Inhibition of TGFβ signaling increases direct conversion of fibroblasts to induced cardiomyocytes.

Ethics Statement

All animal work was conducted under a protocol (804335) approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Primary Cell Isolation

Mouse embryonic fibroblasts

(MEFs, isolated at E14.5) were prepared as previously described [5] . Briefly, embryos were harvested from mice of mixed background at 14.5 dpc followed by decapitation and removal of internal visceral organs, including the heart. The tissue was minced and digested with trypsin and trituration. Cells were resuspended in MEF medium (10% FBS and 2 mM L-Glutamine) and plated onto one 10 cm dish per embryo. After 24 hours, cells were passaged at 1∶3 (passage 1). MEFs were used at passages 3–5 for all reprogramming experiments. Adult mouse cardiac fibroblasts were prepared as previously described [5] . Hearts were removed from mice (8–12 weeks in age) and minced in cold PBS. The tissue was digested in 4 mg mL −1 collagenase IV (Sigma) and 10 U mL −1 deoxyribonuclease I (Worthington Biochemical Corporation) and agitation at 37°C for 10 minutes. Samples were spun down and resuspended in TrypLE (Invitrogen) at 37°C with agitation. After 5 minutes, medium (DMEM supplemented with 15% FBS, 1% NEAA) was added and the resulting solution was plated onto gelatin coated 6-well plates. When confluent, the cells were passaged after filtration through a 40 µM filter at 1∶1 to a gelatin coated 10 cm dishes (passage 1). Cells were then passaged 1∶5 and frozen when confluent. Cardiac fibroblasts were used at passage 3 for reprogramming experiments. Plasmid Information All plasmids were constructed as previously described [5] and can be found on Addgene using the following catalog numbers: Troponin T-GCaMP5-Zeo (46027), tetO-Hand2 (46028), tetO-NKX2.5 (46029), tetO-GATA4 (46030), tetO-MEF2C (46031), tetO-TBX5 (46032). Plasmids that were used from Addgene also include: FUdeltaGW-rtTA (19780), psPAX2 (12260), pMD2.G (12259), and PGK-H2B-mCherry (21217). All plasmids were amplified in STBL3 bacteria (Invitrogen) and prepared with Qiagen MidiPrep Kits.

Lentivirus Preparation

Lentiviral vectors were packaged into Lenti-X 293T cells (Clontech) using Lipofectamine 2000 (Invitrogen) to deliver 12 µg of the lentiviral backbone plasmid, 7.7 µg psPAX2, and 4.3 µg pMD2.G in 3 mL OPTI-MEM (Invitrogen) to ∼90% confluent 10 cm plates of 293T cells with 10 mL of fresh MEF medium. Viral supernatant was collected at 24 and 48 hours post-transfection (total ∼23 mL), filtered using a 0.45 µM filter (Millipore), aliquots were prepared and frozen at −80°C until use. Viral titer was determined using Lenti-X GoStix (Clontech) and only lentiviruses with a minimum titer of 5×10 5 IFU mL −1 were used for reprogramming experiments. Direct Conversion of Fibroblasts to iCMs Direct conversion of MEFs and cardiac fibroblasts was completed using a protocol similar to that previously described [5] , as shown in Figure 1A . Briefly, glass bottom 12-well plates (MatTek) were coated with poly-L-Lysine solution overnight followed by incubation with MEF medium for 1 hour prior to seeding. At Day -2 cells were dissociated using TrypLE and plated at 30k (∼7.5 k cm −2 ) per well with 250 µL of each of the FUdeltaGW-rtTA and Troponin T-GCaMP5-ZEO reporter lentivirus as well as 500 µL MEF medium (1 mL total per well). On Day-1, the culture medium was replaced with 250 µL MEF medium and 250 µL of each tetO-transcription factor lentivirus (1.5 mL total) as well as any candidate small molecules or vehicle controls (see Table 1 ) where indicated. After another 24 hours (Day 0), the media was changed to Reprogramming medium with doxycycline (2 µg mL −1 ) and the PGK-H2B-mCherry lentivirus. Reprogramming Medium consists of AGM (Lonza, CC-3186) without EGF and supplemented with 2 µg mL −1 doxycycline (Sigma). Cells were also transduced with 200 µL of PGK-H2B-mCherry lentivirus for constitutive expression of nuclear-localized mCherry expression. Reprogramming medium was changed every 2–3 days. For microarray analysis, cells were plated into standard tissue-culture treated 12 well plates (BD Falcon) using the same volumes and protocol described above. For immunocytochemistry, the identical protocol was followed, except cells were plated onto 12 mm diameter poly-L-Lysine coated glass coverslips in 24 well plates (1 coverslip per well) and using half of the volumes described above for all steps. 10.1371/journal.pone.0089678.g001 Figure 1 Direct conversion of mouse embryonic fibroblasts to iCMs can be influenced by treatment with small molecules. Schematic of direct reprogramming strategy of MEFs and timeline (A). Percentage of total cells with flashing Troponin T-GCaMP activity for control and small molecule treated MEFs (B). Representative immunocytochemistry images for HNGMT +DMSO (C), or +BIX (D), +CHIR+XAV (E), and +SB (F) treatments for cardiac Troponin T (green) and nuclei (red). * indicates p