Rationale: The lung is an organ essential for survival, however, due to its exposure to the outside environment, it is continuously subjected to multiple injuries. As a result, lung diseases are the leading cause of human death worldwide. The majority of these diseases are chronic and incurable, therefore representing a considerable financial burden. An ideal way in which such diseases could be effectively treated would be via regenerative medicine; however, for such medicine to be available, lung stem/progenitor cells must first be characterized. ❧ Objective: To develop an in vitro/in vivo assay, which would prove to be the simplest, yet most informative, in testing the contribution of given cells to different lung cell lineages. We have composed an adequate organoids system made from dissociated epithelial and mesenchymal embryonic 14.5 lung cells. These organoids were then grafted in vivo in different locations. ❧ Results: We constructed a number of different organoids systems so as to achieve the ideal conditions. These varying approaches included: single cell suspension plated directly atop carbon filters, single cell suspension mixed with matrigel plated atop carbon filters, and hanging droplets. These different systems were then implanted in different locations within 2 month-old NOD/SCID female mice, thus reducing chances of rejection by the host. These implantation sites included: the omentum, subcutaneous, and subcapsular. Via the use of immunohistochemistry, we would determine whether or not the organoids were able to develop and differentiate during implantation. Identification of progenitor cells was done using Sox2, Sox9, and Id2 antibodies. Cell markers include CC10, αSMA, SpC, TTF-1, p63, and T1α. Our results indicate that the subcapsular engrafted hanging droplets are the most promising. All of the aforementioned markers stained positively in their appropriate locations, indicating that the E14.5 cells advanced in the organoids in their differentiation status, generating both proximal- and distal-like airway structures, as well as alveoli-like structures. ❧ Conclusions: The results of the different organoids assays within different implantation sites indicated that the subcapsular hanging droplets would be the ideal approach. Once the conditions for this in vivo system are optimized, it will prove to be an elegant, yet powerful system in determining lung stem-cellness. With the use of different genetically modified mice and additional methods by which to separate the assorted cells in the lung, a powerful assay will have been constructed that may test how different signaling pathways and cell-types effect the construction of mature lung tissue.
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