We report a method to form multifunctional polymer coatings through simple dip-coating of objects in an aqueous solution of dopamine. Inspired by the composition of adhesive proteins in mussels, we used dopamine self-polymerization to form thin, surface-adherent polydopamine films onto a wide range of inorganic and organic materials, including noble metals, oxides, polymers, semiconductors, and ceramics. Secondary reactions can be used to create a variety of ad-layers, including self-assembled monolayers through deposition of long-chain molecular building blocks, metal films by electroless metallization, and bioinert and bioactive surfaces via grafting of macromolecules.Methods for chemical modification of bulk material surfaces play central roles in modern chemical, biological, and materials sciences, and in applied science, engineering, and technology (1-4). The existing toolbox for the functional modification of material surfaces includes methods such as self-assembled monolayer (SAM) formation, functionalized silanes, Langmuir-Blodgett deposition, layer-by-layer assembly, and genetically engineered surfacebinding peptides (5-9). Although widely implemented in research, many available methods have limitations for widespread practical use; specific examples include the requirement for chemical specificity between interfacial modifiers and surfaces (e.g., alkanethiols on noble metals and silanes on oxides), the use of complex instrumentation and limitations of substrate size and shape (Langmuir-Blodgett deposition), or the need for multistep procedures for implementation (layer-by-layer assembly and genetically engineered surface-binding peptides).Development of simple and versatile strategies for surface modification of multiple classes of materials has proven challenging, and few generalized methods for accomplishing this have been previously reported (10). Our approach is inspired by the adhesive proteins secreted by mussels for attachment to wet surfaces (11). Mussels are promiscuous fouling organisms and have been shown to attach to virtually all types of inorganic and organic surfaces (12), including classically adhesion-resistant materials such as poly(tetrafluoroethylene) (PTFE) (Fig. 1A). Clues to mussels' adhesive versatility may lie in the amino acid composition of proteins found near the plaque-substrate interface (Fig. 1, B to D), which are rich in 3,4-dihydroxy-Lphenylalanine (DOPA) and lysine amino acids (13). In addition to participating in reactions

Summary Niches regulate lineage-specific stem cell self-renewal vs. differentiation in vivo and are comprised of supportive cells and extracellular matrix components arranged in a 3-dimensional topography of controlled stiffness in the presence of oxygen and growth factor gradients. Mimicking stem cell niches in a defined manner will facilitate production of the large numbers of stem cells needed to realize the promise of regenerative medicine and gene therapy. Progress has been made in mimicking components of the niche. Immobilizing cell-associated Notch ligands increased the self-renewal of hematopoietic (blood) stem cells. Culture on a fibrous scaffold that mimics basement membrane texture increased the expansion of hematopoietic and embryonic stem cells. Finally, researchers have created intricate patterns of cell-binding domains and complex oxygen gradients.

Mimicking the in vivo stem cell niche to increase stem cell expansion will likely require the presentation of multiple ligands. Presenting ligands in fluid supported lipid monolayers (SLMs) or bilayers (SLBs) allows for ligand diffusion to complement the arrangement of cell receptors, as well as cell-mediated ligand rearrangement and clustering. Cells in tissues interact with ligands presented by other cells and the extracellular matrix (ECM), so it will likely be beneficial to present both cell-associated and ECM-derived ligands. A number of investigators have incorporated cell-membrane-associated ligands within fluid surfaces and several groups have shown that these ligands cluster beneath the cells. However, few studies have investigated cell adhesion to ECM-derived ligands in fluid surfaces. Fibronectin is an important ECM component in many tissues, including the hematopoietic stem cell niche. We examined adhesion of the M07e and THP-1 hematopoietic progenitor cell lines to fibronectin-derived peptide ligands for the α5β1 (cyclic and linear RGD) and α4β1 (cyclic LDV) integrins, as well as the heparin-binding domain (HBD), presented as lipopeptides in fluid and gel SLMs. M07e cells adhered more avidly than THP-1 cells to all of the lipopeptides in fluid and gel surfaces. Adhesion of both cell lines to all peptides was less avid in fluid vs. gel SLMs. Adhesion to cyclic LDV (cLDV) and cRGD was similar on gel SLMs for both cell lines. In contrast, adhesion to cLDV was less extensive than to cRGD in fluid SLMs, especially for M07e cells. Adhesion to linear RGD was less avid than to cRGD or cLDV, and decreased to a greater extent in fluid SLMs. Human aortic endothelial cells adhered to cRGD in fluid SLMs and remained viable for at least 24 h, but did not spread. We also showed additive THP-1 cell adhesion to cLDV and linear RGD lipopeptides presented in a fluid SLM. Although DOPC (dioleoyl phosphatidyl choline) SLMs are not sufficiently stable for long-term cell culture studies, our results and those of others suggest that fluid SLMs are likely to be useful for presenting multiple ligands and for mimicking short-term interactions in the stem cell niche.

