BMP9 (bone morphogenetic protein 9) induces NGF as an autocrine/paracrine cholinergic trophic factor in developing basal forebrain neurons.

Acetylcholine (ACh) synthesis and release from basal forebrain cholinergic neurons (BFCN) innervating the cerebral cortex and hippocampus are essential processes for normal learning, memory and attention. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiation factor in the developing septum that increases ACh synthesis and choline acetyltransferase (Chat) gene expression both in vivo and in vitro. We investigated the possible induction of cholinergic trophic factors by BMP9 in murine septal cells. Nerve growth factor (NGF) protein expression and secretion into the medium was increased in cultured embryonic septal cells treated with BMP9, and partially mediated BMP9-induced acetylcholine production and Chat gene expression. BMP9-induced Ngf gene expression was detected in postmitotic cells, required new protein synthesis and was blocked by BMP type I receptor inhibition. Cholinergic neurons were isolated by fluorescence-activated cell sorting based on either transgenic expression of green fluorescent protein driven by the Chat promoter or NGF receptor (p75) immunostaining. Although both noncholinergic and cholinergic neurons in untreated cultures expressed similar low levels of Ngf, increased Ngf gene expression was restricted to Chat-positive neurons in BMP9-treated cultures. Likewise, similar levels of Ngf mRNA were detected in p75-negative and p75-positive septal cells, yet only p75-positive BFCN increased their Ngf gene expression when treated with BMP9, and only these cells expressed the Alk1 BMP receptor. The data suggest an autocrine/paracrine role for NGF in the development and/or maintenance of BFCN and imply that the stimulation of NGF production and release contributes to the cholinergic-supportive properties of BMP9.

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 µm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 µm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.

Expression of a prototypic anti-colorectal cancer polyclonal antibody library in mammalian cells.

We describe the production of a prototypic polyclonal antibody library (PCAL), a standardized mixture of full-length IgG polyclonal antibodies for which the genes are available. The PCAL was generated by mass transfer of heavy and light chain variable region gene pairs, selected for binding to human colorectal cancer cells, from a Fab phage display vector to a mammalian IgG expression vector. Following transfection of the IgG vector library into Sp2/0 myeloma cells, clones were characterized for IgG expression and binding to the colorectal cancer cells by ELISA, and for diversity by DNA fingerprinting, nucleotide sequencing, and immunoblot analysis. The results showed that 76-84% of the library clones produce IgG and of those 72-79% bind antigen. Furthermore, preliminary analysis showed clonal diversity at both the DNA and antigen-binding levels. When depleted of reactivity to normal tissue, polyclonal antibody libraries to cancer cells may be efficacious for cancer therapy.

BMP9 protects septal neurons from axotomy-evoked loss of cholinergic phenotype.

BACKGROUND: Cholinergic projection from the septum to the hippocampus is crucial for normal cognitive function and degeneration of cells and nerve fibers within the septohippocampal pathway contributes to the pathophysiology of Alzheimer's disease. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiating factor during development both in vivo and in vitro. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether BMP9 could protect the adult cholinergic septohippocampal pathway from axotomy-evoked loss of the cholinergic phenotype, we performed unilateral fimbria-fornix transection in mice and treated them with a continuous intracerebroventricular infusion of BMP9 for six days. The number of choline acetyltransferase (CHAT)-positive cells was reduced by 50% in the medial septal nucleus ipsilateral to the lesion as compared to the intact, contralateral side, and BMP9 infusion prevented this loss in a dose-dependent manner. Moreover, BMP9 prevented most of the decline of hippocampal acetylcholine levels ipsilateral to the lesion, and markedly increased CHAT, choline transporter CHT, NGF receptors p75 (NGFR-p75) and TrkA (NTRK1), and NGF protein content in both the lesioned and unlesioned hippocampi. In addition, BMP9 infusion reduced bilaterally hippocampal levels of basic FGF (FGF2) protein. CONCLUSIONS/SIGNIFICANCE: These data indicate that BMP9 administration can prevent lesion-evoked impairment of the cholinergic septohippocampal neurons in adult mice and, by inducing NGF, establishes a trophic environment for these cells.

Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons.

The activity of the basal forebrain cholinergic neurons (BFCNs) that innervate the cerebral cortex and hippocampus is essential for normal learning and memory. Here, we present a method to isolate and culture BFCNs from the embryonic murine septum that takes advantage of their restricted expression of the nerve growth factor receptor (p75) in conjunction with fluorescence-activated cell sorting. The septal region dissection, cell dissociation and staining process, and cell sorting parameters are described in detail. Sufficient cell yield and optimized cell culture conditions make this protocol suitable for multiple assays including immunocytochemistry, reverse transcriptase PCR, microarray profiling, acetylcholine measurements and electrophysiological assessment. The study of these neurons as a purified population will greatly advance our understanding of factors that influence their development and maintenance.

