ROCK INHIBITOR Y-27632 USED DURING CELL AGGREGATE FORMATION ADVERSELY AFFECTS THE EARLY CARDIAC DIFFERENTIATION OF HUMAN iPS CELLS

Abstract

Rho-associated protein kinase (ROCK) inhibitor, Y-27632, is an indispensable chemical molecule to maintain the viability of single-dissociated human induced pluripotent stem cells (hiPSCs) and to form cell aggregates in floating cultures. In this study, we investigated the effect of Y-27632 on the cardiac differentiation of the cell aggregates of hiPS cell line, 201B7. When Y-27632 was not added to the floating culture, the dissociated hiPSCs died and no cell aggregates were formed. The presence of greater than 10 μM Y-27632 was required to form spherical cell aggregates from the dissociated hiPSCs. However, Y-27632 used in the floating culture of the dissociated hiPSCs to form cell aggregates exhibited an inhibitory effect on cardiac differentiation in the adherent culture of cell aggregates. When 30 μM Y-27632 was added to the floating cultures, the extensibility of outgrowth from the cell aggregates was relatively lowered, and the initial time of contraction (the generation of beating cardiomyocytes) was markedly delayed in the adherent cultures. Moreover, the expression levels of the early cardiac differentiation-related genes of NKX2.5 and TNNT2 were decreased with increasing Y-27632 concentration. These results indicate that Y-27632 applied to the floating cultures for cell aggregate formation adversely affected the early cardiac differentiation in the following adherent cultures, although there was no influence on the final cardiac differentiation levels.

Introduction

Rho-associated protein kinases (ROCKs) are involved in many aspects of cell behavior, such as cell adhesion (both cell-substrate and cell-cell), cell motility, cell proliferation, and apoptosis¹⁾. The ROCK inhibitor Y-27632 has been used to reduce dissociation-induced apoptosis in human pluripotent stem cells (hPSCs), such as human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs)^2,3,4,5,6). Treatment with Y-27632 is considered to be essential in the preparation of singularized hPSC cell suspensions, by enzymatically dissociating their colonies. The presence of Y-27632 was found to be effective to maintain the viability of single-dissociated hPSCs and to agglomerate the dissociated hPSCs into cell aggregates, referred to as embryoid bodies (EBs)⁵⁾. Cell aggregate formation is a key stage in the differentiation process of hPSCs, as the quality of EBs affects the efficiency of differentiation.

hiPSCs provide a powerful tool in cell therapy, drug development, and pathology research because of their ability for self-renewal and pluripotency⁷⁾. Therefore, it is essential to overcome the dissociated-induced apoptosis in hiPSCs and to establish reproducible and efficient hiPSC differentiation protocols. Y-27632 is frequently used in the floating culture to form cell aggregates from dissociated hiPSCs in various differentiation processes, such as for cardiac differentiation⁸⁾ and retinal progenitor derivation⁹⁾.

On the other hand, it is of concern that the continuous presence of Y-27632 during cell aggregate formation may affect the subsequent differentiation of hiPSCs. Since ROCKs play a role in several aspects of cell behavior, the inhibition of ROCKs with Y-27632 may consequently have some effect on cell differentiation. It was reported that ectodermal differentiation was inhibited by the application of Y-27632 to hiPSCs during the pre-culture period, owing to a disruption of actin and E-cadherin organization¹⁰⁾. Eyckmans et al. observed that the inhibition of cytoskeletal contractility with Y-27632 enhanced adipogenic differentiation and discouraged osteogenic differentiation of human periosteum-derived cells¹¹⁾. Furthermore, Sivasubramaiyan et al. reported that Y-27632 reduced trophoblast differentiation in early noncystic EBs, while it enhanced the induction of trophoblasts from late cystic EBs¹²⁾.

Human cardiomyocytes hold great promise for cardiovascular research, cardiotoxicity drug testing, and clinical applications, and a number of protocols for the production of cardiomyocytes from hPSCs have been reported^13,14). Because various concentrations of Y-27632 have been used in the cardiac differentiation process^8,15,16), the effect of Y-27632 at various concentrations on cardiac differentiation should be determined. As for mouse iPS cells, it was reported that Y-27632 promoted the cardiac differentiation¹⁷⁾. However, it is still unclear how Y-27632 affects cardiac differentiation of hiPSCs.

In our previous research, we have revealed the effect of Y-27632 on the formation of cell aggregates of hiPSCs in the floating culture⁵⁾. In this study, we investigated the effect of Y-27632 on the cardiac differentiation of the cell aggregates of hiPS cell line, 201B7.

