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Enhanced Reprogramming Efficiency and Kinetics of Induced Pluripotent Stem Cells Derived from Human Duchenne Muscular Dystrophy.

Teotia P, Mohanty S, Kabra M, Gulati S, Airan B - PLoS Curr (2015)

Bottom Line: Using this method, DMD-iPSCs generated on I-HFF feeders displayed pluripotent characteristics and disease genotype with improved reprogramming efficiency and kinetics over to mouse feeders.Moreover, we were able to maintain disease-specific iPSCs without additional supplementation of bFGF on I-HFF feeders.Our findings offer improvements in the generation of DMD-iPSCs and will facilitate in understanding of pathological mechanisms and screening of safer drugs for clinical intervention.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Facility, All India Institute of Medical Sciences, N. Delhi, India.

ABSTRACT

Unlabelled: The generation of disease-specific induced pluripotent stem cells (iPSCs) holds a great promise for understanding disease mechanisms and for drug screening. Recently, patient-derived iPSCs, containing identical genetic anomalies of the patient, have offered a breakthrough approach to studying Duchenne muscular dystrophy (DMD), a fatal disease caused by the mutation in the dystrophin gene. However, development of scalable and high fidelity DMD-iPSCs is hampered by low reprogramming efficiency, the addition of expensive growth factors and slow kinetics of disease-specific fibroblasts. Here, we show an efficient generation of DMD-iPSCs on bFGF secreting human foreskin fibroblast feeders (I-HFF) by employing single polycistronic lentiviral vector for delivering of transcription factors to DMD patient-specific fibroblast cells. Using this method, DMD-iPSCs generated on I-HFF feeders displayed pluripotent characteristics and disease genotype with improved reprogramming efficiency and kinetics over to mouse feeders. Moreover, we were able to maintain disease-specific iPSCs without additional supplementation of bFGF on I-HFF feeders. Our findings offer improvements in the generation of DMD-iPSCs and will facilitate in understanding of pathological mechanisms and screening of safer drugs for clinical intervention.

Key words: Duchenne Muscular Dystrophy, Reprogramming, Induced pluripotent Stem Cells, Immortalized Human Feeder, Basic Fibroblast Growth Factor, Stem Cell Cassette.

No MeSH data available.


Related in: MedlinePlus

(A) Representative images of cystic embryoid body (EB) in suspension culture (I) and their spontaneous differentiation (II) into three germ layers (magnification 10x); (B) Immunostaining of 21 days old EBs derived from D-iPSC revealed expression of ectodermal (Tuj 1 and Nestin), mesodermal (Cardiac troponin 1 and SMA), and endodermal (AFP and GATA4) marker proteins. Tuj 1, Nestin, Cardiac troponin 1, SMA and GATA4 are conjugated with fluorescein isothiocyanate (FITC); AFP protein is conjugated with Texas red. (Magnification 10x) (C) RT-PCR analysis of various differentiation markers for the three germ layers by EB mediated differentiation; D-iPSC is negative for expression. (D) Quantitative analysis of endogenous gene expression of pluripotent markers in D-iPSC EBs using qRT-PCR. Data is presented relative to D-iPSC clones. The expression is normalized to GAPDH. Error bars represent standard deviation of replicates (n=5). D-iPSC EB1, D-iPSC EB2, and D-iPSC EB3 represent embryoid bodies obtained from three different DMD-iPSCs clones.
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d35e569: (A) Representative images of cystic embryoid body (EB) in suspension culture (I) and their spontaneous differentiation (II) into three germ layers (magnification 10x); (B) Immunostaining of 21 days old EBs derived from D-iPSC revealed expression of ectodermal (Tuj 1 and Nestin), mesodermal (Cardiac troponin 1 and SMA), and endodermal (AFP and GATA4) marker proteins. Tuj 1, Nestin, Cardiac troponin 1, SMA and GATA4 are conjugated with fluorescein isothiocyanate (FITC); AFP protein is conjugated with Texas red. (Magnification 10x) (C) RT-PCR analysis of various differentiation markers for the three germ layers by EB mediated differentiation; D-iPSC is negative for expression. (D) Quantitative analysis of endogenous gene expression of pluripotent markers in D-iPSC EBs using qRT-PCR. Data is presented relative to D-iPSC clones. The expression is normalized to GAPDH. Error bars represent standard deviation of replicates (n=5). D-iPSC EB1, D-iPSC EB2, and D-iPSC EB3 represent embryoid bodies obtained from three different DMD-iPSCs clones.


