title: Impaired adipogenic capacity in induced pluripotent stem cells from lipodystrophic patients with BSCL2 mutations.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Eisaku
authors.initials: E
authors.lastname: Mori
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address: jfuji@kuhp.kyoto-u.ac.jp.
authors.forename: Junji
authors.initials: J
authors.lastname: Fujikura
authors.affiliation: Medical Innovation Center (MIC), Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Noguchi
authors.affiliation: Department of Peptide Research, Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nakao
authors.affiliation: Medical Innovation Center (MIC), Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsubara
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Sone
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Taura
authors.affiliation: Department of Peptide Research, Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Kusakabe
authors.affiliation: Institute for Advancement of Clinical and Translational Science (iACT), Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Ebihara
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Human Health Science, Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Kiminori
authors.initials: K
authors.lastname: Hosoda
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Asaka
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Inagaki
authors.affiliation: Medical Innovation Center (MIC), Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address: nakao@kuhp.kyoto-u.ac.jp.
authors.forename: Kazuwa
authors.initials: K
authors.lastname: Nakao
mesh_terms: D050156:Adipogenesis / Q000235:genetics*; D001706:Biopsy; D002454:Cell Differentiation / Q000235:genetics; D018389:Codon, Nonsense / Q000235:genetics; D058732:Embryoid Bodies; D005347:Fibroblasts / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D052497:Lipodystrophy, Congenital Generalized / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D000070780:Perilipin-2; D012867:Skin / Q000378:metabolism; D013724:Teratoma / Q000235:genetics
chemical_list: C432679:BSCL2 protein, human; D018389:Codon, Nonsense; D044388:GTP-Binding Protein gamma Subunits; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; D000070780:Perilipin-2; C000602983:Plin2 protein, mouse
keywords: Adipocyte; BSCL2/SEIPIN induced pluripotent stem cell; Congenital generalized lipodystrophy