Precise Design Strategy Of Smart Extracellular Matrix Based on CRISPR/Cas9 for Regulating Neural Stem Cell Function

The extracellular matrix (ECM) is the natural microenvironment of stem cells survival, as well as proliferation, differentiation and metastasis, containing a variety of biological molecular complexes secreted by the resident cells in tissues and organs. Heparan sulfate proteoglycans (HSPG) is a major component of ECM, containing one or more covalently attached heparan sulfate chains. The heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains with great importance in development and normal physiology. Herein, lentiviral single-guide RNA vectors were constructed to activate the endogenous HSPG2 expression in mouse embryonic fibroblasts based on CRISPR/Cas9 synergistic activation mediator system, and then HSPG2 functional cell-derived ECM (ECM HSPG2 ) was fabricated. The ECM HSPG2 is capable to enrich cytokines effectively, such as basic fibroblast growth factor (bFGF), which was about 3-flod stronger than binding with negative control ECM. With encapsulating abundant bFGF, ECM HSPG2 could maintain the neural stem cells (NSCs) stemness and promote the NSCs proliferation and differentiation in culture. These findings provide a novel and precise design strategy of  smart and special functional cell-derived ECM for the biomaterials research and regenerative medicine.