Generation of Planar Cell Polarity (PCP) in-Vitro for tissue engineering epithelium Ana C Paz, Alison P McGuigan

Laboratory for Tissue Morphogenesis Engineering, University of Toronto, Toronto, ON, Canada.

Introduction The arrangement of the cells within a tissue is essential for its correct function. Currently, the structure of engineered tissues does not emulate completely the structure of native tissues and hence they have limited function. Achieving appropriate tissue organization is necessary if engineered tissues are to become clinically relevant. During the development of a tissue in the embryo, cells organize in defined patterns due to signaling molecules. Artificially generating the signaling that organizes cells within the tissue offers a novel approach for engineering artificial tissues with appropriate structure and function. Planar cell polarity (PCP) is the organization of cells in the plane of a tissue, perpendicular to the apical-basal axis (1). PCP is established by the asymmetric localization of PCP core proteins (Vang, Pk, Dsh, Fz, dgo, fmi) within the proximal-distal axis due to specific signaling molecules. The objective of this stuffy was to characterize how different topographies, cell sheet size and shape, and apical-basal polarization affect the localization of the PCP core proteins within a sheet of cells with a view to engineering PCP in an epithelial sheet. Materials and Methods We generated epithelial sheets using dog kidney epithelial cells (MDCK) transduced with GFP-tagged lentiviral constructs to produce elevated levels of Vang2, one of PCP core proteins. Sheets were patterned using micro-patterning. We produced lentivirus encoding GFP-tagged Vang2 construct by transfecting 293T human embryonic kidney cells (ATCC, USA) with lentiviral vector, in this case GFP-Vang2 construct, and packaging plasmids, using FugGEN 6 transfection regent (Roche, Germany). We sorted lenti-viral infected MDCK cell to get a population with the same GFP fluorescent levels. Finally, this homogenic GFP-Vang2 MDCK population was seeded onto different substrates: (i) Coverslips, (ii) Transwells Filters, (iii) Polydimethylsiloxane (PDMS) with Nano-grooves and (iv) 200µm squares micropatterns. The 200µm square micropatterns were fabricated using agarose wells microfabrication method (2). Briefly, a master with 200um square array was created using photolithography and a PDMS stamp generated using replica molding. The PDMS stamp was placed on a glass slide and a solution of six parts of 1% agarose with four parts ethanol was poured under the stamp. After agarose polymerization the stamp was removed. Results We generated a population of GFP-Vang2 MDCK cells with homogenic levels of GFP fluorescent. The apical-

basal polarization seems to promote the localization of the Vangl2 protein to the membrane in all the different seeding conditions (Fig1). However, there was not a clear asymmetrical distribution of the protein throughout the entire cell sheet. Cells cultured on micropatterns appeared to have a more diffuse Vangl2 distribution and cell shape was less regular than in large epithelial sheets.

Fig. 1. GFP-Vang2 MDCK seeded on glass. 60X , Green = GFP , Blue=DAPI

Fig. 2. GFP-Vang2 MDCK seeded on 200µm microwells. 20X , Green = GFP , Blue=DAPI Conclusions We have demonstrated it is possible to generate membrane localization of PCP proteins in epithelial sheets in vitro, a first step towards generating asymmetric PCP. Understanding how to generate PCP in vitro will enable us to engineer more functional epithelial tissue in the future. References 1. Bacallao RL, et al. Cystic kidney diseases and planar cell polarity signaling. Clin Genet 2009: 75: 107–117. 2. Celeste M. Nelson, Wendy F. Liu, and Christopher S. Chen. Manipulation of Cell–Cell Adhesion Using Bowtie-Shaped Microwells. Methods in Molecular Biology, vol. 370: Adhesion Protein Protocols, Second Edition Disclosures Authors do not have any disclosure.