Many cell types, including stem cells, require interactions with extracellular matrix (ECM) molecules to support and regulate their survival and proliferation.[1
] In addition to serving as important structural elements in formations such as basement membrane, the ECM exhibits a number of bioactivities via engaging with cellular adhesion receptors such as integrins, thereby activating a number of downstream signaling pathways including crosstalk with growth factor dependent pathways.[3
] As a result, ECM molecules regulate a number of downstream cell behaviors, including adhesion, survival, proliferation, migration, and differentiation.[7
] These many active role of ECM molecules explains why many surfaces do not support the growth of stem cells.[8
Although animal- and human-derived ECM molecules are often necessary for current stem cell culture systems, their use is highly problematic for numerous reasons. Natural ECM molecules are very large (e.g. ~500,000 MW fibronectin and ~850,000 MW laminin), are extremely complex (with numerous isoforms, splice variants, and glycoforms), and have numerous signaling motifs that are not yet fully understood.[10
] In addition, there is considerable lot-to-lot variability in animal- and human-derived ECM because of the many isoforms present and difficulty in purifying such proteins to homogeneity,[11
] and such protein preparations run the risk of being contaminated with pathogens and immunogens.[13
] The development of stem cell culture systems that are robust, reproducible, and scaleable can benefit both scientific studies and clinical therapies.
One promising approach is to design and develop synthetic stem cell culture platforms that mimic the physical and biochemical properties of the natural ECM. For example, materials can be functionalized with short (9–15mer) synthetic peptide ligands designed to engage with cell-surface receptors and thereby functionally replace ECM proteins typically used in stem cell culture.[9
] In particular, it is important to develop surfaces that can engage with integrins—heterodimeric transmembrane receptors that bind ECM molecules, anchor cells to the matrix, and act as bidirectional transducers to transmit both mechanical and chemical stimuli into the cell. [18
] Most ligands for integrins that have been exploited in biomaterials research have employed the canonical Arg-Gly-Asp (RGD) sequence that binds a subset of these adhesion receptors.[21
] The flanking residues to the RGD sequence can affect specificity to the RGD-binding integrins such as the αv
] However, identifying new ligands that bind these and other receptors could aid in engineering biomaterials and production of cell-based therapies for the clinic, as well as in gaining a better understanding of signaling mechanisms that regulate cell function.[23
However, while various cell-binding domains—such as RGD and IKVAV motifs—[29
] have been identified in many ECM proteins, many peptides mimicking those domains are not as active as the full ECM proteins, as evidenced by the fact that few of them support the culture of cells to the extent of the native protein.[12
] In addition, many of the important signaling domains in ECM proteins may not be known, and for example only recently has the αv
binding domain in fibronectin been identified.[37
] Finally, in general there is no guarantee that the optimal peptide ligand for a given receptor exactly matches a portion of the “linear” sequence of its natural ECM ligand, because the three-dimensional structure of proteins that typically contain more than one chain (e.g., laminin, collagen). Therefore, there is a need to develop a robust method to identify new candidate peptides that can be used to modify or create biomaterials to replace ECM proteins.
Due to of the vast number of possible peptide sequences, even for a very short polypeptide, rationally designing such a ligand is very challenging; however, library-based screening and selection methods have potential to address this problem. With library strategies such as bacterial display, many peptides with random sequences can be tested simultaneously and thus can facilitate the discovery of peptides even with little prior mechanistic knowledge of the motifs required for receptor engagement.[38
] While phage display libraries have been used in a number of prior studies, these have focused primarily on finding cell adhesion receptors antagonists, including for integrins, rather than identifying peptides agonists for these receptors.[40
] In addition, newer library systems using bacteria as a display platform offer numerous advantages. For example, they are easier to generate since the libraries involve straightforward plasmid manipulations, do not require bacteriophage production or purification, do not involve viral infection steps for amplification during the selection process, and can readily be analyzed after selection by simple plasmid isolation and sequencing.[38
] In addition, the levels of surface-displayed peptide can in principle be varied to modulate selection stringency, and fluorescent protein expressing bacteria can be used to aid library selection and analysis.[48
In this study, we have used a bacterial display library to identify new peptides that bind adult hippocampal neural stem cells (NSCs). Several of these peptides were examined for their ability to support neural stem cells in two different contexts: adsorbed on tissue culture polystyrene (TCPS) or grafted to an interpenetrating polymer network surface. On both surfaces, neural stem cells retained their ability to self-renew or differentiate into multiple lineages, depending on the soluble factors given to the cells.