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Supramolecular protein hydrogels with tunable properties represent promising candidates for advanced designer extracellular matrices (ECMs). To control cellular functions, ECMs should be able to spatiotemporally regulate synergistic signaling between transmembrane receptors and growth factor (GF) receptors. In this study, we developed genetically engineered temperature-responsive multifunctional protein hydrogels. The designed hydrogel was fabricated by combining the following four peptide blocks: thermosensitive elastin-like polypeptides (ELPs), a polyaspartic acid (polyD) chain to control aggregation and delivery of GFs, a de novo-designed helix peptide that forms antiparallel homotetrameric coiled-coils, and a biofunctional peptide. The resultant coiled-coil unit bound ELPs (CUBEs) exhibit a controllable sol-gel transition with tunable mechanical properties. CUBEs were functionalized with bone sialoprotein-derived RGD (bRGD), and human umbilical vein endothelial cells (HUVECs) were three-dimensionally cultured in bRGD-modified CUBE (bRGD-CUBE) hydrogels. Proangiogenic activity of HUVECs was promoted by bRGD. Moreover, heparin-binding angiogenic GFs were immobilized to bRGD-CUBEs via electrostatic interactions. HUVECs cultured in GF-tethered bRGD-CUBE hydrogels formed three-dimensional (3-D) tubulelike structures. The designed CUBE hydrogels may demonstrate utility as advanced smart biomaterials for biomedical applications. Further, the protein hydrogel design strategy may provide a novel platform for constructing designer 3-D microenvironments for specific cell types.

We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA–enzyme complexes.

There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage. The functionality of these chimeric proteins, referred to as ERE-p20 and E-p20, was assessed by determining their ability to immobilize cells on a hydrophobic polystyrene surface, improve mouse induced pluripotent stem cells (miPSCs) attachment, and promote miPSC differentiation to neural progenitors. ERE-p20 and E-p20 proteins showed hydrophobic binding saturation to the polystyrene plates around 500 nM (2.39 μg/cm2 ) and 750 nM (2.27 μg/cm2 ) protein concentrations, respectively. The apparent maximum cell binding to ERE-p20 and E-p20 was approximately 81% and 73%, respectively, relative to gelatin. For neural precursors, neurite outgrowth was enhanced by the presence of RGD and p20 peptides. The expression levels of neuronal marker protein MAP2 were upregulated approximately 2.5-fold and threefold by ERE-p20 and E-p20, respectively, relative to laminin. Overall, we have shown that elastin-mimetic fusion proteins consisting of p20 with and without RGD peptides are able to induce neuronal differentiation. In conclusion, our newly designed bioengineered fusion proteins allow preparation of specific bioactive matrices or coating/scaffold for miPSCs differentiation.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Self-assembling peptides are attractive materials for tissue engineering applications because of their functionality including high biocompatibility and biodegradability. Modification of self-assembling peptides with functional motifs, such as the cell-adhesive tripeptide sequence RGD leads to functional artificial extracellular matrices (ECMs). In this study, we developed an artificial self-assembling ECM protein tethered with a growth factor via heterotrimer triple-helix (helix A/B/C) formation. The helix A and helix C peptides, which are capable of forming a heterodimer coiled-coil structure, were fused to both ends of a matrix protein composed of the elastin-derived structural unit (APGVGV)12 with an RGD motif. The helix B peptide, which constituents the third helix of the triple-helix structure, was fused with basic fibroblast growth factor (bFGF) for tethering to the artificial ECM proteins. Each recombinant protein exhibited cell adhesion and cell proliferation activities similar to the original, while the designed bFGF-tethered ECM protein exhibited superior cell proliferation activity. These results demonstrate that the approach of creating growth factor-tethered self-assembling proteins via triple-helix formation can be applied to develop functional ECMs for tissue engineering applications.