Monitoring differentiation of human embryonic stem cells using real-time PCR
| Autor: | Neven Zoric, Johan Hyllner, Peter Sartipy, Xianmin Zeng, Mikael Kubista, Mahendra S. Rao, Karin Noaksson, Henrik Semb |
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| Rok vydání: | 2005 |
| Předmět: |
Homeobox protein NANOG
Cellular differentiation Down-Regulation Biology Cripto GPI-Linked Proteins Polymerase Chain Reaction Sensitivity and Specificity Humans RNA Messenger reproductive and urinary physiology Cells Cultured Homeodomain Proteins Reporter gene Membrane Glycoproteins Epidermal Growth Factor Stem Cells Expression index Nanog Homeobox Protein Cell Differentiation Cell Biology Molecular biology Embryonic stem cell Immunohistochemistry Neoplasm Proteins Up-Regulation DNA-Binding Proteins Embryo Research embryonic structures Molecular Medicine Intercellular Signaling Peptides and Proteins alpha-Fetoproteins biological phenomena cell phenomena and immunity Stem cell Octamer Transcription Factor-3 Biomarkers Developmental Biology |
| Zdroj: | Stem cells (Dayton, Ohio). 23(10) |
| ISSN: | 1066-5099 |
| Popis: | There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs). Using light microscopy and immunohistochemistry, we observed that morphological changes of differentiating hESCs precede any major alterations in the expression of several commonly used hESC markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog). In an attempt to quantify the changes during stochastic differentiation of hESCs, we developed a robust and sensitive multi-marker quantitative real-time polymerase chain reaction (QPCR) method. To maximize the sensitivity of the method, we measured the expression of up- and downregulated genes before and after differentiation of the hESCs. Out of the 12 genes assayed, we found it clearly sufficient to determine the relative differentiation state of the cells by calculating a collective expression index based on the mRNA levels of Oct-4, Nanog, Cripto, and alpha-fetoprotein. We evaluated the method using different hESC lines maintained in either feeder-dependent or feeder-free culture conditions. The QPCR method is very flexible, and by appropriately selecting reporter genes, the method can be designed for various applications. The combination of QPCR with hESC-based technologies opens novel avenues for high-throughput analysis of hESCs in, for example, pharmacological and cytotoxicity screening. |
| Databáze: | OpenAIRE |
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