ABSTRACT
Numerous cell culture protocols have been described for the proliferation of multipotent human neural progenitor cells (HNPCs). The mitogen combinations used to expand HNPCs vary, and it is not clear to what extent this may affect the subsequent differentiation of these cells. In this study human foetal cortical tissue was cultured in the presence of either EGF, or FGF-2, or a combination of both using a unique chopping method in which cell to cell contact is maintained. The differentiation potential of neurospheres following mitogen withdrawal was assessed at early (8 weeks) and late (20 weeks) times of expansion, both in vitro and in vivo. In addition, changes in gene expression with time were analysed by microarray experiments. Results show that the presence of FGF-2 was highly predictive of neuronal differentiation after short term culture both in vitro and in vivo. In addition, time in culture had a significant effect on transplant size and neural constituents suggesting that cells have a limited life span and restricted lineage potential. Array analysis confirms that following extensive time in culture cells are entering growth arrest with fundamental expression changes in genes associated with cell cycle regulation, apoptosis and immune functions.
Subject(s)
Cell Differentiation/physiology , Cerebral Cortex/cytology , Gene Expression Regulation, Developmental/physiology , Neurons/physiology , Stem Cells/physiology , Animals , Behavior, Animal , Cell Differentiation/drug effects , Cell Proliferation , Cells, Cultured , Epidermal Growth Factor/pharmacology , Fetus , Fibroblast Growth Factor 2/pharmacology , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/drug effects , Humans , Male , Neurons/drug effects , Oligonucleotide Array Sequence Analysis/methods , Organ Culture Techniques , Rats , Rats, Inbred Lew , Stem Cell Transplantation/methods , Stem Cells/drug effects , Time FactorsABSTRACT
Interleukin-1beta (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1beta on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1beta at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1beta. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1beta were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n = 23) and downregulated (n = 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P < 0.02). Our results have identified genes regulated by IL-1beta, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1beta, were verified using real-time PCR. Novel functions regulated by IL-1beta in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1beta in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.