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CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells – Communications Biology

Human iPSC maintenance

The hiPSC lines (TNNI1-EmGFP reporter, 1390C1, and 409B2) were established in our institute23. The TNNI1-EmGFP reporter and 1390C1 hiPSC lines were maintained on an iMatrix-511 (Nippi)-coated dish in AK02N medium (Ajinomoto) as previously described23. The 409B2 hiPSC line was maintained on SL10 feeder cells (REPROCELL) in Repro Stem medium (REPROCELL) supplemented with 5 ng/mL bFGF (REPROCELL) and penicillin/streptomycin (Sigma), as previously described36. Human iPSC studies were approved by the relevant ethical committee.

Differentiation of hiPSCs into VCMs and ACMs

To initiate cardiac differentiation of the TNNI1-EmGFP reporter and 1390C1 hiPSC lines, we used previously described protocols with some modifications23,24. Briefly, hiPSCs were dissociated into single cells with Accumax (STEMCELL TECHNOLOGIES), and EBs were generated using a 6-well ultra-low attachment plate at 2 × 106 cells/well in 1.5 mL/well StemPro-34 medium (Thermo Fisher Scientific) containing 2 mM L-glutamine (Sigma), 50 μg/mL ascorbic acid (AA, sigma), 0.4 mM monothioglycerol (MTG, Sigma), 150 μg/mL transferrin (Wako), 0.5% Matrigel (Corning), 10 μM ROCK inhibitor Y-27632 (Wako), and 2 ng/mL BMP4 (R&D). On day 1 of ACM differentiation, 1.5 mL StemPro-34 medium with the above supplements (without Y-27632 or Matrigel), 10 ng/mL bFGF (R&D, final 5 ng/mL), 4 ng/mL BMP4 (final 3 ng/mL), and 8 ng/mL Activin A (R&D, final 4 ng/mL) were added into the well for the AIC. For VCM differentiation, 1.5 mL StemPro-34 medium with the above supplements (without Y-27632 or Matrigel), 10 ng/mL bFGF (final 5 ng/mL), 18 ng/mL BMP4 (final 10 ng/mL), and 12 ng/mL Activin A (final 6 ng/mL) was added to the well to initiate VIC. On day 3, AIC-EBs were washed with IMDM (Thermo Fisher Scientific) once and then transferred to 3 mL StemPro-34 medium containing 2 mM L-glutamine, 50 μg/mL AA, 0.4 mM MTG, 150 μg/mL transferrin, 10 ng/mL VEGF (R&D Systems), 1 μM IWP-3 (Stemgent), 5.4 μM SB431542 (Wako), and 1 μM RA (Wako). VIC-EBs were washed once with IMDM and then transferred to 3 mL StemPro-34 medium with the above supplements (without RA) and 0.6 μM Dorsomorphin (Sigma). On day 6, the medium was transferred to 2 mL StemPro-34 medium containing 2 mM L-glutamine, 50 μg/mL AA, 0.4 mM MTG, 150 μg/mL transferrin, and 5 ng/mL VEGF, and the EBs were maintained in this medium, with changes every 2–3 d until analysis. The plate was incubated at 37 °C in a hypoxia environment (5% O2) for the first 10 d and then transferred to a normoxia environment.

For cardiac differentiation of the 409B2 hiPSC line, the hiPSCs were treated with dissociation solution for human ES/iPS cells (REPROCELL) for 5 min at 37 °C and then suspended at 5 × 104 cells/mL on the same day (0) with the above described medium. The single-cell suspension was seeded at 100 μL/well in a 96-well ultra-low attachment plate (Corning) for EB formation. On day 1, the same medium used above for AIC or VIC was added to each well at 100 μL/well. On day 3, EBs were dissociated using Accumax and then reaggregated with 3 mL StemPro-34 medium containing 2 mM L-glutamine, 50 μg/mL AA, 0.4 mM MTG, 150 μg/mL transferrin, 10 ng/mL VEGF, and 1 μM IWP-3 for VIC. AIC-EBs were washed once with IMDM and then transferred to 3 mL StemPro-34 medium containing 2 mM L-glutamine, 50 μg/mL AA, 0.4 mM MTG, 150 μg/mL transferrin, 10 ng/mL VEGF, 1 μM IWP-3, and 0.5 μM RA. On day 6, EBs were collected in 6-well ultra-low attachment plates and cultured for 4 weeks with the same medium and conditions described above.

