Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord.

Recent studies have demonstrated that the umbilical cord (UC) is an excellent source of mesenchymal stromal cells (MSCs). However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP) standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H) followed by culture in human platelet lysate (PL) supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D). Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

Glomus tumors in individuals with neurofibromatosis type 1.

BACKGROUND: Glomus tumors have recently been reported in individuals with the neurofibromatosis type 1 (NF1) cancer disposition syndrome. We compare the clinical and molecular features of these painful hamartomas in a series of sporadic and NF1-associated cases. OBJECTIVE: We sought to evaluate the association of NF1 with glomus tumors and to compare NF1-associated glomus tumors with sporadic glomus tumors. METHODS: We conducted a retrospective cohort study of all individuals with a histopathologic diagnosis of glomus tumor at a large tertiary care center from January 1998 to January 2013. Charts were reviewed for a coexisting diagnosis of NF1. RESULTS: A total of 42 glomus tumors were identified in 34 individuals. Twelve (28.6%) were found in 6 patients with NF1. In 28 individuals with 30 sporadic tumors, there was no coexisting medical condition. Although multifocal tumors (16.7%) and tumor recurrence (33.3%) were more common in association with NF1, these trends did not reach statistical significance. NF1-associated glomus tumors exhibited no neurofibromin immunoreactivity, whereas their sporadic counterparts retained neurofibromin expression. LIMITATIONS: The retrospective design resulted in incomplete data capture. CONCLUSIONS: Detection of glomus tumors should raise suspicion for a concurrent diagnosis of NF1.

F11R is a novel monocyte prognostic biomarker for malignant glioma.

OBJECTIVE: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome. METHODS: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM. RESULTS: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype. SIGNIFICANCE: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.

Conditional KIAA1549:BRAF mice reveal brain region- and cell type-specific effects.

Low-grade brain tumors (pilocytic astrocytomas) that result from a genomic rearrangement in which the BRAF kinase domain is fused to the amino terminal of the KIAA1549 gene (KIAA1549:BRAF fusion; f-BRAF) commonly arise in the cerebellum of young children. To model this temporal and spatial specificity in mice, we generated conditional KIAA1549:BRAF strains that coexpresses green fluorescent protein (GFP). Although both primary astrocytes and neural stem cells (NSCs) from these mice express f-BRAF and GFP as well as exhibit increased MEK activity, only f-BRAF-expressing NSCs exhibit increased proliferation in vitro. Using Cre driver lines in which KIAA1549:BRAF expression was directed to NSCs (f-BRAF; BLBP-Cre mice), astrocytes (f-BRAF; GFAP-Cre mice), and NG2 progenitor cells (f-BRAF; NG2-Cre mice), increased glial cell numbers were observed only in the cerebellum of f-BRAF; BLBP-Cre mice in vivo. The availability of this unique KIAA1549:BRAF conditional transgenic mouse strain will enable future mechanistic studies aimed at defining the developmentally-regulated temporal and spatial determinants that underlie low-grade astrocytoma formation in children.

Reduced microglial CX3CR1 expression delays neurofibromatosis-1 glioma formation.

Although traditional models of carcinogenesis have largely focused on neoplastic cells, converging data have revealed the importance of non-neoplastic stromal cells in influencing tumor growth and progression. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1)-associated optic glioma, we now demonstrate that stromal microglia express the CX3CR1 chemokine receptor, such that reduced CX3CR1 expression decreases optic nerve microglia. Moreover, genetic reduction of Cx3cr1 expression in Nf1 optic glioma mice delays optic glioma formation. Coupled with previous findings demonstrating that microglia maintain optic glioma growth, these new findings provide a strong preclinical rationale for the development of future stroma-directed glioma therapies in children.

Pediatric glioma-associated KIAA1549:BRAF expression regulates neuroglial cell growth in a cell type-specific and mTOR-dependent manner.