Materials and Methods

Maintenance culture of hiPSCs

Human induced pluripotent stem cell line of 201B7 was provided by the RIKEN BRC through the Project for Realization of Regenerative Medicine and the National Bio-Resource Project of the MEXT, Japan⁷⁾. hiPSCs were routinely maintained on mitomycin C-inactivated SNL76/7 feeder cells (ECACC 07032801, DS Pharma Biomedical Co., Osaka, Japan) in medium consisting of DMEM-Ham’s F-12 basal medium (Nacalai Tesque, Kyoto, Japan) supplemented with 20% knockout serum replacement (KSR, Gibco, Grand Island, NY, USA), 1% GLUTAMAX^TM supplement (Gibco), 0.1 mM non-essential amino acids (Life technologies), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO, USA), 25 units/ml penicillin and 25 μg/ml streptomycin (Gibco), and 4 ng/ml basic fibroblast growth factor (bFGF, KATAYAMA CHEMICAL INDUSTRIES Co., Ltd. ​Osaka, Japan). The cells adhered and grew to form colonies at 37°C in a humidified 3% CO₂ atmosphere. The medium was changed daily.

Feeder-free culture of hiPSCs

hiPSC colonies on the feeder cells were dissociated as described previously⁵⁾. Y-27632 (ROCK inhibitor, Nacalai Tesque) was used to reduce dissociation-induced apoptosis. Collagenase-trypsin-KSR (CTK) solution was used to remove the feeder cells. hiPSC colonies were enzymatically dissociated by Accutase^TM solution (Innovative Cell Technologies, San Diego, CA, USA) and broken into single cells by gently pipetting. The dissociated cells were centrifuged, and the resulting pellet was resuspended with serum-free medium containing 10 μM Y-27632. The serum-free medium used was PluriSTEM^TM Human ES/iPS Medium (Merck Millipore, Billerica, MA, USA), and is referred to herein as feeder-free medium. The cell suspension was prepared at a cell density of 1.0 × 10⁵ cells/ml and was seeded onto vitronection (Life technologies)-coated (0.5 μg/cm²) cell culture plates at 1.0 × 10⁴ cells/cm² as the initial cell density. Cultivation was performed at 37°C in a humidified 5% CO₂ atmosphere. After 24 h incubation, the medium was replaced with fresh feeder-free medium without Y-27632. After three passages in feeder-free medium, hiPSC colonies were dissociated with Accutase^TM solution and gently pipetted into single hiPSCs. Singularized hiPSCs were used for the floating culture to form cell aggregates.

Floating culture of dissociated hiPSCs to form cell aggregates

The singularized hiPSCs in feeder-free medium containing various concentrations (0, 2, 10, 20, and 30 μM) of Y-27632 were seeded at a cell density of 3000 cells/200 μl into each well of a low-adherence 96-well round-bottomed plate (Lipidure-coat plate A-U96 (NOF Co., Tokyo, Japan)), and incubated at 37°C in a humidified 5% CO₂ atmosphere. The floating culture was carried out for 8 days to form cell aggregates. On day 4, feeder-free medium was replaced with Medium A containing the same concentration of Y-27632 as in the previous conditions. On day 6, Medium A was replaced with Medium B containing the same concentration of Y-27632 as in the previous conditions. Medium A and Medium B were obtained as part of the PSC Cardiomyocyte Differentiation Kit (Gibco). During floating culture for 8 days, phase-contrast images of cell aggregates were periodically captured from 8 randomly selected wells by a microscope digital camera (DP21, Olympus, Tokyo, Japan), and the medium in each well was replaced daily with the same medium. A schematic of the workflow of the cardiac differentiation process, consisting of floating culture and adherent culture, is shown in Fig. 1. The floating culture for cell aggregate formation was performed in the presence of Y-27632, and the adherent culture for cardiac differentiation was performed without Y-27632.

Fig. 1.

Schematic workflow of the cardiac differentiation process consisting of floating culture and adherent culture of cell aggregates.

Adherent culture of cell aggregates for cardiomyocyte differentiation

After 8 days of floating culture, resulted cell aggregates (24 aggregates) were transferred to a 0.1% gelatin-coated 24-well plate and adherent culture was performed in Maintenace Medium without Y-27632 for another 16 days. Each of the wells contained one cell aggregate. Maintenance Medium was provided as part of the PSC Cardiomyocyte Differentiation Kit (Gibco). The generation of a rhythmically contracting focal area was microscopically determined in the outgrowth from the adherent cell aggregate in each well.

