Engraftment and dispersion of human embryonic stem cell-derived multipotent neural stem cells in experimental stroke models

Cellular therapy for stroke has demonstrated promising functional recovery in experimental stroke models. However, the current sources of stem cells are not amenable for the needs of large-scale production and multi-centered clinical trials. Human embryonic stem cells (hESCs) have the potential to provide an indefinite and renewable source of a wide range of normal cell types. However, the isolation of homogenous and self-renewable sources of neural progenitors capable of differentiating into the principal neural lineages from hESCs remains largely unexplored. We carried out a series of experiments to isolate stable, homogenous and expandable multipotent neural stem-progenitor cell lines (NSPCs) from hESCs. The hESCs were cultured in serum free medium containing epidermal growth factor, basic fibroblastic growth factor and leukemia inhibitory growth factor. After 3 days in vitro (DIV), the cells appeared aggregated in clusters or spheres. These primary spheres were collected and replated in fresh culture media supplemented with factors. Secondary spheres were then generated and expanded for 5 to 6 passages. To ascertain self-renewal ability under clonal conditions, a single cell suspension made from dissociated spheres were plated at clonal density (10 cell/μl). After 48 hrs, plated single cells underwent a first cell division and gave rise to progeny expressing nestin, a neurepithelial stem cell marker. The single cell-derived spheres continued to grow over the 7 DIV. At this stage, all the neurospheres generated were nestin+ and did not express the pluripotency markers SSEA4, OCT4 or Nanog. The NSPCs showed stable growth and a consistent 2.7±0.2 fold increase every 5 to 7 days. The population doubling at each passage averaged at 1.4±0.1. When induced to differentiate, 36.6±2.7 of the cells expressed nestin, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% were GFAP+ astrocytes and 7.1±0.4% were oligodendrocytes and expressed galactocerebroside. The NSPCs were then evaluated in the rat middle cerebral artery occlusion (MCAO) model of stroke. Sprague Dawley rats (n=10) were subjected to 1.5 h MCAO. One week later, immunosuppressed rats were transplanted with NSPCs (2 x 105) into the ischemic boundary zone in the striatum. Animals were sacrificed 1 week later and the brains processed for histo-pathology and immunocytochemistry. Grafted NSPCs, identified with a human-specific nuclear marker, demonstrated a 14.2±3.1% survival rate and 61.2±4.7% migrated at least 200 μm away from the graft core and penetrated an average distance of 806.4±49.3 μm into the stroke-damaged tissue. They extended neurite outgrowth into the host parenchyma and did not show tumor formation. Characterization of the grafted cells demonstrated the absence of pluripotency markers and the presence of neural-specific markers. Transplanted cells expressed nestin and differentiated into TuJ1+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes with 6.5±0.9% of the neurons expressing doublecortin. We have demonstrated that under defined culture conditions, pluripotent hESCs can give rise to homogenous, expandable multipotent NSPCs. Transplantation of NCPSs into infarcted neural tissue resulted in good survival, migration and neural differentiation into the principal CNS cell types. These findings provide preliminary evidence of potential use of hES derivatives in cell therapy for stroke.