title: Profiling stem cells using quantitative PCR protein assays.
abstract: Reprogramming human somatic cells to induced pluripotent stem cells is an important avenue in biological research. Advances in the profiling of human stem cells have identified important pluripotency maintenance factors. The presence and relative expression levels of these essential markers are commonly used to define the pluripotency status and potential of reprogrammed stem cells. We reprogram human dermal fibroblasts with four transcription factors, OCT3/4, SOX2, KLF4, and cMYC delivered by viral vectors. We describe a real-time quantitative PCR methodology to quantify the levels of key protein factors and examine the kinetics during reprogramming as well as comparing protein expression in different iPS clones. This report describes three applications of TaqMan(®) Protein Assays for reprogramming studies: (1) monitoring of reprogramming proteins over the induction time course, (2) characterization of pluripotent cells by protein expression profiles, and (3) identification of potential iPSC colonies in high-throughput screening protocols. This approach is fast, simple, sensitive and generates a pluripotency scorecard for reprogrammed stem cells.
authors.affiliation: Life Technologies, South San Francisco, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Ruff
authors.forename: Pauline T
authors.initials: PT
authors.lastname: Lieu
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation; D018920:Coculture Techniques; D003470:Culture Media; D015342:DNA Probes / Q000235:genetics*; D061252:Feeder Cells; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D012867:Skin / Q000166:cytology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D003470:Culture Media; D015342:DNA Probes; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors