Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.
BACKGROUND
Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.


RESULTS
Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.


CONCLUSIONS
The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.
Burnham Institute for Medical Research, La Jolla, California, United States of America.
Hiroko
H
Kita-Matsuo
Maria
M
Barcova
Natalie
N
Prigozhina
Nathan
N
Salomonis
Karen
K
Wei
Jeffrey G
JG
Jacot
Brandon
B
Nelson
Sean
S
Spiering
René
R
Haverslag
Changsung
C
Kim
Maria
M
Talantova
Ruchi
R
Bajpai
Diego
D
Calzolari
Alexey
A
Terskikh
Andrew D
AD
McCulloch
Jeffrey H
JH
Price
Bruce R
BR
Conklin
H S Vincent
HS
Chen
Mark
M
Mercola
D000328:Adult; D001483:Base Sequence; D002454:Cell Differentiation; D017931:DNA Primers; D004351:Drug Resistance; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005326:Fetal Proteins / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics; D000098908:Brachyury Protein
D017931:DNA Primers; D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein; C485184:enhanced green fluorescent protein