In vitro studies on human first trimester forebrain cells : differentiation and interactions with immunoregulating molecules

Abstract: This thesis has focused on understanding, at the molecular level, growth regulation and differentiation of human first trimester forebrain cells, and the immune signals mediating these processes. Primary cultures were established from human forebrain tissue derived from elective first trimester abortions. Tissues were carefully triturated and plated in serum-containing culture medium without addition of antibiotics or pre-conditioned medium. Human first trimester forebrain cultures were heterogeneous populations of cells and could survive well for at least 6 weeks. The majority was glial cells. The interactions between these cells and immunoregulating molecules were studied. The capacity of these cells at different gestational ages to produce IFN-[gamma] and IL-4 and the effect of IFN-[gamma] on their survival, proliferation and expression of MHC antigens was examined. IFN-[gamma] mRNA expression was recorded at high levels during week 7.5 and 10.5 of gestation. First trimester forebrain cells cultured for 24 h spontaneously produced high amounts of IFN-[gamma], but no measurable levels of IL-4 were detected. In kinetic studies, cultures with and without IFN-[gamma] stimulation did not show MHC antigen induction until day 7 in vitro where incubation with IFN-[gamma] showed expression of MHC class II molecules. Furthermore, natural induction of IL-1ß, IL-4, IL-6, IL-10, TNF-[alpha], IFN-[gamma] and TGF-ß in human first trimester forebrain cells was evaluated. Constitutive cytokine gene expression were detected already at week 5 of gestational age. Expression increased with age and IFN-[gamma] was mainly expressed in the GFAP-positive cells. To investigate the factors that regulate astrocyte chemokine expression, the ability of human first trimester astrocytes to induce ß-family chemokine mRNA, MIP-1[alpha], MIP-1ß, RANTES, and MCP-1 was examined. Human first trimester astrocytes were induced to express the P-family chemokines in a stimulus-dependent fashion. Astrocyte-derived MCP-1, RANTES MIP-1[alpha] and MIP-1ß mRNA were easily induced by LPS, IFN-[gamma] and TNF-[alpha]. UV-inactivated MV, but not CMV, strongly induced expression of these chemokines, especially MCP-1 and MIP-1ß. Moreover, the signalling pathway used by IFN-[gamma] during the process of growth regulation of human astrocytes obtained from the first trimester was investigated. IFN-[gamma] induced significantly higher cell survival compared to that of unexposed cultures. This effect was suppressed by incubation with A47. STAT-1 was translocated into the nucleus upon IFN-[gamma] stimulation, which was also blocked by incubation with A47. The results of these studies provide evidence for communications between immunoregulatory molecules and human first trimester forebrain cells, suggesting momentous roles for cytokines in neuronal and glial cell survival, proliferation and differentiation during brain development.