Mimicking the Niche - the Potential of Cell-Derived Extracellular Matrices to Support Human Bone Marrow Stem Cells

The therapeutic potential of human bone marrow mesenchymal and hematopoietic stem and progenitor cells (MSC and HSPC, respectively) offers great promise for their use in regenerative medicine. To successfully expand these cells ex vivo, both differentiation and stem cell maintenance need to be controlled. One major factor for this control is the extracellular microenvironment. However, a major obstacle in defining the exact role of extracellular matrices (ECM) within the stem cell niche is the lack of suitable methods that recapitulate complex ECM microenvironments in vitro.

Our current work describes a methodology that permits reliable anchorage of decellularized extracellular matrices generated from cultured bone marrow MSC to mimic the bone marrow stem cell microenvironment. Cell-secreted ECM was immobilized to culture carriers via maleic anhydride co-polymer-mediated covalent binding of fibronectin to the culture surface. Fibronectin was selected for its properties to interact with cell-secreted extracellular matrix proteins, so that culture and decellularization of a cell monolayer on top yields in a superior anchorage of cell-secreted ECM with the opportunity to study the decellularized extracellular phenotype of various cell-derived matrices. We validated our approach by fabricating different types of ECM from cultured human bone marrow MSC, human fibroblasts and endothelial cells, and murine embryonic fibroblasts. The ECM preparations were thoroughly characterized to identify their molecular composition, supra-structural features and nano-mechanical properties.

The generated MSC-derived ECM substrates served as in vitro culture environments for human MSC and HSPC. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures, indicating an intense interplay between the cells and the microenvironment. MSC that re-grew into their own matrices have shown significantly elevated proliferation and cytokine secretion levels as well as enhanced differentiation intensity compared to MSC that were cultured on reconstituted Fibronectin or MatrigelTM substrates. Similarly, HSPC have shown significant increase in proliferation over a culture period of 7 days, with up to 3-fold expansion of CD34+ cells on MSC-derived matrices without exhaustion of CD34/CD133 double positive cells, and with the potential for long-term (20 weeks) engraftment into NSG (NOD/SCID/IL2 receptor {gamma} chain (null)) mice.

We demonstrate the unique ability of these cell-derived ECM scaffolds to support expansion and differentiation of bone marrow stem cells in vitro. Beyond that, the established methodology enables deciphering and modulating native-like multicomponent ECMs from various cell sources offering exciting options for the in-depth analysis of stem cell regulation by exogenous cues.

Prewitz, M. C. et al. Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods 10, 788–794 (2013). 10.1038/nmeth.2523.