Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture
Hiroki Takeuchi1, Norio Nakatsuji2, 3, Hirofumi Suemori1*
1Department ofEmbryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
2Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Ushinomiya-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
3Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
*Correspondence should be addressed to: Hirofumi Suemori ()
Supplementary Figure 1 & 2 & 3 & 4 and Table 1 & 2
Supplementary Figure 1. Comparison of the effects of each factor on differentiation to DE cells.
(A)Quantitative PCR analysis of CXCR4, HNF1B and HNF4A at day 4. Expression levels were normalized to TBP expression. mRNA expression was relative to that in untreated cells at day 4. Error bars indicate SD (n=3).
(B)Flow cytometric analysis of the percentage of FOXA2+/SOX17+ cells among induced cells treated with activin A and CHIR99021 or Wnt3a. Error bars indicate SD (n=3). Control, no factors added; Act A, 100 ng/ml activin A; CHIR, 3M CHIR99021; Wort, 100 nM wortmannin; Wnt, 25 ng/ml Wnt3a.
Supplementary Figure 2. Comparison of the effects of each factor on differentiation to pancreatic progenitor cells.
(A)Quantitative PCR analysis of NGN3, NKX6.1 and NKX2.2 at day 10. Expression levels were normalized to TBP expression. mRNA expression was relative to that in control cells at day 10. Control cells were treated with three factors for 4 days as shown in Fig. 4A, and then without factors for 6 days. Error bars indicate SD (n=3).
(B)Cells were treated with FGF10 for 3 or 6 days and then stained with an anti-PDX1 antibody at day 10. Mean ± SD (n=5–10).
(C)Left: cells were treated with each factor for 6 days and then stained with an anti-PDX1 antibody at day 10. Right: the percentage of PDX1+ cells among differentiated cells treated with each factor. 4F: F10, Nog, Dor, and RA. Mean ± SD (n=5–10). F10, 50 ng/ml FGF10; Nog, 50 ng/ml Noggin; Dor, 1M dorsomorphin; RA, 2retinoic acid; FR, 3 FR180204; PD, 0.5M PD0325901; SR, 1M SR11302. Scale bar, 100 m.
Supplementary Figure 3. Comparison of effect of each factor on differentiation into IPCs.
(A) Quantitative PCR analysis of the expression of pancreatic-related genes in cells treated with each factor at day 16 and 22. Expression levels were normalized to TBP expression. mRNA expression was relative to that in control cells at day 16 or 22. Control cells were treated with each factor for 10 days as shown in A, and then without factors for 6 or 12 days in 2D culture. Error bars indicate SD (n=3).
(B) Cells were treated with four factors for 16 days and then stained with antibodies against PDX1, NKX6.1, C-PEPTIDE, INS, GCG and SST. Fsk, 10 M forskolin; Dex, 10 M dexamethasone; Alk5i, 5 M Alk5 inhibitor II; NA, 10 mM nicotinamide. Scale bar, 100 m.
Supplementary Figure 4. Comparison of effect of each factor on differentiation into IPCs in 3D culture.
(A) Quantitative PCR analysis of the expression of pancreatic-related genes in cells treated with each factor in 3D culture at day 16 and 22. Expression levels were normalized to TBP expression. mRNA expression was relative to that in control cells at day 16 or 22. Control cells were treated with each factor for 10 days as shown in A, and then without factors for 6 or 12 days in 3D culture.Error bars indicate SD n=3.
(B) Immunostaining of C-PEPTIDE, GCG, SST and INS proteins in iPS(IMR90)-4 cells treated with each factor in 3D culture at day 22. INS+ cells were 26.0 ± 1.8% of total cells. INS+/GCG+ cells were 2.5 ± 2.2% of total cells.Mean ± SD (n=5). Scale bar, 100 m. Fsk, 10 M forskolin; Dex, 10 M dexamethasone; Alk5i, 5 M Alk5 inhibitor II; NA, 10 mM nicotinamide.
Supplementary Table 1. List of growth factors and small molecules with differentiation.
Supplementary Table 2 Sequence of qPCR primer
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