Life Technologies

Summary We have successfully generated iPSCs from the dermal fibroblasts of three Parkinson's disease donors, one MSA donor, and two age-matched control samples using the CytoTune®iPS Sendai Reprogramming Kit. The reprogramming efficiency varied from line to line, ranging from 0.01% to nearly 0.3%, and younger donor cells were observed to generate more colonies than older donor cells. The iPSCs were fully evaluated and confirmed to be pluripotent by testing via morphology, cytogenetics, immunostaining, FACS analysis, and gene expression profiling. The cells were also able to form embryoid bodies with three germ layers, confirming their ability to differentiate. The fibroblasts used in this study were evaluated for other genetic mutations associated with PD, and none were detected. Clearance of the Sendai virus was detected as early as passage 10 for some lines. However, clearance varied from line to line and from clone to clone within the same line. After the iPSCs had been fully 26 Life Technologies | Parkinson's cell model generated, they were confirmed to be derived from their corresponding parental fibroblasts samples. Given the efficiency, speed, and ease with which we were able to reprogram adult disease fibroblasts, it is clear that the CytoTune®-iPS Sendai Reprogramming Kit represents a significant step forward in the direction of automated, large-scale reprogramming. The establishment of these fully characterized iPSC lines, from samples with known clinical histories, now sets the stage for further disease-relevant studies. For example, MSA is often characterized by profound formation of Lewy bodies, which contain α-synuclein aggregates as a major component. Gene editing technologies, such as those enabled by TAL nuclease fusions, will allow us to knock out the gene coding for the α-synuclein protein. This may allow us to isolate the impact of these protein aggregates on other phenotypes associated with the disease after differentiation of the iPSCs to neural lineages.  References 1.Olanow CW, Stern MB, Sethi, K (2009) The scientific and clinical basis for the treatment of Parkinson disease. Neurology 72(21 Suppl 4):S1–136. 2.Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85:348–362. 3.Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, Nakata H, Tohyama S, Hashimoto H, Kodaira M, Okada Y, Seimiya H, Fusaki N, Hasegawa M, Fukuda K (2010) Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7:11–14. 4.Tucker BA, Anfinson KR, Mullins RF, Stone EM, Young MJ (2013) Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation. Stem Cells Transl Med 2:16–24. 5.Burn DJ, Jaros E (2001) Multiple system atrophy: cellular and molecular pathology. Mol Pathol 54:419–426. 6.ClinVar database. National Center for Biotechnology Information (Bethesda, MD). URL: http://www.ncbi.nlm.nih.gov/clinvar/. Accessed 17 May 2013.

• Cover