We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; prosurfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; a-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-a; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratinpositive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

In vitro replicas of bone marrow can potentially provide a continuous source of blood cells for transplantation and serve as a laboratory model to examine human immune system dysfunctions and drug toxicology. Here we report the development of an in vitro artificial bone marrow based on a 3D scaffold with inverted colloidal crystal (ICC) geometry mimicking the structural topology of actual bone marrow matrix. To facilitate adhesion of cells, scaffolds were coated with a layer of transparent nanocomposite. After seeding with hematopoietic stem cells (HSCs), ICC scaffolds were capable of supporting expansion of CD34+ HSCs with B-lymphocyte differentiation. Three-dimensional organization was shown to be critical for production of B cells and antigen specific-antibodies. Functionality of bone marrow constructs was confirmed by implantation of matrices containing human CD34+ cells onto the backs of severe combined immunodeficiency (SCID) mice with subsequent generation of human immune cells.

Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.