Cells are regulated by a combination of soluble stimuli and adhesive interactions with other cells and the extracellular matrix (ECM). We are developing systems to present ECM and cytokine receptor ligands in a defined manner for applications in hematopoietic stem and progenitor cell (HSPC) culture. We hypothesize that the controlled, immobilized presentation of multiple ligands found in the HSPC in vivo supportive environment will enhance the ex vivo expansion of stem cells compared to presentation of soluble cytokine controls alone. I focused on the surface-immobilization of individual cell adhesion molecule-binding domains from the adhesive protein fibronectin, several glycosaminoglycans (hyaluronic acid, heparin, heparin sulfate (HS), and different sulfation variants of HS), a peptide mimic of the cytokine thrombopoietin, and the cytokine stem cell factor. Ligand presentation strategies included the use of supported lipid membranes, which represent an idealized mimic of a cell membrane and polydopamine coatings, which were inspired by the wet-adhesive secreted by marine mussels. The biocompatibility of these systems was demonstrated by the similar expansion of hematopoietic cell lines and human CD34+ HSPCs on lipid, polydopamine, and control tissue-culture polystyrene surfaces. Ligands were incorporated into dipalmitoyl phosphocholine supported lipid membranes by either direct incorporation (ligand conjugated to a lipid anchor - called a lipopeptide) or through biotin-avidin interaction. Both strategies yielded surfaces with active ligand, which was linked to the surface with a known orientation and tunable ligand concentration. Normal-force cell adhesion and soluble-competition assays confirmed the bioactivity and specificity of the surface-immobilized ligands. Greater than 80% M07e cell adhesion was observed on lipid surfaces doped with the integrin-binding lipopeptides cRGD and cLDV. Maximum levels of M07e cell adhesion to cRGD and cLDV lipopeptides could be reproduced at ligand loadings as low as 0.5 mol%. To achieve lateral lipid diffusivity within the lipid membranes at 37 °C, dimyristoyl phosphocholine or dioleoyl phosphocholine lipids were substituted for the dipalmitoyl phosphocholine lipids. The effects of surface fluidity on cell adhesion were found to be affected by cell type and ligand sequence. M07e cells maintained their ability to bind to cRGD lipopeptide presented from fluid surfaces at all surface concentrations, whereas THP-1 cell adhesion to cRGD lipopeptide was diminished at lower cRGD lipopeptide loadings (45% vs. 10% cell adhesion at 0.5 mol% cRGD lipopeptide). Similar to the lipid surfaces, normal-force cell adhesion and soluble-competition assays were used to confirm the bioactivity and specificity of polydopamine-immobilized glycosaminoglycans. Greater than 90% of M07e cells adhered to polydopamine surfaces modified with a solution of thiolated hyaluronic acid at concentrations greater than 0.1 mg/mL. Sub-maximal adhesion was achieved by reducing the concentration of hyaluronic acid in the coating solution. Unmodified polydopamine coatings did not support M07e cell adhesion. A major benefit of polydopamine coatings is their ability to coat virtually any substrate. M07e cells adhered to hyaluronic acid coatings formed on glass, polystyrene, and indium tin oxide.

Last modified

• 09/14/2018

Creator

• Shara Marie Dellatore

DOI

• https://doi.org/10.21985/N2QT6K

Subject

• Chemical and Biological Engineering

Keyword

• polydopamine
• CD34+ hematopoietic progenitor cell
• lipid membrane
• lipopeptide
• glycosaminoglycan

Date created

• 2008-11-25

Resource type

• Dissertation

Rights statement

• In Copyright