The list of published study on scalable suspension culture without microcarriers for hPSCs.

HiPSCs maintained under suspension-culture conditions undergo spontaneous differentiation.

(A) Schematics representing hiPSCs in suspension-culture conditions. (B) Phasecontrast images of adherent- or suspension-cultured hiPSCs on day 5 (Passage 1 (P1)) and day 10 (Passage 2 (P2)). Scale bars: 400 µm. (C) Gene expression in hiPSCs cultured under adherent or suspension conditions on P2. Bar graphs show the mean ± SE (n=3). P-values were statistically analyzed by Student’s t-test. (D) Phase-contrast and fluorescent images of adherent or suspension-cultured reporter hiPSCs on P2. White arrowheads indicate spontaneous expression of SOX17 and PAX6 in suspension conditions. Scale bars: 400 µm. (E) Quantification of hiPSCs spontaneous differentiation with flow cytometry. (F) Averaged tdTomato-positive cell ratio (%) from flow cytometry data (mean ± SE from n=3). P-values were statistically analyzed by Student’s t-test. (G) An MA plot comparing transcriptomes between suspension- and adherent-culture from RNA-seq data. The representative gene name is shown in the plot. (H) GSEA on the gene sets of suspension-cultured hiPSCs to adherent cultures. Adjusted P-values are shown as blue to red from low to high values. (I and J) GOEA on the gene sets of suspension-cultured hiPSCs to adherent culture. Results are ranked by significance (p-adjusted value) and/or counted gene numbers.

PKC inhibitors suppress spontaneous differentiation of hiPSCs into neural ectoderm in suspension-culture conditions.

(A) Schematics. (B) Phase-contrast pictures of suspension-cultured hiPSCs in the presence of Wnt signaling inhibitors (IWP-2 and IWR-1-endo) or activator (CHIR99021). Scale bars: 400 µm. (C) Gene expression of hiPSCs in suspensionculture conditions with or without IWP2, IWR-1-endo or CHIR99021. RT-qPCR was performed on day 10 samples (P2). Gene expressions were normalized to GAPDH and displayed as relative fold increase to adherent-cultured samples. Bar graphs show the mean ± SE. P-values were statistically analyzed using Dunnett’s multiple comparisons test. (D) Screening of inhibitory activity of candidate molecules on neuroectoderm differentiation in suspension-cultured hiPSCs. Candidate molecules were added in combination as shown. Results are displayed as relative fold increase to suspension-cultured samples without pharmacological treatment. n=1. (E) Phase-contrast images of suspension-cultured hiPSCs on P2 in the presence of PKC inhibitors (Gö6983 or GF109203X) alone, or in combination with IWR-1-endo. Scale bars: 400 µm. (F) Suspension-cultured hiPSCs gene expression in the presence of IWR-1-endo or with combined IWR-1-endo and different doses of PKC inhibitors. Results are displayed as relative fold increase to adherent-culture. Data are presented as mean ± SE (n=3).

The inhibitors of WNT and PKCβ efficiently maintain the self-renewal of hiPSCs in suspension conditions.

(A) Phase-contrast images of suspension-cultured hiPSCs on day 10 (2 passages) in the presence of IWR-1-endo, LY333531, or both. Scale bars: 400 µm. (B) Gene expression of suspension-cultured hiPSCs on P2 in the presence of IWR-1-endo, LY333531, or both. Data are presented as mean ± SE. P-values were statistically analyzed with Dunnett’s test. (C) After suspension culture of 5 hiPSC lines for 10 days (2 passages), WTC11, 1231A3, HiPS-NB1RGB (NB1RGB), 1383D6 and 201B7, in StemFit AK02N medium with or without IWR-1-endo and LY333531, characteristic analysis was performed. Box plots of flow cytometry for TRA-1-60 and SSEA4 double positive cells (%) are shown. Used cell lines are indicated by the different colored circles shown on the right side of the graph (n=2 x 5 cell lines). (D) Box plots of OCT4 positive cells (%) are shown (n=2 x 5 cell lines) (E) Box plots of RT-qPCR data are shown. Differentiation markers (SOX17, T, PAX6), undifferentiated markers (OCT4, NANOG), and naïve markers (KLF2, KLF5) were assessed. Statistical analysis was performed using by one-way Anova and Tukey’s test for all graphs. P values < 0.05 were considered statistically significant. *, ** or *** in the graphs indicate P<0.05, P<0.01 or P<0.001, respectively (n=2 x 5 cell lines).

