Fibroblast transition to an endothelial “trans” state improves cell reprogramming efficiency

Abstract

Fibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p < 0.01). Cardiac fibroblasts treated with ETV2 demonstrated increased endothelial cell markers, and when then treated with GMT yielded greater prevalence of cells expressing cardiomyocyte markers including cTnT than did fibroblasts treated with GMT or ETV2 (10.3% ± 0.2% vs 1.7% ± 0.06% and 0.6 ± 0.03, p < 0.01). Rat cardiac fibroblasts treated with GMT + ETV2 demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes, whereas cells treated with GMT or ETV2 alone failed to contract in co-culture experiments. Human cardiac fibroblasts treated with ETV2 and then GMT likewise demonstrated greater prevalence of cTnT expression than did cells treated with GMT alone (2.8-fold increase, p < 0.05). Cardiac fibroblast transitioning through a trans-endothelial state appears to enhance cardio-differentiation by enhancing fibroblast plasticity.

p63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation

“…For cell contractility co-culture studies, cardiomyocytes were isolated from 0 to 3 day old rat pups under protocol AN-6223, as previously described 28 – 30 . Human cardiac fibroblasts were treated with GFP-labeled reprogramming factors (GMT, shp63+ H/M, p63-TID+ H/M) and one week after treatment as described above, cells were harvested and re-plated onto neonatal rat cardiomyocytes at a ratio of 1:10 and cultured in DMEM/M-199/10% FBS medium 31 .…”

“…Immunofluorescence (IF) staining was performed using cells fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton‐X solution, as previously described 8 – 10 , 28 . After these cells were blocked with 10% goat serum, they were incubated with primary antibodies against cTnT (1:300 dilution; Thermo Fisher Scientific), or α-actinin (1:300 dilution; Sigma-Aldrich) followed by incubation with appropriate Alexa fluorogenic secondary antibodies (Invitrogen™).…”

“…Ca 2+ transients were expressed as the 340/380‐nm ratios of the resulting 510‐nm emissions. Data were analyzed using Felix software (Photon Technology International) 8 , 9 , 28 – 30 .…”

“…Lentivirus vectors each encoding Gata4, Mef2, or Tbx5 (GMT), Hand2/Myocardin (H/M), non-targeting (NT) shRNA, p63 short hairpin RNA (Origene, Rockville, MD), p63 -transactivation inhibitory domain (Vectorbuilder, Chicago, IL) tagged with green fluorescent protein (GFP) or GFP control vectors were prepared from relevant plasmids by the Baylor College Of Medicine Gene Vector Core, as previously described 9 , 10 , 27 , 28 .…”

“…Fluorescence-activated cell sorting (FACS) was performed as previously described 8 – 10 , 27 , 28 . Briefly, cells adherent to culture dishes were first washed with DPBS and trypsinized with 0.25% trypsin/EDTA.…”