Monolayer differentiation into ACMs and VCMs

Monolayer differentiation was performed according to a published protocol37. Briefly, hiPSCs were plated on Matrigel-coated 12-well multi-well plate. Two days after, 6 μM CHIR99021 was added to RPMI supplemented with B27 minus insulin (RPMI-ins, day 0). After 48 h (day 2), the medium was changed to RPMI-ins with 2 μM IWP-1. For AIC, on day3, 1 μM RA was added to the medium for 48 h. The medium was changed to RMPI-ins supplemented with 1 μM RA on day 5, RMPI-ins on day 6, and RPMI supplemented with B27 on day 8. For VIC, the medium was changed to RPMI-ins on day 5 and to RPMI + B27 on day 8. The medium was changed every 2 days during culture. Differentiated cells were used for analyses on days 20–23.

Cell surface marker screening

Day-20 AIC- and VIC-EBs were dissociated using Liberase and Accumax. Dissociated cells were assayed with the Lyoplate Human Cell Surface Marker Screening Panel (BD Biosciences) according to the manufacturer’s instructions. Flow cytometric analysis was performed using FACS Lyric (BD Biosciences).

Flow cytometric analysis

Differentiated EBs were dissociated in the same manner described in the cell surface marker screening section above. For TNNI1-EmGFP reporter hiPSC-CMs, purified mouse anti-human CD151 antibody (BD Biosciences, 1:200) was added as a primary antibody, and CD151 expression was detected using APC-anti-mouse IgG antibody (BD Biosciences, 1:200) or Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, 1:200) as the secondary antibody. 1390C1 and 409B2 hiPSC-CMs were isolated using anti-SIRPa-PE/Cy7 (Biolegend, 1:500) as a cardiomyocyte marker and anti-CD90-APC (BD Biosciences, 1:2500), APC anti-human CD31 (Biolegend, 1:500), AlexaFluor647 anti-CD49a (Biolegend, 1:500), and APC anti-CD140b (Biolegend, 1:500) as non-cardiomyocyte lineage markers24 and directly stained with PE anti-human CD151 (BD Biosciences, 1:200). For Ki-67 staining, fixed cells were stained with anti-human ACTN2 antibody (Creative Diagnostics, 1:200). The cells were then stained with Alexa Fluor 647 anti-mouse/human Ki-67 antibody (BioLegend, 1:400), PE anti-human CD151 (BD Biosciences, 1:200), and Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, 1:200) as a secondary antibody against anti-human ACTN2 antibody. For binucleation analysis, dissociated cells were stained with anti-human ACTN2 antibody (Creative Diagnostics, 1:200), Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, 1:200) as a secondary antibody, and Hoechst (DOJINDO, 1:1000). The cells were detected and sorted using FACS Aria Fusion (BD Biosciences) and analyzed using FlowJo software (v10.7.1). The gating strategies are shown in Fig. S8.

Immunocytochemistry

AIC- and VIC-CD151high/low CMs were sorted and then seeded at 1 × 105 cells/well on a fibronectin-coated 96-well plate (Corning). The cells were fixed after 5–7 d of culture for 20 min with 4% paraformaldehyde, permeabilized for 15 min with 0.1% TritonX100-PBS, and then blocked for 1 h at room temperature with 2% goat serum/0.1% TritonX100-PBS. The fixed cells were stained using anti-MLC-2A (Synaptic systems, 1:100) and anti-MLC-2V (Proteintech, 1:200) overnight at 4 °C. The following day, the cells were washed twice with PBS and stained using Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, 1:500), Alexa Fluor 647 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500), or Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500) as the secondary antibody for 1 h at 4 °C under dark conditions. The cells were washed twice with PBS and stained with Hoechst (DOJINDO) for 5 min at room temperature. Images were captured using a BZ-X710 (KEYENCE) with a 20× objective.