Long-term suspension culture of hiPSCs is maintained by simultaneous suppression of PKCβ and Wnt signals.

(A) The number of hiPSCs at passage during long-term suspension-culture every 5 days. Cell culture was performed in the presence of IWR-1-endo plus Gö6983 or LY333531. (B) Bar graph indicating average cell numbers for 10 passages (mean ± SE). (C) Phase-contrast images of suspension-cultured hiPSCs on passage 10. Scale bars: 400 µm. (D) Immunocytochemistry of OCT4 on passage 10 samples. Scale bars: 100 µm. (E) Bar graph showing the percentages of OCT4 positive cells. Values were calculated from randomly selected three regions from immunofluorescence images. Data are presented as mean ± SE. P- value was statistically analyzed by Student’s t-test. (F) Immunocytochemistry of differentiated cells in EBs from suspension-cultured hiPSCs in the presence of IWR-1-endo; and Gö6983 or LY333531. Anti-TUJ1, -SMA and -AFP antibodies were used to detect ectoderm, mesoderm and endoderm differentiation, respectively. Scale bars:100 µm. (G) Chromosomal copy numbers detected with CNV array analysis (Karyostat assay) of suspension-cultured hiPSCs in the presence of IWR-1-endo; and Gö6983 (Upper panel) or LY333531 (Lower panel) at passage 10.

The inhibitors of WNT and PKCβ in suspension conditions efficiently suppress differentiated gene marker expression in transcriptome analysis.

(A) Hierarchical clustering of adherent and suspension-cultured hiPSCs on P2 using Ward’s method from RNA-seq data (n=3 in each condition). Ad, Adherent; Sus, Suspension; IWR, IWR-1-endo; LY, LY333531; IWRLY, IWR-1-endo and LY333531. (B) Principal component analysis (PCA) plot showing clusters of samples based on similarity. Gene expression variance are displayed as PC1 and PC2. (C) MA plot comparing transcriptomes between IWRLY and Sus. (D) GSEA on the gene sets of IWRLY to Sus from these RNA-seq data. Statistically significant enrichment is shown. P-values are represented in blue to red from low to high values. (E and F) GOEA for the gene sets of IWRLY to Sus from these RNA-seq data. Analysis was performed on down-regulated genes in (E) and up-regulated genes in (F).

Mass suspension-culture of clinical-grade hiPSCs in the presence of PKCβ and Wnt signaling inhibitors.

(A) Schematics. (B) Representative phase-contrast images of these hiPSCs. Scale bar: 1 mm. (C) Cell viability at seeding. Data are presented as mean ± SE (n=3). P-values were statistically analyzed with Dunnett’s multiple comparisons test. (D) Total cell numbers were counted on day 5 after thawing. Data were presented as mean ± SE (n=3). P-values were statistically analyzed with Dunnett’s test. (E) Representative flow cytometry data of pluripotent markers in these hiPSCs. (F) Quantification of flow cytometry data for the pluripotent markers. Data are presented as mean ± SE (n=3). (G) Karyotypes of these hiPSCs. Left: Representative pictures of G-band analysis. Right: Table of karyotype results (n=3). The numbers in brackets indicate the cell numbers examined. (H) In vitro differentiation from hiPSCs was assessed using RT-qPCR (mean ± SE) (n=3). P-value was statistically analyzed by Student’s t-test. (I) Representative flow cytometry data for CORIN. (J) Quantification of CORIN-positive cells (mean ± SE, n=3). (K) Representative flow cytometry data for cardiac Troponin T (cTnT). (L) Quantification of cTnT-positive cells (mean ± SE, n=3). (M) Albumin secretion levels of hepatocytes differentiated from hiPSCs (mean ± SE, n=3).

Establishment of single-cell-sorted hiPSC subclones cultured in suspension conditions supplemented with IWR1-endo and LY3333531.

