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.
Original language | English |
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Article number | 22 |
Journal | Biological Procedures Online |
Volume | 23 |
Issue number | 1 |
DOIs | |
State | Published - Dec 2021 |
Bibliographical note
Funding Information:This work was supported by The Miami Project to Cure Paralysis and the Buoniconti Foundation (autologous hSC clinical trials, University of Miami, to P.V.M.), and the Indiana State Department of Health (grants 33997 and 43547 to P.V.M, Indiana University). G.I.A received support from the International Society for Neurochemistry, Fulbright, Bunge & Born, and Williams Foundations, and CONICET-Argentina. The contents of this article are the responsibility of the authors and do not necessarily represent the official views of the funding agencies.
Publisher Copyright:
© 2021, The Author(s).
Keywords
- Autologous cell therapy
- ErbB/HER
- In vitro culture
- Kinase activation
- Peripheral nerve
- Phosphor- antibodies
- Quality control
- Residual testing
- Schwann cells
- Signal transduction
- Western blot
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (all)