Nanopatterned and biofunctionalized PEG hydrogels.

<p>(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.</p

Antibodies for integrin inhibition assays.

<p>Antibodies for integrin inhibition assays.</p

Regulation of Hematopoietic Stem Cell Behavior by the Nanostructured Presentation of Extracellular Matrix Components

<div><p>Hematopoietic stem cells (HSCs) are maintained in stem cell niches, which regulate stem cell fate. Extracellular matrix (ECM) molecules, which are an essential part of these niches, can actively modulate cell functions. However, only little is known on the impact of ECM ligands on HSCs in a biomimetic environment defined on the nanometer-scale level. Here, we show that human hematopoietic stem and progenitor cell (HSPC) adhesion depends on the type of ligand, i.e., the type of ECM molecule, and the lateral, nanometer-scaled distance between the ligands (while the ligand type influenced the dependency on the latter). For small fibronectin (FN)–derived peptide ligands such as RGD and LDV the critical adhesive interligand distance for HSPCs was below 45 nm. FN-derived (FN type III 7–10) and osteopontin-derived protein domains also supported cell adhesion at greater distances. We found that the expression of the ECM protein thrombospondin-2 (THBS2) in HSPCs depends on the presence of the ligand type and its nanostructured presentation. Functionally, THBS2 proved to mediate adhesion of HSPCs. In conclusion, the present study shows that HSPCs are sensitive to the nanostructure of their microenvironment and that they are able to actively modulate their environment by secreting ECM factors.</p> </div

Adhesion of KG-1a cells and HSPC to nanopatterned, biofunctionalized PEG hydrogels.

<p>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. N_{independent 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.</p

Cell morphology on nanostructured glass substrates.

<p>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.</p

Influence of nanostructured matrices on THBS2 expression by HSPC.

<p>(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. N_{independent 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. N_cells = 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. N_{independent 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.</p

Integrin-mediated HSPC adhesion to FN.

<p>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. N_{independent experiments =}4. (B) HSPC adhesion to FN was significantly reduced when inhibiting either the α₄ integrin chain, the α₅ integrin chain or both. N_{independent 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.</p

Short peptide binding motifs and protein domains used for biofunctionalization of gold NP arrays.

<p>Peptides were purchased from Biosyntan, Berlin, Germany or synthesized by Dr. Hubert Kalbacher (University of Tübingen, Germany).</p

Determination of HSPC differentiation in colony forming assays.

<p>(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 (<i>colony forming unit granulocyte, erythroid, macrophage, megakaryocyte</i>), CFU-GM (<i>colony forming unit granulocyte, macrophage</i>) and BFU-E (<i>burst forming unit erythroid</i>) after 2 weeks. N_{independent 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. N_{independent experiments =}5; TCP = tissue culture plastic, gold = continuous gold film on glass, FNΔRGD is abbreviated with “ΔRGD”.</p

Parameters for producing nanostructured surfaces by BCML.

<p>PS: polystyrene units, P2VP: poly(2-vinylpyridine) units, C: concentration of the polymer, V: substrate retraction velocity, d: distance between gold NPs ± standard deviation.</p