Heregulin Activity Assays for Residual Testing of Cell Therapy Products.
| Autor: | Monje PV; Stark Neurosciences Research Institute, Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA. pmonje@iu.edu., Bacallao K; Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida, USA., Aparicio GI; Stark Neurosciences Research Institute, Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA.; Instituto de Investigaciones Biotecnológicas 'Rodolfo A. Ugalde', Universidad Nacional de San Martín and Consejo Nacional de Investigaciones Científicas y Técnicas (IIBio-UNSAM-CONICET), Buenos Aires, Argentina., Lalwani A; Cell and Gene Therapy CMC and Regulatory Advisor, Boulder, CO, USA. |
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| Jazyk: | angličtina |
| Zdroj: | Biological procedures online [Biol Proced Online] 2021 Nov 12; Vol. 23 (1), pp. 22. Date of Electronic Publication: 2021 Nov 12. |
| DOI: | 10.1186/s12575-021-00157-5 |
| Abstrakt: | 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. (© 2021. The Author(s).) |
| Databáze: | MEDLINE |
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