Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.

Extracellular vesicles (EVs), such as exosomes, are nano-sized vesicles derived from endocytic membranes and contain biomolecules such as proteins, lipids, RNAs, and DNAs for the transfer of signals to recipient cells, playing significant roles in cell-to-cell communication. Discovery of exosomes has attracted attention for possible use as next generation therapies in clinical applications; however, several studies suggest that cells secrete exosomes that perform as mediators in the tumor niche and play several roles in tumorigenesis, angiogenesis, and metastasis. Recently, stem cell-derived exosomes have been suggested as a desirable source for regenerative medicine due to their roles in the promotion of angiogenesis via migratory and proliferative mechanisms. This review is aimed at demonstrating the present knowledge of stem cell-derived exosomes and cell-engineered nanovesicles (CNVs) as proliferative, migratory, and anti-senescent therapeutic biomaterial for use in tissue regeneration; wound healing and anti-ageing are explained. We conclude this review by discussing the future perspectives of stem cell-derived exosomes and CNVs as a platform in therapeutic strategies for treatment of wound damage and skin aging.

Core binding factor β (Cbfβ) is a non‐DNA binding cofactor of Runx2 that potentiates DNA binding. Previously, it has been reported that Cbfβ plays an essential role in osteogenic differentiation and skeletal development by inhibition adipogenesis. Here, we delivered the recombinant Cbfβ protein into human mesenchymal stem cells (MSCs) and triggered osteogenic lineage commitment. The efficient delivery of Cbfβ was achieved by fusing 30Kc19 protein, which is a cell‐penetrating protein derived from the silkworm. After the production of the recombinant Cbfβ‐30Kc19 protein in the Escherichia coli expression system, and confirmation of its intracellular delivery, MSCs were treated with the Cbfβ‐30Kc19 once or twice up to 300 µg/ml. By investigating the upregulation of osteoblast‐specific genes and phenotypical changes, such as calcium mineralization, we demonstrated that Cbfβ‐30Kc19 efficiently induced osteogenic differentiation in MSCs. At the same time, Cbfβ‐30Kc19 suppressed adipocyte formation and downregulated the expression of adipocyte‐specific genes. Our results demonstrate that the intracellularly delivered Cbfβ‐30Kc19 enhances osteogenesis in MSCs, whereas it suppresses adipogenesis by altering the transcriptional regulatory network involved in osteoblast‐adipocyte lineage commitment. Cbfβ‐30Kc19 holds great potential for the treatment of bone‐related diseases, such as osteoporosis, by allowing transcriptional regulation in MSCs, and overcoming the limitations of current therapies.

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The generation of functional osteoblasts from human somatic cells could provide an alternative means of regenerative therapy for bone disorders such as osteoporosis. In this study, we demonstrated the direct phenotypic conversion of human dermal fibroblasts (HDFs) into osteoblasts by culturing them in osteogenic medium supplemented with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. HDFs cultured with the VPA in osteogenic medium exhibited expression of alkaline phosphatase and deposition of mineralized calcium matrices, which are phenotypical characteristics of functional osteoblasts. They also expressed osteoblast-specific genes such as alkaline phosphatase, osteopontin, and bone sialoprotein, which demonstrated their direct conversion into osteoblasts. In addition, co-treatment with VPA and a specific inhibitor for activin-like kinase 5 (ALK5i II) had a synergistic effect on direct conversion. It is considered that the inductive effect of VPA on the conversion into osteoblast-lineage is due to the opening of the nucleosome structure by HDAC inhibitor, which facilitates chromatin remodeling and cellular reprogramming. Our findings provide a novel insight into the direct conversion of human somatic cells into transgene-free osteoblasts with small chemical compounds, thus making bone regeneration using cellular reprogramming strategy more clinically feasible.

Summary Individual cell environment stimulating single cell is a suitable strategy for the generation of sophisticated multicellular aggregates with localized biochemical signaling. However, such strategy for induced pluripotent stem cell (iPSC)-derived embryoid bodies (EBs) is limited because the presence of external stimulation can inhibit spontaneous cellular communication, resulting in misdirection in the maturation and differentiation of EBs. In this study, a facile method of engineering the iPSC membrane to stimulate the inner cell of EBs while maintaining cellular activities is reported. We coated the iPSC surface with nanoscale extracellular matrix fabricated by self-assembly between vitronectin and heparin. This nano-coating allowed iPSC to retain its in vitro properties including adhesion capability, proliferation, and pluripotency during its aggregation. More importantly, the nano-coating did not induce lineage-specific differentiation but increased E-cadherin expression, resulting in promotion of development of EB. This study provides a foundation for future production of sophisticated patient-specific multicellular aggregates by modification of living cell membranes.

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The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.