RNA extraction and qRT-PCR analysis

Total RNA extraction and cDNA synthesis were performed using NucleoSpin RNA (Takarabio, Otsu, Japan) and PrimeScript II 1st strand cDNA Synthesis kits (Takarabio) following the manufacturer’s instructions. The reaction and analysis for qRT-PCR were performed on a Thermal Cycler Dice (Takarabio) using SYBR Premix EX Taq II (Takarabio). The primers used for qRT-PCR were as follows: TATA box binding protein (TBP), 5′-GCTGGCCCATAGTGATCTTT-3′ sense and 5′-CTTCACACGCCAAGAAACAGT-3′ antisense; Homo sapiens NK2 homeobox 5 (NKX2.5), 5′-CCAAGGACCCTAGAGCCGAAA-3′ sense and 5′-ACCCTGGTGAGGGAGACAGA-3′ antisense; troponin T type 2 (TNNT2), 5′-GAGCTGTGGCAGAGCATCTAT-3′ sense and 5′-ATCCTGTTTCGGAGAACATTG-3′ antisense; Homo sapiens myosin, heavy chain 6, cardiac muscle, alpha (MYH6), 5′-CGCTGAGTCCCAGGTCAACA-3′ sense and 5′-TTACAGGTTGGCAAGAGTGAGGTTC-3′ antisense; myosin, light chain 2 (MYL2), 5′-GCAGGCGGAGAGGTTTTC-3′ sense and 5′-AGTTGCCAGTCACGTCAGG-3′ antisense; homo sapiens SRY (sex determining region Y)-box 17 (SOX17), 5′-CTGCAGGCCAGAAGCAGTGTTA-3′ sense and 5′-CCCAAACTGTTCAAGTGGCAGA-3′ antisense; homo sapiens alpha-fetoprotein (AFP); 5′-CAGCCACTTGTTGCCAACTCA-3′ sense and 5′-GGACATATGTTTCATCCACCACCA-3′ antisense; homo sapiens SRY (sex determining region Y)-box 1 (SOX1), 5′-CAGCAGTGTCGCTCCAATTCA-3′ sense and 5′-GCCAAGCACCGAATTCACAG-3′ antisense; Homo sapiens paired box 6 (PAX6), 5′-AATTGATTGCAGAGTGTCGCTTC-3′ sense and 5′-GCTCAGGTGCTCGGGTTCTAA-3′ antisense; homo sapiens POU class 5 homeobox 1 (OCT3/4), 5′-TGAAGCTGGAGAAGGAGAAGCTG-3′ sense and 5′-GCAGATGGTCGTTTGGCTGA-3′ antisense; homo sapiens Nanog homeobox (NANOG), 5′-TCCAACATCCTGAACCTCAGCTA-3′ sense and 5′-AGTCGGGTTCACCAGGCATC-3′ antisense; homo sapiens lin-28 homolog A (LIN28A), 5′-GCGGGCATCTGTAAGTGGTTCA-3′ sense and 5′-TCCATGTGCAGCTTACTCTGGTG-3′ antisense. qRT-PCR was carried out for 50 cycles, with the denaturation steps conducted at 95°C for 5 s. Annealing and elongation was carried out at 60°C for 30 s. Relative gene expressions were calculated using the comparative Ct method after normalization to an endogenous control (TBP).

Statistical analysis

The Jonckheere-Terpstra test was used to determine if there was a statistically significant trend in Y-27632 concentration dependency. Statistical analysis of the Jonckheere-Terpstra test was performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria)¹⁸⁾. All results were presented as mean ± standard deviation (SD).

Results

Effect of Y-27632 concentration on cell aggregation from dissociated hiPSCs

Single-dissociated hiPSCs were seeded into a low-cell-binding 96-well plate at 3000 cells/well and floating culture was carried out for 8 days to form cell aggregates under various Y-27632 concentrations. As shown in Fig. 2, singularized hiPSCs clumped together and formed cell aggregates in the presence of Y-27632. In Y-27632 concentrations of 10–30 μM, hiPSCs similarly cohered and formed spherical cell aggregates by day 4. The cell aggregates increased in size until day 8. In the presence of 2 μM Y-27632, however, the cohesion of cells was insufficient and amorphous cell clusters were formed. In the presence of 10 μM Y-27632, cavitation occurred and a cystic structure was observed in cell aggregates on day 8, as indicated by arrows in Fig. 2-M. When the Y-27632 concentration was increased to 20 μM or 30 μM, cavitation did not occur. In the presence of 30 μM of Y-27632, the amount of debris that appeared out of cell aggregates markedly increased. When the floating culture was performed without Y-27632, no cell aggregate was formed (Fig. 2-A, F, K).

