Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Hypoxia decreases podocyte expression of slit diaphragm proteins.

BACKGROUND: Chronic hypoxia contributes to progressive tubulointerstitial injury and, consequently, renal failure. However, the effect of hypoxia on glomerular podocytes, which are integral to the slit diaphragm complex and responsible for selectivity of the glomerular filtration barrier, has not been completely determined. METHODS: Conditionally immortalized mouse podocyte cells were exposed to hypoxic (1% O(2)) or normoxic (room air) conditions for 24, 48, or 72 hours, after which cell viability was determined by MTT assay. Cells were stained with podocin and phalloidin to determine podocin and intracellular actin distribution. Expression of synaptopodin, CD2-associated protein (CD2AP), NcK, transforming growth factor-ß1 (TGF-ß1), hypoxia-inducible factor (HIF-1α) were evaluated by real-time polymerase chain reaction. RESULTS: Podocytes exposed to hypoxia had significantly reduced viability at 48 (87%) and 72 hours (66%). There was disarrangement of intracellular filament actin by phalloidin staining, a 30% weaker fluorescence intensity by podocin staining, significantly reduced expression of synaptopodin (12%), CD2AP (42%), NcK (38%), and increased expression of TGF-ß1 and P-ERK after hypoxia treatment. CONCLUSION: Podocyte exposure to hypoxia leads to reduced viability and SD protein expression, which may explain persistent and/or increasing proteinuria in patients with progressive renal failure. Increased expression of TGF-ß1 and P-ERK is associated with apoptosis and fibrosis, which could be the link between hypoxia and glomerular injury.

Wnt pathway activity confers chemoresistance to cancer stem-like cells in a neuroblastoma cell line.

Neuroblastoma is the most common solid tumor in infancy. We have shown that the neuroblastoma cell line SK-N-SH contains CD133+ cells that are more resistant than 133- cells to Doxorubicin (DOX), a common chemotherapeutic agent. We hypothesize that activation of wnt signaling pathway in CD133+ cells contributes to their chemoresistance. To test this hypothesis, CD133+ cells were positively selected using magnetic micro-beads. Subsequently, CD133+ and negatively selected CD133- cells were treated with 100 ng/ml of DOX for up to 72 h. Then, cells were either lysed for total RNA extraction or fixed for immunostaining. Wnt "SIGNATURE" PCR Array was used to determine if changes in wnt related gene expression levels occurred and to estimate a pathway activity score. Expression of wnt pathway proteins ß-Catenin and p-GSK3ß (S-9) was determined by immunocytochemistry. Two wnt pathway inhibitors were used to determine the changes in cell viability, using the MTT assay. Results showed that wnt related genes were differentially expressed in CD133+ cells as compared to CD133- cells, both with and without DOX treatment. Pathway activity scores showed that DOX treatment significantly suppressed the wnt pathway activity in CD133- cells. Expression of ß-catenin and p-GSK3ß (S-9) was significantly greater in DOX treated and untreated CD133+ cells. The presence of wnt inhibitors with DOX decreased the number of live cells in CD133+ group and the percentage of live cells in both groups were equal. These data suggest that higher wnt pathway activity could be responsible for the chemoresistance of CD133+ cells in neuroblastoma cell lines.

Ethanol affects differentiation-related pathways and suppresses Wnt signaling protein expression in human neural stem cells.

BACKGROUND: Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder. METHODS: NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single-cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin-coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt-receptor complex proteins (p-LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked-2 (NKD-2), and downstream Wnt proteins (ß-catenin, Tyr-p-GSK3ß, Ser-p-GSK3ß) were analyzed by Western blot. RESULTS: Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF-ß signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p-LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser-p-GSK3ß and ß-catenin. Expression of NKD-2 and DVL3 remained unchanged and the expression of active Tyr-p-GSK3ß increased significantly. CONCLUSIONS: EtOH can significantly alter neural differentiation pathway-related gene expression and suppress Wnt signaling proteins in differentiating human NSC.

Ethanol alters cell fate of fetal human brain-derived stem and progenitor cells.

