Abstract
Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.
Highlights
Neuronal differentiation and morphogenesis have been a subject of intense research during the last decades [1]
PC12 cell proliferation is enhanced under epidermal growth factor (EGF) treatment [2], while mitomycin is known to suppress proliferation
Since all these features were related to the presence of large, partially filled regions, we suggest that the discrimination of the differentiated and the undifferentiated morphology occurs on the basis of how cell growth areas are physically connected
Summary
Neuronal differentiation and morphogenesis have been a subject of intense research during the last decades [1]. Much research in the field of neuronal cell research has focused on characterizing neurite growth of single cells by measuring average neurite length or the number of branching points [5,6]. This leaves out the important question, under which treatment conditions differentiation of the whole cell population occurs. As a model system we use the neuroendocrine PC12 cell line This is a popular substitute to study the processes of neuronal differentiation [7], since study on primary neuron cells is hindered due to the low yield of primary neurons from animal models and the difficulties of primary neuron cell culture. Upon stimulation with nerve growth factor (NGF), PC12 cells change their morphology by flattening and growing neurites, resembling the phenotype of sympathetic ganglion neurons
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
