“Every cell has too many things I want to know. I have been trying to detect more transcription factors, more signaling pathway molecules, more surface markers and more samples – and it’s best to Testing them, this is really the dream of stem cell researchers."
- Stanford University Stem Cell Biology, Dr. Eli Zunder.
Now, the dream of the past has become the daily work of Zunder in reality. A few years ago, Zunder was fortunate enough to work in the laboratory of Professor Garry Nolan of Stanford University, one of the first laboratories to use mass spectrometry. It has long been using the mass spectrometry system to perform physiology at the single cell level. Research on cell function in diseases and diseases.
Since its inception in 2009, mass spectrometry has helped researchers explore many of the fundamental issues in cell biology, immunology, and disease development. Not only does Nolan Labs provide strong technical support for Zunder, but it also provides a creative and rigorous research environment that allows Zunder to freely develop new technologies for some that have until recently been considered “impossibleâ€. Scientific research.
One of Zunder's daily tasks is the molecular mechanism of the early stages of reprogramming of iPSCs (induced pluripotent stem cells). In these studies, single-cell analysis is critical because in a reprogrammed culture, only a small fraction of cells can successfully reach the terminal state of iPSC. Zunder explained that the key to the problem is to observe changes in the cell population during induction of culture, to detect cell surface and internal activity, and then to record these changes to identify possible reprogramming strategies for cells during induction. In order to perform simultaneous detection of many parameters of the cell, Zunder needs to rely on mass spectrometry, in addition to another, his own technological innovation.
Cell Barcode technology makes it possible to combine different parts for labeling, detection and analysis
With the help of other staff, Zunder optimized the metal-based "Mass-tag cell barcording" technology. "Cellular Barcorde" allows cells of different samples to be labeled with different metals, allowing researchers to mix multiple samples into a single tube analysis, simplifying sample preparation and eliminating the need for staining and data collection. Error. Not only does this save time, but it also allows researchers to obtain more accurate data, which is especially important in this large-scale experimental project conducted by Zunder.
At the time, Barcorde technology was not yet used in the mass spectrometry flow field, and Zunder has extensive experience in fluorescence flow Barcorde technology. Using a non-redundant binary coding strategy using a lanthanide metal as a label, he and his colleague Bodenmiller applied this technique to mass spectrometry.
New Barcorde solution based on palladium elements
Recently, Zunder improved Protocol to perform Barcorde on samples using six non-radioactive stable isotopes of palladium. The new Protocol uses a partially redundant ternary encoding that allows researchers to reject dimers formed between different sample cells based on the results. In addition, it is equipped with a semi-automated debarcorde algorithm to facilitate data analysis. These improvements not only increase the available detection channels, but also improve the accuracy of the data, allowing researchers to explore new scientific issues using mass spectrometry.
Using cell Barcorde technology, Zunder uses an experiment to explore the response of the entire biological system to multiple stimuli. He can simultaneously measure near-infinite data points during cell reprogramming, including intracellular signaling pathways, transcription factor networks, and cell surface markers. By analyzing these data, Zunder finds protein molecules that can be used as "signposts" in the reprogramming process. By linking these "roadmaps" with their corresponding time points, the cell population changes over time can be revealed with unprecedented precision. Based on this, Zunder discovered an important early intermediate stage in the process of cell reprogramming.
Zunder uses the iPSC reprogramming "roadmap" depicted by mass spectrometry
“Barcorde's method is very useful because we can combine all the samples collected in the experiment together,†Zunder explained. “ We can confidently say that any changes seen in the cell population are not between samples. Accidental error. This is especially important, especially when analyzing samples with small differences in expression levels."
The debarcode algorithm mentioned above was developed in collaboration with Nolan Labs colleague Rache Finck, which converts the acquired Barcorde data into a separate file corresponding to the original sample. The semi-automated algorithm provides a fast, accurate, and reproducible decoding method that allows researchers to bypass the cumbersome and subjective manual setup process.
In exploratory experiments, it is important for researchers to distinguish single-cell data from cell-dimers. What Zunder is looking for is the change in cell population over time. He doesn't want to treat a cell dimer as a new type of cell that has not been reported. Dana Pe'er of Columbia University came up with the idea of ​​Hamming coding, which inspired Zunder, who improved the original technology and used a redundant coding system, which helped him easily remove nearly 100% of the dimers. data.
Cell Barcorde technology can be easily integrated into any single cell analysis workflow. "Our challenges in stem cell research are equally applicable to other biological systems, so Cytof can also perform similar functions in these areas." Zunder notes, "Experimental conditions that can be tested in complex biological systems. The number is almost limitless. You can consider different processing times, different drug concentrations, and different combinations of conditions. By contrast, you can grasp how the biological system you are studying works.†Mass spectrometry and cell Barcorde The combination of technologies allows researchers to freely access near-infinite data, greatly accelerating the research process.
These advantages of Barcorde technology make it indispensable in Zunder's work. “Now this is a standard practice,†he said. “In our lab, no matter who runs the Cytof sample, the sample will be barcorde.â€
In early 2015, Zunder and his team shared the technology in the Nature Protocols (Vol. 10, No. 2) magazine. Authorized by Stanford University, Fluidigm has carried out extensive standardization and testing work, launched a commercial version of the Zunder protocol and a catalog of Barcorde reagents, which are now readily available to mass spectrometry customers.
Cell Barcorde and Data Debarcorde have become an important part of the CyTOF experimental process
Zunder is very happy with the launch of this product. When he used the Barcorde mark with his colleagues, he had to take various risks. When problems arise, you need to adjust the experiment according to the results. “In the initial optimization of Barcorde protocols and reagents, we need to carefully investigate the cause of the problem and the repeatability of the test system.†Now only the Fluidigm labeling kit is required, and any mass spectrometry lab can use this technology directly. There is no need to repeat their arduous optimization process.
In Zunder's view, the discovery of mass spectrometry and cell Barcorde technology in stem cell biology is exciting, and will further advance stem cell therapy and regenerative medicine in the future. “Everyone knows that single-cell analysis is what we need, but the technology is not always easy to get or use,†recalls Zunder. “Actually, before the advent of CyTOF and Cell Barcorde technology, the experiments I wanted to do were It is impossible."
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