Additional file 1: of Induction of chondrogenesis of human placenta-derived mesenchymal stem cells via heparin-grafted human fibroblast derived matrix

Figure S1. Optical image of decellularized hFDM after hematoxylin staining. It shows a nanofiberous ECM structure without the presence of cells. (DOCX 2570 kb

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly­(l-lactide-<i>co</i>-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5–7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell–cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types

Simple <i>in Vivo</i> Gene Editing <i>via</i> Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment

Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for <i>in vivo</i> applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing <i>in vivo</i>. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) <i>via in vitro</i> and <i>in vivo</i> editing of the <i>KRAS</i> gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening