i iPSC colonies displayed a typical ES
i10984 iPSC colonies displayed a typical ES-like colony morphology (Fig. 1B). Immunocyto-chemical analysis revealed expression of transcription factors OCT4 and SOX2, and surface markers SSEA4 and TRA-1–81, characteristic of pluripotent stem gssg (Fig. 1B). The iPS cells exhibited a normal karyotype (46, XX) by G-Banding analysis (Fig. 1B). The cells also demonstrated differentiation capacity to three germ layers using an embryoid body-based directed differentiation protocol, followed by immunostaining with markers corresponding to the three germ layers i.e. ectoderm (PAX6), mesoderm (FOXC1) and endoderm (GATA4) (Fig. 1C).
Materials and methods
Acknowledgements This work was supported by the Alzheimer Forschung Initiative e.V. (grant agreement 15032).
Introduction Prader-Willi syndrome (PWS) is caused by a loss of paternally-expressed genes in an imprinted region of 15q11.2-q13; the syndrome affects 1 in 25,000 live births (Smith et al., 2003; Angulo et al., 2015). Most instances of PWS (70%) are due to a “large” 4–5Mb deletion in 15q11.2-q13 (Fig. 1A). All large PWS deletions include six imprinted, protein-encoding genes (MKRN3, MAGEL2, NECDIN, C15ORF2, SNURF, SNRPN), seven non-protein coding snoRNA genes (SNORD107, SNORD64, SNORD108, SNORD109A, SNORD116, SNORD115, and SNORD109B), and the long non-coding RNA, IPW (Cassidy and Driscoll, 2009). About 25% of instances of PWS are due to uniparental maternal disomy, while <5% of cases result from unbalanced paternal translocations, imprinting defects, or microdeletions (Angulo et al., 2015; Cassidy and Driscoll, 2009). The clinical phenotypes of PWS include hyperphagic obesity, hypogonadism, low growth hormone (GH) associated with short stature, hyperghrelinemia, and relative hypoinsulinemia (Smith et al., 2003; Angulo et al., 2015; Butler et al., 2006). Although over a dozen mouse models of PWS have been generated, none develop the hallmark obesity associated with PWS. Patient-specific iPSCs could provide a novel model system with which to study the molecular etiology of the disease.
Results and discussion
Materials and methods
Acknowledgements We thank Claudia A. Doege, Liheng Wang, Haiqing Hua, Linshan Shang, and Bjarki Johanessen for valuable discussions. We thank the following foundations/funding agencies for their support of this project: FPWR Research Grants, FPWR/PWSA (Best Idea Grant), Rudin Foundation, NYSCF, Helmsley Foundation, Russell Berrie Foundation, and NIH (RO1DK52431; P30 DK26687 the New York Obesity Research Center).
Resource table. Resource details Peripheral blood was collected from a 11-year-old female patient with Kleefstra syndrome (KS) diagnosed by targeted next generation sequencing (Bock et al., 2016). The diagnosis was confirmed by Sanger sequencing and by clinical evaluation of the patient. The patient phenotype includes autism, normal intellectual performance, developmental delay, childhood hypotonia and facial dysmorphisms. The patient carries a heterozygous, de novo, PTC (Trp1138Ter) mutation in the Euchromatic histone lysine methyltransferase 1 (EHMT1) gene, which leads to haploinsufficiency and causes KS (Bock et al., 2016). To generate the BIOT-0708-EHMT1 iPSC line (Fig. 1A) the PBMCs were reprogrammed by the four “Yamanaka factors” OCT3/4, SOX2, KLF4, and C-MYC using the integration-free Sendai virus gene-delivery method (Yang et al., 2008; Fusaki et al., 2009). The iPSC-like colonies were picked 20–27days post-transduction. The absence/presence of SeV expression in the iPSCs was monitored from passage 5 by SeV-specific RT-PCR (Table 1). After 7 passages, the transgene-free status of the BIOT-0708-EHMT1 iPSC line was confirmed and selected for further analysis (Fig. 1B). Expression of pluripotency markers was examined by alkaline phosphatase staining (AP) and by immunocytochemistry staining (ICC) using antibodies against human OCT3/4, NANOG and E-CADHERIN (Fig. 1A). The in vitro spontaneous differentiation potential of the BIOT-0708-EHMT1 iPSC line towards the three germ layers was demonstrated by the expression of endodermal (GATA4), mesodermal (BRACHYURY) and ectodermal (βIII-TUBULIN) markers (Fig. 1A) (Itskovitz-Eldor et al., 2000; Carpenter et al., 2003).