Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.

Background and Purpose-We sought to evaluate the long-term effects of bone marrow stromal cell (BMSC) treatment on retired breeder rats with stroke. Methods-Female retired breeder rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) followed by an injection of 2ϫ10 6 male BMSCs (nϭ8) or phosphate-buffered saline (nϭ11) into the ipsilateral internal carotid artery at 1 day after stroke. The rats were humanely killed 1 year later. Functional tests, in situ hybridization, and histochemical and immunohistochemical staining were performed. Results-Significant recovery of neurological deficits was found in BMSC-treated rats beginning 2 weeks after cell injection compared with control animals. The beneficial effects of cell transplantation persisted for at least 1 year (PϽ0.01). In situ hybridization for the Y chromosome showed that donor cells survived in the brains of recipient rats, among which 22.3Ϯ1.95% of cells expressed the astrocyte marker glial fibrillary acidic protein, 16.8Ϯ2.13% expressed the neuronal marker microtubule-associated protein 2, and 5.5Ϯ0.42% and Ͻ1% of cells colocalized with the microglial marker IB4 and the endothelial cell marker von Willebrand factor, respectively. Only very few BMSCs, however, were found in peripheral organs such as the heart, lung, liver, spleen, and kidney in recipient rats. BMSCs significantly reduced axonal loss (PϽ0.01), the thickness of the lesion scar wall (PϽ0.01), and the number of Nogo-A-positive cells (PϽ0.05) along the scar border; meanwhile, synaptophysin expression (PϽ0.05) was significantly increased in BMSC-treated ischemic brains compared with control untreated brains. Conclusions-The beneficial effects of BMSCs on ischemic brain tissue persisted for at least 1 year. Most surviving BMSCs were present in the ischemic brain, but very few were found in other organs. The long-term improvement in functional outcome may be related to the structural and molecular changes induced by BMSCs.

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Background and Purpose-Motor recovery after stroke is associated with neuronal reorganization in bilateral hemispheres. We investigated contralesional corticospinal tract remodeling in the brain and spinal cord in rats after stroke and treatment of bone marrow stromal cells. Methods-Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion. Phosphate-buffered saline or bone marrow stromal cells were injected into a tail vein 1 day postischemia. An adhesive removal test was performed weekly to monitor functional recovery. Threshold currents of intracortical microstimulation on the left motor cortex for evoking bilateral forelimb movements were measured 6 weeks after stroke. When intracortical microstimulation was completed, biotinylated dextran amine was injected into the left motor cortex to anterogradely label the corticospinal tract. At 4 days before euthanization, pseudorabies virus-152-EGFP and 614-mRFP were injected into left or right forelimb extensor muscles, respectively. All animals were euthanized 8 weeks after stroke. Results-In normal rats (nϭ5), the corticospinal tract showed a unilateral innervation pattern. In middle cerebral artery occlusion rats (nϭ8), our data demonstrated that: 1) stroke reduced the stimulation threshold evoking ipsilateral forelimb movement; 2) EGFP-positive pyramidal neurons were increased in the left intact cortex, which were labeled from the left stroke-impaired forelimb; and 3) biotinylated dextran amine-labeled contralesional axons sprouted into the denervated spinal cord. Bone marrow stromal cells significantly enhanced all 3 responses (nϭ8, PϽ0.05). Conclusions-Our data demonstrated that corticospinal tract fibers originating from the contralesional motor cortex sprout into the denervated spinal cord after stroke and bone marrow stromal cells treatment, which may contribute to functional recovery.