In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells.
ABSTRACT One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.
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ABSTRACT: Nucleotides are of crucial importance as carriers of energy in all organisms. However, the concept that in addition to their intracellular roles, nucleotides act as extracellular ligands specifically on receptors of the plasma membrane took longer to be accepted. Purinergic signaling exerted by purines and pyrimidines, principally ATP and adenosine, occurs throughout embryologic development in a wide variety of organisms, including amphibians, birds, and mammals. Cellular signaling, mediated by ATP, is present in development at very early stages, e.g., gastrulation of Xenopus and germ layer definition of chick embryo cells. Purinergic receptor expression and functions have been studied in the development of many organs, including the heart, eye, skeletal muscle and the nervous system. In vitro studies with stem cells revealed that purinergic receptors are involved in the processes of proliferation, differentiation, and phenotype determination of differentiated cells. Thus, nucleotides are able to induce various intracellular signaling pathways via crosstalk with other bioactive molecules acting on growth factor and neurotransmitter receptors. Since normal development is disturbed by dysfunction of purinergic signaling in animal models, further studies are needed to elucidate the functions of purinoceptor subtypes in developmental processes.Cellular and Molecular Life Sciences CMLS 01/2011; 68(8):1369-94. · 5.62 Impact Factor
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ABSTRACT: An important challenge in pancreatic islet transplantation in association with type 1 diabetes is to define automatic high-throughput assays for evaluation of human islet function. The physiological techniques presently used are amenable to small-scale experimental samples and produce descriptive results. The postgenomic era provides an opportunity to analyze biological processes on a larger scale, but the transition to high-throughput technologies is still a challenge. As a first step to implement high-throughput assays for the study of human islet function, we have developed two methodologies: multiple automated perifusion to determine islet hormone secretion and high-throughput kinetic imaging to examine islet cellular responses. Both technologies use fully automated devices that allow performing simultaneous experiments on multiple islet preparations. Our results illustrate that these technologies can be applied to study the functional status and explore the pharmacological profiles of islet cells. These methodologies will enable functional characterization of human islet preparations before transplantation and thereby provide the basis for the establishment of predictive tests for beta-cell potency.Cell Transplantation 02/2008; 16(10):1039-48. · 4.42 Impact Factor
Article: Stem cells in cell transplantation.[show abstract] [hide abstract]
ABSTRACT: This commentary documents the increased number of stem cell-related research reports recently published in the cell transplantation field in the journal Cell Transplantation. The journal covers a wide range of issues in cell-based therapy and regenerative medicine and is attracting clinical and preclinical articles from around the world. It thereby complements and extends the basic coverage of stem cell physiology reported in Stem Cells and Development. Sections in Cell Transplantation cover neuroscience, diabetes, hepatocytes, bone, muscle, cartilage, skin, vessels, and other tissues, as well as tissue engineering that employs novel methods with stem cells. Clearly, the continued use of biomedical engineering will depend heavily on stem cells, and these two journals are well positioned to provide comprehensive coverage of these developments.Stem Cells and Development 01/2007; 15(6):963-6. · 4.67 Impact Factor