Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex Masatoshi Suzuki*†, Lynda S. Wright*, Padma Marwah‡, Henry A. Lardy‡, and Clive N. Svendsen*§ *Departments of Anatomy and Neurology and the Waisman Center, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705-2280; †Department of Veterinary Physiology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and ‡Department of Biochemistry and Institute for Enzyme Research, University of Wisconsin, 1710 University Avenue, Madison, WI 53726 Contributed by Henry A. Lardy, December 29, 2003 Dehydroepiandrosterone (DHEA) is a neurosteroid with potential very low (6, 11). Thus, there is some question as to the relevance effects on neurogenesis and neuronal survival in humans. How- of the effect of DHEA on rodents and how this relates to the ever, most studies on DHEA have been performed in rodents, and human situation. there is little direct evidence for biological effects on the human Recent studies have shown that neural precursor cells can be nervous system. Furthermore, the mechanism of its action is derived from the developing brain allowing the direct examina- unknown. Here, we show that DHEA significantly increased the tion of proliferation, differentiation, and migration of these cells growth rates of human neural stem cells derived from the fetal in the culture dish (12, 13). These cells can be maintained in cortex and grown with both epidermal growth factor (EGF) and culture as spherical aggregates termed neurospheres (14–16) leukemia inhibitory factor (LIF). However, it had no effect on that consist of both multipotent stem cells and more restricted cultures grown in either factor alone, suggesting a specific action progenitor cells responsive to the mitogens epidermal growth on the EGF͞LIF-responsive cell. Precursors of DHEA such as preg- factor (EGF) and fibroblast growth factor 2 (FGF-2) (17). nenolone or six of its major metabolites, had no significant effect Neurospheres are regionally specified based on the brain region on proliferation rates. DHEA did not alter the small number (<3%) from which they were isolated (18, 19) and also differ between of newly formed neuroblasts or the large number (>95%) of species (20). However, recently, we have grown neurospheres nestin-positive precursors. However, the number of glial fibrillary from the human fetal cortex for extended periods of time in EGF acidic protein-positive cells, its mRNA, and protein were signifi- and LIF and shown that they represent a fairly homogenous cantly increased by DHEA. We found both N-methyl-D-aspartate population of cells immunoreactive for the stem cell marker and sigma 1 antagonists, but not GABA antagonists, could com- nestin (21, 22). After a full genomic analysis of these cells, we pletely eliminate the effects of DHEA on stem cell proliferation. have shown them to be stable in culture over time and between Finally we asked whether the EGF͞LIF͞DHEA-responsive stem cells lines with regard to global gene expression and termed them had an increased potential for neurogenesis and found a 29% long-term human neural stem cells derived from the cortex increase in neuronal production when compared to cultures grown (ltNSCCTX) (22). in EGF͞LIF alone. Together these data suggest that DHEA is in- Given the species differences in DHEA and stem cell biology, volved in the maintenance and division of human neural stem cells. ltNSCCTX provide an ideal and innovative model to understand Given the wide availability of this neurosteroid, this finding has developmental and molecular mechanisms of neurosteroid ac- important implications for future use. tions in the human central nervous system. We show here that DHEA, but not its derivatives, increased the cell proliferation of ehydroepiandrosterone (DHEA) and its sulfate are the ltNSCCTX in vitro. We also established which receptor signaling Dmost abundant steroids in the blood of young adult humans. mediated this DHEA action, and finally showed that ltNSCCTX Levels of DHEA peak at Ϸ20 years of age and then decline to grown in the presence of DHEA underwent increased neuro- reach values of 20–30% at 70–80 years of age (1). Significant genesis. These results have important implications for brain interest has arisen from the hypothesis that declining DHEA repair and are relevant to understanding the actions of DHEA concentrations in adults may serve as an indicator of a number on the human brain. of conditions including the loss of insulin sensitivity, obesity, diabetes, cardiovascular diseases, stress, and aging (2). In clinical Materials and Methods studies, DHEA levels were found to decline in mental illnesses Neuronal Progenitor Cell Culture. Human embryonic tissue was such as major depressive disorder or in systematic diseases that obtained from the Birth Defects Laboratory at the University of respond to DHEA supplementation (3, 4). Washington, Seattle. Neurospheres were generated from three While DHEA represents the most abundant steroid product of cortex samples, M006 (13 weeks postconception male), M007 (18 the adrenal cortex, it has also been identified as a ‘‘neuroste- weeks postconception male), and M009 (16 weeks postconcep- roid.’’ Neurosteroids are synthesized de novo in the central tion male). The method of collection conformed to the guide- nervous system independent, at least in part, of peripheral organ lines recommended by National Institutes of Health for the activity (5, 6). Neurosteroids possess the ability to affect neurons collection of such tissues and set out by the University of ␥ through activation of -aminobutyric acid (GABA)A, N-methyl- Washington and the University of Wisconsin, Madison. Institu- D-aspartate (NMDA), and sigma receptors (5, 6), although the exact role of each with regard to DHEA is not well established. DHEA has been shown to be neuroprotective after oxidative Abbreviations: DHEA, dehydroepiandrosterone; EGF, epidermal growth factor; GABA, ␥-aminobutyric acid; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; stress in rat hippocampal neuronal cultures (7) and hippocampal ltNSCCTX, long-term cortical neural stem cells; MK801, (ϩ)-5-methyl-10,11-dihydro-5H- damage induced by NMDA (8). DHEA has also been shown to dibenzo[a,d]cyclohepten-5,10-imine hydrogen malate; NMDA, N-methyl-D-aspartate; be a potential signaling molecule in neuronal differentiation TuJ1, ␤-tubulin III. during development (9) and has recently been shown to increase §To whom correspondence should be addressed at: T611 Waisman Center, 1500 Highland neurogenesis in the adult rodent hippocampus (10). In contrast Avenue, Madison, WI 53705. E-mail: [email protected]. to the human brain, levels of DHEA in the adult rat brain are © 2004 by The National Academy of Sciences of the USA 3202–3207 ͉ PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307325101 Downloaded by guest on September 28, 2021 Fig. 1. Schematic describing culture schedules and treatments for ltNSCctx. Neurospheres were generated from three fetal cortex samples, initially grown for 4 weeks in EGF and FGF-2. The cultures were then switched to medium supplemented with EGF alone. After Ͼ10 weeks of growth, the cultures were supplemented with EGF and LIF and maintained for a further 4 weeks. Thirty to 40 neurospheres were cultured in coated T25 flasks with basal medium in the presence or absence of EGF, LIF, and DHEA. After 9 days, neurospheres were pulsed with 0.2 ␮M BrdUrd for 14 h and then dissociated into a single cell suspension with enzyme. Dissociated cells were plated onto poly(L)-lysine͞laminin-coated coverslips for 1 h. To check the number of cells immunostained with BrdUrd or other cell markers, cells were fixed 1 h after plating. For differentiation studies, the plated cells were differentiated for 7 days under serum-free conditions and then visualized with anti-TuJ1, GFAP, and Hoechst-labeled nuclei. tional Review Board approval was obtained for all of these Systems) were used at a final concentration of 10 ng͞ml. The studies. NMDA receptor antagonist (ϩ)-5-methyl-10,11-dihydro-5H- The schematic illustration of the experimental schedule and dibenzo[a,d]cyclohepten-5,10-imine hydrogen malate (MK801) ␮ treatments is summarized in Fig. 1. Cortical progenitors were and GABAA receptor antagonist bicuculline (10 M each in isolated from embryonic brain and induced to proliferate as medium) were purchased fromR&DSystems. Sigma 1 receptor free-floating neurospheres (16). Freshly isolated tissue was antagonists BD1063 or haloperidol (Tocris Cookson, Ellisville, mechanically chopped into small cubes using a McLwain tissue MO) were used at a final concentration of 3 ␮M. chopper (Mickle Laboratory Engineering, Surrey, England) and seeded into T75 flasks at an approximate density of 200,000 cells Immunocytochemistry. Cultures were fixed in ice cold methanol (eight cubes) per ml of basal medium [DMEM͞Ham’s F-12 (7:3) for 20 min or 4% paraformaldehyde (PFA in PBS) at room containing penicillin͞streptomycin͞amphotericin B (PSA, 1%)] temperature and washed in PBS. The cells were then incubated supplemented with B27 (2%; Life Technologies), EGF (20 in 2 M HCl for 20 min at 37°C. The acid was neutralized with 0.1 ng͞ml; Sigma), and FGF-2 (20 ng͞ml;R&DSystems) with M sodium borate and washed several times in PBS. Fixed cells heparin (5 ␮g͞ml). All cultures were maintained in a humidified were blocked in 5% goat serum with 0.3% Triton X-100 and incubator (37°C) and half the growth medium was replenished incubated with anti-BrdUrd antibodies (rat monoclonal, 1:500, every 4–5 days. For passaging of neurospheres, a chopping Accurate Chemical, Westbury, NY) for 30 min. After incubation method was used that does not require trypsin or mechanical with the primary antibodies, either fluorescein or Cy3 (goat dissociation, and cell͞cell contact was continuously maintained anti-rat IgG, 1:1,000, The Jackson Laboratory) was used to (16). Neurospheres were passaged every 14 days by chopping visualize the signal.