Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jan;8(1):68-74.
doi: 10.1039/b712116d. Epub 2007 Nov 2.

A Microfluidic Processor for Gene Expression Profiling of Single Human Embryonic Stem Cells

Affiliations
Free PMC article

A Microfluidic Processor for Gene Expression Profiling of Single Human Embryonic Stem Cells

Jiang F Zhong et al. Lab Chip. .
Free PMC article

Abstract

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.

Figures

Fig. 1
Fig. 1
The setting of the microfluidic device for single hESC mRNA extraction. A. The system includes a microscope, a computer to control air pressure with pressure regulators, and a heating stage to heat the microfluidic chip to desire temperatures. B. A typical microfluidic chip. C. Merged image of immunofluorescent stained (Oct-3/4) and light microscope images from a pluripotent hESC colony. The hESC colony was labeled with mouse α human Oct-3/4 IgG and PE-conjugated rabbit anti-mouse IgG antibodies. Only cells in the center of the hESC colony expressed Oct-3/4. The intensity of the labeling indicates the Oct-3/4 positive cells expressed Oct-3/4 at different levels. The spontaneously differentiated cells around the colony do not express Oct-3/4.
Fig. 2
Fig. 2
Single-cell mRNA extraction microfluidic device filled with food dye for illustration. All flow channels are filled with yellow food dye, multiplexer control channels are filled with red dye, collection and waste channels are in blue. The inserts show enlargements of four important areas of the chip. After loading cell suspension from the cell input inlet, single-cells are captured in cell lysis module (Insert 1) within the flow channels (blue). The pump valves are green. The separation valve is black. The lysis buffer is yellow. A captured single hESC is labeled with a fluorescent dye (green) and shown in Insert 2. Cell lysis is performed by opening the portion valve and pumping to mix lysis buffer (yellow) with the captured cell. The resulting cell lysate is pushed through oligo-dT bead columns for mRNA capture. Oligo-dT beads are stacked into columns by closing the sieve valve while loading bead suspension. Insert 3 shows six stacked oligo-dT bead columns next to the sieve valve. After washing beads with buffers, RT reaction master mix is flown through the bead columns to synthesize cDNA from the captured mRNA at 40 °C. After RT reaction, beads with attached cDNA are pushed to collection wells (Insert 4) by opening the sieve valve. The beads are recovered by cutting the wells off the chips and centrifuging a flipped-well in a microcentrifuge tube.
Fig. 3
Fig. 3
Measuring the absolute molecule number of the three genes in a single hESC with multiplex quantitative PCR. A. Standard curves are generated with known amounts of plasmid DNA containing the full sequence of the genes. The curves cover from 2 to 2 × 106 copies of the respective genes. With our primer design, all the curves overlap each other, and indicate similar PCR efficiency. B. The multiplex quantitative PCR amplification curves obtained from cDNA of hESC colonies are plotted with curves obtained from cDNA of a representative hESC. Because the standard curves of the three genes are very similar, these amplification curves show that the expression ratio of B2M and Nodal is similar in population cDNA and this single-cell cDNA. However, the expression of Fzd4 and Nodal is very similar in this particular single hESC, but very different in the hESC population. Unlike this single hESC, some single hESC do not express all three genes. This result suggests the heterogeneity of hESC and underscores the importance of single-cell analysis.
Fig. 4
Fig. 4
Expression of B2M, Nodal and Fzd4 in single hESCs. A. A single-hESC expresses both Nodal and Fzd4, but does not express B2M in a detectable level. B. A single hESC expresses only Nodal. The mRNAs of B2M and Fzd4 are undetectable. C, D and E shows the distribution of B2M, Fzd4 and Nodal in single hESCs. Among the 54 interrogated single hESCs, 14.8%, 37% and 37% of cells (indicated by black strait pattern) do not express detectable levels (less than 4 copies) of B2M, Fzd4 and Nodal respectively. The distribution pattern of expressions is narrower for B2M compared to the other 2 genes. The discontinuous distribution of Nodal and Fzd4 suggest a high heterogeneity of the 54 cells.

Similar articles

See all similar articles

Cited by 70 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback