fructose 1 6 bisphosphatase The PBMC iPS F cell

The PBMC1-iPS4F1 cell line has been adapted to feeder-free culture conditions and displays a normal karyotype (46, XX) after extensive mechanical and enzymatic transfers (Fig. 1D). The iPSC line was also characterized using a genetic fingerprinting assay by microsatellite analysis. The endogenous expression of the pluripotency associated transcription factors Oct3/4, Sox2, Rex1 and Nanog was evaluated by RT-PCR (Fig. 2A). We also confirmed the protein expression levels of the pluripotency markers Oct3/4, SSEA3, SSEA4, Tra1-60 and Tra1-81 by flow cytometry analysis (Fig. 2B). The in vivo differentiation capacity of the PBMC1-iPS4F1 cell line was demonstrated by teratoma formation. The teratomas showed expression of representative markers of the three germ layers (Glial Fibrilar Acid Protein/GFAP for ectoderm, Vimentin for mesoderm, and Cytokeratin/CK AE1-AE3 for endoderm) (Fig. 3A). In addition, in vitro differentiation capacity was tested by spontaneous embryoid body (EB) differentiation. The fructose 1 6 bisphosphatase inside the EBs were able to differentiate to the three germ layers, demonstrated by the expression of representative markers of each layer (β-III-tubulin for ectoderm, Vimentin for mesoderm and CK AE1-AE3 for endoderm) (Fig. 3B). We also routinely monitor the PBMC1-iPS4F1 cell line to confirm the absence of Mycoplasma contamination.
Materials and methods
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Resource details NBS-iPSCs were derived by retroviral transduction of OCT4, SOX2, c-MYC and KLF4. Both clones expressed the pluripotency associated transcription factors NANOG, OCT4, SOX2 and cell surface markers, TRA-l-60, TRA-1-81 and SSEA-4. Pluripotency was further demonstrated in vitro by embryoid body-based differentiation to cell types representative of the three germ layers endoderm, ectoderm and mesoderm and in vivo by teratoma formation in immunodeficient mice. Chromosomal analysis revealed a karyotype with few aberrations. In addition microarray-based transcriptome analysis confirmed similarity of the derived NBS-iPS-clones to a collection of validated hESCs and iPSCs derived transcriptomes (Müller et al., 2011, http://www.pluritest.org).
Materials and methods
Verification and authentication The fibroblasts used for reprogramming were derived from a patient with confirmed NBS (Charité Berlin). A heterozygous 657del5 mutation in the patient was detected by Sanger sequencing (Fig. 2a). The 657del5 mutation leads to truncation of the wild-type protein (p95 NBN). The wild-type version of the NIBRIN protein was detected by Western blot in control samples of fibroblasts and hPSCs, but not in NBS fibroblasts and NBS-iPSCs (Fig. 2a and b). The NBS-iPSCs expressed pluripotency markers (Fig. 1a and b). Embroid body-based differentiation and teratoma formation generated cell types representative of the three germ layers as detected by immunofluorescence and histological analysis (Fig. 1c and d). Confirmation of pluripotency in the iPCS-lines (Fig. 1c and d) employed Pluritest (Müller et. al., 2011). Karyotype analysis showed a normal karyotype apart from one derivative chromosome 11 [46,XY,der(11)t(5;11)(q12;q25)] in vNBS8-iPS-c1 (Fig. 1g). The second clone, vNBS8-iPS-c2, had two aberrant karyotypes. One, with trisomy 8 and a derivative chromosome 11 with the frequency of 12 in 15 counted metaphases [47,XY,+8,der(11)t(5;11)(q12;q25)]. The second one occurred with a frequency of 3 in 15 counted metaphases and contained an isochromosome of the long arm of chromosome 1, a derivative chromosome 4 with additional material of unknown origin and different banding pattern, a deletion in 13q and additional material of unknown origin at 17p (47,XY+i(1)(q10),add(4)(p15),del(13)(q13),add(17)(p11)).
Acknowledgements We thank Prof. Karl Sperling for providing NBS fibroblasts and JA acknowledges financial support from the Max Planck Society, Medical Faculty of Heinrich-Heine Universität-Düsseldorf, Germany and the BMBF grant number 01GN1005.