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1. Peptoid‐Cross‐Linked Hydrogel Stiffness Modulates Human Mesenchymal Stromal Cell Immunoregulatory Potential in the Presence of Interferon‐Gamma

   https://doi.org/10.1002/mabi.202400111  

   Castilla‐Casadiego, David A; Morton, Logan D; Loh, Darren H; Pineda‐Hernandez, Aldaly; Chavda, Ajay P; Garcia, Francis; Rosales, Adrianne M (April 2024, Macromolecular Bioscience)

   Abstract Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross‐linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein‐1, macrophage colony‐stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN‐γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN‐γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN‐γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN‐γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN‐γ. 

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2. Heparin–collagen I bilayers stimulate FAK / ERK ½ signaling via α2β1 integrin to support the growth and anti‐inflammatory potency of mesenchymal stromal cells

   https://doi.org/10.1002/jbm.a.37614  

   Cifuentes, Said J; Domenech, Maribella (September 2023, Journal of Biomedical Materials Research Part A)

   Abstract Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high‐quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin‐HEP interactions in the advantages of MSC culture. The layer‐by‐layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC₅₀and IC₇₅levels of BTT‐3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow‐derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP‐ending HEP/COL surfaces significantly increased FAK‐ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL‐ending HEP/COL and uncoated surfaces. BTT‐3033 but not Cilengitide treatment markedly affected FAK‐ERK½ activity levels on HEP‐ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT‐3033 treatment on HEP‐ending bilayers reduced MSC‐mediated macrophage inhibitory activity and altered the cytokine profile of co‐cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1‐FAK‐ERK½ signaling mechanism. 

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3. Substrate topographies modulate the secretory activity of human bone marrow mesenchymal stem cells

   https://doi.org/10.1186/s13287-023-03450-0  

   Rosado-Galindo, Heizel; Domenech, Maribella (August 2023, Stem Cell Research & Therapy)

   Abstract BackgroundMesenchymal stem cells (MSCs) secrete a diversity of factors with broad therapeutic potential, yet current culture methods limit potency outcomes. In this study, we used topographical cues on polystyrene films to investigate their impact on the secretory profile and potency of bone marrow-derived MSCs (hBM-MSCs). hBM-MSCs from four donors were cultured on topographic substrates depicting defined roughness, curvature, grooves and various levels of wettability. MethodsThe topographical PS-based array was developed using razor printing, polishing and plasma treatment methods. hBM-MSCs from four donors were purchased from RoosterBio and used in co-culture with peripheral blood mononuclear cells (PBMCs) from Cell Applications Inc. in an immunopotency assay to measure immunosuppressive capacity. Cells were cultured on low serum (2%) for 24–48 h prior to analysis. Image-based analysis was used for cell quantification and morphology assessment. Metabolic activity of BM-hMSCs was measured as the mitochondrial oxygen consumption rate using an extracellular flux analyzer. Conditioned media samples of BM-hMSCs were used to quantify secreted factors, and the data were analyzed using R statistics. Enriched bioprocesses were identify using the Gene Ontology toolenrichGOfrom theclusterprofiler.One-way and two-way ANOVAs were carried out to identify significant changes between the conditions. Results were deemed statistically significant for combinedP < 0.05 for at least three independent experiments. ResultsCell viability was not significantly affected in the topographical substrates, and cell elongation was enhanced at least twofold in microgrooves and surfaces with a low contact angle. Increased cell elongation correlated with a metabolic shift from oxidative phosphorylation to a glycolytic state which is indicative of a high-energy state. Differential protein expression and gene ontology analyses identified bioprocesses enriched across donors associated with immune modulation and tissue regeneration. The growth of peripheral blood mononuclear cells (PBMCs) was suppressed in hBM-MSCs co-cultures, confirming enhanced immunosuppressive potency. YAP/TAZ levels were found to be reduced on these topographies confirming a mechanosensing effect on cells and suggesting a potential role in the immunomodulatory function of hMSCs. ConclusionsThis work demonstrates the potential of topographical cues as a culture strategy to improve the secretory capacity and enrich for an immunomodulatory phenotype in hBM-MSCs. 

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4. Serial Passaging Affects Stromal Cell Mechanosensitivity on Hyaluronic Acid Hydrogels

   https://doi.org/10.1002/mabi.202300110  

   Sumey, Jenna L.; Harrell, Abigail M.; Johnston, Peyton C.; Caliari, Steven R. (January 2024, Macromolecular Bioscience)

   Abstract There is a tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work, a methacrylated hyaluronic acid hydrogel platform is leveraged to study stromal cell mechanotransduction. Hydrogels are first formed through thiol‐Michael addition to model normal soft tissue (e.g., lung) stiffness (E ≈ 1 kPa). Secondary cross‐linking via radical photopolymerization of unconsumed methacrylates allows matching of early‐ (E ≈ 6 kPa) and late‐stage fibrotic tissue (E ≈ 50 kPa). Early passage (P1) human bone marrow mesenchymal stromal cells (hMSCs) display increased spreading, myocardin‐related transcription factor‐A (MRTF‐A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF‐A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models. 

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5. In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells

   https://doi.org/10.3389/fimmu.2021.621744  

   Jeske, Richard; Yuan, Xuegang; Fu, Qin; Bunnell, Bruce A.; Logan, Timothy M.; Li, Yan (March 2021, Frontiers in Immunology)

   null (Ed.)

   Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use. 

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