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Shape memory materials have been successfully applied to minimally invasive implantation of medical devices. However, organ-movement-specific shape programing at a microscale level has never been demonstrated despite significant unmet needs. As vein-to-artery grafting induces vein dilation and stenosis, a polymeric self-enclosable external support (SES) is designed to wrap the vascular out-wall. Its micropores are programmed to increase sizes and interconnections upon dilation. Vessel dilation promotes venous maturation, but overdilation induces stenosis by disturbed blood flow. Therefore, the unique elastic shape-fixity of SES provides a foundation to enable a stable microscale shape transition by maintaining the vein dilation. The shape transition of micropore architecture upon dilation induces beneficial inflammation, thereby regenerating vasa vasorum and directing smooth muscle cell migration toward adventitia with the consequent muscle reinforcement of veins. This game-changer approach prevents the stenosis of vein-to-artery grafting by rescuing ischemic disorders and promoting arterial properties of veins.
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Vasa Vasorum , Enfermedades Vasculares , Constricción Patológica , Dilatación , Humanos , Enfermedades Vasculares/prevención & control , VenasRESUMEN
Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.
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Aterosclerosis/terapia , Células Madre Mesenquimatosas , Nanopartículas , Animales , Arterias Carótidas , Células Endoteliales , Humanos , Ligadura , Ratones , PorcinosRESUMEN
The structural stability of medical devices is established by managing stress distribution in response to organ movement. Veins abruptly dilate upon arterial grafting due to the mismatched tissue property, resulting in flow disturbances and consequently stenosis. Vascular cast is designed to wrap the vein-artery grafts, thereby adjusting the diameter and property mismatches by relying on the elastic fixity. Here, a small bridge connection in the cast structure serves as an essential element to prevent stress concentrations due to the improved elastic fixity. Consequently, the vein dilation is efficiently suppressed, healthy (laminar and helical) flow is induced effectively, and the heathy functions of vein grafting are promoted, as indicated by the flow directional alignment of endothelial cells with arterialization, muscle expansion, and improved contractility. Finally, collaborative effects of the bridge drastically suppress stenosis with patency improvement. As a key technical point, the advantages of the bridge addition are validated via the computational modeling of fluid-structure interaction, followed by a customized ex vivo set-up and analyses. The calculated effects are verified using a series of cell, rat, and canine models towards translation. The bridge acted like "Little Dutch boy" who saved the big mass using one finger by supporting the cast function.
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Células Endoteliales , Venas , Animales , Perros , Ratas , Constricción Patológica , Hemodinámica/fisiologíaRESUMEN
Tissue regeneration requires structural holding and movement support using tissue-type-specific aids such as bone casts, skin bandages, and joint protectors. Currently, an unmet need exists in aiding breast fat regeneration as the breast moves following continuous body motion by exposing the breast fat to dynamic stresses. Here, the concept of elastic structural holding is applied to develop a shape-fitting moldable membrane for breast fat regeneration ("adipoconductive") after surgical defects are made. The membrane has the following key characteristics: (a) It contains a panel of honeycomb structures, thereby efficiently handling motion stress through the entire membrane; (b) a strut is added into each honeycomb in a direction perpendicular to gravity, thereby suppressing the deformation and stress concentration upon lying and standing; and (c) thermo-responsive moldable elastomers are used to support structural holding by suppressing large deviations of movement that occur sporadically. The elastomer became moldable upon a temperature shift above Tm. The structure can then be fixed as the temperature decreases. As a result, the membrane promotes adipogenesis by activating mechanotransduction in a fat miniature model with pre-adipocyte spheroids under continuous shaking in vitro and in a subcutaneous implant placed on the motion-prone back areas of rodents in vivo.
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Cell-cell interactions regulate intracellular signaling via reciprocal contacts of cell membranes in tissue regeneration and cancer growth, indicating a critical need of membrane-derived tools in studying these processes. Hence, cell-membrane-derived nanoparticles (CMNPs) are produced using tonsil-derived mesenchymal stem cells (TMSCs) from children owing to their short doubling time. As target cell types, laryngeal cancer cells are compared to bone-marrow-derived MSCs (BMSCs) because of their cartilage damaging and chondrogenic characteristics, respectively. Treating spheroids of these cell types with CMNPs exacerbates interspheroid hypoxia with robust maintenance of the cell-cell interaction signature for 7 days. Both cell types prefer a hypoxic environment, as opposed to blood vessel formation that is absent in cartilage but is required for cancer growth. Hence, angiogenesis is inhibited by displaying the Notch-1 aptamer on CMNPs. Consequently, laryngeal cancer growth is suppressed efficiently in contrast to improved chondroprotection observed in a series of cell and animal experiments using a xenograft mouse model of laryngeal cancer. Altogether, CMNPs execute a two-edged sword function of inducing hypoxic cell-cell packing, followed by suppressing angiogenesis to promote laryngeal cancer death and chondrogenesis simultaneously. This study presents a previously unexplored therapeutic strategy for anti-cancer and chondroprotective treatment using CMNPs.
