Exploring "Impossible" Science

CyTOF and mass-tag barcoding let Eli Zunder ask more questions and find new answers


“There are so many different things we want to know about every cell,” says Eli Zunder, PhD and stem cell biologist at Stanford. “And I always want to measure more—more transcription factors, more cell signaling molecules, more cell surface proteins, with more samples, simultaneously. That’s really the dream for stem cell biologists.”

What may be a dream for other researchers is an everyday reality for Zunder. He has the good fortune to work in Dr. Garry Nolan’s lab within the Baxter Laboratory for Stem Cell Biology at Stanford School of Medicine. The Nolan Lab was one of the earliest adopters of mass cytometry and, specifically, the Fluidigm® CyTOF® system to explore normal cell function as well as disease progression at the single-cell level.

Since its introduction in 2009, mass cytometry has helped researchers explore fundamental questions of cell biology, immunology and disease progression. Exploring many different questions simultaneously at the single-cell level inspires Zunder, and a lab environment that embraces creative thinking, scientific rigor and powerful technology gives him the freedom to develop new methods for exploring what until recently would have been considered “impossible” science.

Zunder’s daily work involves studying the molecular mechanisms that control the early stages of induced pluripotent stem cell (iPSC) reprogramming. Single-cell analysis is critical for these studies, because only a small percentage of cells in a reprogramming culture successfully reach the desired pluripotent end state. The trick, as Zunder explains it, is to monitor cell populations as they change over time, identify the activity occurring within the cell and at the surface level, and then map those changes to help determine the possible trajectories for cellular reprogramming. To take the complex simultaneous measurements that he needed, Zunder relied on CyTOF—with an extra innovation of his own.

Collaborating with other researchers in the Nolan lab, Zunder optimized CyTOF with mass-tag cell barcoding. Cell barcoding allows researchers to combine multiple cell samples into a single tube, which streamlines sample preparation and eliminates staining and collection variation between samples. This saves time and produces more accurate results, especially with very large and complex datasets such as those produced by Zunder’s experiments.

Barcoding was not readily available to early adopters of mass cytometry, but Zunder had experience using Peter Krutzik’s barcoding method for fluorescent flow cytometry. He worked with colleague Bernd Bodenmiller to adapt this technique for mass cytometry using a nonredundant binary barcode and lanthanide isotopes as reagents.

More recently, Zunder modified the protocol to use palladium reagents with a doublet-filtering partially redundant barcode scheme, along with a semi-automated debarcoding algorithm. These modifications have enabled him to further explore new questions with mass cytometry by increasing the number of available measurement channels, identifying cell doublets for removal from his datasets, and improving the accuracy and reproducibility of barcode sample deconvolution.

Using mass-tag cell barcoding, Zunder can explore an entire biological system’s response to multiple stimuli at one time. He is able to simultaneously measure an almost unlimited number of data points throughout the iPSC reprogramming process, including cell signaling pathways, transcription factor networks and the expression of cell-surface markers. Linking the common sets of reprogramming landmarks with associated timepoints enabled Zunder to map cell populations as they change over time with an unprecedented level of detail, and to characterize a previously unidentified early intermediate stage of cellular reprogramming. With this deeper understanding of cell development and disease progression, Zunder aims to significantly accelerate progress in regenerative medicine and hopes to influence discoveries across a range of disciplines.

“The barcoding method was incredibly useful because we could take all of our samples over the entire reprograming time course and combine them into a single tube for analysis,” Zunder explains. “This let us have very high confidence that any changes we saw in the cell populations over time were not due to tube-to-tube variability, which is especially important when comparing subtle changes in expression level between samples.”

The debarcoding algorithm, developed in collaboration with Nolan Lab colleague Rachel Finck, separates the collected barcoded dataset into individual files corresponding to the original samples. The semi-automated algorithm provides a fast, accurate and reproducible method for sample deconvolution, allowing researchers to bypass the tedious and subjective process of manual gating for Boolean deconvolution.

With exploratory experiments, it is vital for researchers to be able to discriminate single-cell events from potential doublet artifacts in the data. As Zunder is looking for changes within a cell population over time, he does not want to mistake a doublet for a new cell type not previously described or to misidentify a point in cell reprogramming. Using an idea based on Hamming codes from his collaborator Dana Pe’er at Columbia University, Zunder modified his original technique to use a redundant barcoding scheme, which has enabled him to easily filter nearly 100% of doublets from the dataset.

Mass-tag barcoding can easily be integrated into the workflow for any single-cell analysis application. “The challenges we see in stem cell analysis are true for many other biological systems, so the role the CyTOF plays can be the same for these as well,” Zunder observes. “The number of experimental conditions you can test in complex biological systems is virtually infinite, when you consider all of the cell treatments you want to use at different times, with different concentrations, and in different combinations to get a handle on how your system is behaving.” Using mass cytometry in conjunction with mass-tag barcoding gives researchers the freedom to explore an almost unlimited data set, thereby accelerating the rate of discovery.

The advantages of barcoding have made it integral to Zunder’s work. “It's just standard practice now,” he says. “Everybody in our lab who runs CyTOF samples will barcode them.”

Zunder and his team shared the full protocol for this approach in early 2015 in Nature Protocols Vol. 10, No. 2. Under a licensing agreement with Stanford University, Fluidigm standardized, tested and packaged a commercial version of Zunder’s protocol for mass-cell barcoding, along with a catalog of appropriate barcoding reagents, which is now available to its customers.

Zunder is more than pleased with this development. When he and his colleagues first began using barcoding for mass cytometry, they were willing to take risks, have things go wrong and make adjustments based on those results. “We had to do a lot of careful troubleshooting and reproducibility experiments in the beginning to optimize the barcoding protocol and reagents.” With the barcoding kit from Fluidigm, any lab with a mass cytometry instrument can adopt the new technique without the trial and error, delays and expense associated with developing a new protocol.

From Zunder’s perspective, the applications for mass cytometry and barcoding in stem-cell biology are tremendous, with great future potential for stem-cell therapy and regenerative medicine. “Everybody knows that single-cell analysis is what we need, but the technology hasn’t always been easy to use or accessible,” he says. “For all practical purposes, before CyTOF and mass-tag barcoding, the experiments that I wanted to do were impossible.”