Single-Cell Western Analysis of iPSC-derived Neural Progenitors Confirms High Differentiation Efficiency and Population Distribution Dynamics

K. Flynn, D. Galitz, J. Sabat, S. Luhowskyj, S. Stoesz, E. Jabart, J. Cooper, and J. Aho

ABSTRACT

Human pluripotent stem cells, including embryonic (ES) and induced pluripotent stem (iPS) cells, offer an essentially unlimited source of neural cells that can be used to investigate mechanisms of human neurological disease and neural regeneration. A critical step during the derivation of neurons, astrocytes, or oligodendrocytes from pluripotent stem cells is generating a robust and homogeneous neural progenitor cell population, which ultimately impacts the efficiency of downstream differentiation protocols and helps control experimental reproducibility. Understanding population heterogeneity is an important step in the optimization of differentiation protocols, which is challenging with existing methods. This study demonstrates how Single-Cell Western blot analysis can complement traditional verification approaches by providing insights into protein expression both at the population and single-cell level. In this study, human iPS cell lines were differentiated into neural progenitor cells using the standardized protocol and reagents in the StemXVivo® Neural Progenitor Differentiation Kit. Combining our standardized differentiation protocol for neural progenitor cells with Milo Single-Cell Western technology (ProteinSimple), we were able to assess population protein expression dynamics during the differentiation of iPS cells into neural progenitor cells. Using Oct-3/4 as a marker for pluripotent stem cells and Pax6 as a marker for neural progenitor cells we show, at the single-cell level, that during differentiation the cells shift from an exclusively Oct-3/4 expressing population to one that is robustly Pax6-positive. We were also able to identify subpopulations of neural progenitor cells that express Pax6 at high or low levels. Additionally, single-cell analysis also used to characterize the population distribution of terminally-differentiated neural cells following growth factor withdrawal-induced differentiation of iPS-derived neural progenitor cells.