BACKGROUND: Prenatal ethanol (ETOH) exposure can lead to fetal alcohol spectrum disorder (FASD). We previously showed that ETOH alters cell adhesion molecule gene expression and increases neurosphere size in fetal brain-derived neural stem cells (NSC). Here, our aim was to determine the effect of ETOH on the cell fate of NSC, premature glial-committed precursor cells (GCP), and premature neuron-committed progenitor cells (NCP). METHODS: NSC, GCP, and NCP were isolated from normal second-trimester fetal human brains (n = 3) by positive selection using magnetic microbeads labeled with antibodies to CD133 (NSC), A2B5 (GCP), or PSA-NCAM (NCP). As a result of the small percentage in each brain, NSC were cultured in mitogenic media for 72 hours to produce neurospheres. The neurospheres from NSC and primary isolates of GCP and NCP were used for all experiments. Equal numbers of the 3 cell types were treated either with mitogenic media or with differentiating media, each containing 0 or 100 mM ETOH, for 120 hours. Expression of Map2a, GFAP, and O4 was determined by immunoflourescence microscopy and western blot analysis. Fluorescence intensities were quantified using Metamorph software by Molecular Devices, and the bands of western blots were quantified using densitometry. RESULTS: ETOH in mitogenic media promoted formation of neurospheres by NSC, GCP, and NCP. Under control conditions, GCP attached and differentiated, NSC and NCP formed neurospheres that were significantly smaller in size than those in ETOH. Under differentiating conditions, Map2a expression increased significantly in NSC and GCP and reduced significantly in NCP, and GFAP expression reduced significantly in GCP and NCP, and Gal-C expression reduced significantly in all 3 cell types in the presence of ETOH compared to controls. CONCLUSIONS: This study shows that ETOH alters the cell fate of neuronal stem and progenitor cells. These alterations could contribute to the mechanism for the abnormal brain development in FASD.

Resistance of stem-like cells from neuroblastoma cell lines to commonly used chemotherapeutic agents.

BACKGROUND: Cancer stem cell theory suggests that the presence of tumor initiating stem-like cells in cancers may be responsible for cancer progression and relapse. CD133 cell surface maker expression has been used to identify stem-like cells in cancer cell lines. Our goal was to identify such cells in neuroblastoma cell lines and to study the cytotoxicity of common anticancer drugs for those cells. MATERIALS AND METHODS: CD133+ cells from SK-N-SH and SK-N-BE cell lines were isolated using magnetic microbeads. Cytotoxicity of four anticancer drugs was studied on CD133+ and CD133- populations. The percentage of live, apoptotic, and dead cells in each population after drug treatment was estimated by MTT and PI/Annexin-binding assays. Western blot analyses were used to identify differences in the expression of kinases. RESULTS: Eight to 10% of SK-N-SH and 3-5% of SK-N-BE cells were CD133+. These cells were more resistant than CD133- cells to all four chemotherapeutic agents tested in the MTT assay. Decreased apoptosis was observed in CD133+ cells compared to CD133- cells by PI/Annexin V-binding assay. Western blot analysis showed that CD133+ cells expressed less MKP-1. Phosphorylated forms of both ERK and P-38 kinases were expressed at higher levels in CD133+ cells than in CD133- cells. CONCLUSIONS: This study suggests that CD133+ cells are more resistant to anticancer drugs than CD133- cells. Differences in the expression and phosphorylation of kinases could be partially responsible for this difference. Targeting CD133-expressing cells could be a strategy to develop more effective treatments for neuroblastoma.

Ethanol increases fetal human neurosphere size and alters adhesion molecule gene expression.

BACKGROUND: Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). METHODS: Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. RESULTS: Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed. CONCLUSIONS: ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.

HIV-1 RNA concentration and cognitive performance in a cohort of HIV-positive people.

OBJECTIVE: To determine whether higher viral concentrations in the cerebrospinal fluid (CSF) and/or peripheral blood were associated with greater severity of cognitive impairment in HIV-1-seropositive subjects with cognitive-motor impairment. METHODS: Cognitive performance measurements and viral load were obtained from HIV-1-seropositive individuals with cognitive-motor impairment entering a clinical trial before the introduction of highly active antiretroviral therapy (HAART). CSF viral load (UltraSensitive Roche HIV-1 Monitor test with detection limit of 50 copies/ml) was available from 179 patients, and peripheral (plasma or serum) viral load from 111 patients. Of these patients, 62% met the 1993 Centers for Disease Control (CDC) criteria for AIDS, and 19% had clinically significant cognitive impairment (i.e., global deficit score > or = 0.5). Possible associations between viral load and cognitive scores were examined with general linear regression models with and without adjustment for age, education, study site, antiretroviral use, CD4 cell count, and CDC stage. RESULTS: The mean CSF viral load was 2.83 log(10)/ml +/- 0.94 (SD) (undetectable in 19.5%). Mean peripheral viral load was 4.11 log(10)/ml +/- 0.90 (SD). No statistically significant associations emerged between either CSF or peripheral viral load and the global deficit score, or any of the seven cognitive domain deficit scores. CONCLUSIONS: Among these HIV-1-sero-positive individuals with mainly minor HIV-1-associated cognitive deficits and not receiving HAART, no association between CSF or blood concentration of HIV-1 RNA and cognitive performance could be found. These results suggest that the severity of HIV-1-associated cognitive impairment is not directly related to concurrent viral concentration in the CSF or the peripheral blood.

