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While mesenchymal stromal cell (MSC) therapies show promise for treating several indications due to their regenerative and immunomodulatory capacity, clinical translation has yet to be achieved due to a lack of robust, scalable manufacturing practices. Expansion using undefined fetal bovine serum (FBS) or human platelet lysate contributes to MSC functional heterogeneity and limits control of product quality. The need for tunable and consistent media has thus motivated development of chemically defined media (CDM). However, CDM development strategies are often limited in their screening approaches and unable to reliably assess the impact of media on MSC function, often neglecting high-level interactions of media components such as growth factors. Given that MSC morphology has been shown to predict their immunomodulatory function, we employed a high throughput screening (HTS) approach to elucidate effects of growth factor compositions on MSC phenotype and proliferation in a custom CDM. Methods:HTS of eight growth factors in a chemically defined basal medium (CDBM) was conducted via a two-level, full factorial design using adipose-derived MSCs. Media hits were identified leveraging cell counts and morphological profiles. After validating phenotypic responses to hits across multiple donors, MSCs were cultured over three passages in serum-containing medium (SCM) and CDM hits and assayed for growth and immunomodulatory function. Finally, growth factor concentrations in one hit were further refined, and MSC growth and function was assessed. Results: Our HTS approach led to the discovery of several CDM formulations that enhanced MSC proliferation and demonstrated wide ranging impacts on MSC immunomodulation. Notably, two hits showed 4X higher growth compared to SCM over 3 passages without compromising immunomodulatory function. Refinement of one CDM hit formulation reduced growth factor concentrations by as much as 90% while maintaining superior growth and similar function to SCM. Altogether, distinct MSC morphological profiles observed from screening were indicative of differential MSC quality that allowed for development of an effective CDM for MSC expansion. Conclusions: Overall, this highlights how our HTS approach led to the development of CDM formulations for robust MSC expansion and serves as a generalizable tool for improvement of MSC manufacturing processes.
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Development of a Robust Consensus Modeling Approach for Identifying Cellular and Media Metabolites Predictive of Mesenchymal Stromal Cell Potency
Abstract Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine applications due in part to their ability to modulate immune cells. However, MSCs demonstrate significant functional heterogeneity in terms of their immunomodulatory function because of differences in MSC donor/tissue source, as well as non-standardized manufacturing approaches. As MSC metabolism plays a critical role in their ability to expand to therapeutic numbers ex vivo, we comprehensively profiled intracellular and extracellular metabolites throughout the expansion process to identify predictors of immunomodulatory function (T-cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). Here, we profiled media metabolites in a non-destructive manner through daily sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular metabolites at the end of expansion using mass spectrometry (MS). Using a robust consensus machine learning approach, we were able to identify panels of metabolites predictive of MSC immunomodulatory function for 10 independent MSC lines. This approach consisted of identifying metabolites in 2 or more machine learning models and then building consensus models based on these consensus metabolite panels. Consensus intracellular metabolites with high predictive value included multiple lipid classes (such as phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins) while consensus media metabolites included proline, phenylalanine, and pyruvate. Pathway enrichment identified metabolic pathways significantly associated with MSC function such as sphingolipid signaling and metabolism, arginine and proline metabolism, and autophagy. Overall, this work establishes a generalizable framework for identifying consensus predictive metabolites that predict MSC function, as well as guiding future MSC manufacturing efforts through identification of high-potency MSC lines and metabolic engineering.
