3D Human Adipose-Derived Stem Cell Clusters as a Model for In Vitro Fibrosis

Abstract

Excessive extracellular matrix (ECM) deposition is a cause of progressive fibrosis, which ultimately leads to progressive organ dysfunction. The lack of an in vitro fibrosis model and in vitro drug screening tools limits the development of effective antifibrotic drugs. The profibrotic cytokine transforming growth factor-beta 1 (TGF-beta 1), which is secreted by a variety of cells under continuous hypoxic condition, correlates strongly with tissue fibrosis and is largely responsible for the observed increases in ECM deposition in fibrotic diseases. In this study, we established an in vitro fibrosis model in which human adipose-derived stem cells (hASCs) secrete TGF-beta 1 by engineering three-dimensional cell masses (3DCMs) of hASCs on a maltose-binding protein-basic fibroblast growth factor (MBP-FGF2)-immobilized substrate. We found that the hypoxic microenvironment created in the interior of 3DCMs during the early stages of culture leads to activation and synthesis of TGF-beta 1. The gene expression of fibrosis-related molecules such as TGF-beta 1, alpha-smooth muscle actin (alpha SMA), and collagen type I was upregulated in 3DCMs. As culture time increased, overexpression of TGF-beta 1 led to differentiation of hASCs into activated myofibroblasts, which accumulate excessive collagen type I and are characterized by alpha SMA expression. Furthermore, immunofluorescence data verified the increase in collagen type I synthesis in alpha SMA-positive cells. Scanning electron microscopy revealed rigid and compact 3DCMs, probably due to accumulation of ECM components and cross-linking of these components. The advantage of this TGF-beta 1-mediated 3D in vitro fibrosis model is that it opens up new avenues to understand the common mechanism of fibrosis, which will then facilitate the development of broadly effective antifibrotic compounds and the screening of existing antifibrotic agents. To the best of our knowledge, this is the first proper biomimetic 3D in vitro fibrosis model to be developed.