Characterization of the human smooth muscle cell secretome for regenerative medicine.

Smooth muscle cells (SMC) play a central role in maintaining the structural and functional integrity of muscle tissue. Little is known about the early in vitro events that guide the assembly of 'bioartificial tissue' (constructs) and recapitulate the key aspects of smooth muscle differentiation and development before surgical implantation. Biomimetic approaches have been proposed that enable the identification of in vitro processes which allow standardized manufacturing, thus improving both product quality and the consistency of patient outcomes. One essential element of this approach is the description of the SMC secretome, that is, the soluble and deposited factors produced within the three-dimensional (3D) extracellular matrix (ECM) microenvironment. In this study, we utilized autologous SMC from multiple tissue types that were expanded ex vivo and generated with a rigorous focus on operational phenotype and genetic stability. The objective of this study was to characterize the spatiotemporal dynamics of the first week of organoid maturation using a well-defined in vitro-like, 3D-engineered scale model of our validated manufacturing process. Functional proteomics was used to identify the topological properties of the networks of interacting proteins that were derived from the SMC secretome, revealing overlapping central nodes related to SMC differentiation and proliferation, actin cytoskeleton regulation, and balanced ECM accumulation. The critical functions defined by the Ingenuity Pathway Analysis included cell signaling, cellular movement and proliferation, and cellular and organismal development. The results confirm the phenotypic and functional similarity of the SMC generated by our platform technology at the molecular level. Furthermore, these data validate the biomimetic approaches that have been established to maintain manufacturing consistency.

Expansion of the human adipose-derived stromal vascular cell fraction yields a population of smooth muscle-like cells with markedly distinct phenotypic and functional properties relative to mesenchymal stem cells.

Adipose tissue contains a heterogeneous cell population composed of endothelial cells, adipocytes, smooth muscle cells (SMC), and mesenchymal progenitors and stromal cells that meet the criteria put forth by the International Society for Cellular Therapy as defining mesenchymal stem cells (MSC). In this study, we expanded the stromal vascular fraction (SVF) of human adipose tissue and characterized the resulting adherent primary cell cultures by quantitative reverse transcription-polymerase chain reaction, antigen expression, protein fingerprinting, growth kinetics, in vitro tri-lineage differentiation bioactivity, and functional responses to small molecules modulating SMC-related developmental pathways and compared the results to those obtained with functionally validated MSC cultures. SVF-derived initial cultures (P0) were expanded in a defined medium that was not optimized for MSC growth conditions, neither were recombinant cytokines or growth factors added to the media to direct differentiation. The adherent cell cultures derived from SVF expansion under these conditions had markedly distinct phenotypic and biological properties relative to functionally validated MSC cultures. SVF-derived adherent cell cultures retained characteristics consistent with the SMC subpopulation within adipose tissue--phenotype, gene, and protein expression--that were independent of passage number and source of SVF (n=4 independent donors). SVF-derived cells presented significantly less robust in vitro tri-lineage differentiation bioactivity relative to validated MSC. Expanded SVF cells and MSC had opposite responses to the thromboxane A2 mimetic U46619, demonstrating an unambiguous functional distinction between the two cell types. Taken together, these data support the conclusions that SVF cells expanded under the conditions described in these studies are accurately described as adipose-derived SMC and represent a cellular subpopulation of adipose SVF that is separate and distinct from other classes of adipose-derived cells.