Differential modulation of nerve growth factor receptor (p75) and cholinergic gene expression in purified p75-expressing and non-expressing basal forebrain neurons by BMP9.

The synthesis of acetylcholine and its release from basal forebrain cholinergic neurons (BFCN) that innervate the cerebral cortex and hippocampus are considered essential processes for normal learning, memory and attention. We have developed a purification and cell culture method of BFCN in order to examine the regulation of their cholinergic phenotype. Cells isolated from the septal region of late embryonic mice were purified by fluorescence-activated cell sorting based on their expression of the nerve growth factor receptor (p75), a surface marker for mature BFCN. Consistent with previous reports, p75-positive (p75+) cells were enriched in choline acetyltransferase (ChAT) and the high-affinity choline transporter (ChT), as measured by reverse transcriptase PCR. In culture, these cells maintained their gene expression of p75, ChAT and ChT, while p75-negative (p75-) cells had a low expression of these genes. Incubation of the cells with BMP9 not only increased p75 and ChAT gene expression in p75- cells, but also augmented the expression of these genes in p75+ cells. Conversely, BMP9 decreased ChT gene expression in p75+ cells and had no such effect in p75- cells. Immunostaining confirmed that p75 protein expression was modulated by BMP9 in a similar way as p75 mRNA, and also revealed that only a subset of p75- cells respond to BMP9 in this manner. These data suggest that mature BFCN in culture may express their cholinergic phenotype in the absence of exogenous trophic input, but that BMP9 can further modulate this phenotype. Moreover, BMP9 induces the cholinergic phenotype in a set of basal forebrain non-cholinergic neurons or precursor cells.

Induction of cell signaling events by the cholera toxin B subunit in antigen-presenting cells.

Cholera toxin (CT) is one of the most effective and widely studied mucosal adjuvants. Although the ADP-ribosylating A subunit has been implicated in augmenting immune responses, the receptor-binding B subunit (CT-B) has greater immunogenicity and may be a repository of adjuvant activity without potential toxicity. In order to elucidate mechanisms of immune modulation by CT-B alone, primary B cells and macrophages were assessed for responses to CT-B in vitro, as measured by the expression of cell surface markers, cellular signaling events, and cytokine secretion. Increased phosphorylation of multiple signaling molecules, including Erk1/2 and p38, was detected. CT-B also induced transactivation of the transcription elements cyclic AMP-responsive element and NF-kappaB, the latter of which was inhibited by phosphotyrosine inhibition. While specific inhibition of MEK1/2 did not reduce CT-B induction of cell surface marker expression, it did attenuate CT-B-mediated interleukin-6 secretion. These data show that CT-B induces a set of signaling events related to cellular activation, surface molecule expression, and cytokine production that has potential implications for elucidating CT-B adjuvant activity in the absence of enzymatically active holotoxin.

Gestational choline deficiency causes global and Igf2 gene DNA hypermethylation by up-regulation of Dnmt1 expression.

During gestation there is a high demand for the essential nutrient choline. Adult rats supplemented with choline during embryonic days (E) 11-17 have improved memory performance and do not exhibit age-related memory decline, whereas prenatally choline-deficient animals have memory deficits. Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine, a substrate of DNA methyltransferases (DNMTs). We describe an apparently adaptive epigenomic response to varied gestational choline supply in rat fetal liver and brain. S-Adenosylmethionine levels increased in both organs of E17 fetuses whose mothers consumed a choline-supplemented diet. Surprisingly, global DNA methylation increased in choline-deficient animals, and this was accompanied by overexpression of Dnmt1 mRNA. Previous studies showed that the prenatal choline supply affects the expression of multiple genes, including insulin-like growth factor 2 (Igf2), whose expression is regulated in a DNA methylation-dependent manner. The differentially methylated region 2 of Igf2 was hypermethylated in the liver of E17 choline-deficient fetuses, and this as well as Igf2 mRNA levels correlated with the expression of Dnmt1 and with hypomethylation of a regulatory CpG within the Dnmt1 locus. Moreover, mRNA expression of brain and liver Dnmt3a and methyl CpG-binding domain 2 (Mbd2) protein as well as cerebral Dnmt3l was inversely correlated to the intake of choline. Thus, choline deficiency modulates fetal DNA methylation machinery in a complex fashion that includes hypomethylation of the regulatory CpGs within the Dnmt1 gene, leading to its overexpression and the resultant increased global and gene-specific (e.g. Igf2) DNA methylation. These epigenomic responses to gestational choline supply may initiate the long term developmental changes observed in rats exposed to varied choline intake in utero.

Retinoid-dependent restriction of human immunodeficiency virus type 1 replication in monocytes/macrophages.

Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease. Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality. Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription. Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo. We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin. Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible. We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation. In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression. Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages.