Objective: Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2/SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2/SEIPIN mutations.
Methods: Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2/SEIPIN. The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red O and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP.
Results: iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP.
Conclusion: BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation.
Keywords: Adipocyte; BSCL2/SEIPIN induced pluripotent stem cell; Congenital generalized lipodystrophy.
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