Jack Price

Tracking Transplanted Stem Cell Migration Using Bifunctional, Contrast Agent-Enhanced, Magnetic Resonance Imaging

The ability to track stem cell transplants in the brain by in vivo neuroimaging will undoubtedly aid our understanding of how these cells mediate functional recovery after neural transplantation. One major challenge for the development and refinement of stem cell transplantation is to map the spatial distribution and rate of migration in situ. Here we report a method for tracking transplanted stem cells in the ischemia-damaged rat hippocampus by magnetic resonance imaging (MRI). Before transplantation, stem cells were labeled in vitro either with a novel bifunctional contrast agent, gadolinium rhodamine dextran (GRID), identifiable by both MRI and fluorescence microscopy, or with PKH26, visible exclusively under fluorescence microscopy. At different time points following engraftment, the brains were evaluated by both histology and ex vivo MR imaging. Transplanted stem cells were identified by MRI only if prelabeled with GRID, whereas fluorescence microscopy detected transplanted cells using either label. The distribution of GRID-labeled stem cells identified by MRI corresponded to those detected using fluorescence microscopy. These results demonstrate that GRID-enhanced MRI can reliably identify transplanted stem cells and their migration in the brain.

Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study

Preferential migration of stem cells toward the site of a lesion is a highly desirable property of stem cells that allows flexibility in the site of graft implantation in the damaged brain. In rats with unilateral stroke damage, neural stem cells transplanted into the contralateral hemisphere migrate across to the lesioned hemisphere and populate the area around the ischaemic infarct. To date, the migration of neural stem cells in the damaged brain has been mainly inferred from snapshot histological images. In this study, we demonstrate that by pre-labelling neural stem cells with the bimodal contrast agent Gadolinium-RhodamIne Dextran [GRID, detectable by both magnetic resonance imaging (MRI) and fluorescent microscopy], the transhemispheric migration of transplanted neural stem cells contralateral to a stroke lesion can be followed in vivo by serial MRI and corroborated by subsequent histological analyses. Our results indicate that neural stem cells migrated from the injection tract mainly along the corpus callosum within 7 days of transplantation and extensively re-populated the peri-lesion area by 14 days following implantation. In contrast, neural stem cells transplanted into sham controls did not show any substantial migration outside of the injection tract, suggesting that the transcallosal migration observed in the stroke-lesioned animals is due to neural stem cells being attracted by the lesion site. In vivo tracking of the migration of neural stem cells responding to damage will greatly enhance our understanding of optimal transplantation strategies as well as how neural stem cells promote functional and anatomical recovery in neurological disorders.

A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke

Transplantation of neural stem cells into the brain is a novel approach to the treatment of chronic stroke disability. For clinical application, safety and efficacy of defined, stable cell lines produced under GMP conditions are required. To this end, a human neural stem cell line, CTX0E03, was derived from human somatic stem cells following genetic modification with a conditional immortalizing gene, c-mycER(TAM). This transgene generates a fusion protein that stimulates cell proliferation in the presence of a synthetic drug 4-hydroxy-tamoxifen (4-OHT). The cell line is clonal, expands rapidly in culture (doubling time 50-60 h) and has a normal human karyotype (46 XY). In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes. Transplantation of CTX0E03 in a rat model of stroke (MCAo) caused statistically significant improvements in both sensorimotor function and gross motor asymmetry at 6-12 weeks post-grafting. In addition, cell migration and long-term survival in vivo were not associated with significant cell proliferation. These data indicate that CTX0E03 has the appropriate biological and manufacturing characteristics necessary for development as a therapeutic cell line.

