ABSTRACT
Neural stem cells (NSCs) are believed to repair brain damage primarily through cell replacement: i.e., the ability to regenerate lost neurons and glia in a site-specific fashion. The neural stem cell line, MHP36, has been shown to have this capacity, but we have little idea of the molecular mechanisms that control the differentiation of such cells during brain repair. In this study we show that an early event in the differentiation of MHP36 cells, both in vivo and in vitro, is the loss of expression of the intermediate filament protein, nestin. We use a co-culture assay to show that loss of nestin is fast, being detectable after just 1 h and complete in 4 h, and is controlled by proteasome degradation rather than down-regulation of de novo nestin synthesis. We also show that nestin loss is regulated by Notch, and mediated by cell contact.
Subject(s)
Acetylcysteine/analogs & derivatives , Cysteine Endopeptidases/metabolism , Intermediate Filament Proteins/metabolism , Membrane Proteins/physiology , Multienzyme Complexes/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Signal Transduction/physiology , Stem Cells/metabolism , Acetylcysteine/pharmacology , Animals , Cell Count/methods , Cell Differentiation/physiology , Cell Line , Cells, Cultured , Cerebral Cortex/cytology , Coculture Techniques/methods , Cricetinae , Cricetulus , Cysteine Proteinase Inhibitors/pharmacology , Embryo, Mammalian , Fluorescent Dyes , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Green Fluorescent Proteins , Hippocampus/metabolism , Immunohistochemistry/methods , Intermediate Filament Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , Nerve Tissue Proteins/genetics , Nestin , Neuroglia/metabolism , Organic Chemicals , Phosphopyruvate Hydratase/metabolism , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Receptors, Notch , Reverse Transcriptase Polymerase Chain Reaction/methods , Stem Cell Transplantation/methods , Temperature , Time Factors , Transfection/methodsABSTRACT
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.
Subject(s)
Brain Damage, Chronic/physiopathology , Cell Movement/physiology , Dominance, Cerebral/physiology , Image Enhancement/methods , Infarction, Middle Cerebral Artery/physiopathology , Magnetic Resonance Imaging/methods , Neurons/transplantation , Stem Cell Transplantation , Animals , Brain Damage, Chronic/pathology , Cell Differentiation , Cell Line , Contrast Media/administration & dosage , Corpus Callosum/pathology , Dextrans , Gadolinium DTPA , Hippocampus/cytology , Infarction, Middle Cerebral Artery/pathology , Microscopy, Confocal , Microscopy, Fluorescence , Rats , Rats, Sprague-Dawley , RhodaminesABSTRACT
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.