Stem Cell-Derived Extracellular Matrix Enables Survival and Multilineage Differentiation within Superporous Hydrogels

Abstract

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 hours. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73 and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multi lineage potential of hMSCs grown in the PEGDA SPHs whereby differentiation into osteoblasts, chondrocytes and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the “blank” PEGDA environment to a milieu conducive to cell growth and multi-lineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.

The role of laminins in cartilaginous tissues: from development to regeneration

“…For instance, an extensive expression of laminin was observed in vitro following three weeks of chondrogenic culture of bone marrow-derived mesenchymal stem cells (MSCs) in both poly (ethylene glycol) diacrylate (PEGDA) hydrogel (Köllmer et al, 2012) and hyaluronic acidbased hydrogel (Toh et al, 2012). Similarly, Jeng et al (2012Jeng et al ( , 2013 found widespread expression of laminins throughout the ECM, in both engineered cartilage constructs and reparative tissues, following implantation of chondrocyte-seeded constructs in caprine cartilage defects -although the expression of laminin appeared diffused compared to the pericellular staining pattern observed in normal adult cartilage.…”

Surfactant-free CO2-in-water emulsion-templated poly (vinyl alcohol) (PVA) hydrogels

“…In vitro cell culture studies were conducted according to the literature [45,46]. The ability of human embryonic lung diploid fibroblast cells and H9c2 cardiac muscle cells to grow into the hydrogels 3D structure was assessed.…”

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices

“…32,33,45,48 However, this technique is limited in that the user must select a panel of antibodies targeting proteins of interest. Here, we pair shotgun proteomics and immunostaining to circumvent the need for a priori knowledge of what proteins the cells may be secreting.…”