Quantitative transcription factor analysis of undifferentiated single human embryonic stem cells.
BACKGROUND
Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs.


METHODS
We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated.


RESULTS
The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05).


CONCLUSIONS
We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.
Ståhlberg
Anders
A
Lundberg Laboratory for Cancer, Department of Pathology, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden. anders.stahlberg@tataa.com
Bengtsson
Martin
M
Hemberg
Martin
M
Semb
Henrik
H
D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000096:biosynthesis / Q000235:genetics; D051797:Inhibitor of Differentiation Protein 2 / Q000096:biosynthesis / Q000235:genetics; D051796:Inhibitor of Differentiation Proteins / Q000096:biosynthesis / Q000235:genetics; D008957:Models, Genetic; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D018398:Homeodomain Proteins; C497453:ID1 protein, human; C497450:ID2 protein, human; D051798:Inhibitor of Differentiation Protein 1; D051797:Inhibitor of Differentiation Protein 2; D051796:Inhibitor of Differentiation Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; C075640:ID3 protein, human