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Membrana Celular/química , Nanopartículas/química , Receptor Notch1/química , Animales , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Portadores de Fármacos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Nanopartículas/uso terapéutico , Nanopartículas/toxicidad , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Fisiológica/efectos de los fármacos , Tonsila Palatina/citología , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Trasplante HeterólogoRESUMEN
The use of nanoscale materials (NMs) could cause problems such as cytotoxicity, genomic aberration, and effects on human health, but the impacts of NM exposure during pregnancy remain uncharacterized in the context of clinical applications. It was sought to determine whether nanomaterials pass through the maternal-fetal junction at any stage of pregnancy. Quantum dots (QDs) coated with heparinized Pluronic 127 nanogels and polyethyleneimine (PEI) were administered to pregnant mice. The biodistribution of QDs, as well as their biological impacts on maternal and fetal health, was evaluated. Encapsulation of QDs with a nanogel coating produces a petal-like nanotracer (PNt), which could serve as a nano-carrier of genes or drugs. PNts were injected through the tail vein and accumulated in the liver, kidneys, and lungs. QD accumulation in reproductive organs (uterus, placenta, and fetus) differed among phases of pregnancy. In phase I (7 days of pregnancy), the QDs did not accumulate in the placenta or fetus, but by phase III (19 days) they had accumulated at high levels in both tissues. Karyotype analysis revealed that the PNt-treated pups did not have genetic abnormalities when dams were treated at any phase of pregnancy. PNts have the potential to serve as carriers of therapeutic agents for the treatment of the mother or fetus and these results have a significant impact on the development and application of QD-based NPs in pregnancy.
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Portadores de Fármacos/administración & dosificación , Heparina/administración & dosificación , Poloxámero/administración & dosificación , Polietileneimina/administración & dosificación , Puntos Cuánticos/administración & dosificación , Animales , Portadores de Fármacos/farmacocinética , Femenino , Heparina/farmacocinética , Humanos , Cariotipo , Intercambio Materno-Fetal , Células Madre Mesenquimatosas , Ratones Endogámicos ICR , Poloxámero/farmacocinética , Polietileneimina/farmacocinética , Embarazo , Distribución TisularRESUMEN
Epiphora is the overflow of tears typically caused by obstruction or occlusion of the nasolacrimal duct. More attention is required to address this global health issue owing to the increase in air pollution. Implantation of a silicone stent is the preferred treatment for epiphora; however, introducing a silicone stent into a narrow duct with complex geometry is challenging as it requires guidance by a sharp metal needle. Additionally, silicone can cause adverse reactions such as biofilm formation and tear flow resistance due to its extreme hydrophobicity. To overcome these problems, in this study we developed a new type of biocompatible shape memory polymer (SMP) stent with elasticity capacity for self-expansion. First, SMPs in the form of x%poly(ε-caprolactone)-co-y%poly(glycidyl methacrylate) (x%PCL-y%PGMA) were synthesized via ring opening polymerization by varying the molar ratio of PCL (x%) and PGMA (y%). Second, the shape memory and mechanical properties were tuned by controlling the crosslinking degree and concentration of x%PCL-y%PGMA solution to produce a test type of SMP stent. Lastly, this 94%PCL-06%PGMA stent exhibited more standout critical functions in a series of in vitro and in vivo experiments such as a cell growth-supporting level of biocompatibility with nasal epithelial cells without significant inflammatory responses, better resistance to biofilm formation, and more efficient capacity to drain tear than the silicone control. Overall, 94%PCL-06%PGMA can be suggested as a superior alternative to the currently used materials for nasolacrimal stents. STATEMENT OF SIGNIFICANCE: Silicone intubation (stenting) has been widely used to treat nasolacrimal duct obstruction, however, it can cause adverse clinical effects such as bacterial infection; presents procedural challenges because of the curved nasolacrimal duct structure; and shows poor drainage efficiency stemming from the highly hydrophobic nature of silicone. In this work, we describe an innovative shape memory polymer (SMP) as a superior alternative to conventional silicone-based materials for nasolacrimal duct intubation. We demonstrate the clear advantages of the SMP over conventional silicone, including a much higher drainage capacity and superior resistance to bacterial infection.
