Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture

Abstract

Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.

Figure 1

Schematic of biomimetic ligand selection and incorporation into biomaterials. (1) Bacterial libraries were expanded. (2) Co-expression of green fluorescent protein and bacterial outer membrane protein CPX with the displayed peptide are induced with arabinose. (3) Stem cells are added to bacterial libraries in a co-incubation step in which the bacteria can bind to the stem cell surface. (4) Non-adherent bacteria are washed away with low speed co-centrifugation. (5) For third round selections or analysis of bacterial populations, samples of the stem cells are sorted or analyzed on a fluorescence activated cell sorter or flow cytometer. (6) Bacteria populations are frozen or plated for further selection or analysis. (7) Peptides from clones are sequenced. Synthetic versions of these peptides are then conjugated on biomaterials.

Figure 2

Binding capacity of peptide display library populations with neural stem cells. (a) Example histograms of library and clonal populations of the bacterial peptide display libraries binding to neural stem cells: (1) CPX (no peptide), (2) unselected 7C library, (3) 7C library after Round 1, (4) 7C library after Round 2, (5) 7C library after Round 3, and (6) high affinity clone 15-2. (b) Quantification of library populations. Three libraries were tested: (■) 15mer library composed of peptides with the sequence X₁₅, (▨) 7C library composed of peptides with the sequence X₂CX₇CX₂, and (□) a combined library containing both types of peptide clones. All unselected and post round 1 library populations showed similar binding as bacteria expressing CPX, the outer membrane display protein, but no peptide. After rounds 2 and 3, there were significantly more bacteria binding to the neural stems, with the 7C library having the highest amount of binding. The 15mer and in particular the combined libraries exhibited the highest binding. Data represent mean ± standard deviation. Library populations not in the same group (#, $, or ‡) were statistically different from one another (p < 0.05 using ANOVA between groups with Tukey-Kramer significant difference post hoc test).

Figure 3

Neural stem cells on peptide-adsorbed TCPS surfaces. Peptides, including (■) 7C-9(9), (▨) 7C-24(9), (▩) 15-2, (□) bsp-RGD(15), (▤) Laminin, (▥) bsp-RGE(15), and () TCPS alone, were dissolved at 100 μM in synthesis-grade water or DMSO for 15-2, and then peptides were dried on TCPS. (a). Brightfield micrographs of the neural stem cells after 4 days of culture on the adsorbed surfaces exhibited similar attachment and clumping of cells on the surface on the 7C-9(9), 7C-24(9), 15-2, and bsp-RGD(15) surfaces, while the bsp-RGE(15) and TCPS surfaces had significantly fewer cells. The scale bar represents 250 μm. (b) Quantification of the number of cells on the surface with the Cyquant cell counting assay showed similar numbers of cells on all surfaces except the bsp-RGE(15) and TCPS surfaces, which had significantly fewer cells. (c) NSCs grown under differentiating conditions were assessed for expression of GFAP (red), a cytoskeletal marker for astrocytes, and β-Tubulin III (green), a cytoskeletal marker for neurons. Nuclei were stained with DAPI (blue). All peptide surfaces had both astrocytes and neurons under differentiating conditions. All scale bars represent 100 μm. (d) Quantification of differentiation markers, β-Tubulin III and GFAP, on peptide-adsorbed surfaces. All library-selected and bsp-RGD(15) peptide surfaces had similar percentages of neurons and astrocytes compared to laminin, while bsp-RGE(15) and TCPS surfaces had fewer neurons and more astrocytes. (Data represent mean ± standard deviation. Library populations not in the same group (*) were statistically different from one another (p < 0.05 using ANOVA between groups with Tukey-Kramer significant difference post hoc test).

Figure 4

Peptide grafting and cell proliferation on IPN surfaces. (a) Peptide density on interpenetrating polymer network (IPN) surfaces. Peptide densities on IPN surfaces were determined by grafting on a FITC-tagged peptide and digesting the FITC from the peptide with chymotrypsin. Fluorescent measurements then allowed for the calculation of the surface peptide concentration. Three peptides – (●) bsp-RGD(15), (□) 15-2 and (◆) 7C-9(9) – were examined. bsp-RGD(15) and 15-2 exhibited saturation at approximately 25 and 20 pmol/cm², respectively, while the looped 7C-9(9) peptide showed saturation around 8 pmol/cm². (b) Neural stem cells were cultured on interpenetrating polymer networks (IPNs) conjugated with peptides including (■) 7C-9(9), (▨) 7C-24(9), (▩) 15-2, (□) bsp-RGD(15). For comparison, neural stems were also cultured on (▤) laminin. Brightfield images after 4 days illustrated that cells on the 7C-9(9) and 15-2 surfaces either attached in clumps or remained as non-adherent neurospheres. 7C-24(9) and bsp-RGD(15) surfaces showed similar cell morphology and growth to the laminin control surface. The bsp-RGE(15)-conjugated surface showed little cell attachment. All surfaces had peptides at 8 pmol/cm², and the scale bar represents 250 μm. (c) The amount of cells on each surface after 5 days was quantified with Cyquant. 7C-24(9) and bsp-RGD(15) surfaces supported cell proliferation at or above the amount of laminin at all peptide surface concentrations. The 7C-9(9) and 15-2 surfaces had substantially fewer cells than all other surfaces, as anticipated since the cells primarily formed neurospheres rather than attaching to the surface. Results from the bsp-RGE(15) and unconjugated IPN surfaces were below the detection limit of the assay. Data represent mean ± standard deviation. Library populations not in the same group (* or **) were statistically different from one another (p < 0.05 using ANOVA between groups with Tukey-Kramer significant difference post hoc test).

Figure 5

Expression of lineage markers under proliferative and differentiating conditions on peptide-conjugated interpenetrating polymer networks (IPNs). Neural stem cells were cultured on the surfaces for 5 days either under proliferative conditions with 20 ng/mL basic Fibroblast Growth Factor (FGF-2) or with 1% fetal bovine serum and 1 μM retinoic acid. (a). NSCs grown under proliferative conditions were assessed for the expression of nestin (green), a cytoskeletal marker for a neural stem cell, while NSCs grown under differentiating conditions were assessed for expression of GFAP (red), a cytoskeletal marker for astrocytes, and β-tubulin III (green), a cytoskeletal marker for neurons. All cells were stained with DAPI (blue) for the nucleus. All surfaces had most of the cells staining for nestin under proliferative conditions, and all surfaces had astrocytes and neurons under differentiating conditions. All scale bars represent 100 μm. (b) Quantification of differentiation markers, β-tubulin III and GFAP, on (■) 7C-9(9), (▩) 15-2, (▨) 7C-24(9), (□) bsp-RGD(15), or (▤) laminin. Laminin had significantly more cells expressing β-tubulin III than any other surface, but all other surfaces had similar β-tubulin III expression. With GFAP expression there was no significant difference in expression on any surface. Data represent mean ± standard deviation. Library populations not in the same group (*) were statistically different from one another (p < 0.05 using ANOVA between groups with Tukey-Kramer significant difference post hoc test).