trp channel Background green fluorescence was detected in th

Background green fluorescence was detected in the lentiviral reprogrammed MML-6838-CL2 iPSC line and a control episomal (non-GFP) reprogrammed iPSC, indicating silencing (Fig. 1B). Pluripotency was confirmed by detecting expression of SOX2, OCT4, SSEA4, TRA1-61 and TRA1-80 pluripotency-associated markers by flow cytometry and alkaline phosphatase staining (Fig. 1C, D). The spontaneous differentiation potential of MML-6838-CL2 iPSC line was shown in vivo by analyzing teratomas formed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG). Three mice were injected and 2/3 formed teratomas. The commitment of the iPSC line to all three germ layers was confirmed by immunohistochemistry staining in which endodermal (PAN-CYTOKERATIN), mesodermal (α-SMA) and ectodermal (βIII-TUBULIN) markers were detected (Fig. 1E, F). The Genomic integrity of MML-6838-CL2 iPSC line was determined by Giemsa-banding, proving normal diploid 46XY karyotype, without any detectable abnormalities (Fig. 1G).
Materials and methods
Resource table. Resource details Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by trinucleotide repeat (CAG) expansion in the coding region of ATXN3 gene on chromosome 14, which produces an elongated polyglutamine tract, leading to purkinje cell loss (Paulson, 2007). In this report, we successfully generated an iPS cell line, TVGH-iPSC-SCA3-04, from human peripheral blood mononuclear trp channel (PBMC) that were donated from a patient with SCA3. The PBMCs were reprogrammed by co-expressing Yamanaka factors, OCT3/4, SOX2, KLF4, and cMYC through the integration-free Sendai virus gene-delivery method (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Fusaki et al., 2009). Then, embryonic stem cell (ES)-like colonies were picked and cultured for characterization on day 21. The TVGH-iPSC-SCA3-04 at passage 10 featured a complete removal of all exogenous reprogramming factors (Fig. 1A). The resulting iPSCs had a normal karyotype and retained the disease-causing ATXN3 mutation (Fig. 1B & C). The endogenous expression of the pluripotent markers, OCT4, SOX2 and NANOG was evaluated by RT-PCR (Fig. 1D). We also confirmed the protein expression of the pluripotent markers, OCT4, SOX2, NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 by flow cytometry and immunocytochemistry staining (Fig. 1E & F). In vitro spontaneous differentiation potential towards the three-germ layers of the TVGH-iPSC-SCA3-04 cell line was demonstrated by the expression of ectodermal (TUJ1), mesodermal (alpha-smooth muscle actin, a-SMA) and endodermal (alpha-fetoprotein, AFP) markers (Fig. 1G). Typical teratoma-like masses were observed and harvested for histopathological analysis eight weeks post-transplantation. We observed that the teratoma comprised of tissues from all three germ layers, including neural tissue (ectoderm), cartilage (mesoderm), and glandular structure (endoderm) (Fig. 1H). Potentially, the iPSC line, TVGH-iPSC-SCA3-04 could be a useful tool for the investigation of disease mechanisms of SCA3.
Materials and methods
Acknowledgment
Resource table. Resource details We generated human iPSC clones from urine cells of non-affected subjects (iPAI-005-B) to serve as control for iPSCs containing homozygous or heterozygous dinucleotide (TA) insertion mutations within exon 4 of the PAI-1 gene (Fay et al., 1992, 1997) (see companion lab resource articles). iPSC clones were established under feeder-free culture conditions using the Cytotune® iPS Reprogramming Kit (Life Technologies, Carlsbad, CA) which employs the non-integrating Sendai Virus (SeV) to deliver reprogramming factors, Oct3/4, Sox2, Klf4 and cMyc (Takahashi et al., 2007). The morphologic iPAI-005-B iPSC clones displayed typical pluripotent cell morphology (Fig. 2A). The iPSC clones stained positive for the pluripotency markers Oct3/4 and TRA-1-81 (Fig. 2B). The presence of endogenous pluripotency markers Oct3/4 and Sox2 and the absence of exogenous reprogramming transgenes Klf4, cMyc and the SeV was confirmed by RT-PCR (Fig. 2C). Demonstration of three germ layer differentiation capacity was confirmed by immunofluorescence analysis of SOX1, OTX2, BRY and GATA4 to identify ectoderm, mesoderm and endoderm in spontaneously differentiated embryoid bodies (Fig. 2D). The iPSC lines showed normal karyotype (46, XX) (Fig. 2E) and the exon 4 of the PAI-1 gene was confirmed to be the wild-type sequence (Fig. 2F).