RESUMEN
Radiation induces DNA and protein damage and free radical formation, effectively establishing cellular senescence in a variety of models. We demonstrate the effects of two known pleiotropic drugs following gamma radiation damage in neurosphere/cerebral organoid system based on human embryonic stem cells. mTORC1 repression by rapamycin prior to irradiation, or metabolic activation by minocycline after irradiation, partially rescues neuroepithelium integrity, neurite-growing capacity, ventricle formation and extracellular acidification rate as an integral measure of metabolic output. Cerebral organoid model thus provides valid and robust readouts for radiation studies in a complex 3D setting.
Asunto(s)
Encéfalo/citología , Rayos gamma , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de la radiación , Minociclina/farmacología , Organoides/citología , Sirolimus/farmacología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismoRESUMEN
This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cell-derived functional differentiated cells.
Asunto(s)
Dendrímeros/química , Factores de Crecimiento Nervioso/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Condrogénesis/genética , Condrogénesis/fisiología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factores de Crecimiento Nervioso/genética , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Osteogénesis/genética , Osteogénesis/fisiología , Plásmidos/genética , Transfección/métodosRESUMEN
We report the use of polyamidoamine (PAMAM-NH(2)) dendrimers along with other non-viral vehicles for the in vitro transfection of human bone marrow mesenchymal stem cells (hMSCs) and for engineering MSCs to secrete brain-derived neurotrophic factor (BDNF). Different generations of cationic polyamidoamine dendrimers (generations 3-6) were tested on HEK 293T cells. hMSCs were then transfected with PAMAM-NH(2) G4 dendrimers and Lipofectamine 2000, which elicited the expression of GFP reporter in around 6 and 20% of the cells, respectively. Both vehicles were then shown to elicit the expression of BDNF in MSCs from a bicistronic cassette. Non-virally induced neurotrophin expression may be a safe and easy method for adapting autologous stem cells for therapeutic treatment of diseases and neural system injuries.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Técnicas de Cultivo de Célula/métodos , Dendrímeros/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adulto , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular , ADN/metabolismo , Vectores Genéticos/genética , Humanos , Plásmidos/genética , Transfección , Transgenes/genéticaRESUMEN
We report the adaptation of dendrimer-based nonviral expression system for ciliary neurotrophic factor (CNTF) overproduction in human mesenchymal stem cells (hMSCs) embedded into fibrin-based three-dimensional (3D) matrix. Time-restricted neurotrophin expression enables autologous adult stem cells for additional trophic support and increases their therapeutic potential in neuroregeneration applications. Polyamidoamine (PAMAM)-NH(2) dendrimers of fourth generation effectively provided virus-free delivery and expression of CNTF-internal ribosome entry site-green fluorescent protein cassette with a transfection efficiency in hMSCs over 11%. CNTF levels in transfected cultures were 10-fold higher as compared with the control cells. Dendrimer-driven CNTF expression also persisted in hMSCs embedded into fibrin-based 3D matrix, an emerging vehicle for cell delivery or bioartificial organ formation. Nonviral modification of autologous adult stem cells with use of dendrimers is a novel tool perspective in terms of biosafety and technological availability.
Asunto(s)
Factor Neurotrófico Ciliar/biosíntesis , Fibrina/administración & dosificación , Terapia Genética , Células Madre Mesenquimatosas/metabolismo , Biodegradación Ambiental , Células Cultivadas , Dendrímeros/química , Humanos , TransfecciónRESUMEN
Dendriplexes were characterized by ethidium bromide intercalation assay and their transfection efficiency was studied using HEK 293 cells and human mesenchymal stem cells. PAMAM G4 showed a higher transfection efficiency than PAMAM G3-G6, G4-OH, G4-25% or G4-50% dendrimers. Substitution of OH groups for the NH(2) surface groups rendered the dendrimer unable to form dendriplexes and to transfect cells. Partial (25%) substitution of CH(3) groups for the NH(2) groups markedly impaired transfection; 50% substitution decreased the ability of PAMAM G4 to transfect threefold. It was concluded that increased hydrophobicity decreased the ability of dendrimers to transfect. PAMAM G4-50% is highly hydrophobic and forms micelles in solution, which can transfect pGFP. The results of ethidium bromide intercalation assays, ANS fluorescence studies and transfection efficiencies of PAMAM dendrimers were correlated. Subsequently, we constructed a neurotrophin-encoding plasmid and studied its delivery to mesenchymal stem cells using PAMAM G4 dendrimer and Lipofectamine 2000. Lipofectamine 2000 was a more effective carrier (18.5%) than PAMAM G4 dendrimer (1.2%).