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Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1-3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.

Background: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man., Materials and Methods: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss., Results: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI., Conclusion: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.

Background: We have proposed that an increased interaction between monocyte/macrophages and blood vessel endothelium predisposes subjects to strokes. The effect of chronic monocyte activation on the development of cerebral infarcts was thus studied in rats after provocation of a modified local Swartzman reaction, in brain vasculature., Materials and Methods: Two weeks after an IV bolus of bacillus Calmette-Guérin (BCG), we studied spontaneous superoxide production, integrin expression, endothelial adhesion of monocytes and the neurological symptoms, brain histology, and cytokine immunoreactivity after a provocative dose of LPS (30-300 microg/rat i.c.v.)., Results: Monocyte migration into the brain was stimulated by BCG priming. The incidence of paralysis and death in response to LPS was markedly increased in BCG-primed rats. Histological evaluation of the brains of neurologically impaired and moribund animals revealed intravascular thrombosis and pale and hemorrhagic infarcts. Infiltrates of leukocytes expressing immunoreactive IL-1:, IL-6, and TNF-alpha were found around blood vessels, cerebral ventricles, and meninges, and were accompanied by a profound microglial expression of IL1P, endothelial expression of IL-6, and expression of TNF-alpha and TNF-R 1 in glia and neurons of cortex and hippocampus. Treatment (2 x 100 microg/10 ,I, i.c.v.) with recombinant human (rh-)TNF 55kDa receptor completely prevented, and treatment with rh-IL- I receptor antagonist significantly decreased the incidence of paralysis and death in response to BCG + LPS. The improvement of neurological symptoms was accompanied by reduced histological damage and supppression of IL-1P/ expression in the brain tissue., Conclusions: The data demonstrate that chronic monocyte activation predisposes subjects to thrombosis and hemorrhage via an exaggerated release of proinflammatory cytokines.