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Dacriocistorrinostomía , Obstrucción del Conducto Lagrimal , Ensayo de Materiales , Conducto Nasolagrimal , Siliconas , Stents , Animales , Línea Celular , Obstrucción del Conducto Lagrimal/metabolismo , Obstrucción del Conducto Lagrimal/microbiología , Masculino , Ratones , Conducto Nasolagrimal/metabolismo , Conducto Nasolagrimal/microbiología , Conducto Nasolagrimal/cirugía , ConejosRESUMEN
Development of a stable and prolonged gene delivery system is a key goal in the gene therapy field. To this end, we designed and fabricated a gene delivery system based on multiply-clustered gold particles that could achieve prolonged gene delivery in stem cells, leading to improved induction of differentiation. Methods: Inorganic gold nanoparticles (AuNPs) underwent three rounds of complexation with catechol-functionalized polyethyleneimine (CPEI) and plasmid DNAs (pDNAs), in that order, with addition of heparin (HP) between rounds, yielding multiply-clustered gold-based nanoparticles (mCGNPs). Via metal-catechol group interactions, the AuNP surface was easily coordinated with positively charged CPEIs, which in turn allowed binding of pDNAs. Results: Negatively charged HP was encapsulated with the positive charge of CPEIs via electrostatic interactions, making the NPs more compact. Repeating the complexation process yielded mCGNPs with improved transfection efficiency in human mesenchymal stem cells (hMSCs); moreover, these particles exhibited lower cytotoxicity and longer expression of pDNAs than conventional NPs. This design was applied to induction of chondrogenesis in hMSCs using pDNA harboring SOX9, an important chondrogenic transcription factor. Prolonged expression of SOX9 induced by mCGNPs triggered expression of chondrocyte extracellular matrix (ECM) protein after 14 days, leading to more efficient chondrogenic differentiation in vitro and in vivo.
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Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Nanopartículas del Metal/química , Plásmidos/química , Catecoles/química , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , ADN/química , ADN/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Oro/química , Heparina/química , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Plásmidos/genética , Polietileneimina/química , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Electricidad Estática , Transfección/métodos , Adulto JovenRESUMEN
RNA interference (RNAi) plays important roles, and microRNAs (miRNAs) are used as biomarkers and targets in cell therapies. In this study, we fabricated miRNAs and short hairpin RNAs (shRNAs) targeting the osteogenic RUNX2 gene to induce chondrogenesis of human mesenchymal stem cells (hMSCs). pDNA harboring these miRNAs and shRNAs was complexed with nanoparticles composed of dexamethasone and tetramethlyrhodamine-labeled branched polyethyleneimine (RDtNPs). The miRNAs and shRNAs reduced RUNX2 expression in hMSCs at early (12 h) and late (72 h) time points, respectively. Co-delivery of miRNAs and shRNAs resulted in rapid and sustained RUNX2 silencing. Moreover, dexamethasone in the nanoparticles enhanced chondrogenic differentiation. Gene and protein expression of RUNX2 was lower in hMSCs transfected with RDtNPs complexed with pDNA harboring miRNAs plus shRNAs for 72 h than in control hMSCs. Moreover, delivery of these miRNAs and shRNAs increased gene and protein expression of chondrogenic SOX9. These changes in expression facilitated the chondrogenic differentiation of hMSCs, as demonstrated by analysis of markers related to mature chondrocytes. Furthermore, histological and immunohistological analyses detected specific extracellular matrix and cartilage-related proteins in cultures of hMSCs transfected with these miRNAs and shRNAs.
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Células Madre Mesenquimatosas , Nanopartículas , Diferenciación Celular , Condrocitos , Condrogénesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Dexametasona , Humanos , MicroARNs , ARN Interferente PequeñoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12â¯h) and late (36â¯h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72â¯h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells.
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Diferenciación Celular , Células Madre Mesenquimatosas/citología , Interferencia de ARN , Transfección/métodos , Factor de Transcripción Activador 4/genética , Adipogénesis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Línea Celular , Condrogénesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , ADN/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Nanopartículas/química , Osteogénesis , Plásmidos/genética , ARN Interferente Pequeño/genética , Factor de Transcripción SOX9/genéticaRESUMEN
The timing of gene transfection greatly influences stem cell differentiation. Sequential transfection is crucial for regulation of cell behavior. When transfected several days after differentiation initiation, genes expressed at the late stage of differentiation can regulate cell behaviors and functions. To determine the optimal timing of key gene delivery, we sequentially transfected human mesenchymal stem cells (hMSCs). This method can easily control osteogenesis of stem cells. hMSCs were first transfected with RUNX2 and SP7 using poly(lactic-co-glycolic acid) nanoparticles to induce osteogenesis, and then with ATF4 after 5, 7, and 14 days. Prior to transfecting hMSCs with all three genes, each gene was individually transfected and its expression was monitored. Transfection of these genes was confirmed by RT-PCR, Western blotting, and confocal microscopy. The pDNAs entered the nuclei of hMSCs, and RUNX2 and SP7 proteins were translated and triggered osteogenesis. Second, the ATF4 gene was delivered when cells were at the pre-osteoblasts stage. To induce the osteogenesis of hMSCs, the optimal timing of ATF4 gene delivery was 14 days after RUNX2/SP7 transfection. Experiments in 2- and 3-dimensional culture systems confirmed that transfection of ATF4 at 14 days after RUNX2/SP7 promoted osteogenic differentiation of hMSCs.
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Factor de Transcripción Activador 4/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Dexametasona/farmacología , Osteogénesis/efectos de los fármacos , Factor de Transcripción Sp7/genética , Factor de Transcripción Activador 4/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Ácido Láctico/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Nanosferas , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Factor de Transcripción Sp7/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications.
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Células Madre Mesenquimatosas/metabolismo , Nanopartículas/química , Línea Celular , Inestabilidad Genómica/genética , Humanos , Cariotipificación , Células Madre Mesenquimatosas/efectos de los fármacos , Microscopía Confocal , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: During differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage. In this study, using microarray-based technology, gene expression profiling was examined during the process of chondrogenic differentiation of human mesenchymal stem cells (hMSCs). To induce chondrogenic differentiation of hMSCs, the cationic polymer polyethyleneimine (PEI) was coupled with the synthetic glucocorticoid dexamethasone (DEX). DEX/PEI could be polyplexed with anionic plasmid DNAs (pDNAs) harboring the chondrogenesis-inducing factors SOX5, SOX6, and SOX9. These are named differentiation-inducing nanoparticles (DI-NPs). METHODS: A DI-NP system for inducing chondrogenic differentiation was designed and characterized by dynamic light scattering and scanning electron microscopy (SEM). Chondrogenic induction of hMSCs was evaluated using various tools such as reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, confocal fluorescent microscopy, and immunohistochemistry analysis. The gene expression profiling of DI-NP-treated hMSCs was performed by microarray analysis. RESULTS: The hMSCs were more efficiently transfected with pDNAs using DI-NPs than using PEI. Moreover, microarray analysis demonstrated the gene expression profiling of hMSCs transfected with DI-NPs. Chondrogenic factors including SOX9, collagen type II (COLII), Aggrecan, and cartilage oligometric matrix protein (COMP) were upregulated while osteogenic factors including collagen type I (COLI) was downregulated. Chondrogenesis-induced hMSCs were better differentiated as assessed by RT-PCR, Western blotting analyses, and immunohistochemistry. CONCLUSION: DI-NPs are good gene delivery carriers and induce chondrogenic differentiation of hMSCs. Additionally, comprehensive examination of the gene expression was attempted to identify specific genes related to differentiation by microarray analysis.