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Ex vivo expansion of hematopoietic stem cells (HSCs) would greatly facilitate cell and gene therapies. However, HSC division in culture is associated with differentiation. This contrasts with sustained HSC expansion in vivo, and has led to the hypothesis that a stem cell niche supports self-renewal. It is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. We are developing a defined culture surface for the presentation of cytokines and cell adhesion molecule (CAM) ligands that are thought to be in the HSC niche. Peptide mimics of CAM ligands and cytokines conjugated to dipalmitoyl glycerol via a polyethylene glycol tether are incorporated into dipalmitoylphosphatidylcholine (DPPC) vesicles and deposited onto a hydrophobic surface to create a lipid monolayer. We have previously shown that this system effectively presents adhesive peptide ligands (Jensen et al., JACS 126:15223, 2004). The strategy for immobilizing lipopeptides has been extended to the presentation of a peptide mimetic for the hematopoietic growth factor thrombopoietin (TPO). The lipopeptide mimetic of TPO is based on the branched dimer mimic (TPOm) developed by Cwirla et al. (Science 276:1696, 1997). We have synthesized two versions of TPOm lipopeptide, the first linked to a lipid at both of the amine termini (TPOm-2L) and the second is linked by a single lipid at the carboxy terminus (TPOm-1L). This immobilization strategy does not interfere with the bioactivity of the TPOm as evidenced by cell adhesion and signaling assays. Adhesion was measured with a normal force assay at 30g using the TPO-responsive M07e cell line. We observed a dose-dependent increase in adhesion, with <5% adherent cells for DPPC surfaces and a plateau of ~70% adherent cells at 1.0 mol% TPOm-1L. There was much less adhesion to TPOm-2L (a maximum of ~25% adhesion). Selective adhesion to the TPOm lipopeptides persisted after 6 days of culture, both in the presence and absence of serum. Culture surfaces with TPOm lipopeptides elicit similar M07e cell signaling response kinetics via the ERK1,2 and STAT5 pathways as compared to soluble TPOm and recombinant human TPO (rhTPO). It is interesting that surface presentation of TPOm synergizes more extensively with stem cell factor (SCF) for the activation of STAT5 than does soluble TPOm. Experiments with bone marrow (BM) CD34+ cells show that surfaces incorporating TPOm-2L supplemented with SCF and flt-3 ligand (FL) support similar overall expansion and protection from apoptosis as controls of soluble TPOm or rhTPO with SCF and FL. Further, there was no difference in the ability of TPOm to retain CD34+ cells or CD34+Thy1+ cells. Also, BM CD34+ cell cultures supplemented with TPOm-1L alone supported similar megakaryocyte maturation, evidenced by the appearance of polyploid CD41+ cells after 9 and 12 days of culture, as those supplemented with soluble TPOm. An advantage of this presentation strategy is the potential to save on cytokines during long-term culture. Feeding cultures stimulated by TPOm lipopeptides requires only exchange of basal media. In summary, we have developed a method to present immobilized TPOm in an active conformation that supports cell adhesion and signaling as well as the expansion and differentiation of CD34+ cells.