Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Important challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date ac...

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Main Authors: Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh, Anja van de Stolpe
Format: Article
Language:English
Published: Elsevier 2022-05-01
Series:Stem Cell Research
Online Access:http://www.sciencedirect.com/science/article/pii/S1873506122000976
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author Laurent Holtzer
Yvonne Wesseling-Rozendaal
Wim Verhaegh
Anja van de Stolpe
author_facet Laurent Holtzer
Yvonne Wesseling-Rozendaal
Wim Verhaegh
Anja van de Stolpe
author_sort Laurent Holtzer
collection DOAJ
description Important challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date activity of these STPs could not be directly measured on a cell sample.Here we validate a novel assay platform for measurement of activity of developmental STPs (STP) for use in stem cells and stem cell derivatives. In addition to previously developed STP assays, we report development of an additional STP assay for the MAPK-AP1 pathway. Subsequently, activity of Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signaling pathways was calculated from Affymetrix transcriptome data of human pluripotent embryonic (hES) and iPS cell lines under different culture conditions, organ-derived multipotent stem cells, and differentiated cell types, to generate quantitative STP activity profiles.Results show that the STP assay technology enables reliable and quantitative measurement of multiple STP activities simultaneously on any individual cell sample. Using the technology, we found that culture conditions dominantly influence the pluripotent stem cell STP activity profile, while the origin of the stem cell line was a minor variable. A pluripotency STP activity profile (Pluripotency qPAP) was defined (active PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathway, inactive Wnt pathway). Differentiation of hES cells to intestinal progenitor cells resulted in an STP activity profile characterized by active PI3K, Wnt and Notch pathways, comparable to the STP activity profile measured on primary intestinal crypt stem cells. Quantitative STP activity measurement is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture/differentiation, and enable controlled manipulation of pluripotency/differentiation state using pathway targeting compounds.
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spelling doaj.art-da7fca7fbde74de39703fbf02014bd222022-12-22T01:53:05ZengElsevierStem Cell Research1873-50612022-05-0161102748Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cellsLaurent Holtzer0Yvonne Wesseling-Rozendaal1Wim Verhaegh2Anja van de Stolpe3Molecular Pathway Diagnostics, Philips, Eindhoven, The NetherlandsMolecular Pathway Diagnostics, Philips, Eindhoven, The NetherlandsMolecular Pathway Diagnostics, Philips, Eindhoven, The NetherlandsCorresponding author.; Molecular Pathway Diagnostics, Philips, Eindhoven, The NetherlandsImportant challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date activity of these STPs could not be directly measured on a cell sample.Here we validate a novel assay platform for measurement of activity of developmental STPs (STP) for use in stem cells and stem cell derivatives. In addition to previously developed STP assays, we report development of an additional STP assay for the MAPK-AP1 pathway. Subsequently, activity of Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signaling pathways was calculated from Affymetrix transcriptome data of human pluripotent embryonic (hES) and iPS cell lines under different culture conditions, organ-derived multipotent stem cells, and differentiated cell types, to generate quantitative STP activity profiles.Results show that the STP assay technology enables reliable and quantitative measurement of multiple STP activities simultaneously on any individual cell sample. Using the technology, we found that culture conditions dominantly influence the pluripotent stem cell STP activity profile, while the origin of the stem cell line was a minor variable. A pluripotency STP activity profile (Pluripotency qPAP) was defined (active PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathway, inactive Wnt pathway). Differentiation of hES cells to intestinal progenitor cells resulted in an STP activity profile characterized by active PI3K, Wnt and Notch pathways, comparable to the STP activity profile measured on primary intestinal crypt stem cells. Quantitative STP activity measurement is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture/differentiation, and enable controlled manipulation of pluripotency/differentiation state using pathway targeting compounds.http://www.sciencedirect.com/science/article/pii/S1873506122000976
spellingShingle Laurent Holtzer
Yvonne Wesseling-Rozendaal
Wim Verhaegh
Anja van de Stolpe
Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
Stem Cell Research
title Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
title_full Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
title_fullStr Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
title_full_unstemmed Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
title_short Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
title_sort measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells
url http://www.sciencedirect.com/science/article/pii/S1873506122000976
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AT wimverhaegh measurementofactivityofdevelopmentalsignaltransductionpathwaystoquantifystemcellpluripotencyandphenotypicallycharacterizedifferentiatedcells
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