Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application.

In the present study, mouse embryonic stem cells (ESCs) were differentiated into alveolar epithelial type II (AEII) cells for endotracheal injection. These enriched lung-like populations expressed lung epithelial markers SP-A, SP-B, SP-C, and CC10. First we show that rapid differentiation of ESCs requires a dissociated seeding method instead of an embryoid body culture method. We then investigated a two-step differentiation of ESCs into definitive endoderm by activin or A549-conditioned medium as a precursor to lung epithelial cells. When conditioned medium from A549 cells was used to derive endoderm, yield was increased above that of activin alone. Further studies showed that Wnt3a may be one of the secreted factors produced by A549 cells and promotes definitive endoderm differentiation, in part, through suppression of primitive endoderm. Activin and Wnt3a together at appropriate doses with dissociated cell seeding promoted greater endoderm yield than activin alone. Next, fibroblast growth factor 2 was shown to induce a dose-dependent expression of SPC, and these cells contained lamellar bodies characteristic of mature AEII cells from ESC-derived endoderm. Finally, ES-derived lung cells were endotracheally injected into preterm mice with evidence of AEII distribution within the lung parenchyma. This study concludes that a recapitulation of development may enhance derivation of an enriched population of lung-like cells for use in cell-based therapy.

In vivo pulmonary tissue engineering: contribution of donor-derived endothelial cells to construct vascularization.

Intrapulmonary engraftment of engineered lung tissues could provide a potential therapeutic approach for the treatment of pediatric and adult pulmonary diseases. In working toward this goal, we report here on in vivo generation of vascularized pulmonary tissue constructs utilizing the subcutaneous Matrigel plug model. Mixed populations of murine fetal pulmonary cells (FPCs) containing epithelial, mesenchymal, and endothelial cells (ECs) were isolated from the lungs of embryonic day 17.5 fetuses. FPCs were admixed to Matrigel and injected subcutaneously into the anterior abdominal wall of adult C57/BL6 mice to facilitate in vivo pulmonary tissue construct formation. Vascularization was enhanced by placing fibroblast growth factor 2 (FGF2)-loaded polyvinyl sponges into the hydrogel. After 1 week, routine histology and immunohistochemical staining for donor-derived epithelial cells and ECs as well as analysis of patent vasculature in the constructs following tail vein injection of fluorescein isothiocyanate-conjugated dextran were performed. In the Matrigel-only controls, some level of host infiltrate, but no measurable vascularization, was detected. In the presence of FPCs, the constructs contained ductal epithelial structures and patent vasculature. In the absence of FPCs, exogenous FGF2 induced the formation of numerous patent blood vessels throughout the entire constructs; in combination with FPCs, it resulted in enhanced capillary density and abundant interfacing between developing epithelial and vascular structures. The significant findings of this study are that distal pulmonary epithelial differentiation (as assessed by the expression of prosurfactant protein C) can be maintained in vivo and that donor-derived ECs contribute to the formation of patent vessels that interface tightly with ductal epithelial structures.

Novel methods for delivery of cell-based therapies.

BACKGROUND: Pulmonary hypoplasia (PH) is found in 15% to 20% of all neonatal autopsies, accounting for 2850 deaths yearly. Development of engineered tissue substitutes that could functionally restore damaged tissue remains a unique opportunity for biotechnology. Recently, we isolated and characterized murine fetal pulmonary cells (FPC) and engineered 3-D pulmonary tissue constructs in vitro. Our goal is to devise a reliable and reproducible method for delivering FPC into a live animal model of PH. MATERIALS AND METHODS: Three methods of delivery were explored: intraoral, intratracheal, and intrapulmonary injection. Adult Swiss Webster mice were anesthetized and fluorescent labeled microspheres (20 microm diameter) were delivered by intraoral and intratracheal injection. Subsequently, labeled FPC (Cell Tracker, CMTPX; Molecular Probes, Eugene, OR) were delivered by the same methods. In addition, direct transpleural intrapulmonary injection of FPC was performed. Outcome analysis included survival, reproducibility, diffuse versus confined location of the injected substance, and adequacy of delivery. Routine histological examination, fluorescent microscopy, and immunostaining were performed. RESULTS: Microspheres: We demonstrated reproducible, diffuse instillation via tracheotomy into the distal alveoli. Intraoral delivery appeared less reliable compared to direct intratracheal injection. FPC: Intratracheal injection was a reliable method of delivery. Labeled FPC showed transepithelial migration after 7 d of in vivo culture. Intrapulmonary injection led to local accumulation of cells in sites of injection. CONCLUSIONS: We demonstrate that delivery of FPC is feasible with intratracheal injection giving the most reliable, diffuse delivery throughout the lung. This represents the first step toward translational research with site-specific delivery for a cell-based therapeutic approach toward PH and similar pulmonary diseases.