The Chemokine Receptor CCR1 Mediates Microglia Stimulated Glioma Invasion.

Glioblastoma multiforme (GBM) is the most aggressive form of adult brain tumor which is highly resistant to conventional treatment and therapy. Glioma cells are highly motile resulting in infiltrative tumors with poorly defined borders. Another hallmark of GBM is a high degree of tumor macrophage/microglia infiltration. The level of these tumor-associated macrophages/microglia (TAMs) correlates with higher malignancy and poorer prognosis. We previously demonstrated that inhibition of TAM infiltration into glioma tumors with the CSF-1R antagonist pexidartinib (PLX3397) can inhibit glioma cell invasion in-vitro and in-vivo. In this study, we demonstrate an important role for the chemokine receptor CCR1 in mediating microglia/TAM stimulated glioma invasion. Using two structurally distinct CCR1 antagonists, including a novel inhibitor "MG-1-5", we were able to block microglial activated GL261 glioma cell invasion in a dose dependent manner. Interestingly, treatment of a murine microglia cell line with glioma conditioned media resulted in a strong induction of CCR1 gene and protein expression. This induction was attenuated by inhibition of CSF-1R. In addition, glioma conditioned media treatment of microglia resulted in a rapid upregulation of gene expression of several CCR1 ligands including CCL3, CCL5, CCL6 and CCL9. These data support the existence of tumor stimulated autocrine loop within TAMs which ultimately mediates tumor cell invasion.

Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents.

Type 1 diabetes (T1D) presents with two therapeutic challenges: the need to correct underlying autoimmunity and restore ß-cell mass. We harnessed the unique capacity of regulatory T cells (Tregs) and the T cell receptor (TCR) to direct tolerance induction along with tissue-localized delivery of therapeutic agents to restore endogenous ß-cell function. Specifically, we designed a combinatorial therapy involving biomaterials-based poly(lactic-co-glycolic acid) nanoparticles co-loaded with the Treg growth factor, IL-2, and the ß-cell regenerative agent, harmine (a tyrosine-regulated kinase 1A [DYRK1A] inhibitor), conjugated to the surface of Tregs. We observed continuous elution of IL-2 and harmine from nanoparticles for at least 7 days in vitro. When conjugated to primary human Tregs, IL-2 nanoparticles provided sufficient IL-2 receptor signaling to support STAT5 phosphorylation for sustained phenotypic stability and viability in culture. Inclusion of poly-L-lysine (PLL) during nanoparticle-cell coupling dramatically increased conjugation efficiency, providing sufficient IL-2 to support in vitro proliferation of IL-2-dependent CTLL-2 cells and primary murine Tregs. In 12-week-old female non-obese diabetic mice, adoptive transfer of IL-2/harmine nanoparticle-conjugated NOD.BDC2.5 Tregs, which express an islet antigen-specific TCR, significantly prevented diabetes demonstrating preserved in vivo viability. These data provide the preclinical basis to develop a biomaterials-optimized cellular therapy to restore immune tolerance and promote ß-cell proliferation in T1D through receptor-targeted drug delivery within pancreatic islets.

Cognitive impairment persists at least 1 year after juvenile rats are treated with methotrexate.

Methotrexate (MTX) is widely employed for children with cancer, but is also associated with persistent cognitive deficits among survivors. The present study investigated the mechanisms behind long-term cognitive dysfunction after juvenile animals are treated with MTX. Male and female Long-Evans rats were treated with a combination of 6 systemic doses (0.5 mg/kg/dose intraperitoneally) and 4 intrathecal doses (1 mg/kg) beginning at post-natal age 3 weeks, a schedule designed to mimic repeated exposure given to children with leukemia. Behavioral testing was conducted at 60-61 weeks of age, followed by analysis of brain histolopathology. This MTX regimen had no acute toxicity and no effect on growth. The spatial memory and visual memory deficits observed at 13 and 17 weeks of age persisted 1 year after MTX exposure in both females and males. Significantly decreased cell proliferation and increased hippocampal microglial activation were observed in MTX-treated females when compared to the controls, with a similar trend in the male groups. In addition, MTX treatment significantly increased the number of TUNEL positive cells in the periventricular area. Our study demonstrates that a clinically relevant regimen of systemic and intrathecal MTX induces persistent deficits in cognition, lasting approximately 1 year after the last injection. The mechanisms behind MTX-induced deficits are likely multifactorial, including suppression of neurogenesis, microglial activation, and increased brain cell apoptosis. Our study suggests female and male animals differ in susceptibility to MTX-induced neurotoxicity and provides insights for developing therapeutic approaches to prevent treatment related cognitive impairment among children with ALL.

