Polymer- and/or protein-based nanofibers that promote stable cell adhesion have drawn increasing attention as well-defined models of the extracellular matrix. In this study, we fabricated two classes of stimulus-responsive fibers containing gelatin and supramolecular crosslinks to emulate the dynamic cellular microenvironment in vivo. Gelatin enabled cells to adhere without additional surface functionalization, while supramolecular crosslinks allowed for the reversible switching of the Young’s modulus through changes in the concentration of guest molecules in culture media. The first class of nanofibers was prepared by coupling the host–guest inclusion complex to gelatin before electrospinning (pre-conjugation), while the second class of nanofibers was fabricated by coupling gelatin to polyacrylamide functionalized with host or guest moieties, followed by conjugation in the electrospinning solution (post-conjugation). In situ AFM nano-indentation demonstrated the reversible switching of the Young’s modulus between 2–3 kPa and 0.2–0.3 kPa under physiological conditions by adding/removing soluble guest molecules. As the concentration of additives does not affect cell viability, the supramolecular fibers established in this study are a promising candidate for various biomedical applications, such as standardized three-dimensional culture matrices for somatic cells and the regulation of stem cell differentiation.

PDMS (polydimethylsiloxane) is still the most widely used biomaterial for bioengineering studies, which can mimic the tissue stiffness and micro/nanostructure to improve the generation and investigation of human cardiomyocyte (iCM) differentiation from human induced pluripotent stem cells (hiPSCs). Hence, PDMS-based substrates modified to reflect biomechanical—stiffness-related—and biophysical—topography-related—properties, in combination with biochemical cues, can enhance the efficiency of in vitro iCM generation. In this study, human fetal cardiomyocytes (HFCMs) were isolated and their natural geometrical micro/nanotopography was imprinted on PDMS surfaces with different stiffnesses combined with tailored biochemical factors, and the impact of the three factors on the differentiation of hiPSC-derived CM (iCM) was evaluated. The results show that the combination of biophysical, biomechanical, and biochemical factors could improve the expression of iCM differentiation and maturation markers compared to biochemical factors alone. Based on these findings, which can be applied in organ-on-chip studies, by imitating the in vivo environment, cultured cells behave authentically, providing realistic platforms for studying biological systems and ensuring accurate, translatable results.

Manipulating gene expression in a tissue-specific and temporally controlled manner is essential for understanding the function of the focal genes. Still, in many cases, the limited availability of specific promoters to drive ectopic manipulation remains a restricting factor in developing organs, even in Drosophila. Developing external genitalia is one such organ with a complex anatomical structure shaped by a joint regulatory network of many transcription factors. To overcome the restriction, we employed the infrared laser-evoked gene operator system (IR-LEGO), in which infrared laser (1,480 nm) irradiation induces gene expression under the control of a heat shock promoter. Pupal genital structures were irradiated at approximately 24 or 48 h after puparium formation. We tested a range of laser power and depth to the target structure by a reporter assay using green fluorescent protein, which was induced under the control of the heat shock protein 70 promoter (hs-GAL4). In previous studies, the IR-LEGO has been used as a tool to induce ectopic transgene expression. In this study, we attempted to knock down genes such as yellow (y) and odd-paired (opa) ectopically by RNAi using the GAL4/UAS system. The results demonstrated that this technique has a high potential in manipulating transcript abundance levels in small groups of cells in specific genital structures to unravel novel functions of genes involved in the morphogenesis of species-specific and rapidly evolving anatomical structures.