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Series GSE45274 Query DataSets for GSE45274
Status Public on Apr 22, 2013
Title Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success
Organism Mus musculus
Experiment type Expression profiling by array
Summary Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.
 
Overall design Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.
 
Contributor(s) Addis RC, Ifkovits JL, Pinto F, Kellam LD, Esteso P, Rentschler S, Christoforou N, Epstein JA, Gearhart JD
Citation(s) 23591016
Submission date Mar 19, 2013
Last update date Mar 04, 2019
Contact name Russell C Addis
E-mail(s) raddis@mail.med.upenn.edu
Organization name University of Pennsylvania
Department Cell and Developmental Biology
Lab Gearhart Lab
Street address 3400 Civic Center Blvd, Bldg 421, SCTR 9-196
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
Platforms (1)
GPL6246 [MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST Array [transcript (gene) version]
Samples (28)
GSM1100519 0F MEFs, biological rep1
GSM1100520 0F MEFs, biological rep2
GSM1100521 0F MEFs, biological rep3
Relations
BioProject PRJNA193394

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE45274_RAW.tar 128.9 Mb (http)(custom) TAR (of CEL, CHP)
Processed data included within Sample table
Processed data provided as supplementary file
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