Microglia Isolation from Neural Stem Cell-Enriched Regions.

Microglia are the resident immune cell of the central nervous system and are instrumental in detecting and eliminating invading pathogens and debris. They also play key roles in neural development, neurodegeneration, and maintaining microenvironment homeostasis. The relatively low number of microglia that can be isolated from primary dissociates precludes many in vitro assays from being efficiently conducted. Here we describe a method to isolate large numbers of functional microglia in a repeatable fashion using serially expanded cultures derived from neurogenic regions of the brain.

Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model.

Antigen specificity is a primary goal in developing curative therapies for autoimmune disease. Dendritic cells (DCs), as the most effective antigen presenting cells in the body, represent a key target to mediate restoration of antigen-specific immune regulation. Here, we describe an injectable, dual-sized microparticle (MP) approach that employs phagocytosable ∼1 µm and nonphagocytosable ∼30 µm MPs to deliver tolerance-promoting factors both intracellularly and extracellularly, as well as the type 1 diabetes autoantigen, insulin, to DCs for reprogramming of immune responses and remediation of autoimmunity. This poly(lactic-co-glycolic acid) (PLGA) MP system prevented diabetes onset in 60% of nonobese diabetic (NOD) mice when administered subcutaneously in 8 week old mice. Prevention of disease was dependent upon antigen inclusion and required encapsulation of factors in MPs. Moreover, administration of this "suppressive-vaccine" boosted pancreatic lymph node and splenic regulatory T cells (Tregs), upregulated PD-1 on CD4+ and CD8+ T cells, and reversed hyperglycemia for up to 100 days in recent-onset NOD mice. Our results demonstrate that a MP-based platform can reeducate the immune system in an antigen-specific manner, augment immunomodulation compared to soluble administration of drugs, and provide a promising alternative to systemic immunosuppression for autoimmunity.

Clinical Applications of Regulatory T cells in Adoptive Cell Therapies.

Interest in adoptive T-cell therapies has been ignited by the recent clinical success of genetically-modified T cells in the cancer immunotherapy space. In addition to immune targeting for malignancies, this approach is now being explored for the establishment of immune tolerance with regulatory T cells (Tregs). Herein, we will summarize the basic science and clinical results emanating from trials directed at inducing durable immune regulation through administration of Tregs. We will discuss some of the current challenges facing the field in terms of maximizing cell purity, stability and expansion capacity, while also achieving feasibility and GMP production. Indeed, recent advances in methodologies for Treg isolation, expansion, and optimal source materials represent important strides toward these considerations. Finally, we will review the emerging genetic and biomaterial-based approaches on the horizon for directing Treg specificity to augment tissue-targeting and regenerative medicine.

Microglia from neurogenic and non-neurogenic regions display differential proliferative potential and neuroblast support.

Microglia isolated from the neurogenic subependymal zone (SEZ) and hippocampus (HC) are capable of massive in vitro population expansion that is not possible with microglia isolated from non-neurogenic regions. We asked if this regional heterogeneity in microglial proliferative capacity is cell intrinsic, or is conferred by interaction with respective neurogenic or non-neurogenic niches. By combining SEZ and cerebral cortex (CTX) primary tissue dissociates to generate heterospatial cultures, we find that exposure to the SEZ environment does not enhance CTX microglia expansion; however, the CTX environment exerts a suppressive effect on SEZ microglia expansion. Furthermore, addition of purified donor SEZ microglia to either CTX- or SEZ-derived cultures suppresses the expansion of host microglia, while the addition of donor CTX microglia enhances the over-all microglia yield. These data suggest that SEZ and CTX microglia possess intrinsic, spatially restricted characteristics that are independent of their in vitro environment, and that they represent unique and functionally distinct populations. Finally, we determined that the repeated supplementation of neurogenic SEZ cultures with expanded SEZ microglia allows for sustained levels of inducible neurogenesis, provided that the ratio of microglia to total cells remains within a fairly narrow range.