(A) Schematics representing the establishment of single-cell-derived hiPSC subclones from 201B7 line. Single-cell-sorted cells were expanded in the culture medium supplemented with IWR-1-endo and LY333531. Formed colonies were picked on day 14, and expanded by repeating passage every 4-5 days under suspension conditions. Characteristic analysis was performed on day 28 after single-cell sorting. (B) On day 7 after single-cell sorting, formed colonies were counted per well in the 96-well plate. The ratio (%) of the colony formation is shown in a bar graph (mean ± SE) (n=3). Statistical analysis was performed using by one-way ANOVA and Tukey’s tests for all graphs. P values < 0.05 were considered statistically significant. *, ** or *** in the graphs indicate P<0.05, P<0.01 or P<0.001, respectively. (C) Phase-contrast images of Clone #2 on day 28 after single-cell sorting. Scale bars: 500 µm. (D) Represented flow cytometry of TRA-1-60 (Clone #2). (E) Represented immunocytochemistry of OCT4 (Clone #2). Scale bars: 200 µm. (F) Summary table of the characterization of single cell-sorted clones. Total cell numbers on day 28, the ratio of TRA-1-60-positive cells (%), and the ratio of OCT4-positive cells (%) are shown (mean ± SE) (n=7).

Re-suspension from frozen stocks of hiPSCs under suspension culture conditions supplemented with IWR-1-endo and LY333531.

(A) Schematics of re-suspension culture of frozen stocks of single-cells-derived 201B7 clone (Vial 1-3). (B) Represented phase-contrast images on day 10 (Vial 3). Scale bars: 500 µm. (C) Represented flow cytometry of TRA-1-60 (Vial 3). (D) Represented immunocytochemistry of OCT4 (Vial 3). Scale bars: 200 µm. (E) Summary table of the characterization of re-suspension-cultured iPSCs from frozen stocks. Total cell numbers on day 10, the ratio of TRA-1-60-positive cells (%), and the ratio of OCT4-positive cells (%) are shown (mean ± SE) (n=3).

Establishment of hiPSCs in complete suspension conditions using SeVdp.

(A) Schematics. (B) Phase-contrast images of PBMCs on day 56 after infection. Scale bars: 400 µm. (C) Immunocytochemistry of OCT4 on bulk-hiPSCs on day 56. Scale bars: 200 µm. (D) Flow cytometry of TRA-1-60 in bulk-hiPSCs on day 61. (E) Immunocytochemistry of TUJ1, SMA, and AFP on differentiated cells in EBs from bulk-hiPSCs on day 56. Scale bars: 100 µm. (F) HE staining of teratoma sections derived from bulk-hiPSCs. White arrowheads indicate representative tissue structures derived from ectoderm, mesoderm, and endoderm. Scale bars: 100 µm. (G) Chromosomal copy numbers detected with CNV array analysis on bulk-hiPSCs. (H) Phase-contrast image of an established hiPSC clone at passage 7. Scale bars: 400 µm. (I) Immunocytochemistry of established clone, F-10, with anti-OCT4 antibody. Scale bars: 200 µm. (J) The percentage of TRA-1-60-positive cells with flow cytometry on established clone F-10 line. (K) HE staining of teratoma sections derived from F-10 clone line. The details are the same as in (F). (L) Immunocytochemistry in EBs from established F-10 clone. Scale bars: 100 µm. (M) Chromosomal copy numbers of F-10 clone line. (N) Residual SeVdp genomic RNA in established hiPSCs with RT-qPCR.

Characterization of PAX6-tdTomato and SOX17-tdTomato reporter lines.

