Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images |
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| Authors: | Vincenzo Paduano Daniela Tagliaferri Geppino Falco Michele Ceccarelli |
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| Affiliation: | 1. Bioinformatics Lab, Genetic Research Institute “G. Salvatore” (IRGS) c/o BioGeM s.c.a r.l., Ariano Irpino, Avellino, Italy.; 2. Stem Cell Research Lab, Genetic Research Institute “G. Salvatore” (IRGS) c/o BioGeM s.c.a r.l., c.da Camporeale, Ariano Irpino, Avellino, Italy.; 3. Department of Science and Technologies, University of Sannio, via Port''Arsa, Benevento, Benevento, Italy.; Baylor College of Medicine, United States of America, |
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| Abstract: | Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on the control genes showed that they behave as expected. |
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