Electro-optic investigation of the surface trapping efficiency in n-alkanethiol SAM passivated GaAs(001).

The electro-optic characteristics of the semi-insulating and n(+)-type GaAs(001) surfaces passivated with n-alkanethiol self-assembled monolayers were investigated using Kelvin probe surface photovoltage (SPV) and photoluminescence (PL) techniques. Referencing the equilibrium surface barrier height established in an earlier report, SPV measurements demonstrated a significant (>100 mV) increase in the non-equilibrium band-bending potential observed under low-level photo-injection. Modeling of the SPV accounts for these observations in terms of a large (>10(4)) decrease in the hole/electron ratio of surface carrier capture cross-sections, which is suggested to result from the electrostatic potential of the interfacial dipole layer formed upon thiol chemisorption. The cross-section effects are verified in the high-injection regime based on carrier transport modeling of the PL enhancement manifested as a reduction of the surface recombination velocity.

Surface dipole layer potential induced ir absorption enhancement in n-alkanethiol SAMs on GaAs(001).

The work function of n-alkanethiol self-assembled monolayers (SAMs) prepared on the GaAs(001) surface was measured using the Kelvin probe technique yielding the SAM 2D dipole layer potential (DLP). Direct n-dependent proportionality between the DLP values and the C-H stretching mode infrared (IR) absorption intensities was observed, which supports a correspondence of reported IR enhancements with the electrostatic properties of the interface. X-ray photoelectron spectroscopy is also used to verify the work function measurements. In addition, the principal components of the refractive index tensor are shown to be n-invariant in the ordered SAM phase. Our results suggest that a local field correction to the transition dipole moment accounts for the observed increase in IR activity through an increase to the electronic polarizability.

Bromodeoxyuridine induces senescence in neural stem and progenitor cells.

Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating studies and as a means of measuring the proliferative index of various cell populations. In the absence of secondary stressors, BrdU is thought to incorporate relatively benignly into replicating DNA chains. However, we report here that a single, low-dose pulse of BrdU exerts a profound and sustained antiproliferative effect in cultured murine stem and progenitor cells. This is accompanied by altered terminal differentiation, cell morphology, and protein expression consistent with the induction of senescence. There is no evidence of a significant increase in spontaneous cell death; however, cells are rendered resistant to chemically induced apoptosis. Finally, we show that a brief in vivo BrdU regimen reduces the proliferative potential of subsequently isolated subependymal zone neurosphere-forming cells. We conclude, therefore, that BrdU treatment induces a senescence pathway that causes a progressive decline in the replication of rapidly dividing stem/progenitor cells, suggesting a novel and uncharacterized effect of BrdU. This finding is significant in that BrdU-incorporating neural stem/progenitor cells and their progeny should not be expected to behave normally with respect to proliferative potential and downstream functional parameters. This effect highlights the need for caution when results based on long-term BrdU tracking over multiple rounds of replication are interpreted. Conversely, the reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging and neurogenesis.

Bromodeoxyuridine inhibits cancer cell proliferation in vitro and in vivo.

