Barbara Krynska

A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages.

Glial fibrillary acidic protein (GFAP)‐positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP‐positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of βIII‐tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) βIII‐tubulin‐ and nestin‐positive cells and (ii) GFAP‐ and nestin‐positive cells. These cells, when subsequently cultured in serum‐free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down‐regulated and βIII‐tubulin expression became robust. Cells of glial lineage differentiated by down‐regulating nestin expression and up‐regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

BackgroundAlthough features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.MethodsFollowing methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.ResultsIn vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.ConclusionOur results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Reactivation of human neurotropic JC virus expressing oncogenic protein in a recurrent glioblastoma multiforme

Examination of the primary tumor of glioblastoma multiforme and its recurrence for their association with JC virus revealed that, while the viral genome is present in both initial and recurrent tumors, expression of the viral oncoprotein T‐antigen occurs only in the recurrent tumor cells. Accordingly, the level of inducible cellular transcription factors, including the p65 subunit of NF‐κB and YB‐1, which have the ability to stimulate JCV gene expression, was found to be higher in the recurrent tumor cells. These observations suggest that induction of the regulatory factors after resection of the primary tumor may have reactivated JC virus gene expression and led to redevelopment of the tumor in brain. Ann Neurol 2000;48:932–936

Binding and Internalization of Iron Oxide Nanoparticles Targeted To Nuclear Oncoprotein

A targeted nanoconjugate is being developed for non-invasive detection of gene expression in cells expressing the JC virus oncoprotein, T-antigen, which has been associated with medulloblastoma and other cancers. JC virus T-antigen localizes predominantly to the nucleus via a classical monopartite nuclear localization signal (NLS). An antibody fragment which recognizes JC virus T-antigen was attached to cross-linked dextran coated iron oxide nanoparticles. Radiolabeled conjugates were added to mouse medulloblastoma cells expressing the target T-antigen to test their ability to bind to tumor cells and be internalized by the cells. All conjugates containing targeting antibody bound to cells and were internalized, with increasing levels over time. There was no difference in cell binding or internalization among conjugates containing 2, 4, 6 or 8 antibody fragments per nanoparticle. Conjugates with only nonspecific antibody on nanoparticles, or unconjugated nonspecific antibody, had significantly lower total binding and internalization than conjugates with targeting antibody. Unconjugated targeting antibody had equivalent or lower cell uptake compared with targeted nanoparticle conjugates. Specificity of uptake was demonstrated by >80% reduction of nanoconjugate uptake in the presence of 100 fold excess of unconjugated antibody. The presence of a membrane translocation peptide (Tat) on the nanoparticles in addition to targeting antibody did not improve nanoconjugate internalization over the internalization caused by the antibody alone. This antibody nanoconjugate demonstrates feasibility of targeting a nuclear protein and suggests that a minimum number of antibody fragments per nanoparticle are sufficient for achieving binding specificity and efficient uptake into living cells.

JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP‐2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein‐producing CG‐4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP‐Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen‐activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein‐induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions. J. Cell. Physiol. 227: 3119–3127, 2012.

Bone marrow-derived mesenchymal stem cells undergo JCV T-antigen mediated transformation and generate tumors with neuroectodermal characteristics.

There is now accumulating evidence showing that some tumors may arise from transformed stem cells. In this study we demonstrate that adult bone marrow- derived mesenchymal stem cells (MSCs) undergo neoplastic transformation induced by the human polyomavirus JCV, early protein, T-antigen, and are tumorigenic when transplanted into the flanks of Nude mice as compared to non-transformed MSCs. Histologically, the tumors are heterogeneous with mesenchymal and neural crest characteristics as evidenced by expression of the neural crest markers p75, SOX-10, and S-100, with populations of tumor cells exhibiting characteristics of primitive neuroectodermal cells. In addition, a subset of T-antigen positive tumor cells exhibit a high proliferation index as detected by Ki-67 labeling, and co-express CD133, a marker which is expressed on cancer stem cells. These results show that tumors with neuroectodermal characteristics may arise from transformation of MSCs, a globally accessible adult stem cell with multipotent differentiation capacity. In light of earlier reports on the association of JCV with a broad variety of human tumors, our data suggests that T-antigen transformation of adult stem cells with a multipotent capacity can serve as a possible common origin for some of these cancers, and offers a novel model for oncogenesis.

Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model

UNLABELLED Rebuilding of infarcted myocardium by mesenchymal stem cells (MSCs) has not been successful because of poor cell survival due in part to insufficient blood supply after myocardial infarction (MI). We hypothesize that targeted delivery of vascular endothelial growth factor (VEGF) to MI can help regenerate vasculature in support of MSC therapy in a rat model of MI. VEGF-encapsulated immunoliposomes targeting overexpressed P-selectin in MI tissue were infused by tail vein immediately after MI. One week later, MSCs were injected intramyocardially. The cardiac function loss was moderated slightly by targeted delivery of VEGF or MSC treatment. Targeted VEGF+MSC combination treatment showed highest attenuation in cardiac function loss. The combination treatment also increased blood vessel density (80%) and decreased collagen content in post-MI tissue (33%). Engraftment of MSCs in the combination treatment group was significantly increased and the engrafted cells contributed to the restoration of blood vessels. FROM THE CLINICAL EDITOR VEGF immunoliposomes targeting myocardial infarction tissue resulted in significantly higher attenuation of cardiac function loss when used in combination with mesenchymal stem cells. MSCs were previously found to have poor ability to restore cardiac tissue, likely as a result of poor blood supply in the affected areas. This new method counterbalances that weakness by the known effects of VEGF, as demonstrated in a rat model.

Neuronal and astrocytic cells, obtained after differentiation of human neural GFAP-positive progenitors, present heterogeneous expression of PrPc

PrP(c) is a cellular isoform of the prion protein with an unknown normal function. One of the putative physiological roles of this protein is its involvement in cell differentiation. Recently, in vitro and in vivo studies showed that GFAP-positive cells have characteristics of stem/progenitor cells that generate neurons and glia. We used an in vitro model of human neurogenesis from GFAP-positive progenitor cells to study the expression of PrP(c) during neural differentiation. Semi-quantitative multiplex-PCR assay and Western blot analysis revealed a significant increase of PRNP expression level in differentiated cells compared to undifferentiated cell population. As determined by immunocytochemistry followed by a quantitative image analysis, the PrP(c) level increased significantly in neuronal cells and did not increase significantly in glial cells. Of note, glial and neuronal cells showed a very large heterogeneity of PrP(c) expression. Our results provide the basis for studying the role of PrP(c) in cell differentiation and neurogenesis from human GFAP-positive progenitor cells.

A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.