Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

Abstract

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation. Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors

“…We next tested whether different colony sizes may change the proportion of cell fates on micropatterns (Sup Fig 2A). (Kwon et al, 2008; Scheibner et al, 2021; Viotti et al, 2014)Using our established quantitative immunofluorescence pipeline (Blin et al, 2019; Wisniewski et al, 2019), we measured the proportion of each individual population (Sup Fig 2B) and plotted these proportions as a function of the radial distance from the colony edge (Sup Fig 2C). We found that all three domains, i.e SOX17, TBXT/SOX2 and SOX2-only domains, remained located at a consistent distance from the colony edge across all colony diameters except for colonies below 320µm where this rule did not apply as strictly.…”

“…Using our established single-cell-resolution quantitative immunofluorescence pipeline (Blin et al, 2019;Wisniewski et al, 2019), we measured the proportion of each individual population (Fig 1Cii).…”

Predicting pattern formation in embryonic stem cells using a minimalist, agent-based probabilistic model

“…This technique makes it possible to control the biochemical environment of the cells while at the same time providing precise geometrical confinement. A detailed video protocol of the method employed in Blin et al is available at https://www.jove.com/t/59634/mapping-emergent-spatial-organization-mammalian-cells-using 21 . Briefly, we have 186 images for disc micropatterns and 152 images for ellipse micropatterns.…”

Quantitative developmental biology in vitro using micropatterning

“…Multi parametric quantitative data analysis Quantitative experimental design requires control of experimental variables as well as meaningful metrics to monitor the biological process of interest and the effects of eventual perturbations. Here, three examples are provided: (A) front-rear polarity ( Théry et al, 2006 ), (B) fate patterning in embryonic cells ( Ostblom et al, 2019 ; Wisniewski et al, 2019 ) and (C) collective migration ( Jain et al, 2020 ). In these examples, geometric confinement provides relevant cellular cues and facilitates quantification at the same time.…”