Fig. 2.

Effect of Y-27632 concentration on cell aggregation of dissociated hiPSCs in floating culture in a 96-well plate. Scale bar is 300 µm.

Appearance of outgrowth in adherent cell aggregates formed under various Y-27632 concentrations

After 8 days of floating culture under various Y-27632 concentrations, resulted cell aggregates were transferred to adherent culture without Y-27632. Figure 3 shows the appearance of outgrowth from adhered cell aggregates on day 24 of the total duration of culture (day 16 of adherent culture time). The photo image of outgrowth related to 0 μM Y-27632 was not shown because no cell aggregate was formed in the floating culture without Y-27632. As shown in Fig. 3, well-extended outgrowth was observed in the adherent cultures of the cell aggregates formed under 2, 10, and 20 μM Y-27632. The extensibility of outgrowth from the cell aggregates formed under 30 μM Y-27632 was relatively low.

Fig. 3.

Extension of outgrowth from cell aggregates in adherent culture, which were formed under varying Y-27632 concentrations in floating culture. Scale bar is 300 µm.

Generation of contracting focal area in the outgrowth of cell aggregates formed under various Y-27632 concentrations

Figure 4 shows the percent change in the generation of contracting focal area in the outgrowth of cell aggregates. The percentage was calculated as the ratio of the number of wells in which contracting focal area was microscopically observed to the number of wells in which adherent culture was performed. In the early stages of adherent culture of the cell aggregates, from day 10 to day 12 of the total duration of culture, the percentage of contraction was lowered in the concentration-dependent manner of Y-27632 used during floating culture as shown in Fig. 4. There was a statistically significant trend in Y-27632 concentration dependency from day 10 to day 12 of the total duration of culture (Jonckheere-Terpstra test, * P < 0.05). The time at which contraction was first observed was markedly delayed when cell aggregates were formed using 30 μM Y-27632. In the latter stages of adherent cultures, there was no statistically significant difference in the percentage of contraction. The final percentage of contraction reached a plateau of 80–100% regardless of Y-27632 concentration.

Fig. 4.

Time course changes in the generation of contracting focal area in the outgrowth from the adherent cell aggregates. The percentage of contraction represents the ratio of the wells in which contracting focal area was observed to the total wells. * P < 0.05, significance calculated by Jonckheere-Terpstra test.

Effect of Y-27632 concentration on the expression levels of cardiac differentiation-related genes

The expression levels of NKX2.5 and TNNT2, early cardiac differentiation-related genes, were examined in the adherent cultures on day 8. As shown in Fig. 5-A, the gene expression levels of NKX2.5 and TNNT2 were decreased with increasing Y-27632 concentration. In particular, Y-27632 concentration-dependent decrease in the gene expression of TNNT2 was statistically significant (Jonckheere-Terpstra test, * P < 0.05).

Fig. 5.

Expression of cardiac differentiation-related genes in the adherent culture of cell aggregates. Cell aggregates were formed under varying Y-27632 concentrations in floating culture. (A) Expression levels of NKX2.5 and TNNT2 on day 8. (B) The expression levels of MHY6 and MYL2 on days 8 and 24. Error bars represent SD. * P < 0.05, significance calculated by Jonckheere-Terpstra test.

The expression levels of MHY6 and MYL2, cardiac muscle-related genes as matured cardiac markers, were examined in the adherent cultures on days 8 and 24. As shown in Fig. 5-B, the gene expression levels of MHY6 and MYL2 were not influenced by Y-27632 concentration. There was a marked increase in the expression levels of MHY6 and MYL2 during the period from day 8 to day 24.

Effect of Y-27632 concentration on the expression levels of pluripotency, endoderm, and ectoderm-related genes

Figure 6-A and -B show the expression levels of endodermal and ectodermal differentiation-related genes in the adherent cultures on day 8, respectively. 30 μM Y-27632 suppressed the expression levels of all of examined genes, SOX17, AFP, SOX1, and PAX6. The gene expression levels of AFP and SOX1 were decreased with increasing Y-27632 concentration in the statistically significant manner (Jonckheere-Terpstra test, * P < 0.05).

Fig. 6.

