Pro-inflammatory cytokines, IFNgamma and TNFalpha, influence immune properties of human bone marrow and Wharton jelly mesenchymal stem cells differentially

Abstract

Background: Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-gamma (IFNgamma) and Tumor Necrosis Factor-alpha (TNFalpha).

Methodology/principal findings: Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNgamma or TNFalpha, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNgamma primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNgamma inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation.

Conclusion/significance: This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.

Figure 1. Phenotypic changes and tri-lineage differentiation potential of human MSCs on exposure to IFNγ and TNFα.

(A) Phase contrast pictures of MSCs, captured by a Nikon Eclipse TE2000S microscope (magnification 10×10), depicting phenotypic changes on exposure to pro-inflammatory cytokines. (B) Lipid droplets stained with Oil O Red depicting adipogenesis {Inset: FABP4 staining of MSC- derived adipocytes} (magnification 20×10) (C) Osteogenisis depicted by Von Kossa staining of calcium mineralized deposits {Inset: Osteocalcin staining of MSC derived mature osteocytes} (magnification 10×10). (D). Sulfated proteoglycans stained with Safranin O demonstrating chondrogenesis {Inset: Collagen II staining of MSC derived chondrocytes} (magnification 10×10). Control refers to cells unexposed to pro-inflammatory cytokines.

Figure 2. Inflammatory cytokines affect the immune-phenotype of human MSCs derived from bone marrow and Wharton's jelly.

BMMSC's (A) and WJMSC's (B) cultured with or without IFNγ or TNFα for 72 hr were stained with appropriate conjugated antibodies and the surface expression was monitored by flow cytometry. Grey filled histograms represent specific isotype controls. Data is representative of three experiments performed with three independent donor MSCs.

Figure 3. Cytokine secretions from activated lymphocytes are differentially altered upon co-culture with BMMSCs and WJMSCs.

The graphs represent amounts of cytokine released (y axis) from MSC/PHA-activated PBMC co-cultures at different time points of activation (x axis). Panel A and Panel B refers to cytokine secretions in BMMSC and WJMSC co-cultures respectively.

Figure 4. Expression of key activation markers on PHA-stimulated lymphocytes were differentially altered upon co-culture with primed BMMSCs and WJMSCs.

Flow cytometry analysis of CD28 (4A), CTLA4 (4B {i}, 2B {ii}), HLA-ABC (4C) and CD45RO (4D) on PHA-treated PBMCs was performed kinetically during different co-culture conditions. “P+P” is PBMC stimulated with PHA and “+” refers to activation markers on PHA-stimulated PBMCs when co-cultured with primed or unprimed MSCs. Key changes are highlighted. % positive cells depicted within each histogram are calculated using FACSDiva software.

Figure 5. Differences in immune-suppressive gene and protein levels in primed/unprimed BMMSC and WJMSCs.

(A) Delta Cycle Threshold (delta C_T) is obtained after subtracting the endogenous control (GAPDH) Cycle Threshold from the test. ND refers to not detected. (B) IDO activity (reflected by Kynurenine amounts), PGE2 and HGF levels are depicted.