Using similar approach additional mESC lines could

Using similar approach, additional mESC lines could be derived using a Pitx3::GFP reporter mouse (Zhao et al., 2004) and another aSYN mouse model of Parkinson\'s disease, expressing human WT SNCA (Westerlund et al., 2008) (data now shown). These mESC lines (Table 1) can be used to study human SNCA-induced pathology in mouse dopaminergic neurons and oligodendrocytes.
Materials and methods
Contribution
Acknowledgments We are thankful to Professors Anders Bjorklund, Pekka Kallunki, Meng Li and Hideyuki Okano for providing and facilitating access to the mice. This project was supported by the Crafoord foundation, the Bergvall foundation, the Kockska foundation, and the strategic area MultiPark to L.R.; and the Swedish and Chinese Medical Research Councils and the National Natural Science Foundation (81430025) to J.-Y.L.
Resource table. Resource details The study was approved by the ethics committee of 1st and 2nd Hospital of Jilin University, written informed consent was obtained from the patient. 1ml of peripheral blood sample was extracted from a 60-year-old female patient with multiple schwannoma (MS). This patient does not harbour SMARCB1 and NF2 mutations, which was often observed in familial MS cases (Jacoby et al., 1997; Smith et al., 2012). The MS-BP002 iPSC lines were derived using the CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), including four Yamanaka factors Klf-4, c-Myc, Oct-4 and Sox-2 (Ban et al., 2011). The derived hiPSC lines displayed a typical ES cell morphology, a high nucleus/cytoplasm ratio and prominent nucleoli. The identity of derived iPSC lines was confirmed
Materials and methods
Author disclosure statement
Acknowledgments This study was supported by fund from National Medical Research Council (NMRC) of China to SZ (fund number: 30400447) and HW (fund number: 30500548).
Resource table. Resource details The study was approved by the ethics committee of 1st and 2nd Hospital of Jilin University, written informed consent was obtained from the patient. 1ml of peripheral blood sample was extracted from a 72-year-old male patient with late-onset Alzheimer\'s disease (AD). Alzheimer\'s disease includes early onset AD and later onset AD (Cuyvers and Sleegers, 2016; Scheltens et al., 2016). The AD-BP001 iPSC lines were derived using the CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), including four Yamanaka factors Klf-4, c-Myc, Oct-4 and Sox-2 (Ban et al., 2011). The derived hiPSC lines displayed a typical ES cell morphology, a high nucleus/cytoplasm ratio and prominent nucleoli. The identity of derived iPSC lines was confirmed by immunofluorescence staining. After 7–9 passages, the absence of exogenous reprogramming transgenes was checked by RT-PCR. The differentiation capacity of hiPSC lines into three germ layers was investigated by in vivo teratoma formation assay. The derived hiPSC lines showed normal karyotype (46, XY).
Materials and methods
Author disclosure statement
Acknowledgments This study was supported by fund from National Medical Research Council (NMRC) of China to SZ (fund number: 30400447) and HW (fund number: 30500548).
Resource table Resource details Autosomal calcium sensing receptor familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disorder characterised by severe polyuria and polydipsia and caused by variants in the arginine vasopressin (AVP) gene. Previous studies have indicated that it is associated with degeneration of the AVP producing magnocellular neurons in the hypothalamus (Arima et al., 2014; Azuma et al., 2014; Hagiwara et al., 2014; Hayashi et al., 2009; Hiroi et al., 2010; Morishita et al., 2011; Russell et al., 2003) but the pathogenesis remains largely unknown. In order to investigate the pathogenesis leading to the specific degeneration in more details cell lines developed from fibroblasts directly obtained from a patient with adFNDI would be preferable. Therefore, fibroblasts were obtained from a 42-year-old man diagnosed with adFNDI and being heterozygous for the variant g.276_278delTCC (Ser18del) in the AVP gene. The reprogramming was performed using a polycistronic lentiviral vector (pRRL.PPT.SF.hOCT3/4.hKLF4.hSOX2.hMYC.dTOMATO, dubbed LV-OKSM-Tomato) encoding four human Yamanaka factors (OCT4, KLF4, SOX2, and C-MYC), which was packaged into lentiviral particles as previously described (Kang et al., 2015). During the establishment of the induced pluripotent stem cells (iPSCs), expression of the vector encoded red fluorescent marker tdTomato was monitored by microscopy to ensure that transgene expression from the integrated vector decreased to background levels as expected (Warlich et al., 2011) (data not shown). Direct sequencing of AVP-Ser18del-iPSC DNA confirmed the presence of the g.276_278delTCC variant in one of the alleles of the AVP gene (Fig. 1A). Immunostaining of the iPSC colonies revealed expression of the pluripotency markers OCT4, NANOG, SSEA3, SSEA4, and TRA-1-60 (Fig. 1B). A pluripotency analysis using quantitative real-time polymerase chain reactions (qPCR) showed that the endogenous expression of the pluripotency genes SOX2, DPPA4, TDGF1, LIN28A, ZFP42, POU5F1, DNMT3B, NANOG, and SALL4 was between 100 and 100,000 times upregulated compared to fibroblasts in both AVP-Ser18del-iPSCs and iPSCs from a healthy control subject (Fig. 1C). The AVP gene was found to be upregulated in the fibroblasts compared to the iPSCs, especially in the AVP-Ser18del-fibroblasts (Fig. 1C). However, since only very low amounts of AVP mRNA were measured in both cell types, the ratio between them is uncertain. The endogenous expression of the general cell marker KLF4 was increased in the fibroblasts compared to the iPSCs in both AVP-Ser18del-variant and control cells (Fig. 1C), which is well known. The endogenous expression of the general cell marker MYC was slightly upregulated in the AVP-Ser18del-iPSCs while a small upregulation was found in the control fibroblasts (Fig. 1C). Methylation analysis of the OCT4 promoter in both AVP-Ser18del-fibroblasts and -iPSCs revealed the expected de-methylation of all seven CpG units investigated in the stem cells (Fig. 1D). In vitro differentiation followed by immunostaining with the ectodermal marker beta-III-tubulin (TUJ1), the endodermal marker alpha-feto-protein (AFP), and the mesodermal marker smooth muscle actin (SMA) showed that the iPSCs possessed a potential to differentiate into all three germ layers (Fig. 1E). Finally, karyotype analysis revealed that the AVP-Ser18del-iPSCs had no systematic deviation from the normal chromosome number and no large chromosomal abnormalities (Fig. 1F).