Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture

Abstract

Mesenchymal stem cells (MSCs) play important roles in tissue regeneration, and multi-lineage differentiation and immunomodulation are two major characteristics of MSCs that are utilized in stem cell therapy. MSCs in vivo have a markedly different three-dimensional (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. A 3D scaffold is predicted to provide an artificial 3D environment similar to the in vivo environment. Significant changes in MSC differentiation are shown to be occurred when under 3D culture. However, the immunomodulatory characteristics of MSCs under 3D culture remain unknown. In this study, 3D culture systems were constructed using different substrates to evaluate the common immunomodulatory characteristics of MSCs. Compared to the MSCs under 2D culture, the MSCs under 3D culture, which had higher stemness and maintained cell phenotype, showed altered immunophenotypic pattern. Gene expression profile analysis at mRNA and protein level detected by gene chip and protein chip, respectively, further revealed the difference between 3D cultured MSCs and 2D cultured MSCs, which was mainly concentrated in the immunoregulation related aspects. Moreover, the immunoregulatory role of 3D culture was confirmed by our immunosuppressive experiments. These findings demonstrated that the immunomodulatory capacities of MSCs were enhanced by the 3D geometry of substrates.

Fig 1. Proliferation of MSCs on different substrates.

A. Light microscopy and scanning electron microscopy (SEM) images of MSCs on three different scaffolds. Scale bar = 50μm. B. Proliferation assay for MSCs cultured in different scaffolds (n = 3). C. Cell cycle assay of MSCs after 5 days culture on collagen scaffolds (3D-Collagen), chitosan substrates (3D-Chitosan) and PLGA substrates (3D-PLGA). MSCs cultured on conditional 2D surfaces served as the control.

Fig 2. Pluripotency and differentiation of MSCs cultured on three different substrates.

A. qRT-PCR for Oct4, Sox2 and Nanog of MSCs cultured in different substrates. MSCs cultured on conditional 2D surfaces served as the control. B. Immunostaining for Oct4, Sox2 and Nanog in MSCs after 5-day culture on collagen coated 2D surface (Collagen 2D) and 3D scaffolds. Scale bars = 50μm. C and D: q RT-PCR for osteogenic genes (C) and adipogenic genes (D) of MSCs cultured in different substrates after 14 days of osteogenic and adipogenic induction. MSCs cultured on conditional 2D surfaces after 14 days of osteogenic and adipogenic induction served as the control. (*) Indicates statistical significance (p<0.05) compared to the 2D surface group (n = 3).

Fig 3. Effect of different 3D scaffolds on MSC phenotype.

Mesenchymal stromal cells (MSCs) were collected after 5 days of culture in the absence or presence of different 3D scaffolds and stained with the appropriate antibodies. A. MSC-related markers are shown. B. Immunological markers are shown.

Fig 4. Microarray analysis of MSCs cultured on 3D scaffolds and 2D scaffolds.

A. Clustering analysis of MSC whole gene expression after 5-day culture on different 3D substrates and 2D surfaces. The color from shallow to deep indicates that the correlation between samples gradually increased. B. Diagram of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes. C. Clustering analysis with a set of immune related genes. D. qRT-PCR for immune modulatory related genes of MSCs. MSCs cultured on traditional 2D surfaces served as the control. (*) Indicates statistical significance (p<0.05) compared to the 2D surface group (n = 3).

Fig 5. Quantitative proteomics analysis of MSCs cultured on 3D substrates and 2D surfaces.

A. Hierarchical clustering of analysis with differentially expressed proteins from 2D-MSCs and 3D-MSCs. B. The differentially expressed proteins of 2D-MSCs and 3D-MSCs. C. Functional categorization of the up-regulated or down-regulated proteins in 3D-MSCs compared to 2D-MSCs. All up-regulated or down-regulated proteins were categorized according to the first-class function annotated by CyanoBase. The bars represent the number of proteins in each functional category.

Fig 6. Effects of the 3D culture system on MSC proliferation of T lymphocytes.

A. Effects of 3D substrates on the proliferation of T lymphocytes. CFSE-labeled dividing cells after 3 days of culture in 3D substrates and 2D surface are shown. CFSE-labeled T cells from healthy donor used as responders (T1) and un-labeled T cells from another related healthy donor treated with mitomycin C (T2) used as stimulators were cultured in 3D substrates or 2D surface. On day 3, the responder cells were harvested and analyzed by FACS. B. Immune suppressive capacities of MSCs toward T lymphocytes for the 3D culture system and traditional 2D culture system. CFSE labeled responder cells (T1) were stimulated with allogeneic stimulator cells (T2) and co-cultured with 2D-MSCs and 3D-MSCs. After 5 days of co-culture, the cells were harvested and analyzed by FACS.