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Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

ABSTRACT: Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development.

SUBMITTER: Gurvich OL

PROVIDER: S-EPMC7441152 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

Gurvich Olga L OL Puttonen Katja A KA Bailey Aubrey A Kailaanmäki Anssi A Skirdenko Vita V Sivonen Minna M Pietikäinen Sanna S Parker Nigel R NR Ylä-Herttuala Seppo S Kekarainen Tuija T

Scientific reports 20200820 1

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., ...[more]

PMID: 32820219

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Project description:Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

Project description:BackgroundStem cells are increasingly seen as a solution for many health challenges for an ageing population. However, their potential benefits in the clinic are currently curtailed by technical challenges such as high cell dose requirements and point of care delivery, which pose sourcing and logistics challenges. Cell manufacturing solutions are currently in development to address the supply issue, and ancillary technologies such as nanoparticle-based labelling are being developed to improve stem cell delivery and enable post-treatment follow-up.MethodsThe application of magnetic particle (MP) labelling to potentially scalable cell manufacturing processes was investigated in a range of therapeutically relevant cells, including mesenchymal stromal cells (MSC), cardiomyocytes (CMC) and neural progenitor cells (ReN). The efficiency and the biological effect of particle labelling were analysed using fluorescent imaging and cellular assays.ResultsFlow cytometry and fluorescent microscopy confirmed efficient labelling of monolayer cultures. Viability was shown to be retained post labelling for all three cell types. MSC and CMC demonstrated higher tolerance to MP doses up to 100× the standard concentration. This approach was also successful for MP labelling of suspension cultures, demonstrating efficient MP uptake within 3 h, while cell viability was unaffected by this suspension labelling process. Furthermore, a procedure to enable the storing of MP-labelled cell populations to facilitate cold chain transport to the site of clinical use was investigated. When MP-labelled cells were stored in hypothermic conditions using HypoThermosol solution for 24 h, cell viability and differentiation potential were retained post storage for ReN, MSC and beating CMC.ConclusionsOur results show that a generic MP labelling strategy was successfully developed for a range of clinically relevant cell populations, in both monolayer and suspension cultures. MP-labelled cell populations were able to undergo transient low-temperature storage whilst maintaining functional capacity in vitro. These results suggest that this MP labelling approach can be integrated into cell manufacturing and cold chain transport processes required for future cell therapy approaches.

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Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

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Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

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