Induction of dopaminergic neurons from growth factor expanded neural stem/progenitor cell cultures derived from human first trimester forebrain.

Multipotent stem/progenitor cells derived from human first trimester forebrain can be expanded as free-floating aggregates, so called neurospheres. These cells can differentiate into neurons, astrocytes and oligodendrocytes. In vitro differentiation protocols normally yield gamma-aminobutyric acid-immunoreactive neurons, whereas only few tyrosine hydroxylase (TH) expressing neurons are found. The present report describes conditions under which 4-10% of the cells in the culture become TH immunoreactive (ir) neurons within 24h. Factors including acidic fibroblast growth factor (aFGF) in combination with agents that increase intracellular cyclic AMP and activate protein kinase C, in addition to a substrate that promotes neuronal differentiation appear critical for efficient TH induction. The cells remain THir after trypsinization and replating, even when their subsequent culturing takes place in the absence of inducing factors. Consistent with a dopaminergic phenotype, mRNAs encoding aromatic acid decarboxylase, but not dopamine-beta-hydroxylase were detected by quantitative real time RT-PCR. Ten weeks after the cells had been grafted into the striatum of adult rats with unilateral nigrostriatal lesions, only very few of the surviving human neurons expressed TH. Our data suggest that a significant proportion of expandable human neural progenitors can differentiate into TH-expressing cells in vitro and that they could be useful for drug and gene discovery. Additional experiments, however, are required to improve the survival and phenotypic stability of these cells before they can be considered useful for cell replacement therapy in Parkinson's disease.

Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression.

Neural stem cells have emerged as a promising therapeutic tool in CNS disease and injuries. In the clinical setting, cultured human neural stem/progenitor cells (hNSC) are an attractive possibility for transplantation to the damaged brain. However, transplantation of hNSC requires toxic immunosuppressive treatment to avoid rejection. The aim of the current study was to evaluate if shortening the duration of immunosuppression by cyclosporin A would affect hNSC survival and differentiation after transplantation to the site of a focal brain injury in the rat. hNSC were xenografted to the hippocampus and the medial limit of an experimentally induced cortical contusion. The animals received immunosuppression for either 6 or 3 weeks or no immunosuppression. The status of the grafted human cells was analysed by immunohistochemistry. No statistically significant differences were observed between the two immunosuppressed groups regarding graft survival, migration or proliferation at 6 weeks post-transplantation. In contrast, the graft survival was extremely poor in the non-immunosuppressed group. Furthermore, the expression of the differentiation markers nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) in the transplanted cells did not differ significantly between the two immunosuppressed groups. Moreover, a fourth group of eight animals that were immunosuppressed for 3 weeks were allowed to survive for 6 months. Five of these rats demonstrated robust graft survival in the hippocampus and scattered cells in the cortex. This study demonstrates the importance of immunosuppression but also the possibility of shortening immunosuppression without impacting on the phenotype of the grafted hNSC.

MHC expression after human neural stem cell transplantation to brain contused rats.

Human neural stem cells survive and improve motor function after transplantation to the contused brain. However, the transplants might be rejected and that depends on the graft immunogenicity, the host immunological status and the immunosuppression strategy. We transplanted human neural stem cells to rats with brain contusion and analyzed the donor and host MHC antigen expression and the effect of a short-term immunosuppression with cyclosporine. In vitro human neural stem cells expressed only MHC-II antigens. This expression was down-regulated 6 weeks after transplantation. The host response was characterized by an increased MHC-II expression which was down-regulated by a longer term of immunosuppression. These findings are novel and necessary in order to understand the immunogenicity of human neural stem cell grafts.

Neuroprotection by human neural progenitor cells after experimental contusion in rats.

Neural progenitor/stem cells (HNPC) have been suggested to contribute essential trophic factors and promote survival of degenerating neurons after traumatic brain injury. For these reasons we hypothesize that the addition of HNPC to a post-injury region could possibly protect injured neurons. Experimental brain contusions were carried out in 18 rats. Immediately post-injury, rats were injected with 0.1 ml of medium (n=8), dead cells (n=4), or live cells (n=6) in the medial border of the lesion. The rats were sacrificed 6 days post-surgery and evaluated by immunohistochemistry using a human nuclear marker (huN), hematoxylin and Fluoro-Jade (FJ). Human neural stem cells showed engraftment detectable by positive huN staining in 5/6 animals. The non-grafted animal was excluded from further analyses. Those given dead HNPC or medium showed no detectable huN immunoreactivity. A statistical comparison between the numbers of FJ positive degenerating endogenous neurons was made between rats receiving vehicle and dead cells to evaluate whether the presence of human cells would increase neuronal degeneration in comparison to vehicle alone. The rats receiving vehicle had a median of 117.5 FJ positive cells and dead progenitor cell recipients 175.0 per counted section (P<0.05, Mann-Whitney). Consequently, the animals receiving dead human cells were chosen as controls to the animals receiving live progenitor cells. The rats that received live HNPC demonstrated significantly fewer FJ positive cells per counted section than controls (58.0 vs. 175.0, P<0.01, Mann-Whitney). The post-traumatic perilesional environment allowed for the engraftment of live HNPC. The stepwise analysis indicated that host neuronal degeneration was higher in animals transplanted with non-viable human neuronal progenitor cells than in those receiving vehicle, indicating a bystander effect from introducing foreign antigen. In contrast, transplantation of viable progenitor cells attenuated neuronal degeneration, indicating that a potentially beneficial effect in progenitor cell transplantation is not limited to restoration by transplanted cells, but also improving survival of host cells.