Limits...
Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

Kita-Matsuo H, Barcova M, Prigozhina N, Salomonis N, Wei K, Jacot JG, Nelson B, Spiering S, Haverslag R, Kim C, Talantova M, Bajpai R, Calzolari D, Terskikh A, McCulloch AD, Price JH, Conklin BR, Chen HS, Mercola M - PLoS ONE (2009)

Bottom Line: Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes.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.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

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.

Methodology/principal findings: 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.

Conclusion/significance: The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Show MeSH

Related in: MedlinePlus

Electrophysiological properties of Neor, Puror-selected, hESC-derived cardiomyocyte spheroids at day 20 of differentiation.(A) The dominant electrophysiological phenotypes of action potentials (APs) of cardiomyocytes recorded from day 20 CSs. (B) Some cardiomyocytes displayed more hyperpolarized MDP and faster Vmax. Left panels (a and b) show expanded time scale and right panels show 3 APs. (C) The summary table of electrophysiological parameters of action potentials.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2662416&req=5

pone-0005046-g008: Electrophysiological properties of Neor, Puror-selected, hESC-derived cardiomyocyte spheroids at day 20 of differentiation.(A) The dominant electrophysiological phenotypes of action potentials (APs) of cardiomyocytes recorded from day 20 CSs. (B) Some cardiomyocytes displayed more hyperpolarized MDP and faster Vmax. Left panels (a and b) show expanded time scale and right panels show 3 APs. (C) The summary table of electrophysiological parameters of action potentials.

Mentions: We next verified that the drug selection protocol does not adversely affect the electrophysiological phenotypes of the cardiomyocytes. Electrophysiological phenotypes of cardiomyocytes in the Neor, Puror-selected CSs at day 20 of differentiation (8 days after Puromycin treatment of day 12 EBs) were obtained by intra-cellular recording techniques (see Methods). As shown in Figure 8A, the majority of selected cardiomyocytes displayed action potentials (APs) with relatively depolarized maximal diastolic potentials (MDPs, >−45 mV) and slow maximal rate of AP depolarization (Vmax, <5 V/s). Figure 8B shows that 10–20% of cardiomyocytes, however, possess MDP<−45 mV and faster Vmax (>5 V/s). The electrophysiological parameters of these APs are summarized in Figure 8C. Overall, the electrophysiological properties of the CS cardiomyocytes were similar to those of human fetal cardiomyocytes [28].


Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

Kita-Matsuo H, Barcova M, Prigozhina N, Salomonis N, Wei K, Jacot JG, Nelson B, Spiering S, Haverslag R, Kim C, Talantova M, Bajpai R, Calzolari D, Terskikh A, McCulloch AD, Price JH, Conklin BR, Chen HS, Mercola M - PLoS ONE (2009)

Electrophysiological properties of Neor, Puror-selected, hESC-derived cardiomyocyte spheroids at day 20 of differentiation.(A) The dominant electrophysiological phenotypes of action potentials (APs) of cardiomyocytes recorded from day 20 CSs. (B) Some cardiomyocytes displayed more hyperpolarized MDP and faster Vmax. Left panels (a and b) show expanded time scale and right panels show 3 APs. (C) The summary table of electrophysiological parameters of action potentials.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662416&req=5

pone-0005046-g008: Electrophysiological properties of Neor, Puror-selected, hESC-derived cardiomyocyte spheroids at day 20 of differentiation.(A) The dominant electrophysiological phenotypes of action potentials (APs) of cardiomyocytes recorded from day 20 CSs. (B) Some cardiomyocytes displayed more hyperpolarized MDP and faster Vmax. Left panels (a and b) show expanded time scale and right panels show 3 APs. (C) The summary table of electrophysiological parameters of action potentials.
Mentions: We next verified that the drug selection protocol does not adversely affect the electrophysiological phenotypes of the cardiomyocytes. Electrophysiological phenotypes of cardiomyocytes in the Neor, Puror-selected CSs at day 20 of differentiation (8 days after Puromycin treatment of day 12 EBs) were obtained by intra-cellular recording techniques (see Methods). As shown in Figure 8A, the majority of selected cardiomyocytes displayed action potentials (APs) with relatively depolarized maximal diastolic potentials (MDPs, >−45 mV) and slow maximal rate of AP depolarization (Vmax, <5 V/s). Figure 8B shows that 10–20% of cardiomyocytes, however, possess MDP<−45 mV and faster Vmax (>5 V/s). The electrophysiological parameters of these APs are summarized in Figure 8C. Overall, the electrophysiological properties of the CS cardiomyocytes were similar to those of human fetal cardiomyocytes [28].

Bottom Line: Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes.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.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

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.

Methodology/principal findings: 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.

Conclusion/significance: The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Show MeSH
Related in: MedlinePlus