Dirty Surface - Cleaner Cells? Some Observations with a Bio-Assembled Extracellular Matrix

Abstract

Conventional human mesenchymal stem cells (hMSC) cultures are carried out on tissue culture polystyrene (TCPS) surfaces while human embryonic stem (hES) cells are normally propagated on feeder layers or Matrigel™. However, TCPS culture of MSCs lead to senescence [1] and decreased self-renewal [2, 3]; while hES cells cultured on xenogenic feeder layers or MatrigelTM express immunogen which presents a xenogenic risk thus hampering transplantion [4]. Culture of MSCs on TCPS does not emulate the natural stem cell microenvironment, which consists of soluble factors, neighbouring cells and extracellular matrix. The extracellular matrix (ECM) is a complex supramolecular assembly of various proteins, some of which binds growth factors like FGF-2 and TGFβ1 [5]. We have directed ECM deposition by human fibroblasts through application of excluded volume effect (EVE) [6, 7] followed by detergent lysis to achieve a cell-free matrix. The matrix obtained contained proteins such as collagens, fibronectin, glycoproteins, proteoglycans and growth factors, FGF-2 and TGFβ1. Proteins within the matrix were analysed by gel electrophoresis, and the protein profile obtained highlighted its complexity. Matrix propagation was observed to enhance the proliferation rate of hMSCs, resulting in more cells in a shorter time compared to TCPS controls. In addition, hMSCs pre-cultured on matrix prior to adipogenesis showed increased differentiation compared to TCPS control based on flow cytometry and adherent cytometry, indicating better retention of differentiation capabilities despite long term culture. The population doubling rate of hES cells cultured on matrix surpassed those on feeder-free culture on Matrigel™ in defined culture medium in just 3 weeks, while maintaining similar expression of pluripotency marker Oct4 as Matrigel controls. In conclusion, we demonstrate that the application of EVE to obtain a collagen-rich fibroblasts layer followed by detergent lysis results in a cell-rich matrix rich in diverse proteins. Culture of hMSCs on such a matrix increases their proliferation rate while still retaining differentiation potential. hES cells grown on matrix also proliferated faster with retention of stem cell markers.