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, 8 (1), 6816

Therapeutic Delivery Specifications Identified Through Compartmental Analysis of a Mesenchymal Stromal Cell-Immune Reaction

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Therapeutic Delivery Specifications Identified Through Compartmental Analysis of a Mesenchymal Stromal Cell-Immune Reaction

Matthew Li et al. Sci Rep.

Abstract

Despite widespread preclinical success, mesenchymal stromal cell (MSC) therapy has not reached consistent pivotal clinical endpoints in primary indications of autoinflammatory diseases. Numerous studies aim to uncover specific mechanisms of action towards better control of therapy using in vitro immunomodulation assays. However, many of these immunomodulation assays are imperfectly designed to accurately recapitulate microenvironment conditions where MSCs act. To increase our understanding of MSC efficacy, we herein conduct a systems level microenvironment approach to define compartmental features that can influence the delivery of MSCs' immunomodulatory effect in vitro in a more quantitative manner than ever before. Using this approach, we notably uncover an improved MSC quantification method with predictive cross-study applicability and unveil the key importance of system volume, time exposure to MSCs, and cross-communication between MSC and T cell populations to realize full therapeutic effect. The application of these compartmental analysis can improve our understanding of MSC mechanism(s) of action and further lead to administration methods that deliver MSCs within a compartment for predictable potency.

Conflict of interest statement

B.P. is a founder and equity holder of Sentien Biotechnologies, Inc whom have licensed patents pertaining to MSC therapeutics. The authors declare no other competing interests.

Figures

Figure 1
Figure 1
Pharmacological assessment of MSC immunosuppression with perturbation and regression analysis. PBMC proliferation was measured using flow cytometry and CFSE staining after stimulation with ConA and IL-2 for a period of 4 days. (A) Density plot of CFSE dilution; clear definition between proliferative generations (up to 5) is apparent. (B) Dose response curve of MSC suppression of T cell activation; data points represent mean +/− SD of 3 samples. Six ratios of MSCs were co-cultured with 1.5 M PBMCs to generate a full dose response curve (1:5, 1:10, 1:50, 1:100, 1:500, and 1:1000). This curve is fit by a pharmacologic dose response regression (Equation 2) with strong fit (R2 = 0.99). (C) Independent variable assessment was performed by doubling the number of PBMCs; data points represent mean +/− SD of 3 samples. (top) Two distinct curves form using the metric of ratio showing poor universal applicably of this metric with a nearly 3-fold difference between IC50 values. (middle & bottom) Implementing a cells/well and cell/mL approach, we find greatly improved agreement between these curves with a IC50 value differences less than 1.5-fold. (D) Each independent variable was then assessed again several matched studies to determine broad applicability using regression derived from 1C (Equation 2; regression values found in Table S1); each data point represent a distinct value from literature. Pharmacologic curves were derived from a non-linear, 4 parameter regression; correlative lines were generated using a linear regression.
Figure 2
Figure 2
Biochemical profiling of PBMCs in response to MSC immunomodulation. PBMC proliferation was attained through stimulation with ConA and IL2 for a period of 4 days. Cytokines are binned based on their predominate origin: MSC, PBMC, or both. Bar graphs of normalized cytokine secretion versus cell concentration; bar graphs and data points represent mean +/− SD of 3 samples. MSC derived factors (IFNa, PGE2, and IL6) are highly correlated with MSC numbers. We further find that MSCs broadly downregulate secretions of pro-inflammatory cytokines (IFNy, IL17, TNFa) while promoting anti-inflammatory factors (IL4, IL10). Factors secreted by both populations (IL1b and IL1ra) demonstrate a stepwise change with moderate correlation. Correlative lines were generated using a linear regression.
Figure 3
Figure 3
PBMC culture duration with MSCs is critical to overall immunosuppressive effect. PBMC proliferation was attained through stimulation with ConA and IL2 for a period of 4 days. MSC transwell inserts were removed after 1, 2, and 3 days co-culture initiation to time duration. Proliferation was measured through flow cytometry and CFSE staining; bar graphs and data points represent mean +/− SD of 3 samples. (A) Bar graphs of normalized proliferation versus time exposure to MSCs. (B) Bar graphs of normalized cytokine secretion versus time exposure to MSCs. Investigating cytokine profiling, we see concordant associations seen in previous sections. Longer exposure to MSCs results in greater suppression of inflammatory cytokines and vice versa for short exposures. Correlative lines were generated using a linear regression.
Figure 4
Figure 4
Volume is an implicit microenvironment factor driving MSC potency. PBMC proliferation was attained through stimulation with ConA and IL2 for a period of 4 days. Proliferation was measured through flow cytometry and CFSE staining; bar graphs represent mean +/− SD of 3 samples. (A) Bar graphs of normalized proliferation versus culture volume conditions. (B) Bar graphs of normalized cytokine secretion versus culture volume conditions. Two-group significance comparisons were performed with a student’s T-test; n.s., no significance; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Figure 5
Figure 5
MSC:PBMC cross communication is critical as shown by the use of a protein transport inhibitor. PBMCs were treated for 24 hours with Brefeldin A prior to the start of co-culture; +BA indicates with brefeldin A pretreatment, -BA indicates without brefeldin A pretreatment. PBMC proliferation was attained through stimulation with ConA and IL2 for a period of 4 days. Proliferation was measured through flow cytometry and CFSE staining; bar graphs represent mean +/− SD of 3 samples. (A) Bar graphs of normalized proliferation versus Brefeldin A conditions. (B) Bar graphs of normalized cytokine secretion versus Brefeldin A conditions. Two-group significance comparisons were performed with a student’s T-test; n.s., no significance; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Figure 6
Figure 6
System specifications of MSC:PBMC microenvironment for an effective immunomodulatory outcome. We find that minimum MSC and PBMC are required for therapeutic function due to secreted factors and the need for MSC licensing. Furthermore, significant time exposure to MSCs is required. In our hands, we discover that the three most relevant factors for overall effect (from licensing through to overall downregulation) are IFNy, IL17, and TNFa. These findings, while performed in vitro, are quite informative are suggest the need for significant time exposure and an appropriately inflammatory environment in order to coax a therapeutic effect.

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