Figure 1.
Nanopatterned and biofunctionalized PEG hydrogels.
(A–C) Cryo-SEM images of the quasi-hexagonally ordered gold NP patterns on PEG hydrogels with interparticle distances of (A) 36±7 nm, (B) 60±11 nm and (C) 110±18 nm. (D–F) Micrographs of fluorescently labeled, FNRGD-functionalized nanostructured hydrogels. Images of the border between the nanostructured area in the lower part of the micrographs and the unstructured area visible in the upper part are shown. (D) The FNRGD domain on biofunctionalized hydrogels was detected with the help of specific primary antibodies. Controls were produced by (E) substituting nickel with EDTA during functionalization and by (F) omitting the primary antibody during the staining procedure. One representative experiment out of 3 is shown.
Figure 2.
Cell morphology on nanostructured glass substrates.
SEM images of critical point dried KG-1a cells on nanostructured, PEG-passivated, cRGD-functionalized substrates with interparticle distances of 36±7 nm. Magnification increases from A to D.
Figure 3.
Adhesion of KG-1a cells and HSPC to nanopatterned, biofunctionalized PEG hydrogels.
Cells were plated on hydrogels in adhesion media and evaluated by phase contrast microscopy or Cy Quant cell quantification after 1 h of incubation. (A) Microscopic images of KG-1a cells on biofunctionalized PEG hydrogels. Cells appear as bright spots on a gray background. The area in the lower part of each image is nanostructured and the upper area is unstructured (internal control). The name of the applied ligand is given above each image. Cell adhesion to NP arrays with a distance of 36±7 nm or 60±11 nm are shown in the upper and lower row of images, respectively. FNΔRGD is abbreviated with “ΔRGD”. One representative experiment out of 4 is shown. Scale bar = 200 µm. (B) Relative quantification of KG-1a cell adhesion and (C) HSPC adhesion to different ligands on NP arrays with 36±7 nm (filled columns) or 60±11 nm (diagonally striped columns) spacing. On the y-axis the number of adherent cells normalized to the value for adhesion to full-length FN is plotted. The different ligands are indicated on the x-axis. Nindependent experiments = 4, each experiment carried out in technical duplicates; error bars = standard deviation of the mean; * = significant p value <0.05 in Wilcoxon rank sum test.
Figure 4.
Integrin-mediated HSPC adhesion to FN.
HSPCs were preincubated with integrin-specific antibodies or a linear RGD peptide for 1 h and plated onto an adsorbed FN spot. After nonadherent cells were removed, the spots were imaged and the adherent cells counted. (A) Relative HSPC adhesion to FN was either inhibited by preincubation with antibodies blocking the β1 integrin chain or with a linear RGD peptide. The isotype control (set to 100%) shows cell adhesion to FN after preincubation with isotype control antibodies. Nindependent experiments = 4. (B) HSPC adhesion to FN was significantly reduced when inhibiting either the α4 integrin chain, the α5 integrin chain or both. Nindependent experiments = 5. (A, B) Error bars = standard error of the mean; * = significant p value <0.05 in Wilcoxon rank sum test. (C) Representative microscopic images of HSPC adhesion to FN spots. Cells are visible as white spots in the lower part of each image, the dashed black line indicates the border of the FN spot. Scale bar = 200 µm.
Figure 5.
Determination of HSPC differentiation in colony forming assays.
(A) Colony forming units of precultured HSPCs on glass slides biofunctionalized with different ligands. After 7–10 days preculture, colony forming assays were performed in triplicates. Colonies were distinguished into CFU-GEMM (colony forming unit granulocyte, erythroid, macrophage, megakaryocyte), CFU-GM (colony forming unit granulocyte, macrophage) and BFU-E (burst forming unit erythroid) after 2 weeks. Nindependent experiments = 5 in technical triplicates; error bars = standard error of the mean. (B) Box Plot of the total number of colony forming units formed by 1500 precultured HSPCs on glass slides biofunctionalized with different ligands. Nindependent experiments = 5; TCP = tissue culture plastic, gold = continuous gold film on glass, FNΔRGD is abbreviated with “ΔRGD”.
Figure 6.
Influence of nanostructured matrices on THBS2 expression by HSPC.
(A) Relative gene expression of THBS2 in HSPC incubated for 140 min on nanostructured PEG hydrogels biofunctionalized with FNRGD or FNΔRGD (abbreviated with “ΔRGD”). RQ values were normalized to the unstructured hydrogel controls and are plotted on the y-axis. The different functionalized and nanostructured substrates are indicated on the x-axis. Nindependent experiments = 4; error bars = standard error of the mean; * = significant p value <0.05 in Wilcoxon rank sum test. (B) Immunofluorescence staining of THBS2 protein in HSPCs incubated for 13 h on nanostructured, biofunctionalized PEG hydrogels. Fluorescence intensity was measured per cell by applying Image J software and is plotted on the y-axis. Ncells = 60 from 3 donors (3×20); data are presented as box plots overlaid with individual data points (black squares/diamonds); * = significant p value <0.001; # = significant p value <0.001 to cells on hydrogel only; § = significant p value <0.001 to cells on 35±7 nm FNΔRGD (abbreviated with “ΔRGD”) biofunctionalized hydrogels. Two-tailed, unpaired Student’s t-test. (C) HSPC adhesion to full-length FN, BSA or recombinant THBS2 protein in percent of the total applied cell number per well. Nindependent experiments = 5, each experiment carried out in technical triplicates; error bars = standard deviation of the mean; * = significant p value <0.01 in Wilcoxon rank sum test.
Table 1.
Parameters for producing nanostructured surfaces by BCML.
Table 2.
Short peptide binding motifs and protein domains used for biofunctionalization of gold NP arrays.
Table 3.
Antibodies for integrin inhibition assays.