Dataset Information

Analysis of the Intrinsic Self-Organising Properties of Mesenchymal Stromal Cells in Three-Dimensional Co-Culture Models with Endothelial Cells.

ABSTRACT: Mesenchymal stem/stromal cells (MSCs) are typically characterised by their ability to differentiate into skeletal (osteogenic, chondrogenic and adipogenic) lineages. MSCs also appear to have additional non-stem cell functions in coordinating tissue morphogenesis and organising vascular networks through interactions with endothelial cells (ECs). However, suitable experimental models to examine these apparently unique MSC properties are lacking. Following previous work, we have developed our 3D in vitro co-culture models to enable us to track cellular self-organisation events in heterotypic cell spheroids combining ECs, MSCs and their differentiated progeny. In these systems, MSCs, but not related fibroblastic cell types, promote the assembly of ECs into interconnected networks through intrinsic mechanisms, dependent on the relative abundance of MSC and EC numbers. Perturbation of endogenous platelet-derived growth factor (PDGF) signalling significantly increased EC network length, width and branching. When MSCs were pre-differentiated towards an osteogenic or chondrogenic lineage and co-cultured as mixed 3D spheroids, they segregated into polarised osseous and chondral regions. In the presence of ECs, the pre-differentiated MSCs redistributed to form a central mixed cell core with an outer osseous layer. Our findings demonstrate the intrinsic self-organising properties of MSCs, which may broaden their use in regenerative medicine and advance current approaches.

SUBMITTER: Marshall J

PROVIDER: S-EPMC6315484 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Analysis of the Intrinsic Self-Organising Properties of Mesenchymal Stromal Cells in Three-Dimensional Co-Culture Models with Endothelial Cells.

Marshall Julia J Barnes Amanda A Genever Paul P

Bioengineering (Basel, Switzerland) 20181026 4

Mesenchymal stem/stromal cells (MSCs) are typically characterised by their ability to differentiate into skeletal (osteogenic, chondrogenic and adipogenic) lineages. MSCs also appear to have additional non-stem cell functions in coordinating tissue morphogenesis and organising vascular networks through interactions with endothelial cells (ECs). However, suitable experimental models to examine these apparently unique MSC properties are lacking. Following previous work, we have developed our 3D in ...[more]

PMID: 30373192

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Project description:Background:Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. Methods:We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. Results:The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. Conclusion:These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

Project description:BACKGROUND: Transforming growth factor (TGF)-? plays a pivotal role in cancer progression through regulating cancer cell proliferation, invasion, and remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant type of stromal cell, in which TGF-? signaling is activated. Among the strategies for TGF-? signaling inhibition, RNA interference (RNAi) targeting of TGF-? ligands is emerging as a promising tool. Although preclinical studies support the efficacy of this therapeutic strategy, its effect on the tumor microenvironment in vivo remains unknown. In addition, differential effects due to knockdown of various TGF-? ligand isoforms have not been examined. Therefore, an experimental model that recapitulates tumor-stromal interaction is required for validation of therapeutic agents. METHODS: We have previously established a three-dimensional co-culture model of lung cancer, and demonstrated the functional role of co-cultured fibroblasts in enhancing cancer cell invasion and differentiation. Here, we employed this model to examine how knockdown of TGF-? ligands affects the behavior of different cell types. We developed lentivirus vectors carrying artificial microRNAs against human TGF-?1 and TGF-?2, and tested their effects in lung cancer cells and fibroblasts. RESULTS: Lentiviral vectors potently and selectively suppressed the expression of TGF-? ligands, and showed anti-proliferative effects on these cells. Furthermore, knockdown of TGF-? ligands attenuated fibroblast-mediated collagen gel contraction, and diminished lung cancer cell invasion in three-dimensional co-culture. We also observed differential effects by targeting different TGF-? isoforms in lung cancer cells and fibroblasts. CONCLUSIONS: Our findings support the notion that RNAi-mediated targeting of TGF-? ligands may be beneficial for lung cancer treatment via its action on both cancer and stromal cells. This study further demonstrates the usefulness of this three-dimensional co-culture model to examine the effect of therapeutic agents on tumor-stromal interaction.

Dataset Information

Analysis of the Intrinsic Self-Organising Properties of Mesenchymal Stromal Cells in Three-Dimensional Co-Culture Models with Endothelial Cells.

Publications

Analysis of the Intrinsic Self-Organising Properties of Mesenchymal Stromal Cells in Three-Dimensional Co-Culture Models with Endothelial Cells.

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