Tandem duplications involving the BRAF kinase gene have recently been identified as the most frequent genetic alteration in sporadic pediatric glioma, creating a novel fusion protein (f-BRAF) with increased BRAF activity. To define the role of f-BRAF in gliomagenesis, we demonstrate that f-BRAF regulates neural stem cell (NSC), but not astrocyte, proliferation and is sufficient to induce glioma-like lesions in mice. Moreover, f-BRAF-driven NSC proliferation results from tuberin/Rheb-mediated mammalian target of rapamycin (mTOR) hyperactivation, leading to S6-kinase-dependent degradation of p27. Collectively, these results establish mTOR pathway activation as a key growth regulatory mechanism common to both sporadic and familial low-grade gliomas in children.

Neurofibromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner.

Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1(+/-) mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.

Array-based comparative genomic hybridization identifies CDK4 and FOXM1 alterations as independent predictors of survival in malignant peripheral nerve sheath tumor.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with variable patient survival and few known prognostically relevant genomic biomarkers. To identify survival-associated genomic biomarkers, we performed high-resolution array-based comparative genomic hybridization (aCGH) on a large set of MPNSTs. EXPERIMENTAL DESIGN: Candidate gene alterations identified by aCGH in 38 MPNSTs were validated at the DNA, RNA, and protein levels on these same tumors and an independent set of 87 MPNST specimens. RESULTS: aCGH revealed highly complex copy number alterations, including both previously reported and completely novel loci. Four regions of copy number gain were associated with poor patient survival. Candidate genes in these regions include SOX5 (12p12.1), NOL1 and MLF2 (12p13.31), FOXM1 and FKBP1 (12p13.33), and CDK4 and TSPAN31 (12q14.1). Alterations of these candidate genes and several others of interest (ERBB2, MYC and TP53) were confirmed by at least 1 complementary methodology, including DNA and mRNA quantitative real-time PCR, mRNA expression profiling, and tissue microarray-based fluorescence in situ hybridization and immunohistochemistry. Multivariate analysis showed that CDK4 gain/amplification and increased FOXM1 protein expression were the most significant independent predictors for poor survival in MPNST patients (P < 0.05). CONCLUSIONS: Our study provides new and independently confirmed candidate genes that could serve as genomic biomarkers for overall survival in MPNST patients.

Neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth.

Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.

Reduced striatal dopamine underlies the attention system dysfunction in neurofibromatosis-1 mutant mice.

Learning and behavioral abnormalities are among the most common clinical problems in children with the neurofibromatosis-1 (NF1) inherited cancer syndrome. Recent studies using Nf1 genetically engineered mice (GEM) have been instructive for partly elucidating the cellular and molecular defects underlying these cognitive deficits; however, no current model has shed light on the more frequently encountered attention system abnormalities seen in children with NF1. Using an Nf1 optic glioma (OPG) GEM model, we report novel defects in non-selective and selective attention without an accompanying hyperactivity phenotype. Specifically, Nf1 OPG mice exhibit reduced rearing in response to novel objects and environmental stimuli. Similar to children with NF1, the attention system dysfunction in these mice is reversed by treatment with methylphenidate (MPH), suggesting a defect in brain catecholamine homeostasis. We further demonstrate that this attention system abnormality is the consequence of reduced dopamine (DA) levels in the striatum, which is normalized following either MPH or l-dopa administration. The reduction in striatal DA levels in Nf1 OPG mice is associated with reduced striatal expression of tyrosine hydroxylase, the rate-limited enzyme in DA synthesis, without any associated dopaminergic cell loss in the substantia nigra. Moreover, we demonstrate a cell-autonomous defect in Nf1+/- dopaminergic neuron growth cone areas and neurite extension in vitro, which results in decreased dopaminergic cell projections to the striatum in Nf1 OPG mice in vivo. Collectively, these data establish abnormal DA homeostasis as the primary biochemical defect underlying the attention system dysfunction in Nf1 GEM relevant to children with NF1.

Neurofibromatosis-1 regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner.

