Limits...
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

Gene transfer to Pompe disease iPSC. (a) Vector construct. GAA was cloned into downstream of EF-1-α promoter. (b) GAA enzyme assay of transfected iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Transfection is conducted at the multiplicity of infection of 0, 10, 50, and 100. Data were expressed as means ± SEM. (c) Glycogen assay was conducted in iPSCs (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. (d) Immunofluoroscence of transfected iPSCs (Pompe1, Pompe2, and Pompe3). GAA (Alexa568) were stained with DAPI. Scale bar, 100 µm. (e) Immunofluoroscence of transfected iPSCs (Pompe1) GAA (Alexa568) and LAMP-2 (Alexa488) were stained to confirm colocalization. Scale bar, 100 µm. (f) Electron microscopy of iPSCs after lentiviral GAA transfer. Upper scale bar, 5 µm, lower scale bar, 1 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495721&req=5

fig4: Gene transfer to Pompe disease iPSC. (a) Vector construct. GAA was cloned into downstream of EF-1-α promoter. (b) GAA enzyme assay of transfected iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Transfection is conducted at the multiplicity of infection of 0, 10, 50, and 100. Data were expressed as means ± SEM. (c) Glycogen assay was conducted in iPSCs (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. (d) Immunofluoroscence of transfected iPSCs (Pompe1, Pompe2, and Pompe3). GAA (Alexa568) were stained with DAPI. Scale bar, 100 µm. (e) Immunofluoroscence of transfected iPSCs (Pompe1) GAA (Alexa568) and LAMP-2 (Alexa488) were stained to confirm colocalization. Scale bar, 100 µm. (f) Electron microscopy of iPSCs after lentiviral GAA transfer. Upper scale bar, 5 µm, lower scale bar, 1 µm.

Mentions: Third-generation lentiviral vector which express GAA has been generated (Figure 4a). Then we have infected lentiviral vector to Pompe disease iPSCs at the multiplicity of infection of 0, 10, 50, and 100. GAA enzyme activity was increased in dose-dependent manners (Figure 4b). Glycogen contents were significantly decreased by GAA transduction by lentiviral vector only in the highest multiplicity of infection (P < 0.01). Glycogen contents were not normalized in treated iPSCs within 48 hours (Figure 4c).


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)

Gene transfer to Pompe disease iPSC. (a) Vector construct. GAA was cloned into downstream of EF-1-α promoter. (b) GAA enzyme assay of transfected iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Transfection is conducted at the multiplicity of infection of 0, 10, 50, and 100. Data were expressed as means ± SEM. (c) Glycogen assay was conducted in iPSCs (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. (d) Immunofluoroscence of transfected iPSCs (Pompe1, Pompe2, and Pompe3). GAA (Alexa568) were stained with DAPI. Scale bar, 100 µm. (e) Immunofluoroscence of transfected iPSCs (Pompe1) GAA (Alexa568) and LAMP-2 (Alexa488) were stained to confirm colocalization. Scale bar, 100 µm. (f) Electron microscopy of iPSCs after lentiviral GAA transfer. Upper scale bar, 5 µm, lower scale bar, 1 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Gene transfer to Pompe disease iPSC. (a) Vector construct. GAA was cloned into downstream of EF-1-α promoter. (b) GAA enzyme assay of transfected iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Transfection is conducted at the multiplicity of infection of 0, 10, 50, and 100. Data were expressed as means ± SEM. (c) Glycogen assay was conducted in iPSCs (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. (d) Immunofluoroscence of transfected iPSCs (Pompe1, Pompe2, and Pompe3). GAA (Alexa568) were stained with DAPI. Scale bar, 100 µm. (e) Immunofluoroscence of transfected iPSCs (Pompe1) GAA (Alexa568) and LAMP-2 (Alexa488) were stained to confirm colocalization. Scale bar, 100 µm. (f) Electron microscopy of iPSCs after lentiviral GAA transfer. Upper scale bar, 5 µm, lower scale bar, 1 µm.
Mentions: Third-generation lentiviral vector which express GAA has been generated (Figure 4a). Then we have infected lentiviral vector to Pompe disease iPSCs at the multiplicity of infection of 0, 10, 50, and 100. GAA enzyme activity was increased in dose-dependent manners (Figure 4b). Glycogen contents were significantly decreased by GAA transduction by lentiviral vector only in the highest multiplicity of infection (P < 0.01). Glycogen contents were not normalized in treated iPSCs within 48 hours (Figure 4c).

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