March 15, 2011 (Vol. 31, No. 6)

Looking at Individual Cells Can Shed Light on What’s Happening in Their Microenvironment

The analysis of individual cells is now a reality. Single-cell signatures may tell us a lot about the tissue microenvironment, differentiation, aging, or regeneration. For instance, tumor microenvironment contains multiple cell types including immune cells, stroma, cells of blood vessels, and all their chemical signals.

Communication of tumor cells with their microenvironment helps drive tumor progression. Single-cell analysis can explain this communication in great detail, potentially yielding new therapeutic approaches for targeting these signals.

Until recently, the sensitivity of most of the “omics” techniques (genomics, proteomics) was not sufficient enough to trace the contribution of individual components. Many current molecular biology techniques require multiple cells. This inevitably blends cell populations and, therefore, reports the average values.

“This is rapidly changing,” comments Daojing Wang, Ph.D., life sciences division, Lawrence Berkeley National Laboratory. “Single-cell analysis is a new frontier in omics. It will enable systems biology at the level of individual cells.” Single-cell technologies will help to understand the molecular stasis of a particular cell or cell type in a specific biological environment, its interconnections with the surrounding environment, and the roles that individual phenotypes play in healthy and diseased tissue function.

Key cells may be present in small numbers and produce infrequent and weak signals that get diluted and lost during averaging. For example, circulating cancer cells are rare but important, since they play a key role in cancer metastasis. Measuring gene expression within individual circulating cancer cells will hopefully shed light on cancer dissemination and metastasis.

“Single-cell mRNA and genomic analysis have already became a reality,” adds Dr. Wang, who will be speaking at Select Bioscience’s “Single Cell Analysis Congress” in May. “Proteomics and metabolomics experienced more challenges, which may be resolved by combining extremely efficient sample manipulation and highly sensitive detection. In that regard, micro-/nanofluidics interfaced with mass spectrometry (MS) could be a promising combination for single-cell proteomic analysis.”

Dr. Wang and colleagues have developed special multinozzle emitters for MS. Each emitter consists of a parallel silica nozzle array. These emitters could be linked to microfluidic circuits on one side and MS on the other side, thus forming an integrated lab-on-a-chip system with potential for future single-cell proteomics and metabolomics.

qPCR from Single Cells

“A comprehensive understanding of life requires combining molecular data from individual cells and the macro data from the organ/organism,” comments Hideki Kambara, Ph.D., Hitachi. Dr. Kambara’s group participated in a three-year multiprong effort called the “Life Surveyor”. Over 70 scientists from multiple research entities formed workgroups aimed at developing better tools for single-cell analysis.

“While tissues may seem uniform on the macro level, each cell may be individually different,” continues Dr. Kambara. “Our group focused on quantitative PCR as the most accurate method of detecting these differences. We adapted qPCR for single-cell analysis in a cost-efficient, amplification-independent fashion.”

The concept of this method is not new, as it involves using capture of mRNA on magnetic beads followed by reverse transcription into cDNA. However, the adaptation to a single cell required multiple modifications, such as introduction of a special polymer to prevent DNA and RNA adsorbtion to the surfaces.

“Our goal was to reuse this cDNA library multiple times, but we observed that 90 percent of DNA was stripping from beads after only 10 rounds of qPCR,” says Dr. Kambara. “This desorption was caused by thermal decomposition of the bead’s polymer coating. We had to develop a low-temperature PCR reaction, which decreased the desorption rate to only 20 percent per 10 rounds.”

This technology was used to discern whether the lineage of mesenchymal stem cells is predetermined a priori, or if the differentiation is induced by the addition of a chemical agent.

While this research is still ongoing, the preliminary data seems to suggest that the fate of undifferentiated cells is already predetermined. “Our ultimate goal is to develop tools for multiple gene expression at single-cell resolution while preserving the spatial information on cells within the tissues,” says Dr. Kambara. “Such measurements will have a profound impact on understanding the effect of each cell on a tissue or organ.”

Deep Sequencing

“We would like to argue that the most accurate method for transcriptome analysis is sequencing,” notes Kai Lao, Ph.D., principal scientist, genetic systems, Life Technologies. “Recent advances in high-throughput sequencing made it possible to analyze single-cell transcriptomes at high resolution.”

Life Technologies used the SOLiD next-generation sequencing technology for deep sequencing of transcriptomes of mouse oocytes and stem cells. Single-cell RNA-Seq overcomes the typical limitations of microarray analysis while achieving greater accuracy.

To illustrate the accuracy of the method, researchers used genetically modified oocytes lacking only one exon of a single-copy gene (Dicer). Reads that map to exon 23 were entirely absent, whereas the reads from neighboring exons were intact. Overall, the method detects 94% of all expressed genes in a single cell.

