Spreading kinetics for quantifying cell state during stem cell differentiation

Abstract

Major current problems in the stem cell field are how to determine the course of differentiation and how it is influenced by environmental factors. It would be very useful to be able to quantify the differentiation, not least because this would facilitate the discovery of correlations with quantitative environmental features. At present, optical microscopy is the main observational tool. Although it is easy to implement experimentally, quantitative analysis of the results is rather difficult, and some degree of subjectivity in the interpretation is almost inevitable. This paper explores a novel alternative approach to the problem, based on analysis of the rapid adaptive behavioral response of the cell to its arrival on a substratum, namely the transition from sphere to segment, which perturbs evanescent optical fields generated in the substratum and thereby can be monitored in a very straightforward fashion. Parameters characterizing the transition, which we hypothesize are characteristic of the state of differentiation of the cell, are readily extractable from the data. Spreading of a living cell in contact with the surface of a substratum is a basic response of the cell to such contact [1], although among the myriads of cell types and environments, the latter comprising both the nature of the substratum and of the liquid medium bathing cell and substratum, not all combinations thereof lead to spreading (see also, e.g., [2]). Furthermore, the actual temporal course of the spreading (i.e., its kinetics) depends sensitively on cell type and environment [1–4]: We hypothesize that these kinetics are a sensitive indicator of the spreading process. They would therefore be of practical use for identifying differentiation state provided that an appropriately accurate way of measuring them can be found. Our hypothesis is, then, that the rapid spreading1— quantitatively defined by the parameters of an appropriate mathematical function—is characteristic of the state of differentiation of an initially pluripotent stem cell. For the quantitative definition of spreading optical (e.g., [1]) or electron (e.g., [3]) microscopies are not really suitable, partly because of the difficulty of getting good time resolution (although modern techniques of digital photography and image analysis have overcome that to some extent) and partly because the information obtained is typically rather incomplete (e.g., just a cross section, i.e. either a plan or an elevation). Sometimes more complete information can be obtained, e.g. by using confocal microscopy, but mostly with an accompanying disadvantage, such as requiring the cell to be fed with a fluorescent dye, which may alter its physiology in unwanted ways; we note that stem cells are especially sensitive to foreign substances because of their pluripotency. Spreading kinetics for quantifying cell state during stem cell differentiation Amirreza Aref,†, ‡ Robert Horvath†, and Jeremy J. Ramsden†, *