Enhanced Reprogramming Efficiency and Kinetics of Induced Pluripotent Stem Cells Derived from Human Duchenne Muscular Dystrophy.

Teotia P, Mohanty S, Kabra M, Gulati S, Airan B - PLoS Curr (2015)

(A) Representative images of cystic embryoid body (EB) in suspension culture (I) and their spontaneous differentiation (II) into three germ layers (magnification 10x); (B) Immunostaining of 21 days old EBs derived from D-iPSC revealed expression of ectodermal (Tuj 1 and Nestin), mesodermal (Cardiac troponin 1 and SMA), and endodermal (AFP and GATA4) marker proteins. Tuj 1, Nestin, Cardiac troponin 1, SMA and GATA4 are conjugated with fluorescein isothiocyanate (FITC); AFP protein is conjugated with Texas red. (Magnification 10x) (C) RT-PCR analysis of various differentiation markers for the three germ layers by EB mediated differentiation; D-iPSC is negative for expression. (D) Quantitative analysis of endogenous gene expression of pluripotent markers in D-iPSC EBs using qRT-PCR. Data is presented relative to D-iPSC clones. The expression is normalized to GAPDH. Error bars represent standard deviation of replicates (n=5). D-iPSC EB1, D-iPSC EB2, and D-iPSC EB3 represent embryoid bodies obtained from three different DMD-iPSCs clones.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4638229&req=5

d35e569: (A) Representative images of cystic embryoid body (EB) in suspension culture (I) and their spontaneous differentiation (II) into three germ layers (magnification 10x); (B) Immunostaining of 21 days old EBs derived from D-iPSC revealed expression of ectodermal (Tuj 1 and Nestin), mesodermal (Cardiac troponin 1 and SMA), and endodermal (AFP and GATA4) marker proteins. Tuj 1, Nestin, Cardiac troponin 1, SMA and GATA4 are conjugated with fluorescein isothiocyanate (FITC); AFP protein is conjugated with Texas red. (Magnification 10x) (C) RT-PCR analysis of various differentiation markers for the three germ layers by EB mediated differentiation; D-iPSC is negative for expression. (D) Quantitative analysis of endogenous gene expression of pluripotent markers in D-iPSC EBs using qRT-PCR. Data is presented relative to D-iPSC clones. The expression is normalized to GAPDH. Error bars represent standard deviation of replicates (n=5). D-iPSC EB1, D-iPSC EB2, and D-iPSC EB3 represent embryoid bodies obtained from three different DMD-iPSCs clones.
Bottom Line: Using this method, DMD-iPSCs generated on I-HFF feeders displayed pluripotent characteristics and disease genotype with improved reprogramming efficiency and kinetics over to mouse feeders.Moreover, we were able to maintain disease-specific iPSCs without additional supplementation of bFGF on I-HFF feeders.Our findings offer improvements in the generation of DMD-iPSCs and will facilitate in understanding of pathological mechanisms and screening of safer drugs for clinical intervention.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Facility, All India Institute of Medical Sciences, N. Delhi, India.

ABSTRACT

Unlabelled: The generation of disease-specific induced pluripotent stem cells (iPSCs) holds a great promise for understanding disease mechanisms and for drug screening. Recently, patient-derived iPSCs, containing identical genetic anomalies of the patient, have offered a breakthrough approach to studying Duchenne muscular dystrophy (DMD), a fatal disease caused by the mutation in the dystrophin gene. However, development of scalable and high fidelity DMD-iPSCs is hampered by low reprogramming efficiency, the addition of expensive growth factors and slow kinetics of disease-specific fibroblasts. Here, we show an efficient generation of DMD-iPSCs on bFGF secreting human foreskin fibroblast feeders (I-HFF) by employing single polycistronic lentiviral vector for delivering of transcription factors to DMD patient-specific fibroblast cells. Using this method, DMD-iPSCs generated on I-HFF feeders displayed pluripotent characteristics and disease genotype with improved reprogramming efficiency and kinetics over to mouse feeders. Moreover, we were able to maintain disease-specific iPSCs without additional supplementation of bFGF on I-HFF feeders. Our findings offer improvements in the generation of DMD-iPSCs and will facilitate in understanding of pathological mechanisms and screening of safer drugs for clinical intervention.

Key words: Duchenne Muscular Dystrophy, Reprogramming, Induced pluripotent Stem Cells, Immortalized Human Feeder, Basic Fibroblast Growth Factor, Stem Cell Cassette.

No MeSH data available.


Related in: MedlinePlus