Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies

Abstract

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5 days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Lineage mapper: A versatile cell and particle tracker

“…This dataset was segmented using the custom built segmentation BioSeg24. For the third application25, we tracked colonies of pluripotent stem cells, in which colonies move, grow, and fuse (merge) with other colonies. Pluripotent stem cells exist in a privileged developmental state with the potential to form any of the cell types of the adult body.…”

“…These cells grow as isolated colonies, with each colony consisting of tens to thousands of cells. There is an interest in tracking these colonies to understand the temporal relation between gene expression and cell state, in order to potentially engineer the cell state for regenerative medicine applications25. Since an individual colony is larger than the size of a single camera frame, colony tracking can only be done from time-lapse stitched image mosaics.…”

“…Three replicates were created on different dates for a total ≈1 TB of image data. LM tracked these colonies which move, grow, and merge with other colonies25. The tracking performance was validated visually by looking at the movies and checking the fusion lineage for any mistakes.…”

“…This study was approved by NIST and carried out in accordance with the approved guidelines of the NIST Human Subjects Protection Office. A complete description of the set up used to acquire this dataset was published in Bhadriraju25, and briefly described below. Stem cell cultures were maintained at 37 °C and 5% CO 2 with 80% humidity, in a custom built incubation chamber attached to a Zeiss 200 M microscope with a Zeiss 10x, 0.3 NA objective and an automated stage.…”

Opportunities and Challenges in Implementation of Multiparameter Single Cell Analysis Platforms for Clinical Translation

“…Dynamic data can also confirm stability in gene expression, such as in stem cell colonies, and the spatial location of the expressed gene within colonies. Such analysis can also allow detection of rare events 41. Longer‐term time‐dependent measurements of cell populations by imaging or flow cytometry can indicate whether population heterogeneity is the result of stochastic fluctuations within individual cells or is due to genomic differences within the population.…”