Clinically viable magnetic poly(lactide-co-glycolide) particles for MRI-based cell tracking.

PURPOSE: To design, fabricate, characterize, and in vivo assay clinically viable magnetic particles for MRI-based cell tracking. METHODS: Poly(lactide-co-glycolide) (PLGA) encapsulated magnetic nano and microparticles were fabricated. Multiple biologically relevant experiments were performed to assess cell viability, cellular performance, and stem cell differentiation. In vivo MRI experiments were performed to separately test cell transplantation and cell migration paradigms, as well as in vivo biodegradation. RESULTS: Highly magnetic nano (∼100 nm) and microparticles (∼1-2 µm) were fabricated. Magnetic cell labeling in culture occurred rapidly achieving 3-50 pg Fe/cell at 3 h for different particles types, and >100 pg Fe/cell after 10 h, without the requirement of a transfection agent, and with no effect on cell viability. The capability of magnetically labeled mesenchymal or neural stem cells to differentiate down multiple lineages, or for magnetically labeled immune cells to release cytokines following stimulation, was uncompromised. An in vivo biodegradation study revealed that NPs degraded ∼80% over the course of 12 weeks. MRI detected as few as 10 magnetically labeled cells, transplanted into the brains of rats. Also, these particles enabled the in vivo monitoring of endogenous neural progenitor cell migration in rat brains over 2 weeks. CONCLUSION: The robust MRI properties and benign safety profile of these particles make them promising candidates for clinical translation for MRI-based cell tracking.

Serial monitoring of endogenous neuroblast migration by cellular MRI.

Endogenous neural progenitor cell migration in vivo can be monitored using MRI-based cell tracking. The current protocol is that micron sized iron oxide particles (MPIOs) are injected into the lateral ventricle proximal to the neural stem cell niche in the brain. MPIOs are endocytosed and incorporated into the neural progenitor cell population, making them visible by gradient echo MRI. Here this new method is extended to serially quantify cell migration. Initially, in vivo cell labeling methodologies were optimized, as high susceptibility effects from the MPIOs generate substantial signal loss around the injection site, masking early migratory events. Then, using improved labeling conditions, a longitudinal study was conducted over two weeks to quantify the migration of labeled progenitor cells toward the olfactory bulb (OB). By 3 days following injection, we calculated 0.26% of the volume of the OB containing labeled cells. By 8days, this volume nearly doubled to 0.49% and plateaued. These MRI results are in accordance with our data on iron quantification from the OB and with those from purely immunohistochemical studies.

Comparative transduction efficiency of AAV vector serotypes 1-6 in the substantia nigra and striatum of the primate brain.

Vectors derived from adeno-associated virus (AAV) are promising candidates for neural cell transduction in vivo because they are nonpathogenic and achieve long-term transduction in the central nervous system. AAV serotype 2 (AAV2) is the most widely used AAV vector in clinical trials based largely on its ability to transduce neural cells in the rodent and primate brain. Prior work in rodents suggests that other serotypes might be more efficient; however, a systematic evaluation of vector transduction efficiency has not yet been performed in the primate brain. In this study, AAV viral vectors of serotypes 1-6 with an enhanced green-fluorescent protein (GFP) reporter gene were generated at comparable titers, and injected in equal amounts into the brains of Chlorocebus sabaeus. Vector injections were placed in the substantia nigra (SN) and the caudate nucleus (CD). One month after injection, immunohistochemistry for GFP was performed and the total number of GFP+ cells was calculated using unbiased stereology. AAV5 was the most efficient vector, not only transducing significantly more cells than any other serotype, but also transducing both NeuN+ and glial-fibrillary-acidic protein positive (GFAP+) cells. These results suggest that AAV5 is a more effective vector than AAV2 at delivering potentially therapeutic transgenes to the nigrostriatal system of the primate brain.

Novel neuronal phenotypes from neural progenitor cells.