(A) Schematic of construction of reporter hiPSC lines. CRISPR/Cas9 was employed to knock-in the 2A-tdTomato gene and EF1a promoter-driven Zeocin (Bleomycin) resistance gene sequence (TEZ) to the 3’ terminal of SOX17 or PAX6 open reading frame (ORF), respectively. (B) Genotyping of selected clones by genomic PCR. Arrows indicate alleles of wildtype or insertion in the PAX6 (left panel) or SOX17 (right panel) genes. (C) Phase-contrast images of representative colonies and immunocytochemistry of pluripotency markers: OCT4 and NANOG. Left panels: PAX6-TEZ. Right panels: SOX17-TEZ. (D) Flow cytometry of cell surface markers: TRA-1-60 and SSEA4. Left: PAX6-TEZ. Right: SOX17-TEZ. (E) Chromosomal copy numbers detected with CNV array analysis of PAX6-TEZ (top) and SOX17-TEZ (bottom). (F) Immunocytochemistry of differentiated cells in EBs from established bulk-hiPSCs on day 56. Anti-TUJ1, -SMA and -AFP antibodies were used. Scale bars: 100 µm. (G) Differentiation potency of PAX6-TEZ in the neural ectoderm. Left: Flow cytometry of tdTomato using PAX6-TEZ cultured in neuroectodermal differentiation medium for 7 days. Right upper: Overlay images of phase-contrast and tdTomato fluorescence of PAX6-TEZ cultured in neuroectodermal differentiation medium (upper) or maintenance medium (lower). Scale bars: 200 µm. Right lower: Images of tdTomato fluorescence and endogenous PAX6 protein detected with immunocytochemistry with anti-PAX6 antibody. Nuclear staining was performed with DAPI. Scale bars: 200 µm. (H) Differentiation potency of SOX17-TEZ in the endoderm. Left: Flow cytometry of tdTomato using SOX17-TEZ cultured in endodermal differentiation medium for 7 days. Right upper: Overlay images of phase-contrast and tdTomato fluorescence of SOX17-TEZ cultured in endoderm differentiation medium (upper) or maintenance medium (lower). Right lower: Images of tdTomato and endogenous SOX17 protein detected by immunocytochemistry with anti-SOX17 antibody. Nuclear staining was performed with DAPI. Scale bars: 200 µm.

Effects of PKCβ and IWR-1-endo on suspension-cultured hiPSCs at the protein level.

(A) Simple western blotting of adherent- or suspension-cultured hiPSCs (SOX17-TEZ) in the presence or absence of IWR-1-endo. SOX17-TEZ cultured in endoderm differentiation medium (Actinin A + CHIR) was used as a positive control for SOX17 expression. (B) Quantification of SOX17 expression. The protein expression was normalized to GAPDH. Data are presented as mean ± SE (n=3). (C) Simple western blotting of adherent- or suspension-cultured hiPSCs (PAX6-TEZ) in the presence or absence of IWR-1-endo and LY333531. PAX6-TEZ cultured in neurectoderm differentiation medium (SB431542+DMH1) was used as the positive control for PAX6 expression. (D) Quantification of expression of PAX6. The protein expression was normalized to GAPDH. Data are presented as mean ± SE (n=3). (E) Phase-contrast images and representative flow cytometry data of pluripotency marker, TRA-1-60, in adherent- or suspension-cultured hiPSCs (1383D6 line) on day10 (Passage 2). Suspension-culture was performed in the presence or absence of IWR-1-end and LY333531. The percentage of TRA-1-60 positive cells is shown in the right corner. (F) Quantified data for TRA-1-60 positive ratio (%). The percentage of TRA-1-60 positive cells is shown as mean ± SE (n=3).

Activity of different type of PKC inhibitors on spontaneous differentiation of suspension-cultured hiPSCs.

(A) Schematic representing suspension culture of hiPSCs supplemented with PKC inhibitors. Healthy donor-derived hiPSCs (201B7 line) were grown under continuous agitation in the presence of Y-27632, IWR-1-endo and PKC inhibitors. Passage was performed every four days. (B) Phase-contrast images of suspension cultured hiPSCs on day 8 (P2) in the absence or presence of IWR-1-endo and each PKC inhibitors. Scale bars, 500 µm. (C) Cell growth of hiPSCs (201B7). Graph shows live cell numbers counted on day 4 and day 8 in suspension-culture (n=1). The PKC inhibitors added are labelled in the graphs. (D) Gene expression in hiPSCs cultured in adherent or suspension conditions with each PKC inhibitor. RT-qPCR was performed on cDNAs prepared from day 8 (passage 2) samples (n=1). qPCR was performed for pluripotency (OCT4, SOX2, NANOG) and early differentiation markers (PAX6, T, SOX17). Gene expression was normalized to beta-ACTIN.

