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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.

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Related in: MedlinePlus

Pluripotency of clonal hESC reporter lines.(A–C) Outgrowths of differentiated cells from EBs derived from a clonal PGK-H2BmCherry hESC line showed coincident H2BmCherry (A) and DAPI (B) immunostaining visible in the merged fluorescent image (C). (D–I) Clonal PGK-H2BmCherry hESC-derived EBs developed endothelial (D–F) and neuronal (G–I) lineages. (J) Expression of endodermal genes alpha fetoprotein (AFP) and hPDX-1 by day 20 EBs of clonal PGK-H2BmCherry hESCs (lane 2) compared to undifferentiated cells (lane 1) and isolated human pancreatic islet tissue as positive control for hPDX-1 (lane 3) and day 20 parental H9 EBs as control for AFP (lane 3). (K) Pluripotency markers expressed by clonal PGK-H2BmCherry hESCs (lane 2) compared to no reverse transcriptase control (lane 1) and undifferentiated parental H9 cells (lane 3).
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pone-0005046-g004: Pluripotency of clonal hESC reporter lines.(A–C) Outgrowths of differentiated cells from EBs derived from a clonal PGK-H2BmCherry hESC line showed coincident H2BmCherry (A) and DAPI (B) immunostaining visible in the merged fluorescent image (C). (D–I) Clonal PGK-H2BmCherry hESC-derived EBs developed endothelial (D–F) and neuronal (G–I) lineages. (J) Expression of endodermal genes alpha fetoprotein (AFP) and hPDX-1 by day 20 EBs of clonal PGK-H2BmCherry hESCs (lane 2) compared to undifferentiated cells (lane 1) and isolated human pancreatic islet tissue as positive control for hPDX-1 (lane 3) and day 20 parental H9 EBs as control for AFP (lane 3). (K) Pluripotency markers expressed by clonal PGK-H2BmCherry hESCs (lane 2) compared to no reverse transcriptase control (lane 1) and undifferentiated parental H9 cells (lane 3).

Mentions: Engineered clonal hESCs retain pluripotency, as demonstrated by expression of hTERT, nanog, Rex-1 and Oct4 (Figure 4K) and the ability to differentiate into endothelial (Figures 4D–F), neuronal (Figures 4G–I), cardiac (see below) and endodermal (Figure 4J) lineages. Nuclear-localized H2BmCherry fluorescence is visible in each cell (compare Figures 4A,G to B,H). Taken together, these protocols and lentiviral vectors provided uniformly labeled hESCs for quantitative analyses.


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)

Pluripotency of clonal hESC reporter lines.(A–C) Outgrowths of differentiated cells from EBs derived from a clonal PGK-H2BmCherry hESC line showed coincident H2BmCherry (A) and DAPI (B) immunostaining visible in the merged fluorescent image (C). (D–I) Clonal PGK-H2BmCherry hESC-derived EBs developed endothelial (D–F) and neuronal (G–I) lineages. (J) Expression of endodermal genes alpha fetoprotein (AFP) and hPDX-1 by day 20 EBs of clonal PGK-H2BmCherry hESCs (lane 2) compared to undifferentiated cells (lane 1) and isolated human pancreatic islet tissue as positive control for hPDX-1 (lane 3) and day 20 parental H9 EBs as control for AFP (lane 3). (K) Pluripotency markers expressed by clonal PGK-H2BmCherry hESCs (lane 2) compared to no reverse transcriptase control (lane 1) and undifferentiated parental H9 cells (lane 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005046-g004: Pluripotency of clonal hESC reporter lines.(A–C) Outgrowths of differentiated cells from EBs derived from a clonal PGK-H2BmCherry hESC line showed coincident H2BmCherry (A) and DAPI (B) immunostaining visible in the merged fluorescent image (C). (D–I) Clonal PGK-H2BmCherry hESC-derived EBs developed endothelial (D–F) and neuronal (G–I) lineages. (J) Expression of endodermal genes alpha fetoprotein (AFP) and hPDX-1 by day 20 EBs of clonal PGK-H2BmCherry hESCs (lane 2) compared to undifferentiated cells (lane 1) and isolated human pancreatic islet tissue as positive control for hPDX-1 (lane 3) and day 20 parental H9 EBs as control for AFP (lane 3). (K) Pluripotency markers expressed by clonal PGK-H2BmCherry hESCs (lane 2) compared to no reverse transcriptase control (lane 1) and undifferentiated parental H9 cells (lane 3).
Mentions: Engineered clonal hESCs retain pluripotency, as demonstrated by expression of hTERT, nanog, Rex-1 and Oct4 (Figure 4K) and the ability to differentiate into endothelial (Figures 4D–F), neuronal (Figures 4G–I), cardiac (see below) and endodermal (Figure 4J) lineages. Nuclear-localized H2BmCherry fluorescence is visible in each cell (compare Figures 4A,G to B,H). Taken together, these protocols and lentiviral vectors provided uniformly labeled hESCs for quantitative analyses.

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