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- Award ID(s):
- 1648035
- PAR ID:
- 10522586
- Publisher / Repository:
- Oxford Academic
- Date Published:
- Journal Name:
- Stem Cells
- Volume:
- 41
- Issue:
- 8
- ISSN:
- 1066-5099
- Page Range / eLocation ID:
- 792 to 808
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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BackgroundMesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large‐scale expansion while preserving therapeutic potency and maintaining small cell size. Conventional 2D culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution. MethodsWe present an alternating 2D/3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations. Placenta‐derived MSCs were cultured under optimized spheroid conditions, with extracellular matrix supplementation and chemically defined media to enhance viability. To address scalability, we developed RGD-functionalized alginate hydrogel tubes (AlgTubes) that enable dynamic transitions between adherent and spheroid states for continuous culture. ResultsSpheroid culture significantly reduced cell size and enhanced immunomodulatory function. The alternating 2D/3D protocol slowed MSC enlargement and senescence over multiple passages while preserving anti-inflammatory activity. Extracellular matrix supplementation and chemically defined media further improved cell viability. AlgTubes successfully supported the alternating culture strategy in a continuous and scalable format. ConclusionsThe alternating 2D/3D culture system effectively overcomes limitations of conventional MSC expansion by mitigating enlargement, delaying senescence, and preserving both proliferative capacity and immunoregulatory potency. Combined with AlgTube technology, this work demonstrates a promising strategy for MSC manufacturingmore » « less
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Abstract Several investigations have identified volatile organic compounds (VOCs) as potential biomarkers for the detection and identification of microbial contamination of metabolically active mammalian cell cultures. In this study, we showed that emitted VOCs discriminate between uncontaminated mesenchymal stromal cells (MSCs) and those contaminated with the bacteriumStaphylococcus epidermidisor fungusAspergillus Fumigatusseparately,in vitro, using a methodology based on an adapted cell culture and thermal desorption–gas chromatography–mass spectrometry. In addition, we elucidated a set of discriminatory volatile compounds from the MSC cultures and media alone across a time series experiment. Partial least squares–discriminant analysis-variable importance in projection confirmed putative identifications of 18, 16, and 26 VOCs that showed relevant changes in a bacterial, fungal, and universal pathogen model, respectively, with an accuracy of 100% in the fungal model. Among these metabolites, octane, 2,5,6-trimethyl- overlapped between the three groups. Furthermore, a total of 15 VOCs were found most relevant to cell culture expansion over three days based on cluster analysis. This novel study goes a step further in identifying distinct VOC signatures of MSCs contaminated withS. epidermidisorA. fumigatus, and in monitoring MSCs proliferation over time. This pilot study shows preliminary results that indicate that VOC headspace analysis could serve as a suitable, rapid, non-invasive, and non-destructive tool for the metabolic and growth monitoring of MSCs in a dynamic cell culture bioreactor system.more » « less
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Abstract Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promising immunomodulatory properties; however, strategies to enhance their therapeutic potential remain limited. Here, we employed CRISPR activation of the gene TSG-6 in MSCs to evaluate the impact of elevated TSG-6 on EV cargo and immunomodulatory function in an in vitro macrophage model. CRISPR-mediated gene activation was confirmed by RT-qPCR, demonstrating more than an 1800 fold increase in TSG-6 mRNA compared to controls. EVs were isolated from TSG-6 overexpressing MSCs and thoroughly characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, confirming their typical size distribution, morphology, and surface markers. Small RNA sequencing of these EVs revealed 15 differentially expressed miRNAs relative to EVs from control MSCs. When THP-1–derived macrophages were stimulated with LPS and treated with TSG-6-overexpressing MSC-EVs (Standard dosage: 1000 particle/cell,n = 11; Alternative dosages: 500, 1000, or 2000 particles/cell,n = 6), a marked reduction in pro-inflammatory cytokine gene expression (IL-1β, CCL2, CXCL10, and TNF-α) and secreted protein levels (CCL2, TNF-α, CXCL1, and MIP-3α) was observed. Taken together, these findings demonstrate that CRISPR-based TSG-6 activation reprograms MSC-EV miRNA cargo (as well as their protein cargo, as previously shown), which can boost their anti-inflammatory effects. These findings underscore the promise of CRISPR-activation as a novel platform for boosting the bioactive properties of MSC-EVs and enhancing immunotherapeutic efficacy.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1093/stmcls/sxad039
