br Materials and methods br Author disclosure statement

Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the National Institutes of Health K08 grant number HL111148 (J.L.S.) and R01 grant number HL051387 (D.E.V.). We are especially indebted to patients and their families for participating in this study and donating samples.
Resource table. Materials and methods
Differentiation
Conclusion
Acknowledgements This work was funded by the generous support of the Clive and Nancy Runnells Embryonic Stem Cell Research Fund (in memory of Pierce Runnells)—The University of Texas Health Science Center at Houston.
Resource table. Resource details Plasminogen activator Inhibitor-1 (PAI-1) is the physiological inhibitor of plasminogen activation and has a canonical role in fibrinolysis. PAI-1 also contributes to a diverse range of biological effects like physiological aging (Yamamoto et al., 2014; Eren et al., 2014a), replicative senescence (Elzi et al., 2012; Eren et al., 2014a, Wan et al., 2014), proteolysis (Eren et al., 2014b), extracellular matrix interaction (Flevaris and Vaughan, 2016), adhesion (Viswanathan et al., 2006), migration (Kozlova et al., 2015) and other signaling functions. Complete deficiency of PAI-1 in humans is rare but the obvious clinical manifestation is abnormal bleeding after trauma (Fay et al., 1992, 1997; Heiman et al., 2014). The effects of PAI-1 deficiency in its other biological roles have been more elusive to study in the human system. The ability to derive human induced pluripotent stem hiv protease inhibitor (iPSCs) from patients with PAI-1 mutations would allow the production of large numbers of target cells for further investigation. We generated human iPSC clones (iPAI-015-2-F) from urine cells of subjects, heterozygous for a dinucleotide (TA) insertion within exon 4 of the PAI-1 gene (Fay et al., 1992, 1997). 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-015-2-F 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) and endogenous pluripotency markers OCT3/4, and SOX2 were confirmed by RT-PCR (Fig. 2C). The absence of exogenous reprogramming transgenes KLF4, cMYC and SeV in later iPSC passages was confirmed by RT-PCR (Fig. 2C). Differentiation of the iPSCs into three germ layers 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 clone showed normal karyotype (46, XX) (Fig. 2E) and the PAI-1 mutation was confirmed by mutation-site specific SfcI restriction enzyme digestion analysis (Fig. 2F).
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the National Institutes of Health K08 grant number HL111148 (J.L.S.) and R01 grant number HL051387 (D.E.V.). We are especially indebted to patients and their families for participating in this study and donating samples.
Resource table. Resource details The study was approved by the Singhealth ethics committee (protocol number SHSIBC-2014-031), and written informed consent was obtained from the patient. 2ml of peripheral blood sample was obtained from a male 59-year-old Parkinson\'s disease patient carrying a heterozygous R1628P variant in LRRK2 gene. LRRK2 R1628P variant may lower the susceptibility of PD among Asian population (Gopalai et al., 2014; Ross et al., 2011; Tan et al., 2010). PD-BP44-iPSC lines were derived using CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), which carrying the 4 Yamanaka reprogramming factors OCT4, SOX2, cMYC and KLF4 (Ban et al., 2011). This reprogramming followed the previous published protocol (Tan et al., 2014). The derived hiPSC lines displayed a typical round shape ESC-like morphology with small and tightly packed cells, and a high nucleus/cytoplasm ratio and prominent nucleoli (Fig. 1A). The presence of the R1628P variant was confirmed in hiPSC lines by Sanger sequencing (Fig. 1B), and the expression of pluripotency markers was verified by immunofluorescence staining (Fig. 1C). Moreover, the absence of exogenous reprogramming transgenes was observed by RT-PCR after 5-8 passages (Fig. 1D). The differentiation capacity of hiPSC lines into three germ layers was demonstrated by in vivo teratoma formation assay (Fig. 1E). The derived hiPSC lines showed normal karyotype (46, XY) (Fig. 1F).