Hypoxic Extracellular Matrix Preserves Its Competence after Expansion of Human MSCs under Physiological Hypoxia In Vitro.

Tissue-relevant O 2 levels are considered as an important tool for the preconditioning of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine needs. The present study investigated the quality and functions of the extracellular matrix (ECM) of MSCs under low O 2 levels. Human adipose tissue-derived MSCs were continuously expanded under normoxia (20% O 2 , N) or "physiological" hypoxia (5% O 2 , Hyp). Decellularized ECM (dcECM) was prepared. The structure of the dcECM was analyzed using confocal laser and scanning electron microscopy. Collagen, dcECM-N, and dcECM-Hyp were recellularized with MSC-N and further cultured at normoxia. The efficacy of adhesion, spreading, growth, osteogenic potential, and paracrine activity of recellularized MSC-N were evaluated. At low O 2 , the dcECM showed an increased alignment of fibrillar structures and provided accelerated spreading of MSC-N, indicating increased dcECM-Hyp stiffness. We described O 2 -dependent "ECM-education" of MSC-N when cultured on dcECM-Hyp. This was manifested as attenuated spontaneous osteo-commitment, increased susceptibility to osteo-induction, and a shift in the paracrine profile. It has been suggested that the ECM after physiological hypoxia is able to ensure the maintenance of a low-commitment state of MSCs. DcECM, which preserves the competence of the natural microenvironment of cells and is capable of "educating" others, appears to be a prospective tool for guiding cell modifications for cell therapy and tissue engineering.