MCL-1 expression in cardiomyocytes was remained even after treatment with dinaciclib. These multiple mechanisms might synergistically and efficiently induce iPS cell apoptosis with minimum influence on cardiomyocyte viability and cell sheet fabrication.
Several groups including our own have previously reported that fibroblasts are indispensable to fabricate functional bioengineered cardiac tissues, and fibroblasts are wildly used to generate bioengineered cardiac tissues (2, 18, 26, 27). Although various types of fibroblasts are available, we used NHDFs to fabricate cell sheets. One of the merits of using stromal cells for bioengineered tissue fabrication is tissue vascularization upon transplantation (28), which leads to better engraftment. However, angiogenic functions are different among the tissue origins of fibroblasts. Recently, we reported that human cardiac fibroblasts inhibit endothelial cell network formation in co-culture by highly expressing LYPD1, an angiogenesis inhibitory factor, whereas NHDFs with low LYPD1 expression promote endothelial cell network formation (29). Therefore, transplantation of iPS cells with dermal fibroblasts as cell sheets might be suitable for engraftment of iPS cells and evaluating tumorigenicity.
There are some limitations in this study. We used only one iPS cell line to evaluate the iPS cell elimination efficacy (Figs. 1–5). Although iPS cell elimination by methionine-free medium, 42 °C cultivation, and dinaciclib has already been shown in various human iPS cell lines (8-10), further optimization will be necessary according to the cell line used in the clinical setting. We used nude rats to evaluate tumor formation. Although nude rats are useful for transplantation of a certain number of cells and size of tissues, more immunedeficient animals such as NOG mice will be necessary to evaluate tumor-preventive effects for clinical application.
The combined cultivation strategy with methionine-free medium at 42 °C sufficiently eliminated iPS cells in the bioengineered tissues without affecting the viability of 16 cardiomyocytes. Further evidence of tumor prevention using iPS cell elimination strategies including our method will promote the development of regenerative medicine products using iPS cells.

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