Single-Cell Western Blotting to Probe Stem Cell Signaling and Differentiation

Heterogeneity is a hallmark of many biological processes, from immune cell recognition to tumor initiation. Stem cell signaling and differentiation are inherently heterogeneous, as single cells can give rise to diverse progeny during development and stem cell populations in vitro can differentially respond to the same set of cues. To study this cell-to-cell variability, single-cell analytical tools are needed. Recent technologies have successfully examined genomic and transcriptomic heterogeneity at the single-cell level, but proteomic analysis more accurately reflects cell composition and allows for probing of posttranslational modifications. Flow cytometry and immunocytochemistry (ICC) are currently the best tools for single-cell proteomics; however, results can be confounded by antibodies with varying specificities, and sample preparation and analysis can be complicated. To address these problems, we employ western blotting, a standard proteomics technique that electrophoretically separates proteins and uses molecular weight information to identify on- and off-target signals, and apply it to single cells. We term this technique single-cell western blotting (scWB) and have successfully characterized its performance and applied it to study heterogeneity in stem cells.

Our scWB allows for the simultaneous analysis of proteins in ~2,000 cells in less than four hours. The method is performed on a microscope slide coated with a thin photoactivatable polyacrylamide gel micropatterned with an array of microwells. Cells are initially settled into microwells, then lysed in–well, followed by electrophoretically induced migration of proteins into the gel and separation over ~500 μm. UV light-induced photocapture then immobilizes the proteins, allowing for subsequent probing by sequential diffusion of primary and secondary antibodies into the gel. Further multiplexing can be achieved through stripping and re-probing; we have successfully analyzed six targets from a single cell. High sensitivity is a key consideration in single-cell analyses; we have shown a detection threshold of ~30,000 molecules, validating our ability to interrogate much of a single cell’s proteome.

ScWB is ideally suited for stem cell heterogeneity analysis, demonstrated by initial studies of neural stem cell (NSC) signaling and differentiation. We first investigated MAPK signaling dynamics within single NSCs in response to FGF-2 stimulation. Phosphorylated ERK and MEK levels followed trends observed with conventional western blotting, but scWB also allowed quantification of the highly variable responses of individual cells within the NSC population. Of note, parallel high-throughput ICC analysis was restricted by poor phosphorlyated MEK antibody specificity, a limitation overcome by spatial separation of proteins in scWB. We then examined differentiation markers of single cells in a NSC population over a six day period under mixed differentiation conditions. Consistent with ICC studies, NSCs expressing the undifferentiated markers Sox2 and Nestin decreased over the time course of the experiment, while cells expressing the neuronal marker β-III-tubulin or the astrocytic marker GFAP increased. Surprisingly, we observed differential regulation of two Nestin isoforms in single NSCs during differentiation, a previously unreported finding undetectable by conventional proteomic tools. ScWB, therefore, is a novel platform for single-cell analysis that addresses current limitations in the field, and can be broadly utilized to advance our understanding of cell heterogeneity.