We report the first isolation of progenitor cells from the hypothalamus, a derivative of the embryonic basal plate that does not exhibit neurogenesis postnatally. Neurons derived from hypothalamic progenitor cells were compared with those derived from progenitor cultures of hippocampus, an embryonic alar plate derivative that continues to support neurogenesis in vivo into adulthood. Aside from their different embryonic origins and their different neurogenic potential in vivo, these brain regions were chosen because they are populated with cells of three different categories: Category I cells are generated in both hippocampus and hypothalamus, Category II cells are generated in the hypothalamus but are absent from the hippocampus, and Category III is a cell type generated in the olfactory placode that migrates into the hypothalamus during development. Stem-like cells isolated from other brain regions, with the ability to generate neurons and glia, produce neurons of several phenotypes including gabaergic, dopaminergic, and cholinergic lineages. In the present study, we extended our observations into neuroendocrine phenotypes. The cultured neural precursors from 7-week-old rat hypothalamus readily generated neuropeptide-expressing neurons. Hippocampal and hypothalamic progenitor cultures converged to indistinguishable populations and produced neurons of all three categories, confirming that even short-term culture confers or selects for immature progenitors with enough plasticity to elaborate neuronal phenotypes usually inhibited in vivo by the local microenvironment. The range of phenotypes generated from neuronal precursors in vitro now includes the peptides found in the neuroendocrine system: corticotropin-releasing hormone, growth hormone-releasing hormone, gonadotropin-releasing hormone, oxytocin, somatostatin, thyrotropin-releasing hormone, and vasopressin.

Specific chemotaxis of magnetically labeled mesenchymal stem cells: implications for MRI of glioma.

PURPOSE: Glioblastoma multiforme (GBM) is a lethal disease marked by infiltration of cancerous cells into the surrounding normal brain. The dire outcome of GBM patients stems in part from the limitations of current neuroimaging methods. Notably, early cancer detection methodologies are lacking, without the ability to identify aggressive, metastatic tumor cells. We propose a novel approach for tumor detection using magnetic resonance imaging (MRI) based on imaging specific tumor tropism of mesenchymal stem cells (MSCs) labeled with micron-sized iron oxide particles (MPIOs). PROCEDURES: MPIO labeled and unlabeled MSCs were compared for viability, multi-lineage differentiation, and migration, where both chemotactic and chemokinetic movement were assessed in the presence of serum-free medium, serum-containing medium, and glioma-conditioned medium. MRI was performed on agarose samples, consisting of MPIO-labeled single MSCs, to confirm the capability to detect single cells. RESULTS: We determined that MPIO-labeled MSCs exhibit specific and significant chemotactic migration towards glioma-conditioned medium in vitro. Confocal fluorescence microscopy confirmed that MPIOs are internalized and do not impact important cell processes of MSCs. Lastly, MPIO-labeled MSCs appear as single distinct, dark spots on T(2)*-weighted MRI, supporting the robustness of this contrast agent for cell tracking. CONCLUSIONS: This is the first study to show that MPIO-labeled MSCs exhibit specific tropism toward tumor-secreted factors in vitro. The potential for detecting single MPIO-labeled MSCs provides rationale for in vivo extension of this methodology to visualize GBM in animal models.

Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions.

BACKGROUND: Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings. CONCLUSIONS/SIGNIFICANCE: We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells.

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinson's disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on alpha-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.

Development of the neuroendocrine hypothalamus.

The development of the neuroendocrine hypothalamus has been studied using a variety of neuroanatomical and molecular techniques. Here, the major findings that mold our understanding of hypothalamic development are reviewed. The rat hypothalamus is generated predominantly from the third ventricular neuroepithelium in a "lateral early to medial late" pattern dictated perhaps by the medially receding third ventricle. Neuroendocrine neurons seem to exhibit a delayed migrational strategy, showing relatively early birthdates, although they are located in the latest-generated, periventricular nuclei. Several homeobox genes seem to play a role in hypothalamic development, and gene knockout experiments implicate a number of genes of importance in the generation of the neuroendocrine cell type.