Dataset Information

Quantification of the morphological characteristics of hESC colonies.

ABSTRACT: The maintenance of the undifferentiated state in human embryonic stem cells (hESCs) is critical for further application in regenerative medicine, drug testing and studies of fundamental biology. Currently, the selection of the best quality cells and colonies for propagation is typically performed by eye, in terms of the displayed morphological features, such as prominent/abundant nucleoli and a colony with a tightly packed appearance and a well-defined edge. Using image analysis and computational tools, we precisely quantify these properties using phase-contrast images of hESC colonies of different sizes (0.1-1.1?[Formula: see text]) during days 2, 3 and 4 after plating. Our analyses reveal noticeable differences in their structure influenced directly by the colony area [Formula: see text]. Large colonies (A?>?0.6?mm²) have cells with smaller nuclei and a short intercellular distance when compared with small colonies (A?2). The gaps between the cells, which are present in small and medium sized colonies with A???0.6?mm², disappear in large colonies (A?>?0.6?mm²) due to the proliferation of the cells in the bulk. This increases the colony density and the number of nearest neighbours. We also detect the self-organisation of cells in the colonies where newly divided (smallest) cells cluster together in patches, separated from larger cells at the final stages of the cell cycle. This might influence directly cell-to-cell interactions and the community effects within the colonies since the segregation induced by size differences allows the interchange of neighbours as the cells proliferate and the colony grows. Our findings are relevant to efforts to determine the quality of hESC colonies and establish colony characteristics database.

SUBMITTER: Orozco-Fuentes S

PROVIDER: S-EPMC6879623 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Quantification of the morphological characteristics of hESC colonies.

Orozco-Fuentes Sirio S Neganova Irina I Wadkin Laura E LE Baggaley Andrew W AW Barrio Rafael A RA Lako Majlinda M Shukurov Anvar A Parker Nicholas G NG

Scientific reports 20191126 1

The maintenance of the undifferentiated state in human embryonic stem cells (hESCs) is critical for further application in regenerative medicine, drug testing and studies of fundamental biology. Currently, the selection of the best quality cells and colonies for propagation is typically performed by eye, in terms of the displayed morphological features, such as prominent/abundant nucleoli and a colony with a tightly packed appearance and a well-defined edge. Using image analysis and computationa ...[more]

PMID: 31772193

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Project description:BACKGROUND:Currently the combination of molecular tools, imaging techniques and analysis software offer the possibility of studying gene activity through the use of fluorescent reporters and infer its distribution within complex biological three-dimensional structures. For example, the use of Confocal Scanning Laser Microscopy (CSLM) is a regularly-used approach to visually inspect the spatial distribution of a fluorescent signal. Although a plethora of generalist imaging software is available to analyze experimental pictures, the development of tailor-made software for every specific problem is still the most straightforward approach to perform the best possible image analysis. In this manuscript, we focused on developing a simple methodology to satisfy one particular need: automated processing and analysis of CSLM image stacks to generate 3D fluorescence profiles showing the average distribution detected in bacterial colonies grown in different experimental conditions for comparison purposes. RESULTS:The presented method processes batches of CSLM stacks containing three-dimensional images of an arbitrary number of colonies. Quasi-circular colonies are identified, filtered and projected onto a normalized orthogonal coordinate system, where a numerical interpolation is performed to obtain fluorescence values within a spatially fixed grid. A statistically representative three-dimensional fluorescent pattern is then generated from this data, allowing for standardized fluorescence analysis regardless of variability in colony size. The proposed methodology was evaluated by analyzing fluorescence from GFP expression subject to regulation by a stress-inducible promoter. CONCLUSIONS:This method provides a statistically reliable spatial distribution profile of fluorescence detected in analyzed samples, helping the researcher to establish general correlations between gene expression and spatial allocation under differential experimental regimes. The described methodology was coded into a MATLAB script and shared under an open source license to make it accessible to the whole community.

Dataset Information

Quantification of the morphological characteristics of hESC colonies.

Publications

Quantification of the morphological characteristics of hESC colonies.

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