The assay can also be used to discover new transcripts and alternative splicing isoforms. It also facilitates quantitative estimates of RNA abundance by the frequency with which the sequence occurs in the RNA-Seq reads. Recent experiments have already generated the insights into possible pathways for derivation of embryonic stem cells (ESCs) from the inner cell mass of blastocysts.

While the cells of the blastocyst follow a strict developmental program, the stem cells are self-renewing and pluripotent. Sequencing of RNA of single cells at different points in the transition from ICM to ESCs demonstrated multiple and profound changes in the transcriptome affecting epigenetic regulators, microRNAs, and pluripotency gene clusters.

Gene network analysis of inner cell mass (ICM) vs. embryonic stem cells (ESC). The genes in gray changed expression levels less than twofold. The pathway was generated using Ingenuity software.[Life Technologies]

Tracking mRNA in a Living Cell

All cells including stem cells and progenitor cells undergo changes over time, which is reflected in their mRNA and protein profiles. Techniques that allow multiple measurements in the same live cell over a period of time are vital for understanding the influence of individual cells on the cellular systems.

Fluorescent imaging has become sensitive enough to measure levels and location of tagged proteins inside a living cell. However, tracking individual mRNA had remained a challenge until the group from Bar-Ilan University, led by Yaron Shav-Tal, Ph.D., developed a real-time detection system for mRNA.

The technology is based on creating a modified 3´ end of a target gene by incorporating multiple repeats of a binding site for MS2, an RNA-binding protein. In turn, MS2 protein is coupled with the green fluorescent protein (GFP). One mRNA transcript binds to about 30 MS2-GFP proteins generating an easily detectable fluorescent signal.

“This was the first instance of measuring transcription of a single gene in vivo in human cells. Now we are able to detect mRNA translocation from the nucleus to cytoplasm. The signal is visible at 3–5 mRNAs per gene,” says Dr. Shav-Tal.

The first few experiments have already shed light on such important questions as the activity of viral promotors in comparison to native promotors. While the native promoter shut down for about 20 minutes every 200 minutes, the viral promoter remained active for hours.

In the future, this technology may provide the answers to how manipulations of transcription increase or decrease expression of disease-related genes. By introducing specific mutations, scientists will determine their influence on promoter potency and therefore gene expression.

“We will be able to research interactions between replication and transcription machinery,” continues Dr. Shav-Tal. “And how the external signal reaches specific gene promotors and turns them on or off.”

A recent development includes a knockin mouse carrying a target gene coupled with MS2 binding sites. Now every cell of the mouse carries this transcriptional reporter. This in vivo mRNA reporting system opens amazing possibilities for tissue-specific comparisons and for studying the signal transduction effects throughout the whole organism.

Detection of cyclin D1 mRNA molecules in a human cell: A 3-D representation of all cyclin D1 mRNA molecules by RNA fluorescent in situ hybridization. The DNA in the nucleus is marked in red and a single actively transcribing gene (large green dot) can be seen without the nucleus. [Bar-Ilan University/Sharon Yunger]

Infrared Cytology

Infrared spectroscopy enables visualization of gross differences between individual cells based on their absorption spectra. A cell can be arrested at a particular point by formalin fixation, after which the IR spectra can be obtained within just a few minutes.

“Infrared cytology is an objective analysis of the cells and brings a unique angle to cytological examination,” comments Peter Gardner, Ph.D., director of assessment at the School of Chemical Engineering and Analytical Science, The University of Manchester. Dr. Gardner anticipates that IR spectroscopy will become an integral part of diagnostics, helping to identify and grade diseased cells in pathology specimens.

Another opportunity lies in personalized medicine. Instead of searching for specific molecular markers to predict a patient’s response to drug therapy, the same prediction can be made by using IR analysis of the person’s own cells.

Using ovarian cancer cell lines, Dr. Gardner’s group demonstrated clear spectroscopic separation between cell lines responding and not responding to treatment with the cytotoxic drug cisplatin. Moreover, when treated with a new experimental compound, KF101, the cells presented yet another unique spectral signature. This observation indicated that KF101 acts by a mechanism different from that of cisplatin.

“I see another opportunity for IR spectroscopy in studies of stem cells,” continues Dr. Gardner. “Stem cell differentiation induces changes in the spectrum. If stem cell implantation has to happen at a certain differentiation stage, the IR spectral data would provide a robust measure of whether the cells have reached that stage.”

Dr. Gardner says that he and his colleagues have resolved a long-standing issue of spectral distortion. Mid-infrared radiation scatters strongly from the organelles distorting the absorption spectrum. The scientists modified the existing Mie scattering theory, which describes scattering of light from the objects of a size comparable to the illuminating wavelength.

A new mathematical algorithm was able to correct the spectral distortion. This methodology was recently applied to identification of stem cells among a side population of tumor cells. This population is thought to be enriched with stem cells, but their low counts prevent further fractionation and characterization. FTIR spectroscopy was able to record the fingerprints of individual cells, confirming their biochemical differences.

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