Method of obtaining of nonneuronal cultures of astrocytes, derivated from human mesenchymal stem cells

Abstract

Objective. Elaboration of method for obtaining of nonneuronal cultures of astrocytes, derivatives from human mesenchymal stem cells of various origin. Materials and methods. Mesenchymal stem cells were obtained from human peripheral and umbilical blood in accordance to standard procedure, using the immune-magnet separation method and distributed into three groups, depending on primary material of origin and the cellular differentiation technique applied: the cells, which were obtained from the umbilical blood and if our modified technique of differentiation (CBMSC) used, and the cells, which were obtained from peripheral blood and the differentiation technique used was standard (PBMSC-1) and modified (PBMSC-2) protocols. As primary antibodies standard sets β- III-Tubulin (Sigma, USA), GFAP, Nestin (Abcam) served, while as secondary antibodies - іmmunoglobulins G, conjugated by activators CNTF, BMP2/4 and FGF1 (Sigma, USA) in accordance to instruction. For visualization of the results obtained microscope EVOS FL LIFE TECHNOLOGIES was used with obtaining of view under ×40 objective. Results. From majority of cells from the PBMSC-1 group the answer was not obtained, while from approximately 80% of all stimulated cells of the PBMSC-2 group the answer was obtained, and the both lines content consisted of 20 - 30% of GFAP-positive cells. This makes background to consider, that expression of GFAP only is insufficient to identify the mature and functional astrocytes. Conclusion. Astrocytes from group PBMSC-1 have had answered very rarely on ATP-stimulation, while astrocytes from groups CBMSC and PBMSC-2 demonstrated characteristic answer. Thus, there was demonstrated, that morphologically close astrocytic lines and single cells owes different functional profile.

Keywords

• nonneuronal cultures of astrocytes; the immune-magnet separation; mesenchymal stem cells.