Human pluripotent stem cell lines
The hESC lines H9 (WiCell, Madison, WI, USA) and KhES-1 (RIKEN, Tsukuba, Japan), and hiPSC lines 253G1 (RIKEN) and 201B7 (RIKEN) were maintained using the standard enzymatic bulk expansion method. All cells were maintained in E8 medium (TeSR-E8, Stem Cell Technologies or Essential-8, Thermo Fisher Scientific) with laminin 511-E8 (iMatrix-E8, Nippi, Japan) substrate and hypertonic sodium citrate passage solution as previously described34. All cells were confirmed to be negative for mycoplasma contamination every three months with the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland). All experiments using human cells were conducted under the approval of the Institutional Ethics Committee, Kyoto University, Japan. The hESC lines were used in accordance with the Guidelines for the Derivation and Utilization of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Synthesis of CPB scaffold
Polyvinyl alcohol (PVA), Poly[poly (ethylene glycol) methacrylate] (PEGMA) and Polyvinyl butyral (PVB) was provided by Sekisui Chemical Co., Ltd. (Tokyo, Japan). Poly (N-isopropylacrylamide) (PNIPAM) were purchased from Sigma Aldrich (St. Louis, MO). Polyvinyl butyral was synthesized using a slight modification of previously reported procedures47. Acrylic acid-grafted polyvinyl butyral was synthesized by general radical polymerization with polyvinyl butyral and acrylic acid using t-butyl peroxy-2-ethylhexanoate in butanol. Acrylic acid-grafted PVB was cast-coated by a micropipette on a standard tissue culturing polystyrene (TCPS, Corning® Costar® TC-Treated Multiple Well Plates (12-well), Corning Incorporated, New York, USA). Then, peptide-grafted polyvinyl butyral was synthesized by a condensation reaction with acrylic acid-grafted PVB and general RGD motif peptide using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-Hydroxy-7-azabenzotriazole in water at 40 °C overnight. The pre-coated TCPS was vacuum-dried at 60 °C overnight.
Preparation of polyvinyl butyral-based polymer coated cell culturing plate
Typically, Acrylic acid-grafted PVB powder was dissolved in butanol at 60 °C for 10 h. The PVB concentrations were 1.0 wt%. PVB in butanol was cast-coated by a micropipette on a standard TCPS and dried in a vacuum overnight at 60 °C, then sterilized using a UV irradiator. After RGD motif peptide was condensed to PVB-coated surface, hPSCs were seeded onto these dishes. As conventional polymers, PVA, PNIPAM, and PEGMA were dissolved in methanol, butanol and/or water, and were coated by the same procedure.
Characterization of polymer coated surface
The hydrophilicity of the polymer surface was analyzed by the water contact angle using the sessile drop method (DMo-901, Kyowa Interface Science Co., Ltd., Saitama, Japan).
Heat-resistant analysis of CPB scaffold coated tissue culturing polystyrene
CPB scaffold-coated TCPS were incubated in an oven at 25 °C and 60 °C for 1 month. After sterilization by a UV irradiator, these dishes were used for hiPSC culture. As a positive control, CPB scaffold-coated TCPS were directly used for cell culture after the preparation process. After culturing for 5 days, cell proliferation was evaluated using a Cell Counting Kit (CCK-8 kit, Dojindo Laboratories Inc., Kumamoto, Japan) and a microplate reader.
Detachment of human induced pluripotent stem cells
hiPSC colonies on PVB-based polymer-coated TCPS were washed with PBS and immersed in EDTA solution (0.5 mM, in PBS) for 5 min at 25 °C. After the EDTA solution was removed using medium flush (1 mL), pipetting was performed for five cycles. The obtained cells were counted as Cell Count 1 by Image Cytometer (NC3000, Chemometec A/S, Allerod, Denmark). Then, PVB-based polymer-coated TCPS was re-immersed in fresh EDTA solution (0.5 mM, in PBS) for 5 min at 37 °C in order to salvage the remaining cells. These salvaged cells were counted as Cell Count 2.