Association between clozapine response and allelic variation in the 5-HT2C receptor gene

A cysteine to serine substitution at amino acid 23 in the 5-HT2C receptor gene alters the pharmacological properties of the protein. We investigated this polymorphism in subjects with schizophrenia resistant to conventional neuroleptic drugs, and analysed our data for allelic association between the disease state or clinical response to the atypical antipsychotic drug, clozapine. Ninety percent of subjects who had one or more 5-HT2Cser alleles (19/21) were classified as clozapine responders compared with 59% (84/141) without this allele (χ2 = 7.7, p = 0.005), suggesting that this mutation is a predictor of good response to clozapine. There was no association between schizophrenia and the 5-HT2Cser allele, but our results indicate that the 5-HT2C receptor may contain the major site of action through which clozapine mediates its antipsychotic effects.

Interleukin-1β: a new regulator of the kynurenine pathway affecting human hippocampal neurogenesis

Increased inflammation and reduced neurogenesis have been associated with the pathophysiology of major depression. Here, we show for the first time how IL-1β, a pro-inflammatory cytokine shown to be increased in depressed patients, decreases neurogenesis in human hippocampal progenitor cells. IL-1β was detrimental to neurogenesis, as shown by a decrease in the number of doublecortin-positive neuroblasts (−28%), and mature, microtubule-associated protein-2-positive neurons (−36%). Analysis of the enzymes that regulate the kynurenine pathway showed that IL-1β induced an upregulation of transcripts for indolamine-2,3-dioxygenase (IDO), kynurenine 3-monooxygenase (KMO), and kynureninase (42-, 12- and 30-fold increase, respectively, under differentiating conditions), the enzymes involved in the neurotoxic arm of the kynurenine pathway. Moreover, treatment with IL-1β resulted in an increase in kynurenine, the catabolic product of IDO-induced tryptophan metabolism. Interestingly, co-treatment with the KMO inhibitor Ro 61-8048 reversed the detrimental effects of IL-1β on neurogenesis. These observations indicate that IL-1β has a critical role in regulating neurogenesis whereas affecting the availability of tryptophan and the production of enzymes conducive to toxic metabolites. Our results suggest that inhibition of the kynurenine pathway may provide a new therapy to revert inflammatory-induced reduction in neurogenesis.

The support of neural stem cells transplanted into stroke-induced brain cavities by PLGA particles

Stroke causes extensive cellular loss that leads to a disintegration of the afflicted brain tissue. Although transplanted neural stem cells can recover some of the function lost after stroke, recovery is incomplete and restoration of lost tissue is minimal. The challenge therefore is to provide transplanted cells with matrix support in order to optimise their ability to engraft the damaged tissue. We here demonstrate that plasma polymerised allylamine (ppAAm)-treated poly(D,L-lactic acid-co-glycolic acid) (PLGA) scaffold particles can act as a structural support for neural stem cells injected directly through a needle into the lesion cavity using magnetic resonance imaging-derived co-ordinates. Upon implantation, the neuro-scaffolds integrate efficiently within host tissue forming a primitive neural tissue. These neuro-scaffolds could therefore be a more advanced method to enhance brain repair. This study provides a substantial step in the technology development required for the translation of this approach.

Glucocorticoid-Related Molecular Signaling Pathways Regulating Hippocampal Neurogenesis

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. We, therefore, investigated the molecular signaling pathways mediating the effects of cortisol on proliferation, neuronal differentiation, and astrogliogenesis, in an immortalized human hippocampal progenitor cell line. In addition, we examined the molecular signaling pathways activated in the hippocampus of prenatally stressed rats, characterized by persistently elevated glucocorticoid levels in adulthood. In human hippocampal progenitor cells, we found that low concentrations of cortisol (100 nM) increased proliferation (+16%), decreased neurogenesis into microtubule-associated protein 2 (MAP2)-positive neurons (−24%) and doublecortin (Dcx)-positive neuroblasts (−21%), and increased differentiation into S100β-positive astrocytes (+23%). These effects were dependent on the mineralocorticoid receptor (MR) as they were abolished by the MR antagonist, spironolactone, and mimicked by the MR-agonist, aldosterone. In contrast, high concentrations of cortisol (100 μM) decreased proliferation (−17%) and neuronal differentiation into MAP2-positive neurons (−22%) and into Dcx-positive neuroblasts (−27%), without regulating astrogliogenesis. These effects were dependent on the glucocorticoid receptor (GR), blocked by the GR antagonist RU486, and mimicked by the GR-agonist, dexamethasone. Gene expression microarray and pathway analysis showed that the low concentration of cortisol enhances Notch/Hes-signaling, the high concentration inhibits TGFβ-SMAD2/3-signaling, and both concentrations inhibit Hedgehog signaling. Mechanistically, we show that reduced Hedgehog signaling indeed critically contributes to the cortisol-induced reduction in neuronal differentiation. Accordingly, TGFβ-SMAD2/3 and Hedgehog signaling were also inhibited in the hippocampus of adult prenatally stressed rats with high glucocorticoid levels. In conclusion, our data demonstrate novel molecular signaling pathways that are regulated by glucocorticoids in vitro, in human hippocampal progenitor cells, and by stress in vivo, in the rat hippocampus.

