Immunofluorescence: main stages of phenotyping and differential potential of autologous mesenchymal stem cells in vitro

Abstract

The article contains detailed information about possible use of immunofluorescence for studying the main stages of phenotyping and differentiation of autologous mesenchymal stem cells (AMSCs) in vitro. The authors present facts, which concern origination of cells (3-month-old male Wistar rats), methods of their generation in laboratory conditions (isolation from femoral and tibial diaphyses and epiphyses of animals followed by washing with culture medium, inoculation on Petri dishes, explantation, cleaning from blood corpuscles, incubation in a monolayer, reinoculation). Conditions of phenotyping are described (with use of flow cytofluorometry, the received DNA was polymerised with specific primers and finished with a polymerase chain reaction). The reaction products were analysed by the method of electrophoresis in agarose gel. AMSCs were differentiated in the neuron direction after the second culture reinoculation. In order to induce the differentiation, neurotrophin-3 growth factor was used. AMSCs were grown up to a thick monolayer. The obtained derivates were phenotyped by staining cells with antibodies to protein markers of neurons, neuron-specific nuclear protein (NeuN) (Chemicon), neurofibrillary axonal protein (Tau) (Chemicon), myelin/oligodendrocyte-specific protein (MOSP) (Chemicon), glial fibrillary acidic protein (GFAP) (Chemicon). As a result of the study, it was revealed that the number of CD90+ cells (which actually proved to be the population of AMSCs) was 95-97 %. The phenotype of cells, determined on the standard basis by use of superficial markers, did not change during the first four passages. Incubation of the population of AMSCs during these passages proved that the above cells did not lose their ability to differentiate in three main orthodox directions. Thus, the population of AMSCs remained homogenous.