Recent studies have shown that neuroglial progenitor/stem cells (NSCs) from different brain regions exhibit varying capacities for self-renewal and differentiation. In this study, we used neurofibromatosis-1 (NF1) as a model system to elucidate a novel molecular mechanism underlying brain region-specific NSC functional heterogeneity. We demonstrate that Nf1 loss leads to increased NSC proliferation and gliogenesis in the brainstem, but not in the cortex. Using Nf1 genetically engineered mice and derivative NSC neurosphere cultures, we show that this brain region-specific increase in NSC proliferation and gliogenesis results from selective Akt hyperactivation. The molecular basis for the increased brainstem-specific Akt activation in brainstem NSCs is the consequence of differential rictor expression, leading to region-specific mammalian target of rapamycin (mTOR)/rictor-mediated Akt phosphorylation and Akt-regulated p27 phosphorylation. Collectively, these findings establish mTOR/rictor-mediated Akt activation as a key driver of NSC proliferation and gliogenesis, and identify a unique mechanism for conferring brain region-specific responses to cancer-causing genetic changes.

Histopathologic predictors of pilocytic astrocytoma event-free survival.

Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. Most arise in the cerebellum, but they also can develop in the brainstem and optic nerve, where gross total resection (GTR) is not possible. In the absence of GTR, significant variability in both clinical behavior and histology exists. To identify potential markers associated with poor clinical outcome, we retrospectively assessed pathological features in 107 patients with PAs. We identified four pathological features (necrosis, oligodendroglioma-like features, vascular hyalinization, and calcification) that showed a significant correlation with decreased event-free survival (EFS). Similar to previous reports, we also found that PAs involving the optic pathway were associated with worse EFS compared with those arising in other locations. In contrast, mitotic index, p53 immunoreactivity and hyperactivation of several mitogenic signaling pathways (MAPK, CREB, mTOR) did not demonstrate a statistically significant relationship with EFS. Lastly, we did find a statistical trend between EFS and the number of CD68+ cells, suggesting that non-neoplastic elements of the tumor microenvironment may influence subsequent growth and clinical recurrence. Collectively, the identification of specific histopathologic features associated with clinical outcome may improve our ability to determine which PAs are more likely to exhibit clinical progression and require more vigilant observation.

The neurofibromatosis 2 protein, merlin, regulates glial cell growth in an ErbB2- and Src-dependent manner.

Individuals with the inherited cancer predisposition syndrome neurofibromatosis 2 (NF2) develop several central nervous system (CNS) malignancies, including glial cell neoplasms (ependymomas). Recent studies have suggested that the NF2 protein, merlin (or schwannomin), may regulate receptor tyrosine kinase signaling, intracellular mitogenic growth control pathways, or adherens junction organization in non-nervous-system cell types. For this report, we used glial fibrillary acidic protein conditional knockout mice and derivative glia to determine how merlin regulates CNS glial cell proliferation. We show that the loss of merlin in glial cells results in increased proliferation in vitro and in vivo. Merlin regulation of glial cell growth reflects deregulated Src activity, such that pharmacologic or genetic inhibition of Src activation reduces Nf2(-/-) glial cell growth to wild-type levels. We further show that Src regulates Nf2(-/-) glial cell growth by sequentially regulating FAK and paxillin phosphorylation/activity. Next, we demonstrate that Src activation results from merlin regulation of ErbB2 activation and that genetic or pharmacologic ErbB2 inhibition reduces Nf2(-/-) glial cell Src/Src effector activation and proliferation to wild-type levels. Lastly, we show that merlin competes with Src for direct binding to ErbB2 and present a novel molecular mechanism for merlin regulation of ErbB2-dependent Src signaling and growth control.

Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis.

