M.Sc Thesis

M.Sc StudentOved Yaara
SubjectTissue Engineering of a Composite Vascularaized Skin Tissue
Using Stem Cells
DepartmentDepartment of Biotechnology
Supervisor PROF. Shulamit Levenberg


Tissue Engineering is a relatively new expanding area, aimed at replacing damaged, injured, or missing body tissues. Skin is a bi-layered organ whose functions are essential for survival. It provides structure, form, protection and performs regulatory functions. These functions are compromised when the skin is severely damaged.

The aim of the research was to induce epidermal differentiation of human Embryonic Stem Cells (hESCs) to be used as a cellular component in full skin substitute, containing both epidermis and dermis layers. We also aimed to generate vascularized dermis to seed the progenitors onto. The use of hESCs as a cell source will alleviate the shortage of donors and availability issues.

We used Embryoid Bodies (EBs) generated from hESCs that were introduced to differentiation processes in vitro in 3D environment to direct differentiation of epithelial progenitors. EB clusters were seeded on gelatin coated plates using several growth factors, including Retinoic Acid (RA), Bone Morphogenic Factor 4 (BMP-4) and Epidermal Growth Factor (EGF). The 2D results were followed by 3D experiments using biodegradable polymers seeded with hESCs with supplement of BMP-4. We anticipated that 3D environment will support differentiation and organization of the cells. To analyze the differentiation of hESCs cells and derived epithelial progenitors toward skin structures we used immunohistology staining focusing on epithelial markers.

The results revealed that growth factors have an important roll in cells' survival and differentiation. Among them BMP-4 influence of epithelial markers expression was shown. Tissue constructs treated with BMP-4 had better epithelial organization with more lumens and thicker lumens compared to control. For inducing skin vascularization we used three primary cell types; fibroblasts, keratinocytes and endothelial cells (EC). The results indicate that EC and fibroblast from same human origin permitted cells orientation to become blood vessels similar to nature tissue. Using two steps seeding methods with extra cellular matrix induced formation of bilayered vascular skin. In addition, fibroblast derived extra cellular matrix was found to induce hESCs differentiation and co-cultured matrix of EC and fibroblast derived smooth muscle cells.

In summary, this research shows the feasibility of constructing EB derived epithelial cells. We demonstrated 3D differentiation of hESCs derived epidermal components. In addition we proposed dermal replacement generated from biodegradable polymeric scaffold co-cultured with fibroblast and EC.