title: Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors.
abstract: Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and Colleges of Pharmacy and Life Sciences, Nankai University, Tianjin, China.|Department of Molecular Genetics and Microbiology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Fang
authors.initials: F
authors.lastname: Bai
authors.affiliation: Department of Pathology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Chae
authors.initials: C
authors.lastname: Ho Lim
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and Colleges of Pharmacy and Life Sciences, Nankai University, Tianjin, China.|Department of Molecular Genetics and Microbiology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Jingyue
authors.initials: J
authors.lastname: Jia
authors.affiliation: Department of Pathology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Katherine
authors.initials: K
authors.lastname: Santostefano
authors.affiliation: Department of Mechanical &Aerospace Engineering College of Engineering, University of Florida, Gainesville, FL 32611.
authors.forename: Chelsey
authors.initials: C
authors.lastname: Simmons
authors.affiliation: Department of Physiology and Functional Genomics, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
authors.forename: Hideko
authors.initials: H
authors.lastname: Kasahara
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and Colleges of Pharmacy and Life Sciences, Nankai University, Tianjin, China.
authors.forename: Weihui
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Pathology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Terada
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and Colleges of Pharmacy and Life Sciences, Nankai University, Tianjin, China.|Department of Molecular Genetics and Microbiology College of Medicine, University of Florida, Gainesville, FL 32610.
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D064326:MEF2 Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; D014157:Transcription Factors