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Diferenciación Celular/genética , Condrogénesis/genética , Dexametasona/farmacología , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Polietileneimina/farmacología , Factores de Transcripción SOX/genética , Biomarcadores/metabolismo , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrogénesis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Nanopartículas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Nano-sized particles (NPs) of various materials have been extensively used as therapeutic and diagnostic agents, drug delivery systems, and biomedical devices. However, the biological impacts of NP exposure during early embryogenesis on following development and next generations have not been investigated. Here, we demonstrated that polylactic-co-glycolic acid (PLGA)-NPs were not toxic and did not perturb development of preimplantation mouse embryos in vitro. Moreover, subsequent fetal development in vivo after embryo transfer proceeded normally and healthy pups were born without any genetic aberrations, suggesting biosafety of PLGA-NPs during developmental processes. TRITC-labeled PLGA-NPs, named TRITC nano-tracer (TnT) were used to visualize the successful delivery of the NPs into sperms, oocytes and early embryos. Various molecular markers for early embryogenesis demonstrated that TnT treatment at various developmental stages did not compromise embryo development to the blastocyst. mRNA-Seq analyses reinforced that TnT treatment did not significantly affect mRNA landscapes of blastocysts which undergo embryo implantation critical for following developmental processes. Moreover, when 2-cell embryos exposed to TnT were transferred into pseudopregnant recipients, healthy offspring were born without any distinct morphologic and chromosomal abnormalities. TnT treatment did not affect the sex ratio of the exposed embryos after birth. When mated with male mice, female mice that were exposed to TnT during early embryogenesis produced a comparable number of pups as control females. Furthermore, the phenotypes of the offspring of mice experienced TnT at their early life clearly demonstrated that TnT did not elicit any negative transgenerational effects on mammalian development.
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Portadores de Fármacos/química , Desarrollo Embrionario , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Animales , Blastocisto/efectos de los fármacos , Blastocisto/fisiología , Línea Celular , Portadores de Fármacos/toxicidad , Transferencia de Embrión , Desarrollo Embrionario/efectos de los fármacos , Femenino , Iminas/química , Masculino , Ratones , Nanopartículas/toxicidad , Oocitos/efectos de los fármacos , Oocitos/fisiología , Polietilenos/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/toxicidad , EmbarazoRESUMEN
Transfection of a cocktail of genes into cells has recently attracted attraction in stem cell differentiation. However, it is not easy to control the transfection rate of each gene. To control and regulate gene delivery into human mesenchymal stem cells (hMSCs), we employed multicistronic genes coupled with a nonviral gene carrier system for stem cell differentiation. Three genes, SOX5, SOX6, and SOX9, were successfully fabricated in a single plasmid. This multicistronic plasmid was complexed with the polycationic polymer polyethylenimine, and poly(lactic-co-glycolic) acid (PLGA) nanoparticles were coated with this complex. The uptake of PLGA nanoparticles complexed with the multicistronic plasmid was tested first. Thereafter, transfection of SOX5, SOX6, and SOX9 was evaluated, which increased the potential for chondrogenesis of hMSCs. The expression of specific genes triggered by transfection of SOX5, SOX6, and SOX9 was tested by RT-PCR and real-time qPCR. Furthermore, specific proteins related to chondrocytes were investigated by a glycosaminoglycan/DNA assay, Western blotting, histological analyses, and immunofluorescence staining. These methods demonstrated that chondrogenesis of hMSCs treated with PLGA nanoparticles carrying this multicistronic genes was better than that of hMSCs treated with other carriers. Furthermore, the multicistronic genes complexed with PLGA nanoparticles were more simple than that of each single gene complexation with PLGA nanoparticles. Multicistronic genes showed more chondrogenic differentiation than each single gene transfection methods.
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Nanopartículas , Diferenciación Celular , Condrocitos , Condrogénesis , Humanos , Células Madre Mesenquimatosas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Factor de Transcripción SOX9 , Factores de Transcripción SOXDRESUMEN
Polyethylenimine (PEI) has been used as a vehicle to deliver genes to cancer cells and somatic cells. In this study, cationic polymers of PEI were shielded with anionic polymers of hyaluronic acid (HA) to safely and effectively deliver genes into human mesenchymal stem cells (hMSCs). HA interacted with CD44 in the plasma membranes of hMSCs to facilitate the internalization of HA-shielded PEI/pDNA complexes. The HA-shielded PEI/pDNA nanogels were confirmed by size changes, ζ-potential, and gel retardation assays. HA-shielded nanogels were easily internalized by hMSCs, and this was reduced by pretreatment with a specific monoclonal antibody that blocked CD44. By shielding PEI/pDNA complexes with HA, nanogels were easily internalized to hMSCs when it did not blocked by anti-CD44. These shielded nanogels were also easily internalized by HeLa cells, and this was reduced by pretreatment with an anti-CD44 monoclonal antibody. Following internalization of the SOX9 gene, chondrogenesis of hMSCs was increased, as determined by RT-PCR, real-time quantitative PCR, and histological analyses.
