Figure S1. Chemical BET inhibition enhances human iPSC reprogramming, related to Figure 1. (A) Effect of JQ1 dosage on reprogramming efficiency.
Cell Reports, Volume 16
Supplemental Information
Reprogramming by De-bookmarking the Somatic Transcriptional Program through Targeting of BET Bromodomains Zhicheng Shao, Chunping Yao, Alireza Khodadadi-Jamayran, Weihua Xu, Tim M. Townes, Michael R. Crowley, and Kejin Hu
Figure S1. Chemical BET inhibition enhances human iPSC reprogramming, related to Figure 1. (A) Effect of JQ1 dosage on reprogramming efficiency. Reprogramming BJ cells were treated wit JQ1 at concentrations indicated from day 3 to 14. (B) Effect of treatment time on reprogramming. 50 nM JQ1 were used for the indicated time. (C) Distinct BET chemical inhibitors all enhances reprogramming. OSK-transduced BJ cells were treated with JQ1 (50 nM), iBET151 (250 nM), or CPI203 (50 nM) from day 3 to 14. (D) DMSO and the inactive (-)-JQ1 do not exhibit reprogramming activity. (E, F) Chemical BET inhibition impaired the growth of human embryonic stem cells (H1). Shown are growth curve (E) and representative images on day 3 of JQ1 treatment (F). Bars, 200 µM. (G) Combination of three BET chemical inhibitors phenocopied negative effect of high concentration of JQ1 on reprogramming. OSK-transduced BJ cells were treated with JQ1 (50 nM), iBET151 (100 nM), or CPI203 (50 nM), or a combination of JQ1/CPI203/iBET151 from day 3 to 14. A, B, and C are representative images of TRA-160 staining of the reprogramming dishes on day 21.
Figure S2. JQ1 enhances human iPSC reprogramming with 4 factors, Related to Figure 1. (A) Reprogramming fold changes relative to OSK reprogramming. Fold changes were calculated based on numbers of TRA-1-60+ colonies on day 21 or 30 of reprogramming. JQ1 was used from day 3 to 14 of reprogramming at 50 nM. n=3; ***, p < 0.001. (B) Representative images of the reprogramming wells stained for TRA-1-60.
Figure S3. Synergistic effect of JQ1 and OSK in downregulation of fibroblast transcriptions, Related to Figure 3. (A) Venn diagram showing downregulation of 510 fibroblast transcriptions by OSK overexpression for two days. The black circle encloses genes with higher expression levels in human fibroblasts compared to expression levels in human embryonic stem cells. Red circle contains genes downregulated by OSK overexpression in BJ fibroblasts for 48 hours. (B) Venn diagram showing the overlap of genes in BJ fibroblasts downregulated by OSK expression and by OSK plus JQ1 treatment (50 nM, 48 hours). Red, genes downregulated by OSK; Cyan, fibroblast genes downregulated by OSK expression plus JQ1 treatment. (C) Ladder plots showing synergistic effect of JQ1 and OSK in downregulation of the 104 fibroblast transcriptions identified in B. (D) Heat map presentation of the same results in C. (E) Venn diagram showing total number of overlapping genes downregulated by JQ1 in reprogramming cells (black) and naïve cells (red). (F) Venn diagram showing number of fibroblastspecific genes downregulated in reprogramming cells (black) and in naïve cells (red).
Figure S4. BET inhibitions result in a loss of fibroblast morphology, Related to Figure 3. Images were BJ cells overexpressing reprogramming factors OSK treated with the indicated chemicals and concentrations for 5 days. Please note the predominant polygonal cells in JQ1, I-BET151 and CPI203 treatments, while the majority of cells in ()-JQ1 treatments remain spindle in shape. Many cells even become round (arrow heads) in reprogramming dishes with BET inhibitions. Bars, 100 µM.