Up-regulation of PKC genes in suspension-cultured hiPSCs.

(A) RNA expression of conventional PKC isoform genes in hiPSCs as detected by RT-qPCR. cDNA was prepared from adherent- or suspension-cultured hiPSCs on day 5. The gene expression was normalized to GAPDH. Results are displayed as relative fold increase to adherent-culture. Data are presented as mean ± SE (n=3). P-values were statistically analyzed by Student’s t-test. (B) Detection of phosphorylated PKCβ protein (pPKCβ) in suspension cultured hiPSCs on day 5 using an automated capillary western blot (Simple Western) assay. Adherent-cultured hiPSCs were used as a control. (C) Quantification of pPKCβ expression. The protein expression was normalized to GAPDH. Data are shown as mean ± SE (n=3). P-value was statistically analyzed using Student’s t-test. Scale bars, 200 µm.

Cell death of suspension cultured iPSCs after the treatment with LY333531 and IWR-1-endo.

(A) Phase-contrast images of suspension-cultured hiPSCs (WTC11 line) on day 10 (Passage 2). Cells were culture in StemFit AK02N medium with no supplement (left), with LY333531 + IWR-1-endo (middle) for 10 days, with Staurosporine for 2 hours. Scale bars: 200 µm. (B) Evaluation of apoptotic cells by flow cytometry using Annexin V (Alexa Fluor 680) conjugates. (C) Bar-graph indicating Annexin V-positive cells (%) in each culture conditions. (D) Bar-graph indicating double positive cells for Annexin V and DAPI (%) in each culture conditions. Data are presented as mean ± SE (n=3). Statistical analysis was performed using by one-way ANOVA and Tukey’s tests for all graphs. P values < 0.05 were considered statistically significant. ** in the graphs indicate P<0.01.

Generality of the inhibitory effects of IWR-1-end and LY333531 on spontaneous differentiation of hiPSCs cultured in suspension condition. (A)

Schematic representing suspension culture of hiPSCs using StemScale medium (Thermo Fisher Scientific) as the basic culture medium. hiPSCs were grown under continuous agitation (90 rpm) in the presence or absence of IWR-1-endo and LY333531. Passage was performed every four days according to the manufacture’s protocol. cDNAs were prepared from day 8 (P2) and used for RNA seq. (B) Phase-contrast images of suspension cultured hiPSCs on day 8 in the presence or absence of IWR-1-endo and LY333531. Scale bars, 400 µm. (C) RNA expression of pluripotency and early differentiated genes in hiPSCs, as detected by RT-qPCR. cDNA was prepared from suspension-cultured hiPSCs on day 8 in the presence or absence of IWR-1-endo and LY33353. The gene expression was normalized to GAPDH. Data are presented as mean ± SE (n=3). (D) Heat map of marker genes for pluripotency and differentiation to three germ layers from RNA-seq data in suspension-cultured iPSCs without compounds (Sus_1, Sus_2 and Sus_3) or with IWR-1-endo and LY333531 (IWRLY_1, IWRLY_2 and IWRLY_3). The listed genes were classified as pluripotent, mesendoderm, mesoderm, endoderm, and ectoderm, as defined in the Scorecard Assay (Thermo Fisher Scientific).

Suspension culture of multiple hiPSC lines in mTeSR1 medium supplemented with or without IWR-1-endo and LY333531.

(A) Schematics of suspension culture of WTC11, 1231A3, HiPS-NB1RGB (NB1RGB) and 201B7 hiPSC lines. After suspension culture of these 4 hiPSC lines in mTeSR1 medium with or without IWR-1-endo and LY333531, characteristic analysis was performed on day10 (Passage 2). Suspension culture was performed twice in each cell lines (n=2 each from 4 cell lines). (B) Box plots of flow cytometry for TRA-1-60 and SSEA4 double positive cells (%). Used cell lines are indicated by the different colored circles shown on the right side of the graph. (C) Box plots of OCT4 positive cells (%). (D) Box plots of RT-qPCR. Differentiation markers (SOX17, T, and PAX6), undifferentiated markers (OCT4 and NANOG), and naïve pluripotency markers (KLF2 and KLF5) were assessed. Statistical analysis was performed using by one-way Anova and Tukey’s test for all graphs. P values < 0.05 were considered statistically significant. *, ** or *** in the graphs indicate P<0.05, P<0.01 or P<0.001, respectively.

