Journal of molecular and cellular cardiology · 2017
Characteristics of induced pluripotent stem cells from clinically divergent female monozygotic twins with Danon disease.
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Yoshida Shohei, Nakanishi Chiaki, Okada Hirofumi, Mori Masayuki, Yokawa Junichiro, Yoshimuta Tsuyoshi, Ohta Kunio, Konno Tetsuo, Fujino Noboru, Kawashiri Masa-Aki, Yachie Akihiro, Yamagishi Masakazu, Hayashi Kenshi
Аннотация
RATIONALE: Induced pluripotent stem cells (iPSCs) have been generated from patients with various forms of disease, including Danon disease (DD); however, few reports exist regarding disease-specific iPSCs derived from clinically divergent monozygotic twins.
OBJECTIVE: We examined the characteristics of iPSCs and iPSC-derived cardiomyocytes (iPSC-CMs) generated from clinically divergent monozygotic female twins with DD.
METHODS AND RESULTS: We generated iPSCs derived from T-cells isolated from clinically divergent, 18-year-old female twins with DD harboring a mutation in LAMP2 at the intron 6 splice site (IVS6+1_4delGTGA). Two divergent populations of iPSCs could prepare from each twin despite of their clinical divergence: one with wild-type LAMP2 expression (WT-iPSCs) and a second with mutant LAMP2 expression (MT-iPSCs). The iPSCs were differentiated into iPSC-CMs and then autophagy failure was observed only in MT-iPSC-CMs by electron microscopy, tandem fluorescent-tagged LC3 analysis, and LC3-II western blotting. Under these conditions, X-chromosome inactivation (XCI) was determined by PCR for the (CAG)n repeat in the androgen receptor gene, revealing an extremely skewed XCI pattern with the inactivated paternal wild-type and maternal mutant X-chromosomes in MT-iPSCs and WT-iPSCs, respectively, from each twin.
CONCLUSION: Regardless of their clinical differences, we successfully established two sets of iPSC lines that expressed either wild-type or mutant LAMP2 allele from each monozygotic twin with DD, of which only the populations expressing mutant LAMP2 showed autophagic failure.
OBJECTIVE: We examined the characteristics of iPSCs and iPSC-derived cardiomyocytes (iPSC-CMs) generated from clinically divergent monozygotic female twins with DD.
METHODS AND RESULTS: We generated iPSCs derived from T-cells isolated from clinically divergent, 18-year-old female twins with DD harboring a mutation in LAMP2 at the intron 6 splice site (IVS6+1_4delGTGA). Two divergent populations of iPSCs could prepare from each twin despite of their clinical divergence: one with wild-type LAMP2 expression (WT-iPSCs) and a second with mutant LAMP2 expression (MT-iPSCs). The iPSCs were differentiated into iPSC-CMs and then autophagy failure was observed only in MT-iPSC-CMs by electron microscopy, tandem fluorescent-tagged LC3 analysis, and LC3-II western blotting. Under these conditions, X-chromosome inactivation (XCI) was determined by PCR for the (CAG)n repeat in the androgen receptor gene, revealing an extremely skewed XCI pattern with the inactivated paternal wild-type and maternal mutant X-chromosomes in MT-iPSCs and WT-iPSCs, respectively, from each twin.
CONCLUSION: Regardless of their clinical differences, we successfully established two sets of iPSC lines that expressed either wild-type or mutant LAMP2 allele from each monozygotic twin with DD, of which only the populations expressing mutant LAMP2 showed autophagic failure.