Abstract
Investigating cohesive tissue sheets in controlled cultures still poses a challenge since the complex intercellular interactions are difficult to mimic in in vitro models. We used supported lipid membranes functionalized by the adhesive part of the extracellular domain of the cell adhesion molecule cadherin-11 for the immobilization of pluripotent tissue sheets, the animal cap isolated from Xenopus laevis blastula stage embryos. Cadherin-11 was bound via histidine tag to lipid membranes with chelator head groups. In the first step, quantitative functionalization of the membranes with cadherin-11 was confirmed by quartz crystal microbalance and high energy specular X-ray reflectivity. In the next step, animal cap tissue sheets induced to neural crest cell fate were cultured on the membranes functionalized with cadherin-11. The adhesion of cells within the cohesive tissue was significantly dependent on changes in lateral densities of cadherin-11. The formation of filopodia and lamellipodia in the cohesive tissue verified the viability and sustainability of the culture over several hours. The expression of the transcription factor slug in externally induced tissue demonstrated the applicability of lipid membranes displaying adhesive molecules for controlled differentiation of cohesive pluripotent tissue sheets.
Highlights
Biological membranes are key components of all living systems, forming the outer boundary of living cells or of internal cell compartments
It should be noted that the suppression of the fluctuation of cell-substrate distance and the formation of filopodia and lamellipodia were observed only for induced tissues on membranes functionalized with Xenopus cadherin-11 (Xcad-11)
We demonstrated the immobilization of pluripotent animal caps isolated from embryos of Xenopus laevis on supported membranes quantitatively functionalized with recombinant Xcad-11 without disrupting the intercellular connections in tissue sheets
Summary
Biological membranes are key components of all living systems, forming the outer boundary of living cells or of internal cell compartments (organelles). They consist largely of a lipid bilayer that imparts a fluid character. Supported membranes can readily be functionalized either by spreading vesicles incorporating transmembrane proteins (proteoliposomes) or by incorporating ‘anchor’ molecules for engineered proteins. This method is a powerful tool for creating complex experimental cell-surface models that can be investigated in a quantitative manner [1,5,6,7,8,9]. As synthetic analogues to GPI anchors, lipid anchors with biotin head groups [11,12] and those with nitrilotriacetic acid (NTA) head groups [13,14] have been used to couple various recombinant proteins and carbohydrates with biotin and histidine tags to the membrane surfaces, respectively
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