RESUMO
We report development and characterization of cell-engineered nanovesicles made from mesenchymal stem cells (MSCNVs), which have more than 300 times higher productivity than natural extracellular vesicles (EVs). MSCNVs had similar morphological characteristics to MSCEVs but have molecular characteristics that more resemble MSCs than MSCEVs. In vitro MSCNV treatment increased the proliferation and migration of primary skin fibroblasts and showed better effects than treatment using natural MSCEVs. Quantitative real-time PCR analysis showed increased expression of growth factors in MSCNV-treated skin fibroblasts. Intraperitoneal injection of MSCNVs into syngeneic mice induced mild local inflammation, which resulted in recruitment of immune cells to the injection site. In vivo MSCNV treatment of a mouse skin wound accelerated its healing; this acceleration by MSCNVs may occur by promoting blood vessel formation at the wound site. These results indicate the promise of MSCNVs as agents for regenerative medicine.
RESUMO
Heterotypic interactions between cells are crucial in various biological phenomena. Particularly, stimuli that regulate embryonic stem cell (ESC) fate are often provided from neighboring cells. However, except for feeder cultures, no practical methods are identified that can provide ESCs with contact-dependent cell stimuli. To induce contact-dependent cell stimuli in the absence of living cells, a novel method that utilizes cell-engineered nanovesicles (CNVs) that are made by extruding living cells through microporous membranes is described. Protein compositions of CNVs are similar to their originating cells, as well as freely diffusible and precisely scalable. Treatment of CNVs produced from three different stromal cells successfully induces the same effect as feeder cultures. The results suggest that the effects of CNVs are mainly mediated by membrane-associated components. The use of CNVs might constitute a novel and efficient tool for ESC research.
Assuntos
Comunicação Celular , Engenharia Celular/métodos , Nanopartículas/química , Animais , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Células-Tronco Embrionárias , Hematopoese , Camundongos Endogâmicos C57BL , Neurônios/citologiaRESUMO
Extracellular vesicles can enhance cell proliferation by stimulating signal transduction and delivering genetic materials, and thus may have applications in regenerative medicine and other therapeutic applications. The processes employed to isolate extracellular vesicles, however, are complex and achieve low yield. To overcome these obstacles, a large-scale, micropore device for generating extracellular vesicle-mimetic nanovesicles that have characteristics similar to those of extracellular vesicles is fabricated. The nanovesicles are generated through the self-assembly capability of cell membrane fragments in an aqueous solution. The nanovesicles enhance the proliferation of murine mesenchymal stem cells (MSCs), stimulate the signal pathway related to cell proliferation, and do not influence the characteristics of murine MSCs. Therefore, these nanovesicles could provide stable MSCs for regenerative medicine and other therapeutic applications.
Assuntos
Células da Medula Óssea/fisiologia , Medula Óssea/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nanotecnologia/métodos , Medicina Regenerativa/métodos , Transdução de Sinais/fisiologiaRESUMO
The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.
Assuntos
Células 3T3/metabolismo , Meios de Cultivo Condicionados/farmacologia , Hepatócitos/efeitos dos fármacos , Cultura Primária de Células/métodos , Albuminas/biossíntese , Albuminas/genética , Animais , Sobrevivência Celular , Células Cultivadas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Meios de Cultura Livres de Soro/farmacologia , Feminino , Hepatócitos/metabolismo , Camundongos , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Células Estromais , Ureia/metabolismoRESUMO
We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (SixNy) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ~100-300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.
Assuntos
Nanoestruturas , Animais , Membrana Celular/metabolismo , Células-Tronco Embrionárias/citologia , Camundongos , Microfluídica/instrumentação , Microesferas , Poliestirenos/químicaRESUMO
Extracellular vesicles (Exosomes and microvesicles) have drawn wide attentions in both diagnostic and therapeutic applications, since they are considered to shuttle biological signals intercellularly. However, further research on exosomes is limited by their rarity and heterogeneity even after lengthy isolation processes. In particular, these limitations are challenging in therapeutic applications. To meet these demands, cell-derived nanovesicles that mimic exosomes were generated by extruding living embryonic stem cells through micro-filters. These nanovesicles have an enclosed lipid bilayer and contain cellular contents. The present study investigated the ability of these nanovesicles to improve proliferation by treating primary murine skin fibroblasts with the nanovesicles. The treated skin fibroblasts showed higher expression levels of mRNA, VEGF-α, protein levels of TGF-ß collagen I, PCNA, and Ki-67, as well as enhanced cell proliferation rate and number, compared to non-treated cells. The results indicate that treatment with the nanovesicles could potentially contribute to recovery or wound healing process of tissues.
Assuntos
Proliferação de Células , Células-Tronco Embrionárias/citologia , Nanoestruturas/química , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Exossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , CicatrizaçãoRESUMO
Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferring biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both. Therefore exosomes are being considered for use as RNA and protein delivery vehicles for various therapeutic applications. However, living cells in nature secrete only a small number of exosomes, and procedures to collect them are complex; these complications impede their use in mass delivery of components to targeted cells. We propose a novel and efficient method that forces cells through hydrophilic microchannels to generate artificial nanovesicles. These mimetic nanovesicles contain mRNAs, intracellular proteins and plasma membrane proteins, and are shaped like cell-secreted exosomes. When recipient cells are exposed to nanovesicles from embryonic stem cells, mRNAs of Oct 3/4 and Nanog are transferred from embryonic stem cells to the target cells. This result suggests that mimetic nanovesicles can be used as vehicles to deliver RNA. This nanovesicle formation method is expected to be used in exosome research and to have applications in drug and RNA-delivery systems.