Detachment ratio defined as
$$mathrm{Detachment,ratio },(mathrm{%})=frac{Cell,count 1}{Cell,count 1+Cell,count 2}times 100$$
Cell culture on peptide-polyvinyl butyral-based polymer
For the evaluation of CPB scaffold in comparison with other culture substrates, the dissociated cells were suspended in Essential-8 medium and seeded on 12-well plates coated with Vitronectin-N terminal fragment (VTN-N, Life Technologies), vitronectin (Vitronectin XF, Stem Cell Technologies), Synthemax (Synthemax II-SC Substrate, Corning, Corning, NY, USA), Laminin-511 E8 fragment (Laminn-E8, iMatrix, Nippi, Tokyo, Japan), and Laminin-511 (Laminin-5, ReproCell, Kanagawa, Japan). After 5 days of culture, cells were dissociated with 0.5 mM EDTA/PBS and the cell number was counted. For the evaluation of CPB scaffold with various culture media, the cells were dissociated with hypertonic sodium citrate solution as previously described, suspended with Essential-8 (Thermo Fisher Scientific), StemFit AK02 (Ajinomoto, Japan), StemFlex (Thermo Fisher Scientific) or mTeSR1 (Stem Cell Technologies) medium supplemented with ROCK inhibitor (10 μM, Y-27632), and seeded on CPB scaffold coated 12-well plates at a concentration of 1.25 × 104 cells/well. The ROCK inhibitor was removed 24 h after seeding, and the medium was changed daily.
For long-term culture, the cells were maintained in Essential-8 medium on CPB scaffold-coated 12-well plates. VTN-N-coated plates were used as a control/reference. The medium was changed daily, and the cells were passaged with hypertonic sodium citrate solution every 3–4 days.
Characterization of human pluripotent stem cells maintained on CPB scaffold34
Cell confluency (%) was calculated using IncuCyte® software by phase contrast images. Phase contrast imaging was performed using the IncuCyte® S3 Live-Cell Analysis System (Essen Bioscience, Ann Arbor, MI, USA). Cells were scanned approximately every 24 h from 1 to 5 days post seeding.
To assess pluripotency, hPSCs were fixed with 4% paraformaldehyde for 10 min, and ALP activity was determined with a Vector Blue Alkaline Phosphatase Substrate Kit. For immunostaining, the fixed cells were permeabilized with 0.2% Triton X-100 in PBS, and then incubated with primary antibodies against OCT4 (clone C10, Santa Cruz), SOX2 (Y17, Santa Cruz), NANOG (D73G4, Cell Signaling Technology), SSEA-3 (clone 631, Santa Cruz), SSEA-4 (clone MC813, Santa Cruz), TRA-1-60 (sc-21705, Santa Cruz), and TRA-1-81 (sc-21706, Santa Cruz), followed by Dylight 488-conjugated (Jackson Labs, Bar Harbor, ME, USA), Alexa Fluor 488-conjugated (Life Technologies), or 594-conjugated (Life Technologies) secondary antibodies. Marker expression was visualized by fluorescence microscopy (BZ-X800, Keyence, Osaka, Japan).
To examine the marker-positive population, the cells were dissociated with 0.05% TrypLE (Life Technologies), fixed with 2% paraformaldehyde, and immunostained with SSEA-4 and TRA-1-60 followed by Alexa Fluor 488-conjugated antibodies (Life Technologies). The cells were then analyzed by flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA, USA). To normalize and overlay the charts, the number of cells in each bin (the numerical ranges for the parameter on the fluorescence intensity in the X-axis) were divided by the number of cells in the bin that contained the largest number of cells in FlowJo software (TreeStar, Ashland, OR, USA).
For karyotype analysis, hPSCs were incubated in colcemid (KaryoMAX, Life Technologies), dissociated with trypsin/EDTA, treated with a hypotonic solution, and then fixed with Carnoy’s fixative solution. The G-banding karyotype of 50 randomly selected mitotic metaphase nuclei was analyzed by Nihon Gene Research Laboratories (Sendai, Japan).
Cell adherent and cytoskeletal molecule analysis
For real time quantitative PCR (RT-qPCR) of integrins, the cells were cultured in 12-well plates that were pre-coated with VTN-N or CPB scaffold for 3–4 days and then harvested for RNA extraction using RNeasy Mini Kit (QIAGEN). RNA was then reverse transcribed into cDNA using the SuperScript™ III First-Strand Synthesis System (18080051, Life Technologies). The primers for RT-qPCR is listed in Table S2. PCR data were analyzed using the AB7900 system and RQ Manager software (Applied Biosystems). Marker expression was normalized to cyclophilin A expression.
