(602b) Illustrating the Mechanisms of the Immunosuppressive Enhancement of hMSCs Initiated By Col/Hep Polyelectrolyte Layer By Layer Coatings

Statement of Purpose: In this study, we aim to demonstrate how collagen and Heparin (COL/HEP) multilayered coatings enhance human mesenchymal stem cell (hMSC) response to soluble Interferon Gamma (IFN) and increase their therapeutic capacity. By investigating the mechanisms behind these coatings and their impact on hMSC function, particularly in response to IFN, the goal is to improve hMSC manufacturing processes, enhance therapeutic efficiency, and reduce costs for patients and hospitals. The study focuses on maximizing the treatment potential of hMSCs by pre-activating them with IFN while maintaining efficient cell manufacturing. The COL/HEP coatings offer a promising method for culturing hMSCs with immunomodulatory abilities without significantly affecting cell proliferation. By identifying the signaling pathways enhanced by these coatings in IFN pre-activation, we aim to design modified coatings for applications in regenerative medicine, such as bioreactors and bioimplants. Our specific objective is to understand these mechanisms to advance both the scientific and economic aspects of hMSC therapies. (1-3)

Methods: The hMSCs were purchased from RoosterBio, and sourced from several donors. Coatings (1) used 6.5 bilayers made up of sodium heparin from Celsius Laboratories, Inc., lyophilized type I collagen sponges from Integra Lifesciences Holdings Corp., and Poly(ethylene imine) (PEI) solution from Sigma-Aldrich, each in sodium acetate buffers at pH 5, 4 and 5 respectively. Proteomics experiments were conducted to confirm preliminary data by other researchers in our lab, analyze protein content, and assess the impact of the coatings on the proteome composition. Cells were cultured per their conditions, flash-frozen, and sent to UAMS for analysis. The Presto Blue viability assay was performed to determine cell viability post-treatment. Cells were exposed to different experimental conditions, and the Presto Blue reagent was added to assess metabolic activity and cell viability. Absorbance readings were taken to quantify the viability of cells under various treatment conditions to narrow down the best concentrations of drugs for subsequent experiments. Western blot experiments were carried out to confirm preliminary data and protein content, focusing on the effect of Interferon Gamma (IFN) on the expression of Indoleamine-pyrrole 2,3-dioxygenase (IDO) and Interferon Regulatory Factor (IRF). Protein samples were separated by gel electrophoresis, transferred to a membrane, and probed with specific antibodies to detect the target proteins. The IDO colorimetric assay was utilized to measure the activity of IDO, an enzyme involved in immunosuppressive responses. This assay involved the conversion of L-tryptophan to N-formyl-kynurenine by IDO in cell supernatants. The reaction products were quantified using a spectrophotometer to assess the immunosuppressive properties of the cells under different experimental conditions.

Results:

(Attached)

Proteomics analysis confirmed preliminary findings from other researchers, evaluated protein content, and investigated the impact of the coatings on the proteome composition. The Presto Blue viability assay assessed cell viability post-treatment. Western blot experiments validated initial data and protein content, specifically focusing on the influence of Interferon Gamma (IFN) on the expression of Indoleamine-pyrrole 2,3-dioxygenase (IDO) and Interferon Regulatory Factor (IRF). The IDO colorimetric assay quantified the activity of IDO, an enzyme crucial for immunosuppressive responses. Furthermore, the study revealed that collagen and Heparin (COL/HEP) multilayered coatings enhanced the response of human mesenchymal stem cells (hMSCs) to IFN, potentially boosting their therapeutic efficacy.

Conclusions: Several preliminary conclusions were reached. It has been seen that HEP/COL LbL coatings show an increase in IDO activity that is likely to be, in part, achieved through the JAK 1/2 pathway. Also, it can be concluded that there are other pathways incorporated in the changes in IDO expression. Some of which become more active when the Jak Pathway is inhibited. More experiments are currently in progress to further investigate and confirm the mechanisms and extent to which the LbL coatings enhance immunosuppressive properties, and the compensatory mechanism seen, and are expected to be completed before the 2024 meeting.

References:

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(Castilla, D. ACS Biomater. Sci. Eng. 2020, 6, 6626−6651)

(Haseli, M. Materials Today Bio. 2022, 13, 100194)