Gene expression (qPCR)

The total RNA content was prepared using a miRNeasy Micro Kit (QIAGEN), and purified RNA was reverse transcribed into cDNA using a SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific). Next, qPCR was performed on QuantStudio 7 Flex (Thermo Fisher Scientific) using Taqman probes (Applied Biosystems). The TaqMan probes used are listed (Supplementary Table 2).

Patch-clamp electrophysiology

AIC- and VIC-CD151high/low CMs sorted by flow cytometry were plated on fibronectin-coated cover glasses (3 mm × 7 mm) in a single well of a 24-well plate (2 × 104 cells). The CMs were cultured for 24 h in the same medium used on day 6 of cardiac differentiation described above with 10 μM Y-27632 and maintained in the same medium without Y-27632 with a medium change every 3 d until analysis. The CMs were used for patch-clamp measurements 1319 d after plating, using an Axopatch 200B amplifier (Molecular Devices) at 5 kHz in the current-clamp mode. APs of spontaneously beating single cells were recorded using the gap-free protocol, and signals were digitized at 10 kHz using Digidata 1322 A. The data were analyzed using pCLAMP 9.2 or 10.7 software (Molecular Devices) and an inverted microscope equipped with differential interface optics (Olympus). The CMs were perfused with Gey’s buffer salt solution (Sigma) and kept at 3537 °C in the chamber. Patch pipettes were made from glass capillaries using a micropipette puller (P-97/IVF, Sutter Instruments) with tip resistances of 3–5 MΩ when filled with the pipette solution. The pipette solution comprised 130 mM KOH, 20 mM KCl, 1 mM MgCl2, 5 mM NaCl, 130 mM L-Aspartic acid, 10 mM HEPES, 10 mM EGTA, and 5 mM Mg-ATP and was adjusted to pH7.2 with KOH. APs were recorded and analyzed using Clampfit 10.7 software (Axon Instruments), and MDP, APA, APD, and Vmax were calculated based on the average AP of 10 consecutive and stable waves. APD was corrected using Bazett’s correction38.

CD151 KO hiPSC line generation

CD151 KO hiPSCs were generated from the TNNI1-EmGFP hiPSC line utilizing the CRISPR-Cas9 system. Gene editing was performed according to a published protocol39, using a gRNA (target sequence: 5’-CAGGTTCCGACGCTCCTTGA-3’). Briefly, to form gRNA, an equimolar amount of crRNA and tracrRNA (IDT) was hybridized for 5 min at 95 °C and then cooled to 20 °C using a thermal cycler with a ramp rate setting of −0.1 °C/s. RNP complexes were formed with 61 pmol of gRNA and Cas9 nuclease each and transfected to TNNI1-EmGFP hiPSCs by electroporation. Transfected hiPSCs were cultured and analyzed by flow cytometry. CD151 KO hiPSCs were purified by sorting of CD151-negative populations. To validate them at the genome level, genomic DNA was isolated from TNNI1-EmGFP hiPSCs and CD151 KO hiPSCs. We then sequenced the regions around the target site of CD151 and five off-target genes predicted by CRISPOR40 using primers listed in Supplementary Table 3. The Sanger sequencing results were analyzed to identify the constitution of indels and the gene-editing efficiency using DECODR (https://decodr.org/).

Western blots for CD151 expression

Proteins were extracted from cells with RIPA buffer, with protein concentrations measured using the Pierce BCA Protein Assay Kit. Proteins were separated by SDS-PAGE using Criterion TGX Precast Gel (BioRad) and transferred to PVDF membrane. CD151 monoclonal antibody (2A8G8) (Thermo Fisher SCIENTIFIC, 1:2000) and anti-Actin monoclonal antibody (MAB1501) (Merck Millipore, 1:5000) were used as primary antibodies. ECL peroxidase-labeled anti-mouse antibody (NA931) (GE Healthcare, 1:5000) was used as the secondary antibody.

