RESUMO
Transforming growth factor ß (TGF-ß) plays a pivotal role in cartilage differentiation and other functions of mesenchymal stem cells (MSCs). In this study, we investigated the therapeutic potential of TGF-ß1 overexpressing amniotic MSCs (AMMs) generated using gene editing in a mouse model of damaged cartilage. The TGF-ß1 gene was inserted into a safe harbor genomic locus in AMMs using transcription activator-like effector nucleases. The chondrogenic properties of TGF-ß1-overexpressing AMMs (AMM/T) were characterized using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR, and histological analysis, and their therapeutic effects were evaluated in mouse model of collagen-induced arthritis (CIA). AMM/T expressed cartilage-specific genes and showed intense Safranin O and Alcian blue staining. Furthermore, injecting AMM/T attenuated CIA progression compared with AMM injection, and increased the regulatory T (Treg) cell population, while suppressing T helper (Th)17 cell activation in CIA mice. Proinflammatory factors, such as interleukin-1ß (IL-1ß), IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α were significantly decreased in AMM/T injected CIA mice compared with their AMM injected counterparts. In conclusion, genome-edited AMMs overexpressing TGF-ß1 may be a novel and alternative therapeutic option for protecting cartilage and treating inflammatory joint arthritis.
Assuntos
Artrite Experimental/terapia , Edição de Genes , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta1/genética , Âmnio/citologia , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Condrogênese , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Imunomodulação , Articulações/patologia , Masculino , Camundongos , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Recently, minicircle (MC)-based cell therapy has been emerging as a novel technology for nonviral genetic modification. In this study, we investigated the characteristics of granulocyte chemotactic protein-2 (GCP-2)-overexpressing fibroblasts (GCP-2/MC) using MC microporation technology, as well as its therapeutic mechanism in wound healing. GCP-2 parent plasmid and MC containing GCP-2 were generated. Human dermal fibroblasts (HDF) were transfected with MC containing GCP-2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), scratch wound assay, and in vivo wound healing assay were performed. Gene and protein expression analysis revealed that GCP-2/MC highly expressed epithelialization growth factor, epidermal growth factor (EGF), chemokines, GCP-2, interleukin (IL)-8, as well as wound healing-associated genes such as insulin growth factor (IGF)-1 and hepatocyte growth factor (HGF). An in vitro scratch wound closure and matrigel tube formation assays demonstrated that the culture medium derived from GCP-2/MC substantially accelerated the wound closure and matrigel network formation. Wounds in nude mice were created by skin excisions followed by injections of GCP-2/MC. Results showed high cell survival potential and that GCP-2/MC transplantation highly accelerated skin wound closure by increasing reepithelialization, capillary density, and enhancing angiogenic factors, suggesting direct benefits for cutaneous closure. Taken together, these data suggest that MC-based GCP-2 overexpression could be a promising alternative strategy for promoting wound healing.
Assuntos
Derme/metabolismo , Fibroblastos , Terapia Genética , Proteínas Associadas aos Microtúbulos , Cicatrização , Ferimentos e Lesões , Animais , Fibroblastos/metabolismo , Fibroblastos/transplante , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Ferimentos e Lesões/genética , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/terapiaRESUMO
Mesenchymal stem cells (MSCs) are known for their ability to repair liver damage. However, their therapeutic potential still needs to be enhanced. In the present study, we produced genome-edited MSCs that secrete interleukin 10 (IL-10) and evaluated their therapeutic potential in a liver fibrosis model. Multiple copies of the IL-10 gene were inserted into a safe harbor genomic locus in amniotic mesenchymal stem cells (AMMs) using transcription activator-like effector nucleases (TALENs). The IL-10 gene-edited AMMs (AMM/I) were characterized by reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), and microarray. The effects of AMM/I-conditioned cell medium (CM) on the activation of hepatic stellate cells (HSC) were analyzed in vitro and in vivo therapeutic assays were performed on a mouse liver fibrosis model. The engineered AMM/I expressed high levels of IL-10. AMM/I-CM inhibited the activation of HSC (in vitro) and TNF-α expression of T cells/macrophage derived from fibrotic liver. In addition, human IL-10 was detected in the serum of the mice transplanted with AMM/I and transplantation of AMM/I significantly inhibited thioacetamide (TAA)-induced liver fibrosis and ameliorated abnormal liver function. Furthermore, a high number of human albumin-expressing AMM/I were successfully engrafted into the liver of recipient mice. Overall, genome-edited AMMs overexpressing anti-fibrotic IL-10 might be a promising alternative therapeutic option for the treatment of liver cirrhosis.
Assuntos
Interleucina-10/metabolismo , Cirrose Hepática/terapia , Transplante de Células-Tronco Mesenquimais , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Âmnio/citologia , Animais , Transdiferenciação Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Edição de Genes , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Interleucina-10/análise , Cirrose Hepática/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Even though mesenchymal stem cells (MSCs) have angiogenic property, their cytokine secretory capacity is limited to treat ischemic vascular disorders. In present study, we produced genome-edited MSCs that secreted dual chemokine granulocyte chemotactic protein-2 (GCP-2) and stromal-derived factor-1α (SDF-1α) and determined their therapeutic potential in the context of experimental ischemia. METHODS: GCP-2 and SDF-1α genes were integrated into safe harbor site at the safe harbor genomic locus of amniotic mesenchymal stem cells (AMM) via transcription activator-like effector nucleases (TALEN). GCP-2 and SDF-1α gene-edited AMM (AMM/GS) were used for quantitative (q)-PCR, Matrigel tube formation, cell migration, Matrigel plug assays and in vivo therapeutic assays using hindlimb ischemia mouse model. RESULTS: AMM/GS-derived culture media (CM) induced significantly higher tube lengths and branching points as compared to AMM/S CM and AMM CM. Interestingly, Matrigel plug assays revealed that significantly higher levels of red blood cells were found in AMM/GS than AMM/S and AMM Matigel plugs and exhibited micro-vascular like formation. Cells was transplanted into ischemic mouse hindlimbs and compared with control groups. AMM/GS injection prevented limb loss and augmented blood perfusion, suggesting that enhances neovascularization in hindlimb ischemia. In addition, transplanted AMM/GS revealed high vasculogenic potential in vivo compared with transplanted AMM/S. CONCLUSION: Taken together, genome-edited MSCs that express dual chemokine GCP-2 and SDF-1α might be alternative therapeutic options for the treatment of ischemic vascular disease.