To detect cell adhesion and cytoskeletal molecules, cells were cultured in an 8-well chamber (SCS-N08, Matsunami) that was pre-coated with VTN-N or CPB scaffold for 3–4 days, then fixed for 15 min at room temperature in 4% paraformaldehyde in PBS. After fixation, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. Cells were then rinsed three times with PBS and immersed in blocking buffer for 30 min. The blocking buffer consisted of 2% BSA and 3% of the animal serum from which the secondary antibody was derived. The primary antibody was diluted in blocking buffer and incubated at 4 °C overnight. Cells were rinsed three times before binding with the secondary antibody for 30 min at room temperature. Finally, the cells were rinsed four times with PBS and mounted with Prolong Gold anti-fade reagent with DAPI (P36935, Life Technologies). Cells were observed under a confocal microscope (FV10i).
The first antibodies used for studying the adherent and cytoskeletal molecules include: ITGA6 (sc-19622, Santa Cruz), ITGAV (sc-9969, Santa Cruz), ITGB1 (100562-T46, Sino Bio), FAK (sc-557, Santa Cruz), p-FAK (sc-11765-R, Santa Cruz), E-cadherin (sc-21791, Santa Cruz), Cx43 (14-4759-80, eBioscience), VINCULIN (sc-25336, Santa Cruz), ZO-1 (33–9100, Thermo Fisher Scientific), CK18 (sc-6259, Santa Cruz). The secondary antibodies included Alexa Fluor 488-conjugated (Life Technologies) or 594-conjugated (Life Technologies).
Particularly for phalloidin staining, cells were permeabilized for 5 min, then stained directly with phalloidin conjugated with iFluor 594 (20553, CAY) in one step.
Differentiation of human induced pluripotent stem cells into neuronal cells, hepatic cells, and cardiomyocytes on CPB scaffold
For targeted differentiation, the cells were cultured following the dual SMAD inhibition neural differentiation protocol40,44, sequential hepatic differentiation protocol42, and chemically defined cardiac differentiation protocol43. The details are as follows: for the control culture of neuronal differentiation, we prepared 12-well plates that were incubated with Poly-D-Lysine (1 μg/cm2) and Poly-D-Ornithine (1 μg/cm2) for 30 min at room temperature, and washed three times with PBS + (containing Ca and Mg). After that, 12-well plates were coated with iMatrix511 (1 μg/cm2). Culture medium of 253G1 cells cultured on CPB scaffold or VTN-N was replaced with PBS + . Cell colonies were dissected into 0.3–0.5 mm square pieces using a 27 gauge needle, and then gently transferred onto CPB scaffold or iMatrix511-coated 12-well plates containing Neural Induction Medium (NIM), supplemented with SB431542 (10 μM) and LDN193189 (100 nM). The NIM contained equal amounts of Neurobasal medium and DMEM/F12, supplemented with 0.3% glucose, 2 mM L-glutamine, 1 × N-2, 0.5 × B27, 1 × ITS-A, and 0.5 × penicillin/streptomycin. The NIM was changed every other day, and the cells were cultured for 7 days. After day 8, the NIM supplemented with FGF2 (20 ng/ml) was changed every other day, and the cells were cultured for 12 days.
For the control culture of hepatic differentiation, the 12-well plates were coated with standard (growth factor-reduced) Matrigel (1:50). 253G1 cells were dissociated into single cells with 1 mM EDTA, and 3.3 × 104 cells per well were cultured in TeSR-E8 medium on 12-well plates. The next day, the cells were cultured in an incubator at 4% O2, 5% CO2, and 37 °C. After 48 h, the medium was replaced with RPMI1640 containing 1 × GlutaMAX, B27 without insulin, 1% NEAA, 1% penicillin/streptomycin, 100 ng/ml activin A, 50 ng/ml BMP4, 3 μM CHIR99021, and cells were cultured in an incubator at 20% O2, 5% CO2, and 37 °C. After 24 h, the medium was replaced with RPMI1640 containing GlutaMAX, B27 without insulin, 1% NEAA, 1% penicillin/streptomycin, activin A, and BMP4. Thereafter, the medium was changed daily. From day 8 to day 10, the cells were cultured in an incubator at 5% O2, 5% CO2, and 37 °C. The DE medium (RPMI1640 containing GlutaMAX, B27 without insulin, 1% NEAA, 1% penicillin/Streptomycin, activin A, and BMP4) supplemented with FGF2 (10 ng/ml) and HGF (10 ng/ml) was changed daily.
