title: Single-cell heterogeneity in suppression of PC12 differentiation by direct microinjection of a differentiation inhibitor, U0126.
abstract: Phenotypic and genomic heterogeneity among single cells in a cell population leads to inaccuracy and obscuration in research about mammalian cell differentiation. In order to address the problems regarding bulk analysis on heterogeneous cell populations, it is necessary to accurately regulate and analyze changes in differentiating cells at the single-cell level. To investigate the single-cell changes in PC12 neuronal differentiation that occur when inhibited by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), we directly injected the chemical into individual target cells and analyzed the outcomes (neurite outgrowth) at the single-cell level. As a result, we could accurately regulate the quantity of U0126 being introduced into each target cell, which was previously not possible using the common method of simply adding the inhibitor to the culture medium. It was possible to analyze the inhibitive effect of U0126 even when the injected quantity was lower than the lower limit for inhibition when added to culture medium (0.1 μM, identical to 1.2 × 10(8) molecules per cell on dish). In particular, injection of 1.5 × 10(7) molecules into each cell resulted in a 59% decrease of the mean total neurite length. Time-course analysis of neurite outgrowth at the single-cell level using fluorescence staining method showed that the changes in neurite length of differentiating PC12 cells were not homogeneous, but were largely variable across individual target cells.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Jihye
authors.initials: J
authors.lastname: Chung
authors.forename: Natsuko
authors.initials: N
authors.lastname: Miura
authors.forename: Akio
authors.initials: A
authors.lastname: Ito
authors.forename: Munetaka
authors.initials: M
authors.lastname: Sawada
authors.forename: Shigemichi
authors.initials: S
authors.lastname: Nishikawa
authors.forename: Kouichi
authors.initials: K
authors.lastname: Kuroda
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Ueda
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008845:Microinjections; D009570:Nitriles / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats; D059010:Single-Cell Analysis
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126
keywords: PC12; automated microinjection; cell differentiation; quantitative single-cell analysis