Körner A et al. (2013), Cell differentiation of pluripotent tissue shee...

XB-ART-46695

PLoS One 2013 Jan 01;82:e54749. doi: 10.1371/journal.pone.0054749.

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Cell differentiation of pluripotent tissue sheets immobilized on supported membranes displaying cadherin-11.

Körner A , Deichmann C , Rossetti FF , Köhler A , Konovalov OV , Wedlich D , Tanaka M .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cad cdh11 fzd7 gap43 slc12a3 snai2

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	Figure 2. Formation and functionalization of supported membranes.Formation of a supported membrane containing 5 mol% DOGS-NTA (I), specific binding of Xcad-11 EC1-3 SNAP His12 (II) and decoupling of Xcad-11 (through incubation with EDTA-solution (III) and subsequent washing (IV)) monitored by QCM-D. The membrane deposition, protein binding and protein decoupling are detected by changes in the resonance frequency Δf and dissipation ΔD.
	Figure 3. Fine structure of supported membranes probed by X-ray reflectivity.(A) Specular X-ray reflectivity curves of the supported membrane with 5 mol% DOGS-NTA before (black) and after (red) the binding of Xcad-11. The experimental errors are within the symbol size. The solid lines represent the best model fits to the data. (B) The corresponding scattering length density (SLD) profiles demonstrated that the Xcad-11 anchored on the surface at a high density can be treated as a “layer”.
	Figure 4. Animal cap tissue incubated on supported membranes: Impact of lateral density of Xcad-11.Xenopus embryos were injected in one blastomere of 2-cell stage with 300 pg tBR and 500 pg XFz7 to induce neural crest cell fate (induced animal cap tissue) or left non-induced (wildtype animal cap tissue). Additionally 500 pg GAP43-mCherry was injected as membrane tracer, and 150 pg slug-promoter-GFP as read-out for neural crest induction process. Animal cap explants were taken at stage 9 and cultivated on pure SOPC membranes (0 mol% DOGS-NTA, A) and on Xcad-11-functionalized membranes (2 mol% DOGS-NTA, B, C). Animal caps were placed on the surface and imaged after 4 hours of cultivation. Activation of slug-promoter-GFP reporter (green) indicates successful neural crest induction. NCC induced tissue started to dissociate and cells lost cell-cell contact (A, gaps are highlighted by asterisks) on pure SOPC membranes. Induced explants on Xcad-11 functionalized surface (B) consist of a cohesive tissue that shows close cell-cell contacts. A clear expression of the slug transcription factor was detected by the positive GFP signal. Wildtype animal cap tissue disintegrated and started dying (C) on functionalized membranes.
	Figure 5. Time evolution of contacts between induced animal cap tissues and supported membranes displaying Xcad-11.With the aid of microinterferometry (RICM), the region of tight cell-surface contacts can be identified as the darker patches (A, B, D). It should be noted that not all the cells in the tissue sheet adhered to the surface, although the animal cap retained a connective structure. (A, B) Animal caps on membranes containing 2 mol% DOGS-NTA (<d>∼5.7 nm) after 1 h and 4 h incubation, respectively. Panel (B’) shows the enlargement of the blue rectangle in panel B and panel (C) shows the intensity profile along a green line in the panel B’, reflecting the accumulation of adhesion patches towards the cell center. (D) Animal cap on a membrane containing 1 mol% DOGS-NTA (<d>∼8.1 nm) after 4 h. (E) The number density of cells in tissue sheets with adhesion patches larger than 30 µm2 within an area of 20000 µm2 showed a clear increase with increasing molar ratio of DOGS-NTA and time, suggesting that the adhesion of animal caps on membranes displaying Xcad-11 is specifically mediated by Xcad-11 and develops as a function of time. At least 3 explants per condition were used to make the histograms. Small black spots in A, B, B’ and D are pigment granules inside ectoderm cells and therefore negligible
	Figure 6. Induced and wildtype animal cap tissues on supported membranes with/without Xcad-11 (t = 4 h).The animal cap cells were labeled with GAP43-GFP for visualization of the membrane. (A–C) Induced tissue did not adhere on non-functionalized SOPC membranes, showing no adhesion patch (A) and no filopodia (B). Image B is take at z∼2 µm above image A. (D–F) Wildtype tissue cultivated on a supported membrane functionalized with Xcad-11 EC1–3 at <d>∼5.7 nm. Although some cells in tissue sheets adhered to the supported membrane, the intensity fluctuations near the cell center are still remarkable (D). The tissue exhibited membrane budding (E, F), which is characteristic for disintegrated tissues. Images D and E are taken at the same z position. (G–I) NCC induced animal cap on a Xcad-11 functionalized membrane (<d>∼5.7 nm). As presented in panel (G), the fluctuation of cell-surface distance was strongly damped, compared to wildtype tissues on the same surface (D). The formation of filopodia and lamellipodia characteristic for neural crest cells (H and I, indicated by arrows) demonstrate that induced tissues remain viable and can properly shape the cells on membranes functionalized with Xcad-11. Images G and H are taken at the same z position.
	Figure 1. Supported membrane functionalized with Xcad-11 EC1-3 SNAP His12.As lipid anchors (DOGS-NTA) are uniformly mixed in matrix lipids (SOPC), the average distance between proteins <d> can be controlled within nm accuracy simply by adjusting the molar fraction of DOGS-NTA.

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