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. 2016 Mar 1;96:85-96.
doi: 10.1016/j.ymeth.2015.11.012. Epub 2015 Nov 25.

A High-Content Platform to Characterise Human Induced Pluripotent Stem Cell Lines

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Free PMC article

A High-Content Platform to Characterise Human Induced Pluripotent Stem Cell Lines

Andreas Leha et al. Methods. .
Free PMC article

Abstract

Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components. Here we report the establishment of a high-content platform for phenotypic analysis of human iPSC lines. In the described assay, cells are dissociated and seeded as single cells onto 96-well plates coated with fibronectin at three different concentrations. This method allows assessment of cell number, proliferation, morphology and intercellular adhesion. Altogether, our strategy delivers robust quantification of phenotypic diversity within complex cell populations facilitating future identification of the genetic, biological and technical determinants of variance. Approaches such as the one described can be used to benchmark iPSCs from multiple donors and create novel platforms that can readily be tailored for disease modelling and drug discovery.

Keywords: Cell based assays; High content; Human pluripotent stem cells; Induced pluripotent stem cells; Phenotype screening; iPSCs.

Figures

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Fig. 1
Fig. 1
A screen of 74 conditions to visualise single iPS cells. List of tested substrate conditions from two combined arrays (Orla and custom made). Columns indicate an arbitrary reference number, name concentration, motifs or residues and source are detailed. Yield refers to number of single cells observed by visual inspection indicative of assay quality. The insets show examples of suboptimal substrates for iPSCs. Very few cells attach when plated on laminin α1 IKVAV (Table 1, n. 11, representative of conditions 1–48) and many clumps and very few single cells are observed when cells are plated on vitronectin PQVTRGDVFTM (Table 1, n. 49, representative of conditions 49–64).
Fig. 1
Fig. 1
A screen of 74 conditions to visualise single iPS cells. List of tested substrate conditions from two combined arrays (Orla and custom made). Columns indicate an arbitrary reference number, name concentration, motifs or residues and source are detailed. Yield refers to number of single cells observed by visual inspection indicative of assay quality. The insets show examples of suboptimal substrates for iPSCs. Very few cells attach when plated on laminin α1 IKVAV (Table 1, n. 11, representative of conditions 1–48) and many clumps and very few single cells are observed when cells are plated on vitronectin PQVTRGDVFTM (Table 1, n. 49, representative of conditions 49–64).
Fig. 1
Fig. 1
A screen of 74 conditions to visualise single iPS cells. List of tested substrate conditions from two combined arrays (Orla and custom made). Columns indicate an arbitrary reference number, name concentration, motifs or residues and source are detailed. Yield refers to number of single cells observed by visual inspection indicative of assay quality. The insets show examples of suboptimal substrates for iPSCs. Very few cells attach when plated on laminin α1 IKVAV (Table 1, n. 11, representative of conditions 1–48) and many clumps and very few single cells are observed when cells are plated on vitronectin PQVTRGDVFTM (Table 1, n. 49, representative of conditions 49–64).
Fig. 2
Fig. 2
Assay development for cell density and time before fixation. (A) The panels show representative microphotographs for 3000 (Top) or 6000 (bottom) cells plated on different fibronectin concentrations (Fn1, Fn5, Fn25). Note that the majority of cells when 6000 cells are plated appear in clumps. Blue, DAPI. Green, EdU. Red, CellMask. One field of view per well is shown here. (B) Live image movies were derived and inspected of cells plated as 3000 cells on the three fibronectin conditions. Timepoints 1 h, 4 h, 12 h and 24 h after seeding are shown here. Adhering cells can be readily observed from 4 h onwards and spreading has occurred in most cells by 24 h.
Fig. 3
Fig. 3
Workflow diagram detailing image acquisition and analysis. (A) Layout of the 96-well plates for phenotype assays showing fibronectin concentration per column (blue). Different experiments present randomised patterns (i.e. Fn1-Fn5-Fn25, Fn1-Fn25-Fn5, Fn5-Fn25-Fn1, Fn5-Fn1-Fn25, Fn25-Fn1-Fn5, Fn25-Fn5-Fn1). Step-by-step experimental conditions for the assay set-up are detailed on the right. KOSR = KnockOut Serum Replacement, RT = room temperature, BSA = bovine serum albumin. (B) Image analysis pipeline detailed in Section 3.3 is summarised here. Input images are segmented to identify nuclei and cytoplasm. Border objects and artefacts are discarded via morphology and intensity assessment on nuclei and on cells. The modify population module is employed to identify clumps based on cell-to-cell proximity and capture the number of cells in each clump as a context feature.
Fig. 4
Fig. 4
Phenotypic features analysed and their aggregation. Cell-based measurements are aggregated in each well to well-based measurements. (A) Histogram of cell areas with highlighted sample mean and standard deviation (in blue). (B) The EdU median intensity is aggregated using a Gaussian distribution fitting to the main peak of cells, which is considered the EdU negative cells population. The area under the curve that is not explained by that main peak is considered the fraction of EdU positive cells (in blue). (C) Distribution of clump sizes in well and the fit of a geometric distribution (in blue). Clump size refers to the number of cells in a clump. The inverse mean is the parameter of that fit and is used to describe the tendency of the cells to form clumps. (D) Shows detail of all phenotypic features for single cells and all cells. μ = mean σ = standard deviation.
Fig. 5
Fig. 5
Phenotypic features vary in single cells and cells in clumps. Values of phenotypic features for single cells (blue) and cells in clumps (red) surrounded by at least one neighbouring cell, examined across the fibronectin concentrations in a series of replicate experiments. (A) Shows the fraction of single cells obtained by the assay. Over one third of cells (37%) appear as single cells. Min–Max range is shown. (B–F) morphological cell features for single cells and cells in clumps. Cell area, roundness and cell width to length display a wider range in single cells versus cells in clumps. This is suggestive of constraint from neighbouring cells limiting the shape of cells in clumps. (D) Cell roundness and (E) cell width to length are plotted versus cell area. These data are shown as examples of the validity of this method to capture features emerging upon cell–cell contact.
Fig. 6
Fig. 6
Fibronectin conditions affect several phenotypic features. Phenotypic features were interrogated in a series of replicate experiments for each of three fibronectin concentrations in all cells (Fn1; green, Fn5; grey, Fn25, red). (A) Number of cells per well. Min–Max range is shown. (B) Fraction of EdU positive cells over the three fibronectin concentrations. Min–Max range is shown. (C) Inverse mean clump size, an indicator of the propensity of cells to clump. (D) Cell roundness plotted over cell area. Note that cells in higher concentration of fibronectin present a wider range of values. (E–G) Density plots for nucleus area, nucleus roundness and nucleus width to length. The y axe shows the relative fraction of cells. Min–Max range is shown. In all graphs: green, Fn1; grey, Fn5; Orange, Fn25.
Fig. 7
Fig. 7
Fibronectin conditions mediate distinct phenotypic responses. (A) Principal component analysis is performed on the described phenotypic features in a series of replicate experiments. Green, Fn1; Grey Fn5; Orange Fn25. The ellipses represent the higher dimensional space defined by 68% of samples. (B) Directionality of the contributions of each phenotypic features to the first two components. (C) Percentage of variance explained by each component. Phenotypic features extracted from cells exposed to different fibronectin concentrations segregate in a high dimensional space.

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