The thymidine analog bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and has been shown to increase the susceptibility of incorporating cells to ionizing radiation. However, in the absence of secondary stressors, BrdU is thought to substitute relatively benignly for thymidine and is commonly used to "birth-date" proliferative cells. We report a novel antiproliferative effect of BrdU on cancer cells, which is independent of its role in radiosensitization. A single, brief in vitro exposure to BrdU induces a profound and sustained reduction in the proliferation rate of all cancer cells examined. Cells do not die but variably up-regulate some senescence-associated proteins as they accumulate in the G1 phase of the cell cycle. Bromodeoxyuridine also impairs the proliferative capacity of primary tumor-initiating human glioma cells and may therefore represent a means of targeting cancer stem cells. Finally, conservative in vivo BrdU regimens--in the absence of any other treatment--significantly suppress the progression of gliomas in the highly aggressive, syngeneic RG2 model. These results suggest that BrdU may have an important role as an adjunctive therapeutic for a wide variety of cancers based on new insights into its effect as a negative regulator of cell cycle progression.

Subventricular zone microglia possess a unique capacity for massive in vitro expansion.

Microglia, the resident immune cells of the brain, have recently been hypothesized to play a role both in neuronal diseases and age-related neurogenic decline, and are theorized to be modulators of adult neurogenesis. Current methods for the isolation of microglia from cultured primary brain tissue result in relatively poor yield, requiring a large tissue sample or multiple specimens to obtain a sufficient number of microglia for cell and molecular analysis. We report here a method for the repetitive isolation of microglia from established glial monolayer cultures from which it is possible to expand the initial population of microglia roughly 10,000-fold. The expanded population expresses appropriate microglial morphology and phenotype markers, and demonstrates functionally normal phagocytosis, thus providing a high-yield assay for the investigation and analysis of microglia from a single initial dissection of primary tissue. Furthermore, this massive expansion is limited to microglia derived from the subventricular zone as the fold expansion of isolatable microglia was found to be up to 20 times greater than cultures from other brain regions, indicating unique properties for this persistently neurogenic region.

Production of neurospheres from CNS tissue.

The relatively recent discovery of persistent adult neurogenesis has led to the experimental isolation and characterization of central nervous system neural stem cell populations. Protocols for in vitro analysis and expansion of neural stem cells are crucial for understanding their properties and defining characteristics. The methods described here allow for cell and molecular analysis of individual clones of cells--neurospheres--derived from neural stem/progenitor cells. Neurospheres can be cultivated from a variety of normal, genetically altered, or pathological tissue specimens, even with protracted postmortem intervals, for studies of mechanisms underlying neurogenesis, cell fate decisions, and cell differentiation. Neurosphere-forming cells hold great promise for the development of cell and molecular therapeutics for a variety of neurological diseases.

Using the neurosphere assay to quantify neural stem cells in vivo.

Since their initial description in 1992, neurospheres have appeared in some aspect of more than a thousand published studies. Despite their ubiquitous presence in the scientific literature, there is little consensus regarding the fundamental defining characteristics of neurospheres; thus, there is little agreement about what, if anything, the neurosphere assay can tell us about the relative abundance or behavior of neural stem cells in vivo. In this review we will examine some of the common features of neurospheres, and ask if these features should be interpreted as a proxy for neural stem cells. In addition, we will discuss ways in which the neurosphere assay has been used to evaluate in vivo treatment/manipulation, and will suggest appropriate ways in which neurosphere data should be interpreted, vis-à-vis the neural stem cell. Finally, we will discuss a relatively new in vitro approach, the Neural-Colony Forming Cell Assay, which provides a more meaningful method of quantifying bona fide neural stem cells without conflating them with more growth-restricted progenitor cells.

Microglia instruct subventricular zone neurogenesis.

Microglia are increasingly implicated as a source of non-neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self-renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia-conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self-renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.

Fusion of neural stem cells in culture.

