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Figures
- Fig. 1.
CKI-7 and SB-431542 promote neural differentiation by blocking Wnt and Nodal signaling. (A-D) Wnt and Nodal signaling during SFEB culture. Levels of Wnt1, Wnt3, Nodal and Cripto were normalized to those on day 0. *P<0.05, **P<0.01, ***P<0.001, compared to day 0 (Dunnett's test). (E) CKI-7 inhibits Wnt signaling during SFEB culture. (F) SB-431542 inhibits Nodal signaling during SFEB culture. (G-I) Effects of CKI-7 and SB-431542 on neural differentiation. SFEB/CS treatment increases the expression of the early neuroectodermal marker Sox1 (G) and the number of cells positive for neural markers Nestin (green) and βIII-tubulin (red; H, SFEB treatment; I, SFEB/CS treatment). **P<0.01, compared with SFEB (unpaired t-test, G). Scale bar, 100 μm (H,I).
- Fig. 2.
Retinal specification of human ES cells by CKI-7 and SB-431542. (A) Schematic diagram of the culture procedure for retinal differentiation. (B,C) CKI-7 and SB-431542 decrease the expression of NANOG and OCT3/4, markers of the undifferentiated state. *P<0.05, **P<0.01, compared with SFEB (unpaired t-test). (D) Phase-contrast image of human ES cells treated with CKI-7 and SB-431542. (E-G) Human ES cells treated with CKI-7 and SB-431542 express the neural markers NES (red) and βIII-tubulin (green) on day 40. (H-M) Regional characterization of SFEB/CS-treated neural tissues derived from human ES cells. Fold expression is the ratio of expression in differentiated versus undifferentiated ES cells. (N) Multi-step commitment in the development of retinal cells. Pluripotent stem cells derived from the inner cell mass (blastocyst) differentiate into retinal progenitors corresponding to those in the eye primordium (optic vesicle/optic cup) that give rise to RPE and photoreceptors (adult retina). (O) RX+ and MITF+ retinal progenitor cells develop from human ES cells under SFEB/CS culture conditions. (P) Effect of CKI-7 and SB-431542 on the percentage of MITF+ colonies. **P<0.01, ***P<0.001, compared with SFEB. NS, not significant (Tukey's test). (Q) Formation of rosette-like clusters positive for PAX6. Scale bars: 300 μm (D), 100 μm (G,Q), 30 μm (O).
- Fig. 3.
Differentiation of RPE from SFEB/CS-treated human ES cells. (A) SB-431542 and CKI-7 treatment induced MITF+ (green)/PAX6+ (red) RPE progenitors from human ES cells. (B) Generation of polygonal pigment cells. (C) Effect of SB-431542 and CKI-7 on the percentage of pigment cells. ***P<0.001, compared with SFEB. NS, not significant (Tukey's test). (D) Tight junction formation of SFEB/CS-treated cells, as shown by anti-ZO1 antibody staining (red). (E) Maturity of human ES-cell-derived pigment cells. RPE65, retinal pigment epithelium-specific protein 65 kDa; CRALBP, cellular retinaldehyde-binding protein. (F) Quantitative RT-PCR analysis of RPE65 in SFEB-CS-treated human ES cells. (G) Induced pigment cells have phagocytic function. Phalloidin-stained polygonal cells (green) incorporated latex beads (red). Scale bars: 30 μm (A,B,D), and 10 μm (G).
- Fig. 4.
Differentiation of photoreceptors from SFEB/CS-treated human ES cells. (A,B) Quantitative PCR analysis for the photoreceptor precursor marker CRX (A) and the mature photoreceptor marker RCVRN (B). Fold expression is the ratio of expression in differentiated versus undifferentiated human ES cells. SFEB/CS-cultured human ES cells were treated with retinoic acid and taurine (RA/T). (C,D) Immunostaining for the photoreceptor marker RHO. An outer process (arrows) and inner process (arrowheads) are present in SFEB/CS+RA/T-treated cells (D). Scale bars: 30 μm (C,D). (E) Effect of CKI-7 and SB-431542 on the percentage of RHO+ cells. Treatment with retinoic acid and taurine (RA+T). **P<0.01, ***P<0.001, compared with SFEB+RA/T. NS, not significant (Tukey's test). (F) RT-PCR analysis of human ES cells treated with SFEB/CS+RA/T. Expression of photoreceptor markers and phototransduction genes on days 100 and 140.
- Fig. 5.
Retinal specification of human iPS cells by CKI-7 and SB-431542. (A,B) Expression of pluripotent cell markers NANOG and TRA-1-60 in human iPS cells. (C) Neural induction of human iPS cells by SFEB/CS treatment. (D-I) Regional characterization of SFEB/CS-treated iPS cells. Fold expression is ratio of expression in differentiated versus undifferentiated iPS cells. *P<0.05, compared with SFEB (unpaired t-test). (J-O) Time-course analysis of the expression of markers of the undifferentiated state, NANOG (J) and OCT3/4 (K) and retinal progenitor markers PAX6 (L), RX (M), MITF (N) and CHX10 (O) during SFEB/CS culture. Fold expression is ratio of expression on each day compared to day 0. *P<0.05, **P<0.01, ***P<0.001, compared to day 0 (Dunnett's test). (P) Differentiation of RX+/PAX6+ neural retina progenitors from SFEB/CS-treated human iPS cells. (Q) Differentiation of MITF+/PAX6+ RPE progenitors from SFEB/CS-treated iPS cells. (R) Effect of CKI-7 and SB-431542 on the percentage of MITF+ colonies. ***P<0.001, compared with SFEB alone (unpaired t-test). (S) Formation of rosette-like clusters positive for PAX6. Scale bar, 100 μm (A-C), 30 μm (P,Q), and 300 μm (S).