Expression of pluripotency, endoderm, and ectoderm-related genes in the adherent culture of cell aggregates. Cell aggregates were formed under varying Y-27632 concentration in floating culture. (A) The expression levels of SOX17 and AFP on day 8. (B) The expression levels of SOX1 and PAX6 on day 8. (C) The expression levels of OCT 3/4, NANOG, and LIN28 on days 0, 8, and 24. Error bars represent SD. * P < 0.05, significance calculated by Jonckheere-Terpstra test.

Figure 6-C shows the expression levels of pluripotecy-related genes in the adherent cultures on days 0, 8, and 24. The gene expression levels of OCT3/4, NANOG, and LIN28, were decreased in the time-dependent manner, regardless of Y-27632 concentration.

Discussion

The presence of Y-27632 was essential for dissociated hiPSCs to form cell aggregates in the floating cultures. When no Y-27632 was added to floating culture, no cell aggregate was formed. This result could be attributed to dissociated-induced apoptosis¹⁹⁾. The concentration of Y-27632 affected the morphological aspect of cell aggregates as shown in Fig. 2. The cohesiveness and sphericity of aggregates were insufficient in the presence of 2 μM Y-27632. The presence of more than 10 μM Y-27632 was required to form spherical cell aggregates. In most of the previous papers on differentiation induction with cell aggregate formation, 10 or 20 μM Y-27632 was added to the medium^3,9,12,20). Since Y-27632 is involved in cell-cell adhesion¹⁾ and cell clustering¹¹⁾, 10 or 20 μM Y-27632 was considered to promote the cell aggregation.

As can be seen in Fig. 3, the extension area of outgrowth from a cell aggregate in the adherent cultures was decreased when 30 μM of Y-27632 was used in the floating culture. This result implies that the Y-27632 concentration used during the floating culture for cell aggregate formation affected the extensibility of outgrowth from cell aggregates in the adherent culture. In other words, Y-27632 used in the previous stage continuously affected the cell behavior of hiPSCs in the next stage of culture. As can be seen in Fig. 2, however, the increase in size of cell aggregates was observed even in the presence of 30 μM Y-27632, which was similar to that in the presence of 20 μM Y-27632. This indicates that 30 μM Y-27632 was not a crucial inhibitory factor in cell aggregate formation. Eyckmans et al. have noted that Y-27632 was not toxic to human periosteum-derived cells¹¹⁾.

For cardiac differentiation, Y-27632 played an inhibitory role in the adherent cultures of cell aggregates. When 30 μM of Y-27632 was added to the floating cultures, the initial time of contraction was markedly delayed in the adherent cultures (Fig. 4). The gene expression levels of NKX2.5 and TNNT2 were decreased in the concentration-dependent manner of Y-27632 in the early period of cardiac differentiation on day 8. These results indicate that Y-27632 applied to the floating cultures for cell aggregate formation adversely affected the early cardiac differentiation in the following adherent cultures. Zhao et al. have reported that inhibition of ROCKs reduced the expression of cardiac proteins and the formation of cardiomyocytes in adipose-derived stromal cells²¹⁾. ROCKs play a critical role in the regulation of cellular functions including cell migration, actin cytoskeletal organization, and stress fiber contraction. Therefore, inhibition of ROCKs will loosen the stress fibers in the cytoplasm, and may eventually reduce the generation of contracting focal area in the outgrowths of cell aggregates. For endodermal and ectodermal differentiation, the gene expression levels of AFP and SOX1 were decreased in the concentration-dependent manner of Y-27632 in the early period of adherent cultures on day 8 (Fig. 6-A, B). However, the inhibitory effect of Y-27632 was not decisive in hiPSCs. As shown in Figs. 4 and 5-B, the inhibitory effect of Y-27632 was gradually diminished by replacing the medium with fresh one without Y-27632 in the adherent cultures. In the latter period of cardiac differentiation on day 24, there was no inhibitory effect of Y-27632 on the generation of contracting focal area and the expression levels of cardiac muscle-related genes. The expression levels of pluripotency-related genes were sufficiently decreased until day 24, regardless of Y-27632 concentration (Fig. 6-C).

Y-27632 is an indispensable chemical molecule to maintain the viability of single-dissociated hiPSCs and to form cell aggregates. Therefore, when using a high concentration of Y-27632 in the differentiation process of hiPSCs, it will be required to minimize the adverse effects of Y-27632.

Acknowledgments

This was supported by JSPS KAKENHI Grant Number JP26420794.

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