An important issue in stem cell biology relates to mechanisms of cellular plasticity. Specifically, could any observed multipotency of, e.g., adult stem cells arise from true transdifferentiation or as a result of cell-cell fusion? We studied this issue using a culture paradigm of astrocyte monolayers and multipotent neurospheres generated from neonatal cerebellar cortex and the subventricular zone (SVZ). Based on fluorescence in situ hybridization (FISH), cells from these cultures were found to contain an abnormal number of sex chromosomes, suggesting that cellular fusion is a common in vitro occurrence. A Cre/lox recombination method was also exploited to further confirm the evidence of fusion. Next, we assessed the potential of fusogenic microglial involvement by combining CD11b immunolabeling with FISH sex chromosome analysis. Differentiating neurospheres were also studied from the PU.1 knockout mouse that lacks cells of myeloid origin, presumed to be a source of central nervous system microglia. Very few cells immunopositive for the microglial marker CD11b were found to be aneuploid, and there was no difference in fusion frequency between PU.1+/+ and PU.1-/- neurospheres. These results, together, suggest that stem and/or progenitor cells that generate neurons and glia in culture possess the ability to generate fused polyploidal cells, but microglial participation is not a requirement for fusion to occur. In addition to caution that should be exerted during the interpretation of in vitro neural cell plasticity, the data also suggest that novel therapeutic treatments could be designed that exploit cellular fusion in rescue paradigms for degenerating neuronal populations.

In vitro-derived "neural stem cells" function as neural progenitors without the capacity for self-renewal.

Hematopoietic stem cells have been defined by their ability to self-renew and successfully reconstitute hematopoiesis throughout the life of a transplant recipient. Neural stem cells (NSCs) are believed to exist in the regenerating regions of the brain in adult mice: the subependymal zone (SEZ) of the lateral ventricles (LVs) and the hippocampal dentate gyrus. Cells from the SEZ can be cultured to generate neurospheres or multipotent astrocytic stem cells (MASCs), both of which demonstrate the stem cell qualities of multipotency and self-renewal in vitro. Whether neurospheres and MASCs possess the true stem cell quality of functional self-renewal in vivo is unknown. The definitive tests for this unique capability are long-term engraftment and serial transplantation. Both neurospheres and MASCs transplanted into the LVs of C57BL/6 mice resulted in short-term engraftment into the recipient brain, with donor-derived migratory neuroblasts visible in the rostral migratory stream and olfactory bulb after transplantation. To test in vivo expansion/self-renewal of the transplanted cells, we attempted to reisolate donor-derived neurospheres and MASCs. Even when rigorous drug selection was used to select for rare events, no donor-derived neurospheres or MASCs could be reisolated. Furthermore, donor-derived migratory neuroblasts were not observed in the rostral migratory stream (RMS) for more than 1 month after transplantation, indicating a transient rather than long-term engraftment. Therefore, in vitro-derived neurospheres and MASCs do not function as NSCs with long-term, self-renewal capabilities in vivo but instead represent short-term neural progenitor cells as defined by an in vivo functional assay.

Ionizing radiation enhances the engraftment of transplanted in vitro-derived multipotent astrocytic stem cells.

The subependymal zone (SEZ) is a region of persistent neurogenesis in the adult mammalian brain containing a neural stem cell (NSC) pool that continuously generates migratory neuroblasts that travel in chains through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate and functionally integrate into existing neural circuitry. NSCs can be isolated from the SEZ and cultured to generate either neurospheres (NSs) or multipotent astrocytic stem cells (MASCs), with both possessing the stem cell characteristics of multipotency and self-renewal. NSs and MASCs home to the SEZ after transplantation into the lateral ventricle (LV) and contribute to neuroblast migration, with minimal engraftment into the OB observed in the adult mouse. Recent studies have compared the relatively uncharacterized NSC with the more established hematopoietic stem cell (HSC) in an effort to determine the level of stemness possessed by the NSC. Depletion of native HSCs in the bone marrow by lethal irradiation (LI) is necessary to maximize functional engraftment of donor HSCs. Our data show that the NSC pool and neuroblasts in the SEZ can be significantly and permanently depleted by exposure to LI. Attenuation of donor-derived migratory neuroblast engraftment into the OB is observed after transplantation of gfp+ MASCs into the LV of LI animals, whereas engraftment is significantly enhanced after transplantation into animals exposed to sublethal levels of ionizing radiation. By increasing receptiveness of the NSC niche through depletion of indigenous cells, the adult SEZ-RMS-OB can be used as a model to further characterize the NSC.