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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.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

In this study, we developed a protein nanoparticle-based immunoassay to detect cancer biomarkers using a bioluminescent fusion protein. This method relies on the use of protein nanoparticles comprised of genetically-engineered elastin-like polypeptides (ELPs) fused with poly-aspartic acid tails (ELP-D), previously developed in our lab. The sizes of the self-assembled ELP-D nanoparticles can be regulated at the nanoscale by charged repulsion of the poly-aspartic acid chains. To improve the sensitivity of enzyme-linked immunosorbent assays (ELISAs), we herein demonstrate the multivalent display of NanoLuc® (Nluc) luciferase and a biotin acceptor peptide (BAP) on the surfaces of ELP-D nanoparticles, and demonstrate the sensitivity of these multivalent nanoparticles as detection probes. The fusion protein comprised of ELP-D and Nluc-BAP (ELP-D-Nluc-BAP) was found to form nanoparticles with Nluc and BAP displayed multivalently on their surfaces. Moreover, the use of the nanoparticles in ELISA resulted in a detection sensitivity for α-fetoprotein (AFP) about 10 times higher than that of an assay relying on the use of the monomeric version of the fusion protein. Taken together, ELP-D-based nanoparticles displaying multivalent luciferases on their surfaces enable the construction of an ELISA with enhanced sensitivity.

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.

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform, combined with image cytometry, enables the systematic analysis of multiple simultaneously detected marker expression in individual cells, for screening of various chemically defined media across hPSC lines, and the study of phenotypic responses.

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening.

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.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.