Global gene expression of suspension-cultured hiPSCs with IWR-1-endo and LY333531 in comparison to adherent-cultured hiPSCs.

(A) MA plot (log2 fold change versus mean average expression) comparing the transcriptomes between adherent- and suspension-cultured hiPSCs treated with IWR-1-endo and LY33353 (IWRLY). Transcripts with log2 fold change ≧ 2 or ≦ -2 (FDR <0.01) are highlighted with red or blue dots, respectively. The names of the representative genes are shown in the plot. (B) GSEA of the gene sets of suspension-cultured hiPSCs with IWRLY compared to adherent-cultured hiPSCs. Analysis was performed on both up-regulated and down-regulated genes. Statistically significant enrichment is shown. Adjusted P-values are illustrated from blue to red as low to high. (C and D) GOEA of the gene sets of suspension-cultured hiPSCs with IWRLY compared to adherent-cultured hiPSCs. Analysis was performed on up-regulated genes in (C) and down-regulated genes in (D), respectively. Results are ranked by significance (adjusted P values) and/or counted gene numbers.

Comparison of expression on naïve markers between adherent and suspension-cultured hiPSCs.

HiPSCs (WTC11 line) were culture in continuous suspension culture in StemFitAK02N in the presence of IWR-1-endo, LY333531, or both. RNA-seq was performed using day 10 samples. The gene expression of naïve pluripotency markers (OCT4, NANOG, KLF2, KLF4, KLF5, and DPPA3) was extracted based on the TPM (Transcripts per million) values. Bar graphs show the mean ± SE (n=3). P-values were statistically analyzed with Dunnett’s test to the samples of adherent conditions. P values < 0.05 were considered statistically significant. *, ** or *** in the graphs indicate P<0.05, P<0.01 or P<0.001, respectively.

Suspension-culture of hiPSCs supplemented with IWR-1-endo and LY333531 using bioreactors with continuous agitation.

(A) Schematic representing suspension culture of hiPSCs in a 30 mL bioreactor. Healthy donor-derived hiPSCs (1383D6 lines) were grown with continuous agitation in the presence or absence of IWR-1-endo and LY333531. Passages were performed every 3 -4 days. (B) Graphs showing population doubling levels from the seeding cell numbers on day 3. (C) Flow cytometric analysis of of differentiation marker proteins, PAX6 and SOX17. Positive cells (%) are shown.

Suspension-culture of hiPSCs supplemented with IWR-1-endo and LY333531 using bioreactors with continuous agitation.

(A) Schematic representing suspension culture of hiPSCs in a 30 mL bioreactor. Healthy donor-derived hiPSCs (1231A3 and 1383D6 lines) were grown with continuous agitation in the presence or absence of IWR-1-endo and LY333531. Passages were performed every three days. (B) Graphs showing cell numbers at each passage (n=1). Upper: suspension-culture of 1231A3 and 1383D6 lines. Lower: adherent-culture of 1231A3 and 1383D6 lines. (C) Graphs showing glucose concentration in the culture medium at each culture day. Upper: suspension-culture of 1231A3 and 1383D6 lines. Lower: adherent-culture of 1231A3 and 1383D6 lines. (D) Graphs showing lactic acid concentration in the culture medium at each culture day. Upper: suspension-culture of 1231A3 and 1383D6 lines. Lower: adherent-culture of 1231A3 and 1383D6 lines. (E and F) RT-qPCR analysis of hiPSCs cultured in 30 mL bioreactor or adherent conditions. cDNA samples were prepared from passage 3 and 5 of the 1231A3 in (E) and 1383D6 in (F). qPCR was performed for OCT4 and NANOG (pluripotent), CDX2 (mesoderm), SOX17 (endoderm) and PAX6 (neuroectoderm). The gene expression was normalized to β-ACTIN. Bar graph showing the average gene expressions of passage 3 and 5 (mean ± SE). (G) Flow cytometric analysis of suspension- or adherent-cultured 1231A3 and 1383D6 lines. Positive cells (%) of pluripotency marker proteins: NANOG, OCT4, and SOX2 are shown. (H) Karyotyping of hiPSCs cultured in 30 mL bioreactor. Cell samples were prepared on passage 5, and used for G-band karyotyping analysis. A short deletion on chromosome 9 of 1231A3 (indicated with arrow) was derived from the original line.

