Heregulin Activity Assays for Residual Testing of Cell Therapy Products

Abstract Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential saf...

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Autores principales:	Paula V. Monje, Ketty Bacallao, Gabriela I. Aparicio, Anil Lalwani
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Schwann cells Peripheral nerve In vitro culture Autologous cell therapy Residual testing Quality control Medicine (General) R5-920 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/58a034fad7b24306836d56db96b3ce90
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spelling	oai:doaj.org-article:58a034fad7b24306836d56db96b3ce902021-11-14T12:07:24ZHeregulin Activity Assays for Residual Testing of Cell Therapy Products10.1186/s12575-021-00157-51480-9222https://doaj.org/article/58a034fad7b24306836d56db96b3ce902021-11-01T00:00:00Zhttps://doi.org/10.1186/s12575-021-00157-5https://doaj.org/toc/1480-9222Abstract Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.Paula V. MonjeKetty BacallaoGabriela I. AparicioAnil LalwaniBMCarticleSchwann cellsPeripheral nerveIn vitro cultureAutologous cell therapyResidual testingQuality controlMedicine (General)R5-920Biology (General)QH301-705.5ENBiological Procedures Online, Vol 23, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Schwann cells Peripheral nerve In vitro culture Autologous cell therapy Residual testing Quality control Medicine (General) R5-920 Biology (General) QH301-705.5
spellingShingle	Schwann cells Peripheral nerve In vitro culture Autologous cell therapy Residual testing Quality control Medicine (General) R5-920 Biology (General) QH301-705.5 Paula V. Monje Ketty Bacallao Gabriela I. Aparicio Anil Lalwani Heregulin Activity Assays for Residual Testing of Cell Therapy Products
description	Abstract Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.
format	article
author	Paula V. Monje Ketty Bacallao Gabriela I. Aparicio Anil Lalwani
author_facet	Paula V. Monje Ketty Bacallao Gabriela I. Aparicio Anil Lalwani
author_sort	Paula V. Monje
title	Heregulin Activity Assays for Residual Testing of Cell Therapy Products
title_short	Heregulin Activity Assays for Residual Testing of Cell Therapy Products
title_full	Heregulin Activity Assays for Residual Testing of Cell Therapy Products
title_fullStr	Heregulin Activity Assays for Residual Testing of Cell Therapy Products
title_full_unstemmed	Heregulin Activity Assays for Residual Testing of Cell Therapy Products
title_sort	heregulin activity assays for residual testing of cell therapy products
publisher	BMC
publishDate	2021
url	https://doaj.org/article/58a034fad7b24306836d56db96b3ce90
work_keys_str_mv	AT paulavmonje heregulinactivityassaysforresidualtestingofcelltherapyproducts AT kettybacallao heregulinactivityassaysforresidualtestingofcelltherapyproducts AT gabrielaiaparicio heregulinactivityassaysforresidualtestingofcelltherapyproducts AT anillalwani heregulinactivityassaysforresidualtestingofcelltherapyproducts
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Heregulin Activity Assays for Residual Testing of Cell Therapy Products

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