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Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient.

Sato Y, Kobayashi H, Higuchi T, Shimada Y, Era T, Kimura S, Eto Y, Ida H, Ohashi T - Mol Ther Methods Clin Dev (2015)

Bottom Line: Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC.The efficacy of gene therapy is maintained following the cardiomyocyte differentiation.Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Jikei University School of Medicine , Tokyo, Japan ; Division of Gene Therapy, Research Center for Medical Sciences, The Jikei University School of Medicine , Tokyo, Japan.

ABSTRACT
Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

No MeSH data available.


Related in: MedlinePlus

Cardiomyocyte differentiation of Pompe disease iPSC. (a) Differentiation protocol. (b) Phase contrast microscopy of derived cardiomyocyte from Control and Pompe-1 iPSC. Scale bar, 100 µm. (c) RT-PCR of differentiated cardiomyocyte. MLC-2A, MLC-2V, NKX2.5, MYH6, MYH7, Troponin I, and GAPDH were analyzed in cardiomyocyte (Control, Pompe1, Pompe2, and Pompe3). (d) Immunofluoroscence of differentiated cardiomyocte (Control and Pompe1). Cardiac troponin T (Alexa488) and DAPI were stained after 4% PFA fixation. Scale bar, 100 µm. (e) Electron microscopy of cardiomyocyte (Control and Pompe1). Black arrow is indicating sarcomeric cardiac fiber connected with gap junction and white arrow is indicating enlarged lysosome. Scale bar, 1 µm.
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fig3: Cardiomyocyte differentiation of Pompe disease iPSC. (a) Differentiation protocol. (b) Phase contrast microscopy of derived cardiomyocyte from Control and Pompe-1 iPSC. Scale bar, 100 µm. (c) RT-PCR of differentiated cardiomyocyte. MLC-2A, MLC-2V, NKX2.5, MYH6, MYH7, Troponin I, and GAPDH were analyzed in cardiomyocyte (Control, Pompe1, Pompe2, and Pompe3). (d) Immunofluoroscence of differentiated cardiomyocte (Control and Pompe1). Cardiac troponin T (Alexa488) and DAPI were stained after 4% PFA fixation. Scale bar, 100 µm. (e) Electron microscopy of cardiomyocyte (Control and Pompe1). Black arrow is indicating sarcomeric cardiac fiber connected with gap junction and white arrow is indicating enlarged lysosome. Scale bar, 1 µm.

Mentions: We have differentiated iPSCs into cardiomyocyte according to the differentiation protocol shown in Figure 3a. Robust differentiation was also possible in healthy control and Pompe disease iPSCs (Figure 3b). Beating cardiomyocyte was observed around 10 days after the differentiation in healthy control and patient specific iPSCs (Supplementary Movies S1 and S2).


Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient.

Sato Y, Kobayashi H, Higuchi T, Shimada Y, Era T, Kimura S, Eto Y, Ida H, Ohashi T - Mol Ther Methods Clin Dev (2015)

Cardiomyocyte differentiation of Pompe disease iPSC. (a) Differentiation protocol. (b) Phase contrast microscopy of derived cardiomyocyte from Control and Pompe-1 iPSC. Scale bar, 100 µm. (c) RT-PCR of differentiated cardiomyocyte. MLC-2A, MLC-2V, NKX2.5, MYH6, MYH7, Troponin I, and GAPDH were analyzed in cardiomyocyte (Control, Pompe1, Pompe2, and Pompe3). (d) Immunofluoroscence of differentiated cardiomyocte (Control and Pompe1). Cardiac troponin T (Alexa488) and DAPI were stained after 4% PFA fixation. Scale bar, 100 µm. (e) Electron microscopy of cardiomyocyte (Control and Pompe1). Black arrow is indicating sarcomeric cardiac fiber connected with gap junction and white arrow is indicating enlarged lysosome. Scale bar, 1 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495721&req=5

fig3: Cardiomyocyte differentiation of Pompe disease iPSC. (a) Differentiation protocol. (b) Phase contrast microscopy of derived cardiomyocyte from Control and Pompe-1 iPSC. Scale bar, 100 µm. (c) RT-PCR of differentiated cardiomyocyte. MLC-2A, MLC-2V, NKX2.5, MYH6, MYH7, Troponin I, and GAPDH were analyzed in cardiomyocyte (Control, Pompe1, Pompe2, and Pompe3). (d) Immunofluoroscence of differentiated cardiomyocte (Control and Pompe1). Cardiac troponin T (Alexa488) and DAPI were stained after 4% PFA fixation. Scale bar, 100 µm. (e) Electron microscopy of cardiomyocyte (Control and Pompe1). Black arrow is indicating sarcomeric cardiac fiber connected with gap junction and white arrow is indicating enlarged lysosome. Scale bar, 1 µm.
Mentions: We have differentiated iPSCs into cardiomyocyte according to the differentiation protocol shown in Figure 3a. Robust differentiation was also possible in healthy control and Pompe disease iPSCs (Figure 3b). Beating cardiomyocyte was observed around 10 days after the differentiation in healthy control and patient specific iPSCs (Supplementary Movies S1 and S2).

Bottom Line: Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC.The efficacy of gene therapy is maintained following the cardiomyocyte differentiation.Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Jikei University School of Medicine , Tokyo, Japan ; Division of Gene Therapy, Research Center for Medical Sciences, The Jikei University School of Medicine , Tokyo, Japan.

ABSTRACT
Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

No MeSH data available.


Related in: MedlinePlus