Supplemental Experimental Procedures Cell culture Human fibroblasts (BJ, ATCC, CRL-2522) were cultured in fibroblast medium: DMEM, 10% heat-inactivated FBS, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM MEM NEAA and 4 ng/ml human bFGF. Human ESCs (H1 and H9, WiCell, Wisconsin) and iPSCs were maintained in E8 medium on Matrigel-coated tissue culture vessels. E8 medium contained DMEM/F12, 64 mg/L L-ascorbic acid 2-phosphate sesquimagnesium, 13.6 µg /L sodium selenium, 1.7 g/L NaHCO3, 1 g/L sodium chloride, 4 µg/L FGF2, 20 mg/L insulin, 10.7 mg/L, transferrin and 2 µg/L recombinant hTGFβ1. Lentiviral particle preparation Lenti-XTM 293T cells (Clontech, Cat#, 632180) were evenly seeded at 1×107 per 150-mm dish coated with Collagen I (5 µg/cm2, BD Bioscience, cat#, 354236). The cells were cultured for 24 hours in 25 ml of 293T medium [DMEM, Gibco, Cat#, 12800-058, 10% FBS (Gibco, Cat#, 10437 or 26140), 4 mM L-Glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco, Cat#, 15140-122), 0.1 mM MEM NEAA (Gibco, Cat#, 11140050)]. At 24 hour, the medium was replaced 1-3 hours prior to transfection with 20 ml of pre-warmed fresh 293T medium. Sixty micrograms of total plasmid DNA (10.5 µg pMD2.G envelop plasmid, 19.5 µg psPAX2 packaging plasmid, 30 µg transfer plasmid) were added in 1.5 ml of 0.25 M calcium chloride solution, and mixed well. The plasmid DNA in calcium solution was mixed with an equal volume (1.5 ml) of 2×HBS (pH 7.07), and was mixed by gently pipetting 15-25 times using a 5-ml pipette. The DNA complex was immediately added into the culture media drop by drop, and the plate was swirled to disperse the DNA over the dish. The cells were incubated for 16-18 hours at 37 °C in 5% CO2. The supernatant was removed, and 25 ml of fresh DMEM containing sodium butyrate (10 mM) was gently added to the dish. Cells were cultured at 37 °C, 5% CO2. Virus-containing supernatant was collected between 48 hours to 72 hours for virus concentration. Concentration of lentiviruses The reprogramming viruses (OCT4, SOX2, and KLF4) were concentrated, and were stored at -80 °C till use. The collected lentiviral supernatant was centrifuged at 3,000×g for 10 minutes at 4 °C to remove the cell debris. Thirty ml of the viral supernatant was transferred into a sterile 50-ml tube. Added to the each viral tube, were 7 ml of 50% PEG-6000 stock solution (final concentration of 8.5%) and 4.1 ml of 4 M NaCl stock solution (final concentration of 0.4 M). Viral particles were precipitated at 4 ℃ for 3-5 hours with intermittent mixing every 20–30 min. Viral particles were collected by centrifugation at 4,000×g for 30 min at 4 ℃. Liquid was carefully decanted. Viral particles were re-suspended with Tris-HCl buffer (50 mM, pH 7.4) at 1/100 to 1/150 the volume of the original viral supernatant. Re-suspended viral particles were aliquoted and stored at -80 ℃. Viral titers were determined with flow cytometry based on GFP expression in Hela cells transduced with viral stocks. Immunocytochemistry and Microscopy Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. The fixed cells were then blocked with 0.1% Triton X-100, 1% BSA in PBS at room temperature for 30 min. After washed 3 times with PBS, cells were incubated with primary antibody overnight at 4 °C. Unbound primary antibody was washed away 3 times, and cells were incubated with appropriate secondary antibody at room temperature in the dark for one hour. After washed with PBS, cell nuclei were stained with DAPI (2 µg/ml) at room temperature for 5-10 min. Fluorescence microscopy was carried out on Olympus IX51 equipped with CellSens software for image acquisition. Antibodies used are TRA-1-60 (Millipore, MAB4360), TRA-1-81 (Millipore, MAB4381), human NANOG (BD, 560109), SOX2 (BD, 561469), and OCT3/4 (BD, 561555). TRA-1-60-HRP staining of hES/iPS cells Cells in reprogramming wells were fixed with 4% PFA or cold methanol for 10-15 min at room temperature, and then washed 3 times with PBS. The fixed cells were immunostained with biotin-conjugated anti-human TRA-160 (eBioscience, Cat#, 13-8863-82) at 1:200 dilutions in staining buffer (DPBS, 3% FBS, 0.3% triton) (5 ml
will cover a 10-cm plate), at 4 °C overnight. After washed 5 times with PBS for 2 min each wash, cells were incubated with streptavidin–HRP (Biolegend Cat#, 405210) diluted 1:500 in staining buffer, at room temperature for 2 hours. Stained cells were washed 5 times with PBS for 2 min each wash. Color development was performed using Vector Laboratories DAB kit (Cat#, SK-4100) for around 15 min. The stained cells were then washed 3 times with PBS before imaging and colony counting. Teratoma Assays UAB Institutional Animal Care and Use Committee (IACUC) approved the use of mice, and the animal protocols comply with ethical regulations .The iPSC lines were cultured on matrigel-coated vessels with E8 medium. At 80% confluence, iPSCs were harvested using the EDTA method. iPSCs were re-suspended at 106 cells in 100 µl of cold E8 media containing 30% Matrigel. iPSCs were injected subcutaneously into one flank of a mouse. After 6 to 8 weeks, teratomas were harvested and fixed in formaldehyde. Histology was performed at UAB Comparative Pathology Laboratory.
