Dynamic transcriptional and epigenomic reprogramming from pediatric nasal epithelial cells to induced pluripotent stem cells Hong Ji, PhD, Xue Zhang,

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Dynamic transcriptional and epigenomic reprogramming from pediatric nasal epithelial cells to induced pluripotent stem cells Hong Ji, PhD, Xue Zhang, PhD, Sunghee Oh, PhD, Christopher N. Mayhew, PhD, Ashley Ulm, BA, Hari K. Somineni, MS, Mark Ericksen, BS, James M. Wells, PhD, Gurjit K. Khurana Hershey, MD, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 1, Pages 236-244 (January 2015) DOI: 10.1016/j.jaci.2014.08.038 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Reprogramming of pediatric NECs. A, Timeline and key steps for reprogramming. B, Representative morphology of NECs before transduction and ESC-like colonies after transduction. hESCs and NEC-iPSCs before (top; scale bar = 500 μm) and after (bottom; scale bar = 100 μm) transition to feeder-free culture are also shown. C, SPT cocktail enhances reprogramming efficiency. Purple color indicates alkaline phosphatase staining. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 NEC-iPSCs express pluripotency markers through downregulation of promoter methylation. A, Nuclear expression of Oct4 and Tra-1-60 in NEC-iPSCs. DAPI, 4′-6-Diamidino-2-phenylindole dihydrochloride. B, Expression of NANOG and CK19 in ESCs, NEC-iPSCs, and nasal epithelial samples (NE). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. C, Promoter methylation of OCT4 and NANOG in samples from Fig 2, B. Data represent means ± SDs from 3 independent experiments of 2 biological samples. One-way ANOVA: **P < .01 and ****P < .001. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 NEC-iPSCs can differentiate into 3 germ layers in vitro and in vivo. A, Expression of pluripotency and germ layer markers in NEC-iPSCs (Undiff.) and embryonic bodies (EB). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. B, In vivo differentiation of NEC-iPSCs in teratomas. Tissues were derived from 3 embryonic germ layers: glandular epithelia (g; endoderm), cartilage (c; mesoderm), and pigmented neuroepithelium (ne; ectoderm). C, G-banded karyotype analysis of NEC-iPSCs. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 NEC-iPSCs have similar transcriptomes compared with ESCs. A, Hierarchic clustering of ESCs, NEC-iPSCs, cNECs, and nasal epithelial samples (NEs). B, Correlation map of samples shown in Fig 4, A. Pearson coefficients between samples were plotted. C, Overlap between the differentially expressed genes from indicated comparisons. D and E, Quantitative RT-PCR of KRT5 and SPRR2A. Data represent means ± SDs of indicated samples. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. One-way ANOVA: **P < .01 and ****P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 NEC-iPSCs have a similar methylome compared with that of ESCs. A, Hierarchic clustering of the same samples shown in Fig 4. B, Reprogramming of airway-specific markers KRT5, ARG2, and ANO3. Data represent means ± SDs of indicated samples. NE, Nasal epithelial samples. One-way ANOVA: ****P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Epigenetic memory of parental tissue persists in NEC-iPSCs. A, DNA methylation at 20 CpG sites with epigenetic memory related to epithelial function. B-D, DNA methylation at CpG sites located within the RPTN, SPRR2A, and CAT promoters. Data represent means ± SDs of duplicate experiments for 2 or more biological samples for each indicated cell type. One-way ANOVA: *P < .05, **P < .01, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 236-244DOI: (10.1016/j.jaci.2014.08.038) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions