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    • The original XO karyotype Fig of

    2018-10-24

    The original 45 XO karyotype (Fig. 1) of the donor CVCs was maintained in TS ViPSCs even after prolonged maintenance (passage 25) indicating that the process of reprogramming with episomal vectors and in vitro culture conditions did not alter this iPSC line chromosomally. The iPSC line established in this study was confirmed to be identical with the original CVCs by STR analysis of 16 genetic loci including the gender determining marker AMELOGENIN by PCR amplification of genomic DNA (Fig. 1). This experiment amplified only the X chromosome allele of AMELOGENIN further confirming the absence of Y chromosome related genes in the cells. The TS ViPSCs expressed the pluripotency markers Alkaline Phosphatase, NANOG, OCT4, SOX2, TRA 1-60, TRA 1-81, SSEA4, E CADHERIN and peripheral BETA CATENIN (Fig. 2A). The TS ViPSCs also exhibited the characteristic lipid droplet derived retinyl ester blue fluorescence associated with human pluripotent stem nebivolol hcl (Muthusamy et al., 2014) (Fig. 2A). TS ViPSCs expressed transcripts of the pluripotency genes OCT3/4, NANOG, SOX2, E CADHERIN, REX1 and the DNA methyl transferases upregulated in pluripotency DNMT1, DNMT3a and DNMT3b (Fig. 2B). These transcripts were either absent or faintly present in the donor CVCs. No Ori P or EBNA 1 DNA was detected in TS ViPSCs after 20 passages as shown by genomic DNA PCR (Fig. 2C). This confirmed that this cell line is transgene free. As also previously reported (Li et al., 2012) the TS ViPSCs differentiated into cell types of 3 germ layers in vitro which were morphologically indistinguishable from cell types differentiated from other euploid iPSCs. They differentiated into cells expressing the ectodermal marker Microtubule Associated Protein 2 (MAP2), the mesodermal marker Smooth Muscle Actin (SMA) and the endoderm marker SRY Box 17 (SOX 17) (Fig. 2D)
    Materials & methods
    Author contribution
    Acknowledgement Financial support for the above research was provided by a CSIR grant (#27(293)/13 EMR II) Characterization of the line was conducted partially in the laboratory of M. M. Panicker at NCBS. We thank Dr. Sri Devi Hegde, HOD, Department of Genetics, Manipal Hospital for providing the sample and Smitha Chander of Anand Diagnostic Laboratory for assistance with karyotyping. We thank Dr Krishna Murthy DS for his comments on the karyotype. We also thank Life Forensics Laboratory for their assistance with STR profiling.
    Resource table Resource details We report the derivation of multiple mESC lines generated from the crossing between heterozygous transgenic mice expressing GFP under the control of the rat tyrosine hydroxylase (TH) promoter (Sawamoto et al., 2001), and homozygous aSYN mice expressing human mutant SNCAA53T under the control of the mouse prion promoter (MoPrP), also known as M83 (Giasson et al., 2002), or non-transgenic wild type (WT) mice expressing endogenous mouse Snca (Fig. 1, panel A). The mESC lines are referred to as TH::GFP, TH::GFP;PrP::hSNCAA53T, PrP::hSNCAA53T and WT. At the time of breeding, the mice were under C57BL/6 (B6) background. Blastocysts aged embryonic day 3.5 (E3.5; panel B) were isolated from pregnant female mice, and cultured separately on CF1 mouse irradiated feeders until the inner cell mass formed a primitive colony (panels C and D). Each colony was then passaged until the lines could be established (panel E). For each line derived, expression of the pluripotency marker Oct4 was confirmed by immunocytochemistry (panel F); the presence of GFP was confirmed by PCR (panel G). TH::GFP neurons could be identified when TH::GFP and TH::GFP;PrP::hSNCAA53T mESC lines were differentiated on a monolayer of PA6 stromal cells (panel H), using previously published protocols (Ganat et al., 2012; Kawasaki et al., 2000). Immunostaining for GFP in midbrain FoxA2-positive cultures revealed co-expression with β-III-tubulin (70±11%) and with TH (59±10%) on the day of analysis (D14+6 DIV). Generated TH::GFP neurons could be purified by fluorescent-activated cell sorting (FACS) for GFP, and recovered in culture after sorting (data not shown). Transgenic expression of human aSYN was identified in TH::GFP;PrP::hSNCAA53T and PrP::hSNCAA53T lines by immunocytochemistry (panel J and data not shown). Oligodendrocytes generated from human aSYN mESC lines also expressed human mutant SNCA (panel K).