Cargando…

Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells

BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a w...

Descripción completa

Detalles Bibliográficos
Autores principales: Eggerschwiler, Benjamin, Canepa, Daisy D., Pape, Hans-Christoph, Casanova, Elisa A., Cinelli, Paolo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390603/
https://www.ncbi.nlm.nih.gov/pubmed/30808403
http://dx.doi.org/10.1186/s13287-019-1170-8
_version_ 1783398172958130176
author Eggerschwiler, Benjamin
Canepa, Daisy D.
Pape, Hans-Christoph
Casanova, Elisa A.
Cinelli, Paolo
author_facet Eggerschwiler, Benjamin
Canepa, Daisy D.
Pape, Hans-Christoph
Casanova, Elisa A.
Cinelli, Paolo
author_sort Eggerschwiler, Benjamin
collection PubMed
description BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations. METHOD: MSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared. RESULT: Quantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification. CONCLUSION: Our approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1170-8) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-6390603
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-63906032019-03-11 Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells Eggerschwiler, Benjamin Canepa, Daisy D. Pape, Hans-Christoph Casanova, Elisa A. Cinelli, Paolo Stem Cell Res Ther Method BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations. METHOD: MSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared. RESULT: Quantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification. CONCLUSION: Our approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1170-8) contains supplementary material, which is available to authorized users. BioMed Central 2019-02-26 /pmc/articles/PMC6390603/ /pubmed/30808403 http://dx.doi.org/10.1186/s13287-019-1170-8 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Method
Eggerschwiler, Benjamin
Canepa, Daisy D.
Pape, Hans-Christoph
Casanova, Elisa A.
Cinelli, Paolo
Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title_full Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title_fullStr Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title_full_unstemmed Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title_short Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
title_sort automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390603/
https://www.ncbi.nlm.nih.gov/pubmed/30808403
http://dx.doi.org/10.1186/s13287-019-1170-8
work_keys_str_mv AT eggerschwilerbenjamin automateddigitalimagequantificationofhistologicalstainingfortheanalysisofthetrilineagedifferentiationpotentialofmesenchymalstemcells
AT canepadaisyd automateddigitalimagequantificationofhistologicalstainingfortheanalysisofthetrilineagedifferentiationpotentialofmesenchymalstemcells
AT papehanschristoph automateddigitalimagequantificationofhistologicalstainingfortheanalysisofthetrilineagedifferentiationpotentialofmesenchymalstemcells
AT casanovaelisaa automateddigitalimagequantificationofhistologicalstainingfortheanalysisofthetrilineagedifferentiationpotentialofmesenchymalstemcells
AT cinellipaolo automateddigitalimagequantificationofhistologicalstainingfortheanalysisofthetrilineagedifferentiationpotentialofmesenchymalstemcells