Larry Denner

Production of stem cells with embryonic characteristics from human umbilical cord blood

Abstract.  When will embryonic stem cells reach the clinic? The answer is simple – not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipients immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the worlds first reproducible production of cells expressing embryonic stem cell markers, – cord‐blood‐derived embryonic‐like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (105 cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3‐ligand 50 ng/ml, c‐kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA‐1‐60, TRA‐1‐81, SSEA‐4, SSEA‐3 and Oct‐4, but not SSEA‐1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity–bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c‐kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin‐18, α‐foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Nuclear Factor-κB and Cell Death After Experimental Intracerebral Hemorrhage in Rats

Background and Purpose—Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-κB in relation to cell death surrounding intracerebral hemorrhage (ICH). Methods—Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-κB protein, Western blot was performed on brain extract. We determined NF-κB activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-κB. DNA fragmentation was detected with terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL) staining. Results—Western blot analysis of the ...

Two forms of rat brain glutamic acid decarboxylase differ in their dependence on free pyridoxal phosphate

Abstract: There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal‐5′‐phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45°C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonie, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl‐trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of γ‐aminobutyric acid (GABA) level is also discussed.

Inhibition of smooth muscle cell growth in vitro by an antisense oligodeoxynucleotide released from poly(DL-lactic-co-glycolic acid) microparticles.

We fabricated poly(DL-lactic-co-glycolic acid) (PLGA) 50:50 microparticles loaded with an antisense (AS) oligodeoxy-nucleotide (ODN) against the rat tenascin mRNA and determined the effect in vitro of the AS-ODN released on smooth muscle cell (SMC) proliferation and migration. AS-ODN was entrapped using a double-emulsion-solvent-extraction technique with high efficiency. Release of AS-ODN was characterized by a small initial-burst effect followed by a period of controlled AS-ODN release for up to 20 days. SMC proliferation studies exhibited dose-dependent growth inhibition with AS-ODN-loaded microparticles. Microparticles loaded with scrambled (SC) ODN showed less growth inhibition than AS-ODN. Moreover, only the AS-ODN-loaded microparticles inhibited migration. These results demonstrate the feasibility of entrapping an AS-ODN to rat tenascin in PLGA microparticles for controlled delivery to inhibit SMC proliferation and migration.

Directed engineering of umbilical cord blood stem cells to produce C-peptide and insulin

Abstract.  Objectives: In this study, we investigated the potential of umbilical cord blood stem cell lineages to produce C‐peptide and insulin. Materials and methods: Lineage negative, CD133+ and CD34+ cells were analyzed by flow cytometry to assess expression of cell division antigens. These lineages were expanded in culture and subjected to an established protocol to differentiate mouse embryonic stem cells (ESCs) toward the pancreatic phenotype. Phase contrast and fluorescence immunocytochemistry were used to characterize differentiation markers with particular emphasis on insulin and C‐peptide. Results: All 3 lineages expressed SSEA‐4, a marker previously reported to be restricted to the ESC compartment. Phase contrast microscopy showed all three lineages recapitulated the treatment‐dependent morphological changes of ESCs as well as the temporally restricted expression of nestin and vimentin during differentiation. After engineering, each isolate contained both C‐peptide and insulin, a result also obtained following a much shorter protocol for ESCs. Conclusions: Since C‐peptide can only be derived from de novo synthesis and processing of pre‐proinsulin mRNA and protein, we conclude that these results are the first demonstration that human umbilical cord blood‐derived stem cells can be engineered to engage in de novo synthesis of insulin.

Heterogeneous apoptotic responses of prostate cancer cell lines identify an association between sensitivity to staurosporine-induced apoptosis, expression of Bcl-2 family members, and caspase activation

The goal of this work was to identify mechanisms for the inability of metastatic prostate cancer cells to engage the apoptotic pathway following hormonal or cytotoxic therapy.