To study the role of the neurofibromatosis-1 (NF1) gene in mammalian brain development, we recently generated mice in which Nf1 gene inactivation occurs in neuroglial progenitor cells using the brain lipid binding protein (BLBP) promoter. We found that Nf1(BLBP)CKO mice exhibit significantly reduced body weights and anterior pituitary gland sizes. We further demonstrate that the small anterior pituitary size reflects loss of neurofibromin expression in the hypothalamus, leading to reduced growth hormone releasing hormone, pituitary growth hormone (GH) and liver insulin-like growth factor-1 (IGF1) production. Since neurofibromin both negatively regulates Ras activity and positively modulates cAMP levels, we examined the signaling pathway responsible for these abnormalities. While BLBP-mediated expression of an activated Ras molecule did not recapitulate the body weight and hypothalamic/pituitary defects, treatment of Nf1(BLBP)CKO mice with rolipram to increase cAMP levels resulted in a partial restoration of the body weight phenotype. Furthermore, conditional expression of the Ras regulatory GAP domain of neurofibromin also did not rescue the body weight or Igf1 mRNA defects in Nf1(BLBP)CKO mice. Collectively, these data demonstrate a critical role for neurofibromin in hypothalamic-pituitary axis function and provide further insights into the short stature and GH deficits seen in children with NF1.

Expression profiling identifies a molecular signature of reactive astrocytes stimulated by cyclic AMP or proinflammatory cytokines.

Specialized glia, termed reactive astrocytes, accompany numerous pathologic conditions affecting the central nervous system, including stroke, multiple sclerosis, and neoplasia. To better define this important cell type, we employed high-density microarray gene expression profiling using two in vitro models of reactive gliosis (stimulation with dbcAMP or IL-1beta/IFNgamma). We identified 44 differentially expressed transcripts common to both in vitro models and demonstrated that a subset of these genes are also differentially expressed in response to experimental autoimmune encephalomyelitis and focal cerebral ischemia in vivo. Moreover, this pattern of differential gene expression is not observed in hyperproliferating or neoplastic glia.

Distinct genetic signatures among pilocytic astrocytomas relate to their brain region origin.

Pilocytic astrocytomas (PAs) are the most common glioma in children. Whereas many PAs are slow-growing or clinically indolent, others exhibit more aggressive features with tumor recurrence and death. To identify genetic signatures that might predict PA clinical behavior, we did gene expression profiling on 41 primary PAs arising sporadically and in patients with neurofibromatosis type 1 (NF1). Whereas no expression signature was found that could discriminate clinically aggressive or recurrent tumors from more indolent cases, PAs arising in patients with NF1 did exhibit a unique gene expression pattern. In addition, we identified a gene expression signature that stratified PAs by location (supratentorial versus infratentorial). Lastly, we also identified a gene expression pattern common to PAs and normal mouse astrocytes and neural stem cells from these distinct brain regions as well as a gene expression pattern shared between PAs and another human glial tumor (ependymoma) arising supratentorially compared with those originating in the posterior fossa. These results suggest that glial tumors share an intrinsic, lineage-specific molecular signature that reflects the brain region in which their nonmalignant predecessors originated.

Neurofibromatosis-1 regulates neuronal and glial cell differentiation from neuroglial progenitors in vivo by both cAMP- and Ras-dependent mechanisms.

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Ab initio molecular orbital and density functional studies on the solvolysis of sarin and O,S-dimethyl methylphosphonothiolate, a VX-like compound.

[reaction: see text] Potential energy surfaces for the alkaline hydrolysis of sarin and O,S-dimethyl methylphosphonothiolate, a VX model compound, and the perhydrolysis of the latter have been computed at the MP2/6-31+G(d)//mPW1K/MIDI! level of theory. The effect of aqueous solvation was accounted for via the integral equation formalism polarizable continuum model (IEF-PCM) at the HF/6-31+G(d) level. Excellent agreement with the experimental enthalpy of activation for alkaline hydrolysis of sarin was found. For the alkaline hydrolysis of O,S-dimethyl methylphosphonothiolate, it was found that the P-O and P-S bond cleavage processes are kinetically competitive but that the products of P-S bond cleavage are thermodynamically favored. For the perhydrolysis of O,S-dimethyl methylphosphonothiolate, it was found that P-O bond cleavage is not kinetically competitive with P-S bond cleavage. In both cases, the data support initial formation of trigonal bipyramidal intermediates and demonstrate kinetic selectivity for nucleophilic attack on the face opposite the more apicophilic methoxide ligand.