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...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2021-11-01
|
| Series: | Biological Procedures Online |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12575-021-00157-5 |
| _version_ | 1819045547836702720 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-21T10:30:19Z |
| format | Article |
| id | doaj.art-58a034fad7b24306836d56db96b3ce90 |
| institution | Directory Open Access Journal |
| issn | 1480-9222 |
| language | English |
| last_indexed | 2024-12-21T10:30:19Z |
| publishDate | 2021-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Biological Procedures Online |
| spelling | doaj.art-58a034fad7b24306836d56db96b3ce902022-12-21T19:07:13ZengBMCBiological Procedures Online1480-92222021-11-0123111410.1186/s12575-021-00157-5Heregulin Activity Assays for Residual Testing of Cell Therapy ProductsPaula V. Monje0Ketty Bacallao1Gabriela I. Aparicio2Anil Lalwani3Stark Neurosciences Research Institute, Department of Neurological Surgery, Indiana University School of MedicineInterdisciplinary Stem Cell Institute, University of Miami Miller School of MedicineStark Neurosciences Research Institute, Department of Neurological Surgery, Indiana University School of MedicineCell and Gene Therapy CMC and Regulatory AdvisorAbstract 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.https://doi.org/10.1186/s12575-021-00157-5Schwann cellsPeripheral nerveIn vitro cultureAutologous cell therapyResidual testingQuality control |
| spellingShingle | Paula V. Monje Ketty Bacallao Gabriela I. Aparicio Anil Lalwani Heregulin Activity Assays for Residual Testing of Cell Therapy Products Biological Procedures Online Schwann cells Peripheral nerve In vitro culture Autologous cell therapy Residual testing Quality control |
| title | 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_short | Heregulin Activity Assays for Residual Testing of Cell Therapy Products |
| title_sort | heregulin activity assays for residual testing of cell therapy products |
| topic | Schwann cells Peripheral nerve In vitro culture Autologous cell therapy Residual testing Quality control |
| url | https://doi.org/10.1186/s12575-021-00157-5 |
| work_keys_str_mv | AT paulavmonje heregulinactivityassaysforresidualtestingofcelltherapyproducts AT kettybacallao heregulinactivityassaysforresidualtestingofcelltherapyproducts AT gabrielaiaparicio heregulinactivityassaysforresidualtestingofcelltherapyproducts AT anillalwani heregulinactivityassaysforresidualtestingofcelltherapyproducts |