Cognitive Enhancement with Rosiglitazone Links the Hippocampal PPARγ and ERK MAPK Signaling Pathways

We previously reported that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (RSG) improved hippocampus-dependent cognition in the Alzheimers disease (AD) mouse model, Tg2576. RSG had no effect on wild-type littermate cognitive performance. Since extracellular signal-regulated protein kinase mitogen-activated protein kinase (ERK MAPK) is required for many forms of learning and memory that are affected in AD, and since both PPARγ and ERK MAPK are key mediators of insulin signaling, the current study tested the hypothesis that RSG-mediated cognitive improvement induces a hippocampal PPARγ pattern of gene and protein expression that converges with the ERK MAPK signaling axis in Tg2576 AD mice. In the hippocampal PPARγ transcriptome, we found significant overlap between peroxisome proliferator response element-containing PPARγ target genes and ERK-regulated, cAMP response element-containing target genes. Within the Tg2576 dentate gyrus proteome, RSG induced proteins with structural, energy, biosynthesis and plasticity functions. Several of these proteins are known to be important for cognitive function and are also regulated by ERK MAPK. In addition, we found the RSG-mediated augmentation of PPARγ and ERK2 activity during Tg2576 cognitive enhancement was reversed when hippocampal PPARγ was pharmacologically antagonized, revealing a coordinate relationship between PPARγ transcriptional competency and phosphorylated ERK that is reciprocally affected in response to chronic activation, compared with acute inhibition, of PPARγ. We conclude that the hippocampal transcriptome and proteome induced by cognitive enhancement with RSG harnesses a dysregulated ERK MAPK signal transduction pathway to overcome AD-like cognitive deficits in Tg2576 mice. Thus, PPARγ represents a signaling system that is not crucial for normal cognition yet can intercede to restore neural networks compromised by AD.

Rosiglitazone reversal of Tg2576 cognitive deficits is independent of peripheral gluco-regulatory status.

Converging lines of evidence associate gluco-regulatory abnormalities and peroxisome-proliferator-activated receptor (PPAR) gamma function with increased risk for Alzheimers disease (AD). In this study, we used the Tg2576 AD mouse model to test the hypothesis that cognitive improvement following 1 month of PPAR gamma agonism with rosiglitazone (RTZ) correlates with peripheral gluco-regulatory status. We assessed cognition and peripheral gluco-regulatory status of Tg2576 mice following 1 month treatment with RTZ initiated prior to, coincident with, or after, the onset of peripheral gluco-regulatory abnormalities (4, 8, and 12 months of age, respectively). Whereas 5 months old (MO) and 13 MO Tg2576 did not gain cognitive improvement after 1 month treatment with RTZ, 9 MO Tg2576 mice exhibited reversal of associative learning and memory deficits. Peripheral gluco-regulatory abnormalities were improved in 9 and 13 MO Tg2576 with RTZ treatment; RTZ treatment had no effect on the normal glucose status of 5 MO Tg2576 mice. These findings suggest that RTZ-mediated cognitive improvement does not correlate with peripheral gluco-regulatory abnormalities per se, but reflects the age-dependent mechanistic differences that underlie cognitive decline in this mouse model.

Diabetes-Induced Activation of Canonical and Noncanonical Nuclear Factor-κB Pathways in Renal Cortex

Evidence of diabetes-induced nuclear factor-κB (NF-κB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-κB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-κB–regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-κB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-κB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of κB kinase-α and several fold increases in NF-κB–inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-κB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-κB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-κB pathway proteins.

A neutralizing antibody against receptor for advanced glycation end products (RAGE) reduces atherosclerosis in uremic mice

Chronic renal failure markedly accelerates atherogenesis in apolipoprotein E-deficient (apoE(-/-)) mice. To study the putative role of receptor for advanced glycation end products (RAGE) in development of uremic atherosclerosis, apoE(-/-) mice received intraperitoneal injections thrice weekly of a neutralizing murine RAGE-antibody (RAGE-ab) (n=21) or an isotype-matched control antibody (placebo-ab) (n=23). Treatment was started 4 weeks after surgical 5/6 nephrectomy in 16 weeks old mice and continued for 12 weeks. The RAGE-ab did not affect blood pressure, plasma cholesterol or measures of uremia. However, the aortic plaque area fraction was reduced by 59% in RAGE-ab compared with placebo-ab-treated mice (0.016 +/- 0.002 versus 0.039 +/- 0.005, P<0.001). In plasma, the RAGE-ab reduced concentrations of oxidized phospholipid neo-epitopes in plasma as detected by the specific monoclonal antibody EO6 (P<0.05) and titers of IgG antibodies against oxidized low-density lipoprotein (P<0.001). In the aorta of treated mice, the RAGE-ab did not affect the mRNA expression of eight selected genes associated with inflammation. The results suggest that blockade of RAGE reduces the proatherogenic effects of uremia, possibly through a systemic decrease in oxidative stress.