Monitoring differentiation of human embryonic stem cells using real-time PCR.
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.
Noaksson
Karin
K
Cellartis AB, Arvid Wallgrens Backe 20, 413 46 Göteborg, Sweden.
Zoric
Neven
N
Zeng
Xianmin
X
Rao
Mahendra S
MS
Hyllner
Johan
J
Semb
Henrik
H
Kubista
Mikael
M
Sartipy
Peter
P
D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D033041:Embryo Research; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D058851:GPI-Linked Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology*; D015854:Up-Regulation; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D004268:DNA-Binding Proteins; D058851:GPI-Linked Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; C070636:TDGF1 protein, human; D000509:alpha-Fetoproteins; D004815:Epidermal Growth Factor