Optical recording of APs with 4-AP and CCh

AIC-CMs and LY411575/CD151low ACMs were sorted on day 20 and plated as 5 μL cell suspensions (5 × 104 cells) on a fibronectin-coated glass-bottom dish to form CM sheets, with the medium added 2 h later. The medium was changed every 23 d during CM culture. The CMs were treated with FluoVolt dye (Thermo Fisher Scientific) following the manufacturer’s instructions. One hour after setting the cells in a stage top incubator (TOKAI HIT) at 37 °C (5% CO2, 95% air), fluorescence measurements were acquired with an ECLIPSE Ti-E inverted fluorescence microscope (Nikon), objective Fluor 10×/0.45 (Nikon), X-Cite TURBO (Excelitas Technologies), EM-CCD camera ImagEM (Hamamatsu Photonics), and AQUACOSMOS 2.6 software (Hamamatsu Photonics) that set the excitation wavelength at 490 nm and bandwidth at 10 nm. The X-Cite TURBO was set at 5% output. CMs cultured for 7 d were used to record the subarray images every 5 ms. Binning was set to 1 × 1. The regions of interest (ROIs) were defined as whole 512 × 64 pixels in the monolayers. The CMs were paced at 4 Hz with 25 ms depolarizing pulses at 10 V using a pulse stimulator (Master-9, A.M.P.I., Jerusalem, Israel) with an interelectrode distance of 12 mm (Intermedical, Osaka, Japan). The effects of drugs were measured 10 min after exposure to 50 μM 4-AP. Waveform traces were superimposed using OriginPro 2021 (OriginLab, Northampton, MA, USA). 4-AP (Sigma) was prepared as a 50 mM stock solution in Gey’s buffer salt solution, with pH adjusted to 7.4, and stored at −20 °C. CCh (Sigma) was prepared as a 10 mM stock solution in Gey’s buffer salt solution and stored at −20 °C.

Library preparation and RNA-seq

Total RNA was prepared with a miRNeasy Micro Kit. RNA-seq libraries were generated using TruSeq Stranded Total RNA (Illumina) according to the manufacturer’s instructions. RNA-seq libraries were sequenced on a NextSeq 500 using High Output Kit v2.0 with single-end reads and 75 cycles (Illumina). RNA-seq reads were mapped to the human reference genome (hg38) using STAR41 and normalized using DESeq242. Genes with less than 10 normalized counts in an average of 12 samples were excluded, and 17142 genes were finally included in the data sets for the analysis. DEGs were identified using Wald’s test in DESeq2. P values of the genes were adjusted via the Benjamini-Hochberg procedure, and adjusted p values < 0.05 were considered significant. PCA was performed using the prcomp package in R (v4.0.3) with the normalized gene expression data. GO and Reactome pathway enrichment analyses were performed using the geneXplain® platform (geneXplain) for DEGs. Hierarchical clustering was performed using Spearman’s distance and Ward’s algorithm, and heatmaps were generated using heatmap.2 in gplots (v3.1.1) R package.

Single-cell RNA sequence analysis

To assess the tissue-specific expression patterns of CD151 in the developing human and mouse heart, we utilized publicly available single-cell RNA sequencing (scRNAseq) datasets30,31 (Human: GSE106118, Mouse: GSE193346) across distinct developmental stages. For human samples, transcriptomic data from left atrial (LA) and ventricular (LV) tissues at 7, 13, and 20 gestational weeks were analyzed. Violin plots were generated at each stage to compare expression profiles, and statistical significance was evaluated using the Mann–Whitney U test. A parallel methodology was applied to mouse samples, focusing on embryonic days 10.5 (E10.5) and 18 (E18), and postnatal day 5 (P5) with corresponding statistical analysis.

Notch inhibitor treatment

EBs were treated with 3 μM LY411575 (Selleck Chemicals), a gamma-secretase inhibitor, from days 820. On day 20, the EBs were dissociated and sorted in the same way as explained above in the flow cytometric analysis section.

Statistics and Reproducibility

GraphPad Prism (Graph Pad Software Inc.) was used to perform statistical analyses and create graphs and bar plots. Quantitative data are shown as the mean ± standard error of the mean (SEM), as indicated in the figure legends. Sample sizes (n) used in each experiment are indicated in the figure legends. Statistical significance was determined using Student’s t test (unpaired or paired, two-tailed) and one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) test or Dunnett’s test, unless specified otherwise.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.