Development and differentiation of multipotent human neural cells in vitro.

Abstract

Many metabolic, genetic, and inflammatory discovers of the central nervous system are characterized by dysfunction and degeneration of a large number of neurons. Transplantation of nervous cells is a promising approach to treatment of these neurodegenerative diseases. Modern neurobiological studies distinctly demonstrate that brain stem cells characterized by a broad spectrum of differentiation potentials are the best candidates for the replacement therapy [1, 2]. Cell populations in the central nervous system are formed from stem cells located in the germinal areas of the developing brain, namely, the ventricular and subventricular zones. Stem and multipotent cells reside in these areas not only in the embryonic and early postnatal periods [2‐5], but throughout the life of mammals and humans [6‐10]. At present, researchers are focusing on isolation, multiplication and preservation of nervous stem cells [7, 11, 12], as well as analysis of their differentiation in culture and brain transplants [13, 14]. We studied development and differentiation of human neural stem cells cultured in vitro. This study was the first to be aimed at obtaining a panel of cytofluorimetric characteristics of human stem cells that might lay basis for standardization of cells for subsequent transplantation. Human embryonic brain tissues were obtained from 8- to 12-week-old embryos isolated by means of clinical abortion. The fetuses used for this research were absolutely nonviable and represented dead abortive material from which surviving brain cells were isolated. To isolate the fetal brain, the membranes were removed; tissue fragments were suspended by means of thorough pipetting in the F12 medium and centrifuged. Cells were counted, and their viability was tested using trypan blue and propidium iodide. The cells were phenotyped using flow cytofluorimetry. The cytofluorimetry was performed using antibodies against the following markers: nestin, an intermediate-filament protein of neuroepithelial stem cells; vimentin, a protein expressed in progenitors of neuroand glioblasts and radial glial cells; NeuN, a nuclear marker of developing brain neurons; GFAP, a protein of glial filaments in astrocytes; PLP, a basic membrane protein of the myelin of oligodendrocytes in the CNS; β -tubulin III, a protein expressed in neuroblasts; CD56, an adhesion protein expressed in neurons; N-Cad, an intercellular adhesion protein expressed after initiation of the neural tube; OB-Cad (cadherin), a mesenchimalcell adhesion protein; HLA-ABC, a molecule of the major histocompatibility complex class I expressed by nuclear cells; HLA-DR, a molecule of the major histocompatibility complex class II expressed by antigenpresenting cells; and CD34, which is expressed by hematopoietic progenitors, endothelium, and tissue fibroblasts. To identify intracellular markers (nestin, vimentin, and NeuN), fixation and permeabilization of cells were performed. The cells were incubated with monoclonal antibodies (MonAb) labeled with fluorescein isothiocyanate (FITC) and ficoerythrin (FE). After washing them from unbound MonAb by centrifuging, the samples were analyzed using a fluorimeter. To assess the nonspecific binding of MonAb, the corresponding isotypic controls were used (Bekton Dickinson, United States) labeled with FITC and FE. The studies were performed by the direct bi-chromatic immunofluorescence method using a flow cytofluorimeter (Bio-Rad, Italy).