High-throughput discovery of synthetic surfaces that support proliferation of pluripotent cells

Abstract

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors, the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors, however, may be needed to guide cell growth and differentiation. To provide access to such new materials, we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait, approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested, and seven cell-binding peptides were identified. Of these, six sequences possess EC cell-binding ability. Specifically, when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold, they mediate cell adhesion. The corresponding soluble peptides block this adhesion, indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR), they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells.

Figure 1

(A) A 1:1 mixture of peptide-presenting (library) and peptide-free (wt) phage was used to optimize the screen for NCCIT-binding phage clones. (B) The amount of peptide-bearing (“blue”) and wt (“white”) phage was monitored at each panning and washing step (see Supporting Information for details). Through multiple panning steps, a steady increase in the blue/white ratio is observed. (C) The phage population that remained associatd with cells after all washing steps (denoted as “R2 BR pellet” in B) was sub-cloned; the clones were amplified and tested using the cell suspension ELISA (CS-ELISA) to identify those that bind to NCCIT cells. (D) Testing of 92 phage clones (sub-library 1, left scale) and 276 phage clones (sub-library 2, right scale) using the CS-ELISA yielded 2 and 5 clones, respectively, that bind to NCCIT cells significantly better than wt phage. The results are averages from two independent measurements. Error bars indicate one standard deviation.

Figure 2

(A) Method for fabrication of surface arrays of peptides.27 SAMs composed of perfluorinated alkanethiol on gold were photopatterned. Solutions that contain peptide-AT, glucamine-AT or mixtures of the two were spotted onto the exposed gold regions. The ratio of peptide-AT and non-cell-adhesive glucamine-AT in solution was altered to vary the surface concentration of peptide. (B) Route for the synthesis of the peptide-AT starting from the AT resin.37 (C) Structures of the glucamine-AT and perfluoro-AT.27

Figure 3

Arrays were prepared by spotting solutions containing mixtures of peptide-AT and glucamine-AT (1 mM total concentration) onto patterned perfluoro-AT SAM (see Figure 2). The percentages are derived from the mole fraction of the peptide-AT in the solution from which the array element was generated. NCCIT cells were incubated with arrays for 1 h in serum-free media. The cells were then fixed with formaldehyde and stained with Hoechst 33342 to visualize cell nuclei. (B) The number of cells on each array element was determined by counting nuclei using ImageJ software. Data are average of 3–6 measurements from three independent experiments; error bars indicate one standard deviation. (C) The short-term cell adhesion of NCCIT cells to monolayers presenting LTTAPKLPKVTR was inhibited with soluble LTTAPKLPKVTR peptide. Another phage-derived peptide that also supports cell adhesion, TVKHRPDALHPQ, had little effect. (D) The ability of the soluble peptides to inhibit binding was quantified using ImageJ software. Data presented are the average of six measurements; error bars are one standard deviation; (*) p<0.05 and (**) p<0.01 as determined by two-tailed unequal variance t-test. (A) and (B) represent mosaic images obtained with a fluorescent microscope with 6.4x objective. An inverted image of the blue-fluorescent channel is presented.

Figure 4

(A) Monolayers that present phage-derived peptides were tested for their ability to support prolonged growth of human ES cells in a defined medium. Human ES cells (line H9) were plated in mTeSR1 media in the presence of ROCK inhibitor (Y-27632) atop SAMs displaying the indicated peptide sequence. Cells were allowed to proliferate for five days; the media was exchanged daily. At day 5, the cells were fixed and stained with anti-Oct4 goat IgG and anti-SSEA-4 mouse IgG, followed by Alexa 488 anti-goat-IgG and Alexa 555 anti-mouse-IgG, and Hoechst 33342. Inverted images of each fluorescent channel (Hoechst, Oct4, SSEA-4) are presented. Arrows (Hoechst, second row) point to groups of cells (B) Quantification reveals that two of five peptides support the proliferation of undifferentiated human ES cells. SAMs presenting the sequence LSTIGMPKLLA support cell growth but induce differentiation. The cell count in each fluorescent channel was estimated by integrating the area of the fluorescent signal. The acquisition time and signal threshold used for the quantification of each fluorescent channel were set to identical levels for all images. For additional images see Supporting Information Figure S3.

Figure 5

Images depicting human ES cells proliferating on SAMs that present the peptide sequences LTTAPKLPKVTR or TVKHRPDALHPQ (abbreviated as “LT…R” and “TV…Q”). (A) Human ES cells were plated onto the SAMs in mTeSR1 media supplemented with 5 µM Y27632 ROCK-1 inhibitor (mTeSR-ROCK). On day 5, the cells were fixed and stained with anti-Oct4 and anti-SSEA4 antibody to visualize markers associated with pluripotency. Scale bar 500 µm. (B) Cells were cultured on peptide-substituted SAMs in mTeSR-ROCK medium for 20 days (three passages). The cells were then harvested and stained with anti-Oct4 PE-conjugate, and their staining was compared to cells proliferated on Matrigel (positive control) and those proliferated on Matrigel in a differentiation-inducing medium (negative control). (C) Inhibition of human ES cell adhesion: A suspension of human ES cells containing 10 mM EDTA or 5 mg/mL heparin was added to wells coated with Matrigel, poly-lysine, or wells with gold-coated peptide-SAM substrates. Cells were allowed to bind for 1 h, and the number of adherent cells was analyzed using CellTiter-Glo. Neither heparin nor EDTA treatment exhibits significant effect on short-term human ES cell adhesion to peptide SAM-presenting substrates.