Re-suspension from frozen stocks of hiPSCs under suspension culture conditions supplemented with IWR-1-endo and LY333531 in mTeSR1 medium.

(A) Schematics of re-suspension culture of frozen stocks of WTC11, 1231A3, HiPS-NB1RGB (NB1RGB) and 201B7 hiPSC lines. After suspension culture in mTeSR1 medium supplemented with IWR-1-endo and LY333531, expanded iPS cells were cryopreserved. Each frozen stocks were expanded again in the mTeSR1 medium supplemented with IWR-1-endo and LY333531 by repeating passage every 5 days. Characteristic analysis was performed on day10. (B) Representative flow cytometry of TRA-1-60 and SSEA4. (C) Representative immunocytochemistry of OCT4. DAPI indicates nuclear staining. Scale bars: 400 µm. (D) Summary of the characterization of re-suspension-cultured hiPSCs from frozen stocks. Total cell numbers on day 10, the ratio of double positive cells for TRA-1-60 and SSEA4 (%), and the ratio of OCT4-positive cells (%) are shown (mean ± SE; n=4).

Establishment of hiPSCs in completed suspension conditions using episomal vectors.

(A) Flow chart representing the establishment of hiPSCs from PBMCs by electroporation of episomal vectors. (B) Phase-contrast images of transfected cells on day 36 after electroporation of episomal vectors in the absence or presence of IWR-1-endo and LY333531. Scale bars: 400 µm. (C) Flow cytometry of TRA-1-60 and SSEA4. TRA-1-60 and SSEA4-psotive cells were sorted as the bulk fraction and further suspended in the absence or presence of IWR-1-endo and LY333531. The percentages of TRA-1-60 and SSEA4-psotive cells are shown in the right corner. (D) Phase-contrast images of suspension-cultured cell aggregates after bulk sorting on passage 5. Scale bars: 400 µm. (E) Flow cytometry of TRA-1-60 and SSEA4. The percentage of TRA-1-60- and SSEA4-psotive cells are shown in the right corner. (F) Immunocytochemistry of OCT4 on sorted-cells cultured in the absence (top) or presence (bottom) of IWR-1-endo and LY333531 for 5 passages. Scale bars: 400 µm. (G) Quantified data for OCT4-positive cell percentage. Bar graph indicating average OCT4-positive cell percentages (mean ± SE) (n=3). P-value was statistically analyzed by Student’s t-test. (H) Single-cell sorting and cloning of TRA-1-60 positive cells. Flow cytometry was performed for cell fraction that was cultured in the presence of IWR-1-endo and LY333531. TRA1-60-positive cells were collected as single cells in a 96-well plate and expanded in the culture medium supplemented with Y-27632, IWR-1-endo and LY333531. (I) Representative phase-contrast images of single-sorted cell aggregates on day 5 and 12. Scale bars, 400 µm. (J) Phase-contrast images of established clones (B-10, E-4, and E-5). Scale bars: 400 µm. (K) Immunocytochemistry of OCT4 and NANOG in these clones. The percentage of positive cell are shown. Scale bars: 100 µm. (L) Flow cytometry of TRA-1-60 in these clones. TRA-1-60 positive percentages are shown in the right corner. (M) Immunocytochemistry of differentiated cells in EBs from these clones. Anti-TUJ1, -SMA and -AFP antibodies were used. Scale bars: 100 µm. (N) Hematoxylin-Eosin staining of paraffin-embedded sections of teratoma derived from these clones. White arrowheads indicate representative tissue structures of the ectoderm, mesoderm, or endoderm. Scale bars, 100 µm. (O) Chromosomal copy numbers detected by CNV array analysis (Karyostat assay) of these clones.

The list of chemicals and cytokines used in the screening assay.

The list of Taqman probes and primer sets used in this study.

The list of primary antibodies used in this study.

The list of secondary antibodies used in this study.

Software used in this study.