- Fig. 6.
Generation of RPE and photoreceptors from both three- and four-factor human iPS cells. (A) Generation of pigment cells from human iPS cells in SFEB/CS cultures. (B) Phalloidin staining shows the polygonal shape of the pigment cells. (C) RT-PCR analysis for markers of the mature RPE, RPE65 and CRALBP in two lines of human iPS cells. (D,E) Generation of RCVRN+ (D) and RHO+ (E) photoreceptors from human iPS cells in SFEB/CS + RA/T culture. (F) RT-PCR analysis of phototransduction genes in two lines of human iPS cells. (G) Comparison of the differentiation of one human ES cell line (khES-1) and two human iPS cell lines (253G1 and 201B7). The 253G1 line was generated by three-factor induction (OCT3/4, SOX2, and KLF4) and the 201B7 line by four-factor induction (OCT3/4, SOX2, KLF4 and MYC). Scale bars: 30 μm (A,D,E) and 10 μm (B).
- Table 1.
Primers used for real-time PCR
Primer sequence (5′-3′) Gene Forward Reverse Mouse Cripto GCCTATGGGATTCCCTTCC ACAGCGGGATACAGGGACT Hes1 ACACCGGACAAACCAAAGAC CGCCTCTTCTCCATGATAGG Hes5 GATGCTCAGTCCCAAGGAGA AGCTTCAGCTGCTCTATGCTG Hesr1 CATGAAGAGAGCTCACCCAGA CGCCGAACTCAAGTTTCC Hesr2 GTGGGGAGCGAGAACAATTA GTTGTCGGTGAATTGGACCT Hesr3 CTGAATTGCGACGATTGGT GCAAGACCTCAGCTTTCTCC Nodal CCAACCATGCCTACATCCA CACAGCACGTGGAAGGAAC Sox1 GTGACATCTGCCCCCATC GAGGCCAGTCTGGTGTCAG Wnt1 TACTGGCACTGACCGCTCT CTTGGAATCCGTCAACAGGT Wnt3 CTCGCTGGCTACCCAATTT GAGGCCAGAGATGTGTACTGC β-actin CTAAGGCCAACCGTGAAAAG ACCAGAGGCATACAGGGACA Human BF1 TACTACCGCGAGAACAAGCA TCACGAAGCACTTGTTGAGG CHX10 GCTGGACACCAGCCAGAC GCAGATTTGGACATTTTTCGAT CRALBP AGATCTCAGGAAGATGGTGGAC GAAGTGGATGGCTTTGAACC CRX CACCAGGCTGTGCCCTAC CTTCCAGCTCCTCCAGTTG GBX2 AAAGAGGGCTCGCTGCTC ATCGCTCTCCAGCGAGAA HOXB4 TGGATGCGCAAAGTTCAC GCTGGACACCAGCCAGAC IRX3 AAAAGTTACTCAAGACAGCTTTCCA GAAATTCCTTCTCCAGCTCCA MITF AGAGTCTGAAGCAAGAGCACTG TGCGGTCATTTATGTTAAATCTTC NANOG ATGCCTCACACGGAGACTGT AGGGCTGTCCTGAATAAGCA OCT3/4 GCAAAACCCGGAGGAGGAGTC CCACATCGGCCTGTGTATATC PAX6 TCACCATGGCAAATAACCTG CAGCATGCAGGAGTATGAGG RCVRN TAACGGGACCATCAGCAAG CCTCGGGAGTGATCATTTTG RPE65 CAATGGGTTTCTGATTGTGGA CCAGTTCTCACGTAAATTGGCTA RX GGCAAGGTCAACCTACCAGA CTTCATGGAGGACACTTCCAG SIX3 CCGGAAGAGTTGTCCATGTT CTCCTCCAGCGTCTCACAG β-actin ATTGGCAATGAGCGGTTC GGATGCCACAGGACTCCA - Table 2.
Primers used for RT-PCR
Primer sequence (5′-3′) Gene Forward Reverse CRALBP AGATCTCAGGAAGATGGTGGAC GAAGTGGATGGCTTTGAACC CRX GCCCCACTATTCTGTCAACG CTTCCAGCTCCTCCAGTTG NRL GAGCCCAGAGGAGACAGGA TTTAGCTCCCGCACAGACAT PDC TCAAAGGAACGAGTCAGCAG CTGCTGCAAGGCATGTTAAA PDE6b CAGTGATGAACACCGACACC ATTTGACCAGGTCCAGTTCG PDE6c CTGAGGTGGCCTCTAGGTTG GCTGGTGTGATGAAGCCTTAG RCVRN TAACGGGACCATCAGCAAG CCTCGGGAGTGATCATTTTG RHO CACCAGGCTGTGCCCTAC GCCTCATCGTCACCCAGT GRK1 GGACTGGTTCCTGGACTTCA AAGCCAGGGTTCTCCTCATT RPE65 CAATGGGTTTCTGATTGTGGA CCAGTTCTCACGTAAATTGGCTA SAG CTGATCCGCAAAGTACAGCA TCAGCGTCTTGGTCAAAGTG GAPDH ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA
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