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The extracellular matrix (ECM) is a multicomponent mixture of fibrillar and adhesive proteins, glycoproteins and proteoglycans, as well as signaling molecules and matrix-associated vesicles. The ECM creates tissue structure and generates mechanical forces that influence differential gene expression in associated cells. In addition, the tissue-specific composition of the ECM, which is different in maintaining homeostasis and in pathology, influences the processes of cell differentiation. When studying cell differentiation in vitro, modeling conditions often include the addition of soluble factors, but overlook such a component of the microenvironment as ECM. Modeling the microenvironment using individual ECM components (collagens, fibronectin, etc.) has obvious limitations, since it does not reproduce the multicomponent composition and stiffness of ECM tissue. We used the technology of decellularization of multilayer cell sheets obtained from different types of human stromal cells, including mesenchymal stromal/stem cells (MSCs) and fibroblasts from different tissues. The resulting dECM retained a multicomponent composition (type I collagen, fibronectin, EDA-fibronectin) and complex architecture, which was studied by scanning electron microscopy. We have previously shown that ECM obtained from human adipose tissue MSCs leads to increase of induced differentiation of multipotent stem cells into osteocytes, adipocytes and chondrocytes. Presumably, dECM, through an ERK-dependent mechanism, stimulated the proliferation of progenitor cells and potentiated differentiation upon the addition of appropriate stimuli. At the same time, dECM obtained from MSCs isolated from human dental pulp had the ability to accelerate the induced differentiation of multipotent cells in the osteogenic direction, compared with dECM obtained from MSCs of adipose tissue and skin fibroblasts. This observed effect could be associated with an increase in the basal expression of the RUNX2 gene, the master gene of osteogenic differentiation, in multipotent cells cultured on dental pulp MSCs dECM. Moreover, in each case, dECM itself did not stimulate differentiation without additional induction. In profibrotic in vitro model, cultivation only on dECM obtained from human skin fibroblasts led to rapid (within 12 hours) growth of the fibroblast activation protein (FAPa), but did not stimulate the formation of myofibroblasts, which indicates the transition of fibroblasts to an activated state on dECM. Myofibroblast formation was observed only 72 hours after TGF-β1 induction of fibroblasts. Thus, in vitro modeling of the cellular microenvironment using dECM obtained by decellularization of cell sheets can recreate more relevant conditions for cell differentiation. The data presented indicate that cultivation on dECM puts cells in a state susceptible to further differentiation stimuli, which is an integral part of the differentiation process.