Cerebrospinal and peripheral human immunodeficiency virus type 1 load in a multisite, randomized, double-blind, placebo-controlled trial of D-Ala1-peptide T-amide for HIV-1-associated cognitive-motor impairment.

D-Ala1-peptide T-amide (DAPTA) has shown neuroprotection in vitro against gp120-induced loss of dendritic arborization and is promulgated as a CCR5 antagonist. A multisite, randomized, double-blind clinical trial of DAPTA versus placebo prior to combination antiretroviral therapy conducted with human immunodeficiency virus (HIV)-1 seropositive participants having cognitive impairment showed no overall cognitive effect, though subgroups with greater impairment and CD4 cell counts of 201 to 500 cells/mm3 at baseline showed significant improvement. The objective of this study was to examine whether intranasal administration of DAPTA at a dose of 2 mg three times per day (tid) was associated with a reduction of cerebrospinal fluid (CSF) and peripheral (plasma and serum) viral load among a subgroup of participants completing 6 months of treatment. Baseline and 6-month CSF (n = 92) and peripheral (plasma n = 33; serum n = 24) viral load were measured by the Roche Ultrasensitive assay, version 1.5, with reflexive use of the AMPLICOR assay and preservation of the blind. A DAPTA treatment indicator variable was tested using generalized linear models on change in viral load. Peripheral load (combined plasma and serum) was significantly reduced in the DAPTA-treated group. No group differences in CSF viral load were found. This retrospective study on a limited subgroup of the original trial sample indicated that DAPTA treatment may reduce peripheral viral load without concomitant CSF effects. Future studies should be undertaken to confirm the existence of this result and the CSF-periphery dissociation observed with respect to HIV-1-associated cognitive-motor impairment.

Human neural stem cells are more sensitive than astrocytes to ethanol exposure.

BACKGROUND: Exposure to ethanol (EtOH) can be deleterious to the developing central nervous system. The mechanisms by which EtOH exposure induces neural pathology in utero remain unclear. However, EtOH-induced increases in protein kinase C (PKC) have been associated with apoptosis in human primary cell cultures. Although the toxic effects of EtOH on differentiated neural cells have been studied in laboratory animal models, the susceptibility of the human neural stem cells (NSCs) that predominate in the central nervous system during embryonic development has not been addressed. METHODS: For this study, fetal human brain cells, which satisfied the criteria for NSCs by being CD133-positive, nestin-positive, and differentiated glial fibrillary acidic protein-positive human astrocytes, were studied. The cytotoxic potential of EtOH in NSC and astrocyte cultures was studied by using morphological and biochemical methods. In addition, membrane and cytosolic fraction PKC activity for each cell type was assessed. RESULTS: NSC showed a dose-dependent increase in EtOH-induced toxicity as estimated by terminal transferase-mediated dUTP nick end labeling (TUNEL) stain and viability assays. TUNEL staining indicating DNA degradation consistent with programmed (apoptotic) cell death was detectable in 90% of NSC 16 hr after 2 hr exposure to 10 mM EtOH. NSC also showed a concentration-dependent increase in membrane, but not cytosol, PKC activity over the same EtOH dose range. By contrast, astrocytes showed no cytotoxic effects at any concentrations of EtOH used (0-10 mM). PKC activity of both the membrane and cytosolic fragments from astrocytes also was unaffected by this range of doses. CONCLUSIONS: This study demonstrates the susceptibility of human NSCs, compared with astrocytes, to EtOH and indicates that alterations in PKC signal transduction in NSC may play a role in EtOH-induced neuropathological processes.

Differential responses of human neural and hematopoietic stem cells to ethanol exposure.