Emx2 Promotes Symmetric Cell Divisions and a Multipotential Fate in Precursors from the Cerebral Cortex

Distinct sets of precursor cells generate the mammalian cerebral cortex. During neurogenesis most precursors are specified to generate a single cell type and only few are multipotent. The cell-intrinsic molecular determinants of these distinct lineages are not known. Here we describe that retroviral transduction of the transcription factor Emx2 in precursors from the cerebral cortex results in a significant increase of large clones that are generated mostly by symmetric cell divisions and contain multiple cell types, comprising neurons and glial cells. Thus, Emx2 is the first cell-intrinsic determinant able to instruct CNS precursors towards a multipotential fate. To evaluate the role of endogenous Emx2 in cortical precursors, we examined cell division in Emx2-/- mice. These analyses further supported the role of endogenous Emx2 in the regulation of symmetric cell divisions in the developing cortex.

Role for the kinase SGK1 in stress, depression, and glucocorticoid effects on hippocampal neurogenesis

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms mediating these effects are poorly understood. Here we identify the glucocorticoid receptor (GR) target gene, serum- and glucocorticoid-inducible kinase 1 (SGK1), as one such mechanism. Using a human hippocampal progenitor cell line, we found that a small molecule inhibitor for SGK1, GSK650394, counteracted the cortisol-induced reduction in neurogenesis. Moreover, gene expression and pathway analysis showed that inhibition of the neurogenic Hedgehog pathway by cortisol was SGK1-dependent. SGK1 also potentiated and maintained GR activation in the presence of cortisol, and even after cortisol withdrawal, by increasing GR phosphorylation and GR nuclear translocation. Experiments combining the inhibitor for SGK1, GSK650394, with the GR antagonist, RU486, demonstrated that SGK1 was involved in the cortisol-induced reduction in progenitor proliferation both downstream of GR, by regulating relevant target genes, and upstream of GR, by increasing GR function. Corroborating the relevance of these findings in clinical and rodent settings, we also observed a significant increase of SGK1 mRNA in peripheral blood of drug-free depressed patients, as well as in the hippocampus of rats subjected to either unpredictable chronic mild stress or prenatal stress. Our findings identify SGK1 as a mediator for the effects of cortisol on neurogenesis and GR function, with particular relevance to stress and depression.

Evidence that retroviruses integrate into post-replication host DNA.

We have studied the question of whether a retrovirus integrates into the chromosomal DNA of the host cell before or after the DNA is replicated during the S phase of the cell cycle. We have infected single NIH‐3T3 cells with BAG, a replication‐incompetent retroviral vector which encodes the lacZ gene, then observed the clones derived from these cells to discover whether all the cells carry a copy of the proviral DNA. We have discovered that only half of the progeny of an infected cell carries a copy of the provirus. This indicates that the virus only integrates into post‐replication DNA. We discuss the implications of this result for applications of retroviruses, such as gene therapy and cell lineage, which use them as vehicles for gene transfer into stem cells.