Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.

In light of the increasing need for differentiated primary cells for cell therapy and the rapid dedifferentiation occurring during standard in vitro cultivation techniques, there is an urgent need for developing three-dimensional in vitro systems in which expanded cells display in vivo-like differentiated phenotypes. It is becoming clear that the natural microenvironment provides the optimal conditions for achieving this aim. To this end, we prepared natural decellularized scaffolds of microscopic dimensions that would allow appropriate diffusion of gases and nutrients to all seeded cells. Scaffolds from either the lung or the liver were analyzed for their ability to support growth and differentiation of progenitor alveolar cells and hepatocytes. We observed that progenitor alveolar cells that have been expanded on plastic culture and thus dedifferentiated grew within the lung-derived scaffolds into highly organized structures and regained differentiation markers classical for type I and type II alveolar cells. The cells generated proper alveolar structures, and only 15%-30% of them secreted surfactant proteins in a localized manner for extended periods. Vice versa, liver-derived scaffolds supported the differentiation state of primary hepatocytes. We further demonstrate that the natural scaffolds are organ specific, that is, only cells derived from the same organ become properly differentiated. A proteomic analysis shows significant different composition of lung and liver scaffolds, for example, decorin, thrombospondin 1, vimentin, and various laminin isoforms are especially enriched in the lung. Altogether, our data demonstrate that complex interactions between the seeded cells and a highly organized, organ-specific stroma are required for proper localized cell differentiation. Thus, our novel in vitro culture system can be used for ex vivo differentiation and organization of expanded primary cells.

Skin-derived micro-organs induce angiogenesis in rabbits.

We have recently reported an alternative cell therapy approach to induce angiogenesis. The approach is based on small organ fragments--micro-organs (MOs)--whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. We have shown that lung-derived MOs, when implanted into hosts, transcribe a wide spectrum array of angiogenic factors and can induce an angiogenic response that can rescue experimentally induced ischemic regions in mice. From a clinical perspective, skin-derived MOs are particularly appealing as they could readily be obtained from a skin biopsy taken from the same target patient. In the present work we have investigated the angiogenesis-inducing capacity of rabbit and human skin-derived micro-organs in vitro and in vivo. Rabbit skin MOs were implanted into homologous adult rabbits and human skin MOs were encapsulated and implanted into xenogenic mice. Skin-derived MOs, as lung-derived MOs, were found to secrete a whole array of angiogenic factors and to induce a powerful angiogenic response when implanted back into animals. We believe the approach presented suggests a novel, efficacious and simple approach for therapeutic angiogenesis.

Epithelial-mesenchymal interactions allow for epidermal cells to display an in vivo-like phenotype in vitro.

We here report that preservation of the basic epithelial-mesenchymal interactions allows for highly complex ex vivo function of epidermal cells. The approach taken is based on the preparation of organ fragments that preserve the basic epithelial/mesenchymal interactions but also ensure appropriate diffusion of nutrients and gases to all cells. Human and mice keratinocytes in such organ fragments, remain viable, proliferate and express epidermal-specific gene products when cultured in serum-free medium without added growth factors, for several weeks in vitro. When implanted into syngeneic animals they remain viable, become vascularized and continue to function and transcribe tissue-specific gene products for several months. Such fragments allow primary cells ex vivo to preserve most of the functional attributes of the in vivo system. Clearly, the effect of the extracellular matrix is critical in this system in order for the cells to proliferate and differentiate ex vivo. We are not aware of any other system which allows for localized expression of epidermal-specific genes ex vivo for significant periods in culture in defined serum-free medium.

A cell-based multifactorial approach to angiogenesis.

We here propose an alternative cell therapy approach to induce angiogenesis. We prepared small organ fragments whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. Fragments derived from lung are shown to behave as fairly independent units, to undergo a marked upregulation of angiogenic factors and to continue to function for several weeks in vitro in serum-free media. When implanted into hosts, they transcribe a similar array of angiogenic factors that specifically induce the formation of a potent vascular network. The angiogenic induction capacity of these fragments was also tested in a mouse and rat model of limb ischemia. We report that such fragments, when implanted in the vicinity of the ischaemic area, induce an angiogenic response which can rescue the ischaemia-induced damage. The approach presented differs from single factor application, gene therapy and other cell therapy methods in that it exploits the complex behaviour of autologous cells in their near to normal environment in order to achieve secretion of a whole range of angiogenic stimuli continuously and in an apparently coordinated fashion.