The mechanisms underlying fetal developmental defects caused by maternal ethanol (EtOH) consumption remain unclear. The symptoms of fetal alcohol syndrome (FAS) include neurological and immunological dysfunctions that are linked to cell reduction in these systems. Neural (NSC) and hematopoietic stem cells (HSC) may be targets for the cytotoxic effects of EtOH. Furthermore, protein kinase C (PKC) signal transduction systems of these stem cells may be involved in EtOH-induced cell death. Purified CD34+ human fetal liver hematopoietic stem cells (HSC) and CD133+/nestin+ human neural stem cells (NSC) were exposed to 0.1-10 mM EtOH. A range of indices of cell damage indicated that these doses of EtOH were deleterious to NSC, but had no observable effects on HSC. Furthermore, the colony-forming ability of NSC was completely inhibited by 5 mM EtOH treatment, whereas HSC were unaffected by even 20 mM EtOH. These results suggest that NSC are much more sensitive to EtOH than HSC. Classic and novel PKC isozyme protein expressions in the membrane fraction of cells were differentially affected by EtOH exposure across the two stem cell types. Concentrations of EtOH capable of inducing NSC, but not HSC, death also changed apoptosis-associated PKC isozyme expression in the membrane of NSC, but not HSC. Therefore, PKC expression may mediate the susceptibility of NSC to EtOH-induced cytotoxicity via cell signal transduction pathways. The toxic effect of EtOH on NSC may lead to the decreased neural cell number observed in FAS patients. The comparable immunity of HSC to the deleterious effects of EtOH exposure indicates that the susceptibility of NSC is not simply due to their being stem cells and also may explain the relative lack of hematopoietic problems associated with FAS.

Increased prevalence of G1P[4] genotype among children with rotavirus-associated gastroenteritis in metropolitan Detroit.

The G and P genotypes of rotavirus stool isolates from 100 children were determined by reverse transcription-PCR and nucleotide sequencing. G1P[4] was the most prevalent genotype(41%), followed by G1P[8] (16%) and G4P[4] (14%). The G genoypes detected were G1 (73%), G4 (17.4%), G9 (6.3%), and G2 (2.8%). The P genotypes were P[4] (71%) and P[8] (29%). Coinfection with more than one G genotype occurred in 12 patients, and coinfection with more than one P genotype occurred in 11 patients.

Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro.

In some rodent models, there is evidence that hematopoietic stem cells (HSC) can differentiate into neural cells. However, it is not known whether humans share this potential, and, if so, what conditions are sufficient for this transdifferentiation to occur. We addressed this question by assessing the ability of fetal human liver CD34(+)/CD133(+)/CD3(-) hematopoietic stem cells to generate neural cells and astrocytes in culture. We cultured fetal liver-derived hematopoietic stem cells in human astrocyte culture-conditioned medium or using a method wherein growing human astrocytes were separated from cultured, nonadherent hematopoietic stem cells by a semipermeable membrane in a double-chamber co-culture system. Hematopoietic stem cell cultures were probed for neural progenitor cell marker expression (nestin and bone morphogenic protein-2 [BMP-2]) during growth in both culture conditions. RT-PCR, western blotting, and immunocytochemistry assays showed that cells cultured in either condition expressed nestin mRNA and protein and BMP-2 mRNA. HSC similarly cultured in nonconditioned medium or in the absence of astrocytes did not express either marker. Cells expressing these neural markers were transferred and cultured on poly-D-lysine-coated dishes with nonconditioned growth medium for further study. Immunocytochemistry demonstrated that these cells differentiated into astrocytes after 8 days in culture as indicated by their morphology and expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and S100, as well as by their rate of proliferation, which was identical to that of freshly isolated fetal brain astrocytes. These findings demonstrate that neural precursor gene expression can be induced when human hematopoietic stem cells are exposed to a suitable microenvironment. Furthermore, the neural stem cells generated in this environment can then differentiate into astrocytes. Therefore, human hematopoietic stem cells may be an alternative resource for generation of neural stem cells for therapy of central nervous system defects resulting from disease or trauma.

Cryopreserved fetal human liver hematopoietic progenitor cells for repopulating immune-deficient mouse bone marrow.

BACKGROUND: Experimental and clinical protocols are being developed for the cryopreservation of human hematopoietic progenitor cells. However, the effect of these procedures on the potential for HPC to repopulate bone marrow is unknown. OBJECTIVES: To examine the effect of cryopreservation on the ability of fetal human liver HPC, which include CD34+ cells and long-term culture-initiating cells, to repopulate immunodeficient non-obese diabetic/severe combined immunodeficiency mouse bone marrow. METHODS: Groups of sublethally irradiated NOD/SCID mice were injected intravenously with cryopreserved or freshly isolated fetal human liver HPC. RESULTS: Seven weeks after transplantation, flow cytometric analysis of bone marrow samples showed that mice that received the transplanted cells (either cryopreserved or freshly isolated) demonstrated both lymphoid and myeloid differentiation as well as the retention of a significant fraction of CD34+ cells. CONCLUSIONS: Cryopreserved fetal human liver-derived HPC appear to be capable of initiating human cell engraftment in NOD/SCID mouse bone marrow and open the possibility of using cryopreserved fetal human liver HPC for gene manipulation, gene transfusion therapy, and transplantation purposes.