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Geles/química , Terapia Genética/métodos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/farmacología , Células Madre Mesenquimatosas/metabolismo , Polietileneimina/química , Diferenciación Celular , Células Cultivadas , ADN Recombinante/administración & dosificación , ADN Recombinante/efectos adversos , Geles/farmacología , Células HeLa , Humanos , Ácido Hialurónico/química , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Plásmidos/administración & dosificación , Plásmidos/efectos adversos , Unión Proteica , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Adulto JovenRESUMEN
During cell differentiation for tissue regeneration, several factors, including growth factors and proteins, influence cascades in stem cells such as embryonic stem cells and mesenchymal stem cells (MSCs). In this study, transforming growth factor (TGF)-ß3 and SOX9, which is an important protein in chondrocytes, were used to generate mature chondrocytes from human MSCs (hMSCs). For safe and effective delivery of bioactive molecules into hMSCs, biodegradable poly-(d,l-lactide-co-glycolide) (PLGA) microspheres (MSs) were coated with TGF-ß3 and loaded with SOX9. Instead of SOX9 protein, release of the model protein FITC-bovine serum albumin (BSA) from PLGA MS was evaluated in vitro and in vivo by confocal laser microscopy and Kodak imaging. The bioactivities of TGF-ß3 and SOX9 were evaluated by assessing α-helical formation using circular dichroism. PLGA MS loaded with FITC-BSA easily entered hMSCs without causing cytotoxicity. To confirm that internalization of PLGA MSs harboring TGF-ß3 and SOX9 induced chondrogenesis of hMSCs, we performed several molecular analyses. By analysis, the specific marker gene expression levels in hMSCs adhered onto PLGA MSs coated with TGF-ß3 and loaded with SOX9 were more than 3-5 times that of the control group both in vitro and in vivo. This result revealed that PLGA MS uptake and subsequent release of SOX9 induced chondrogenesis of hMSCs was enhanced by coating PLGA MSs with TGF-ß3.
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Condrogénesis , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Células Madre/citología , Andamios del Tejido/química , Animales , Cartílago , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Dicroismo Circular , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Regeneración , Factor de Transcripción SOX9/metabolismo , Albúmina Sérica Bovina/química , Células Madre/metabolismo , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
Several factors are involved in angiogenesis. To form new blood vessels, we fabricated vehicles carrying an angiogenesis-related peptide (apelin) and gene (vascular endothelial growth factor (VEGF)165) that were internalized by human mesenchymal stem cells (hMSCs). These non-toxic poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) easily entered hMSCs without cytotoxicity. The negatively charged outer surface of PLGA NPs can be easily complexed with highly positively charged polyethylenimine (PEI) to deliver genes into cells. PLGA NPs complexed with PEI could be coated with negatively charged VEGF plasmid DNA and loaded with apelin. The physical characteristics of these PLGA NPs were determined by size distribution, gel retardation, and morphological analyses. Transfection of VEGF-coated apelin-loaded PLGA NPs resulted in the differentiation of hMSCs into endothelial cells and vascular formation in Matrigel in vitro. Following injection of hMSCs transfected with these PLGA NPs into an ischemic hind limb mouse model, these cells differentiated into endothelial cells and accelerated neovascularization.
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Ácido Láctico/administración & dosificación , Células Madre Mesenquimatosas/citología , Nanopartículas , Neovascularización Fisiológica , Péptidos/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Transfección , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Transfection of specific genes and transportation of proteins into cells have been a focus of stem cell differentiation research. However, it is not easy to regulate codelivery of a gene and a protein into cells. For codelivery into undifferentiated cells (human mesenchymal stem cells (hMSCs)), we used biodegradable carriers loaded with Runt-related transcription factor 2 (RUNX2) protein and coated with bone morphogenetic protein 2 (BMP2) plasmid DNA (pDNA) to induce osteogenesis. The released gene and protein were first localized in the cytosol of transfected hMSCs, and the gene then moved into the nucleus. The levels of internalized PLGA nanoparticles were tested using different doses and incubation durations. Then, transfection of BMP2 pDNA was confirmed by determining mRNA and protein levels and acquiring cell images. The same techniques were used to assess osteogenesis of hMSCs both in vitro and in vivo upon internalization of PLGA NPs carrying the BMP2 gene and RUNX2 protein. Detection of specific genes and proteins demonstrated that cells transfected with PLGA NPs carrying both the BMP2 gene and RUNX2 protein were highly differentiated compared with other samples. Histological and immunofluorescence analyses demonstrated that transfection of PLGA nanoparticles carrying both the BMP2 gene and RUNX2 protein dramatically enhanced osteogenesis of hMSCs.