ABSTRACT
Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.
Subject(s)
Neurotrophin 3/metabolism , Schwann Cells/metabolism , Transfection/methods , Animals , Cells, Cultured , Dendrimers , Female , HEK293 Cells , Humans , Nerve Regeneration , Polypropylenes , Rats , Rats, Inbred Lew , Sciatic Nerve/injuries , Sciatic Nerve/physiology , Skin/cytology , Stem Cell TransplantationABSTRACT
Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.
Subject(s)
Dendrimers/chemistry , Mesenchymal Stem Cells/metabolism , Neurons/metabolism , Polyamines/chemistry , Transfection/methods , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Gene Expression , Genes, Reporter , Green Fluorescent Proteins , Humans , Mesenchymal Stem Cells/cytology , Neurons/cytology , Plasmids/genetics , Polyelectrolytes , Polyethyleneimine/chemistry , TransgenesABSTRACT
We studied the potential of neurogenic induction of human bone marrow mesenchymal stem cells in fibrin-based 3D matrix. The best results were obtained after incubation of mesenchymal stem cells in fibrin 3D matrix in the presence of neurogenic medium containing EGF and bFGF growth factors. Under these conditions, most cells formed numerous branching processes and expressed neural cell marker ßIII-tubulin. Optimal combination of 3D matrix and neurogenic factors of the medium can provide new insight into neurogenic potential of mesenchymal stem cells, which can be used for the therapy of neural traumas and neurodegenerative diseases.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Mesenchymal Stem Cells/metabolism , Neurons/cytology , Bone Marrow Cells/metabolism , Culture Media , Epidermal Growth Factor/pharmacology , Fibrin , Fibroblast Growth Factors/pharmacology , Humans , Tissue Scaffolds , Tubulin/metabolismABSTRACT
Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate.
ABSTRACT
Human bone marrow MSC cultured in neurogenic medium containing EGF and FGFbeta demonstrated alteration of the phenotype and expression of neuronal precursor/early neuron markers nestin and NSE. Signals of expression of neuronal and oligodendroglial markers MAP-2, dm-20, and MBP were detected after prolongation of incubation in neurogenic medium to 2 weeks. Cells with neuronal morphology were immunopositive to early neuronal marker beta-III-tubulin. Replacement of neurogenic medium for alpha-MEM with 10% fetal calf serum induced reversion of the phenotype to that typical for human MSC. This indicates high plasticity of the phenotype and expression profile of neuronal markers in MSC cultured under neurogenic conditions or possibility of dedifferentiation of MSC reaching the stage of neuronal precursors/early neurons.
Subject(s)
Bone Marrow Cells/physiology , Mesenchymal Stem Cells/physiology , Neurons/physiology , Cell Differentiation/physiology , Culture Media , Flow Cytometry , Fluorescence , Humans , Immunohistochemistry , Neurogenesis/physiology , Phenotype , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We compared the efficiency of transduction with lentivectors based on HIV-1 and adeno-associated viruses serotype 2 and stability of transgene expression in human mesenchymal bone marrow stem cells.
