Engineering Novel Peptide and Polymer Systems for Human Pluripotent Stem Cell Self-Renewal and Differentiation

Developing synthetic materials that promote the expansion and differentiation of human pluripotent stem cells (hPSCs) has been a long-standing challenge.  Unfortunately, most synthetic biomaterials either (1) lack bioactive motifs, resulting in low viability and premature stem cell differentiation, or (2) rely on a small subset of peptide motifs that promote few cell fates.  To reliably guide hPSCs down their full repertoire of cell fates, including self-renewal, materials targeting a wider class of surface receptors are required. However, the development of such materials has been hindered by our limited understanding of stem cell surface receptors and their structural interactions with the extracellular matrix (ECM)./nIn Chapter 2, we used an unbiased bacterial display strategy to circumvent the dearth of structural information about receptor-ECM interactions and identify ligands for the highly expressed hPSC surface receptor, α6β1 integrin. Using human embryonic stem cells (hESCs) as bait, theoretical library sizes of 10 trillion random yet unique 15mers were panned to identify 30 sequence-similar peptides that bind α6 integrin. Characterization of adhesion via flow cytometry and centrifuge-based assays suggested that seven of our screened cyclic and linear peptides bound hPSCs with sub-micromolar dissociation constants similar to full-length ECM proteins like laminin. However, as identified in the literature, hPSCs rely on a plurality of motifs to mediate binding and maintain pluripotency over several passages. With this in mind, we were curious whether our novel peptides could interact synergistically with canonical binding motifs for αVβ3 integrin and syndecan-1 to enhance stem cell self-renewal. To that end, we adapted a high-throughput microculture platform to print self-assembled monolayers of peptides (SAMs) and screen combinations of both novel and canonical motifs for their capacity to maintain short-term hiPSC culture. By measuring each SAMs’ capacity to promote cell proliferation, pluripotency and adhesive morphology, we were able to identify an optimal combination of three integrin- and syndecan-binding motifs that supported the self-renewal and expansion of hESCs and hiPSCs over a single passage./nIn Chapter 3, we then asked if some of these peptides identified through screening and selection could be used to create scalable, thermoreversible polymer-peptide systems for multi-passage hPSC culture. Although several alginate, hyaluronic acid and agarose scaffolds have been generated in the past for the long-term culture of hPSCs, these gels have lacked degradable elements requisite for scalable passaging as well as adhesive motifs that promote hPSC adhesion. To develop 3D materials more amenable to large-scale culture, we instead generated a hydrogel blend that was both thermoreversible and functionalizable. Using acrylated hyaluronic acid as the backbone, our screened integrin- and syndecan-binding peptides were conjugated via Michael addition chemistry. Conjugates were characterized via proton NMR for polymer acrylation and BCA assay for peptide conjugation. Peptide-decorated hyaluronic acid was then blended with a previously developed thermoreversible HyA-NIPAAm graft co-polymer to generate bioactive, scalable and defined 3D scaffolds for hPSC culture. Using these scaffolds, we were able to qualitatively show that blends incorporating our α6-integrin-binding cyclic peptides could support 12- to 15-fold expansion over a single passage./nWhile our results in Chapter 2 and 3 suggested the development of 2D and 3D peptide-based scaffolds for hPSC self-renewal, there remain many challenges and open questions regarding these targeted peptide-scaffolds and the utilization of high-throughput screening in other tissue engineering applications. In Chapter 4, we considered some of these challenges and alluded to future applications of display techniques for the generation of defined, synthetic extracellular matrices and purification agents.

Advisor: Healy, Kevin E
Schaffer, David