RESUMO
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.
Assuntos
Neurotrofina 3/metabolismo , Células de Schwann/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , Dendrímeros , Feminino , Células HEK293 , Humanos , Regeneração Nervosa , Polipropilenos , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Pele/citologia , Transplante de Células-TroncoRESUMO
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.
Assuntos
Dendrímeros/química , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Poliaminas/química , Transfecção/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Plasmídeos/genética , Polieletrólitos , Polietilenoimina/química , TransgenesRESUMO
Conditions of human BM and umbilical cord blood MSC in vitro differentiation in the hepatogenic direction were studied. Changes in cell morphology, phenotype, acquisition of the capacity to produce albumin and accumulate glycogen, express cytokeratin, alkaline phosphatase, and albumin mRNA indicated that BM and umbilical cord blood MSC differentiated in vitro into immature hepatocyte-like cells.
Assuntos
Células da Medula Óssea/citologia , Sangue Fetal/citologia , Hepatócitos/citologia , Células-Tronco Mesenquimais/citologia , Albuminas/biossíntese , Fosfatase Alcalina/biossíntese , Antígenos CD/biossíntese , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/metabolismo , Citometria de Fluxo , Glicogênio/biossíntese , Hepatócitos/metabolismo , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Queratinas/biossíntese , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/biossínteseRESUMO
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.