For cardiac differentiation, 253G1 cells (4 × 105 cells per well) were seeded in TeSR-E8 medium containing 5 μM Y27632 on 12-well plates coated with CPB scaffold or iMatrix511. The medium was changed daily. Four days later, the medium was changed to 2 mL per well of RPMI1640 containing B27 without insulin and 12 μM CHIR99021 to initiate differentiation. After exactly 24 h, the medium was replaced with 2 mL of RPMI1640 containing B27 without insulin per well. After 48 h (72 h after addition of CHIR99021), 2 μM XAV939 was added to 2 mL RPMI1640/B27 without insulin. Forty-eight hours later, the medium was replaced with RPMI1640/B27 without insulin and cultured until day 7 of differentiation. On day 7 of differentiation, medium was changed to RPMI1640/B27 with insulin every other day.
At 7 days of differentiation, differentiated neuronal, hepatic, or cardiac cells were analyzed by RT-qPCR. For RT-qPCR, total RNA was isolated from hPSCs and reverse transcribed as previously described. RT-qPCR was performed with the following gene-specific primer/probe mixes: OCT4 (POU5F1, Hs01895061_u1), SOX2 (Hs01053049_s1), NANOG (Hs02387400_g1), PAX6 (cat#Hs01088112_m1), NCAM1 (Hs00941821_m1), NES (Hs00707120_s1), NEUROG2 (Hs00702774_s1), CXCR4 (Hs00607978_s1), GATA4 (Hs00171403_m1), GATA6 (Hs00232018_m1), FOXA2 (Hs00232764_m1), HNF4A (Hs00230853_m1), T (Hs00610080_m1), and Cyclophilin A (PP1A, Hs99999904_m1) (TaqMan Gene Expression Assays, Applied Biosystems, Foster City, CA, USA), TaqMan 2 × Master Mix, and ABI Prism 7900 Sequence Detection System (Applied Biosystems) according to the manufacturer’s protocol. To detect NKX2.5 and TNNT2 expression, RT-qPCR amplifications were carried out in 10 μl reactions using the SYBR Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primer for RT-qPCR48 is listed in Table S3. PCR data were analyzed by the Δ/ΔCT method and normalized to cyclophilin A expression with RQ Manager software (Applied Biosystems).
The differentiated 253G1 cells on day 14 (cardiomyocytes), day 10 (hepatoblasts), day 12 (neural cells) were dissociated with TrypLE express (Life Technologies) and then fixed with 2% paraformaldehyde for 2 min at room temperature. The fixed cells were permeabilized with 0.1% Triton-X in 0.5% BSA/PBS at room temperature for 5 min. The cells were stained with the antibodies against TroponinT-C (cTnT, CT3, sc-20025, Santa Cruz) for cardiomyocytes, FOXA2 (EPR4466, ab108422, ABCAM LIMITED) for hepatoblasts, β-III tubulin (Tuj1, MAB1195, R&D Systems) for neural cells. Alexa Fluor 488-conjugated anti-mouse IgG2A secondary antibody (A21131, Life Technologies), Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (A21206, Life Technologies) and Alexa Fluor 647-conjugated anti-mouse IgG secondary antibody (A32787, Life Technologies) was used for cTNT, FOXA2 and β-III tubulin, respectively. The population was analyzed by flow cytometry (FACS Canto II, BD Biosciences, or SA3800, SONY). To normalize and overlay the chart, the number of cells in a bin (the numerical ranges for the parameter on the fluorescence intensity in the X-axis) were divided by the number of cells in the bin that contained the largest number of cells in FlowJo software (TreeStar).
Statistical analysis
Experiments were repeated at least three times. Statistical significance was determined by Student’ s t test for pairwise comparison or one-way analysis of variance followed by Dunnett’s test using JMP pro version 15 (SAS Institute, Inc., Cary, NC, USA) for multiple comparisons. P-values of less than 0.05 were considered statistically significant.
