Electromagnetic Fields Mediate Efficient Cell Reprogramming Into a Pluripotent State

Transcription

1 Electromagnetic Fields Mediate Efficient Cell Reprogramming Into a Pluripotent State Soonbong Baek, 1 Xiaoyuan Quan, 1 Soochan Kim, 2 Christopher Lengner, 3 Jung- Keug Park, 4 and Jongpil Kim 1* 1 Lab of Stem Cells and Cell Reprogramming, Department of Biomedical Engineering, Dongguk University, Seoul, , Korea 2 Department of Electrical and Electronic Engineering, Hankyong National University, Kyonggi-do, , Korea 3 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania 4 Dongguk University Research Institute of Biotechnology, Dongguk University, Seoul , Korea Supplementary data

2 Figure S1.

3 Supplementary Figure 1. EL-EMF exposure enhances cell reprogramming. (A) Screening of optimal frequency of EL-EMF for efficient reprogramming. The number of alkaline phosphatase (AP) positive colonies were counted 15days after infection under the different frequencies. Equal numbers of 4 factor infected cells were plated but treated with the different conditions. Three independent experiments of three sets each were performed. Data represent mean±sem. Scale Bars: 100µm. (B) RT-PCR analysis of pluripotency markers, Esrrb, Fbx15, Rex1 and Zfp296 of OSKM infected fibroblasts in the presence and absence of EL-EMF 15 days after dox induction. Three independent experiments of three sets each were performed. Data represent mean±sem. (C) FACS analysis for Nanog-eGFP positive ips cells derived from Nanog-eGFP KI fibroblasts in the absence and presence of EL-EMF exposure 15 days after dox induction. (D) EL-EMF derived dox independent ips cells (E-iPSCs). After 50 passages, GFP-positive colonies were maintained. Scale Bars: 100µm. (E) Expression of pluripotency genes, Oct4, Sox2 and Nanog in established ipsc lines by 4 factor combined with EL-EMF exposure correspond to those of mescs. Two independent experiments of three sets each were performed. Data represent mean.

4 Figure S2.

5 Supplementary Figure 2. Characterization of EL-EMF induced ipscs (A) Bisulfite sequencing of Oct4 and Nanog gene promoters in fibroblasts, mes cells, control ipsc and EL-EMF derived ipscs. (B) The representative image and number of AP positive colonies from 4 factor reprogramming combined with different conditions: VPA, Vitamin C and EL-EMF exposure. Equal numbers of 4 factor infected cells were plated but treated with the different conditions, EL-EMF exposure result in the highest induction of AP+ cells at day 15. Three independent experiments of three sets each were performed. Data represent mean±sem. Scale Bars: 100µm. (C) Teratoma assay indicates E-iPSC can differentiate to ectoderm, mesoderm and ectoderm. (D) Germ line transmission of EL-EMF induced ipscs. Mating of offspring from chimera to a C57BL/6J female generate agouti pups and germline transmission confirmed by Oct4-eGFP PCR analysis.

6 Figure S3.

7 Supplementary Figure 3. Characterization of Oct4 & EL-EMF induced ipscs. (A) Phase contrast of Oct4/Sox2 infected reprogramming in the absence and presence of EL-EMF exposure 30 days after dox treatment. (B) Immunofluorescence staining of Oct4 and H3K27me3 at 30 days and 45 days after infection. Scale Bars: 100µm. (C) Quantitative RT-PCR for pluripotency genes Ecat1, Fbx15, Gdf3, and Zfp296 in Oct4 and EL-EMF induced ipscs. Three independent experiments of three sets each were performed. Data represent mean±sem. (D) Chimera derived from the Oct4 and EL-EMF induced ipscs.

8 Figure S4.

9 Supplementary Figure 4. Histone modifications by EL-EMF exposure during reprogramming. (A) The time course of mrna expressions for Mll2 and Oct4 during reprogramming under normal condition (control) and EL-EMF exposure. Three independent experiments of three sets each were performed. Data represent mean±sem. (B) The expression of Mll2 in fibroblasts in the EL-EMF exposure. Three independent experiments of three sets each were performed. Data represent mean±sem. (C) Western blot for Mll2 and H3K4me3 in fibroblasts after 4days EL- EMF exposure without OSKM. (D) The number of positive cells shown in Figure 4E in control fibroblasts, and secondary fibroblasts in the presence and absence of EL- EMF exposure. Three independent experiments of three sets each were performed. Data represent mean±sem. Data represent mean±sem. Student t-test, **p<0.01. (E) Representative image of Oct4-eGFP positive colonies at 10 and 15 days after OSKM infection. Scale Bars: 100µm.

10 Figure S5.

11 Supplementary Figure 5. Mll2 facilitates efficient cell reprogramming. (A) Protein level for Mll2 and H3K4me3 of secondary fibroblasts, control shrna, shmll2 and Mll2 overexpression infected cell at day 6 after dox treatment. (B) The number of Nanog positive colonies from OSKM infected fibroblasts in the overexpression and inhibition of Mll2. Three independent experiments of three sets each were performed. Data represent mean±sem. (C) Representative image of Nanog positive colonies from reprogrammed cell under p53 Knockout, EL-EMF exposure and Mll2 knockdown. Scale Bars: 100µm.

12 Figure S6.

13 Supplementary Figure 6. EMF cancellation system. (A) Design of the 3 axes helmholtz coil to generate zero electromagnetic field. (B) Distance between two coils maintains equal to radius of coil in order to have the uniform magnetic field distribution inside of coil. (C) Implemented 3-axis magnetic cancellation system. 3- axis sensor in the center of a helmholtz coil and a 3-dimensional EMF generator generated reverse magnetic fields to keep a magnetic field-free space by adjusting the voltage across the coil with a power supply.

14 Figure S7.

15 Supplementary Figure 7. Somatic cell reprogramming under EMF free system. (A) Morphology and immunofluorescence staining of pluripotency markers, Oct4 and Nanog of mescs (V6.5) in the presence and absence of earth s magnetic field. No change observed between two conditions. Scale Bars: 100µm. (B) Quantitative RT- PCR analysis for Oct4 expression in ES cells cultured in the EMF free conditions. (C) The number of fibroblasts in the presence and absence of EMF. No differences between the cells in normal and EMF free condition. Three independent experiments of three sets each were performed. Data represent mean±sem. (D) qrt-pcr analysis of cell cycle related genes, Ccnd1, Cyr61, Dazac1, Id4, and Nat5 in the reprogramming under EMF free condition. Expression levels were normalized to GAPDH. Data are presented as mean ± SEM; n = 3.

16 Figure S8. Supplementary Figure 8. Schematic representation of EL-EMF mediated reprogramming. (A) EL-EMF exposure promotes cell reprogramming via the enrichment of H3K4me3 histone modification through activation of Mll2.