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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

Characterization of iPS cell lines. (a) Reverse transcription polymerase chain reaction of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, DPPA4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were analyzed. (b) Immunofluoroscence of iPS cell lines (Pompe-1, Pompe2, Pompe3, and control). ALP stain, SSEA-4 (Alexa488), Tra-1–60 (Alexa488), and Tra-1–81 (Alexa488) were analyzed. Scale bar, 100 µm. (c) RT-PCR of embryoid body from iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). PAX6, MAP2, Brachury, MSX1, FOXA2, AFP, and GAPDH were analyzed. (d) Immunofluoroscence of directed differentiated three germ layers. Otx2 (Alexa488), Brachury (Alexa488), and Sox17 (Alexa488) were analyzed. Scale bar, 100 µm.
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fig1: Characterization of iPS cell lines. (a) Reverse transcription polymerase chain reaction of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, DPPA4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were analyzed. (b) Immunofluoroscence of iPS cell lines (Pompe-1, Pompe2, Pompe3, and control). ALP stain, SSEA-4 (Alexa488), Tra-1–60 (Alexa488), and Tra-1–81 (Alexa488) were analyzed. Scale bar, 100 µm. (c) RT-PCR of embryoid body from iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). PAX6, MAP2, Brachury, MSX1, FOXA2, AFP, and GAPDH were analyzed. (d) Immunofluoroscence of directed differentiated three germ layers. Otx2 (Alexa488), Brachury (Alexa488), and Sox17 (Alexa488) were analyzed. Scale bar, 100 µm.

Mentions: Three iPSC clones from one late-onset PD patient and one clone from one normal control were analyzed. Pluripotency markers, Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, and DPPA4, were analyzed by reverse transcription polymerase chain reaction (RT-PCR). All of the pluripotency markers were expressed in both PD and control iPSC almost equally except relatively low expression of GDF3 in control iPSC (Figure 1a).


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)

Characterization of iPS cell lines. (a) Reverse transcription polymerase chain reaction of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, DPPA4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were analyzed. (b) Immunofluoroscence of iPS cell lines (Pompe-1, Pompe2, Pompe3, and control). ALP stain, SSEA-4 (Alexa488), Tra-1–60 (Alexa488), and Tra-1–81 (Alexa488) were analyzed. Scale bar, 100 µm. (c) RT-PCR of embryoid body from iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). PAX6, MAP2, Brachury, MSX1, FOXA2, AFP, and GAPDH were analyzed. (d) Immunofluoroscence of directed differentiated three germ layers. Otx2 (Alexa488), Brachury (Alexa488), and Sox17 (Alexa488) were analyzed. Scale bar, 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Characterization of iPS cell lines. (a) Reverse transcription polymerase chain reaction of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, DPPA4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were analyzed. (b) Immunofluoroscence of iPS cell lines (Pompe-1, Pompe2, Pompe3, and control). ALP stain, SSEA-4 (Alexa488), Tra-1–60 (Alexa488), and Tra-1–81 (Alexa488) were analyzed. Scale bar, 100 µm. (c) RT-PCR of embryoid body from iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). PAX6, MAP2, Brachury, MSX1, FOXA2, AFP, and GAPDH were analyzed. (d) Immunofluoroscence of directed differentiated three germ layers. Otx2 (Alexa488), Brachury (Alexa488), and Sox17 (Alexa488) were analyzed. Scale bar, 100 µm.
Mentions: Three iPSC clones from one late-onset PD patient and one clone from one normal control were analyzed. Pluripotency markers, Oct3/4, Sox2, Klf4, Myc, Nanog, Gdf3, Rex1, DPPA2, and DPPA4, were analyzed by reverse transcription polymerase chain reaction (RT-PCR). All of the pluripotency markers were expressed in both PD and control iPSC almost equally except relatively low expression of GDF3 in control iPSC (Figure 1a).

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