Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinson's disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH؉ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on ␣-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ͉ dopamine ͉ Parkinson's disease ͉ synuclein ͉ tyrosine hydroxylase D egeneration of dopamine (DA) neurons in the substantia nigra (SN) and the consequent deficit of DA release in the striatum and other target areas appear to be responsible for the characteristic manifestations of Parkinson's disease (PD). Although substantial improvements result from the systemic administration of the DA precursor L-DOPA or DA agonists, such pharmacological replacement does not address the etiology of the disease, provide a permanent redress of the pathophysiology, or forestall progression of the degenerative process. It does, however, support the idea that DA provided by exogenous replacement cells might be therapeutic, a notion verified in rodents (1-3) and monkeys (4 -6), where grafts of fetal DA neurons led to improvements in biochemical and behavioral indices of DA deficiency. However, in graft studies, the improvements in Parkinsonism have been limited and variable (see review in ref. 7). Therefore, we hypothesized that, in addition to DA replenishment, PD treatment should also restore functional equilibrium in the host SN-striatal system. A clinically relevant strategy might be to implant human neural stem cells (hNSCs) and progenitor cells constitutively capable of multiple actions, including neural differentiation and cytokine secretion, and allow them to develop within the PDaffected brains of nonhuman ...

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.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genomewide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote longterm pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epitheliumderived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.secreted proteins | human embryonic stem cells | pigment epitheliumderived factor | automated protein expression | high throughput M any physiological processes are regulated by secreted proteins, making the extracellular proteome a rich source of molecules for understanding basic pathways involved in normal physiological function, development, and human disease. Although many examples of bioactive secreted proteins have been identified, recent technologic advances allow us to directly screen this proteomic diversity for new factors influencing important biological pathways. Efforts to survey the extracellular proteome using conditioned media from transiently transfected cell lines have shown that novel factors can be identified in a prospective screening approach. Lin et al. (1) evaluated ∼3,400 genes encompassing predicted secreted proteins or extracellular domains of single-pass transmembrane proteins. Each gene of interest was expressed by transient transfection using liposomal reagents in 293 T cells, and relative quantitation was determined by ELISA using an affinity tag. Conditioned media from this set was used to screen for biological activity in various assays. However, such screens are complicated by secondary metabolites, the release of cytoplasmic proteins by cell lysis, and variable and undefined levels of extracellular proteins. To overcome these challenges, we have created a collection of purified and physically characterized extracel...

Clinically, cerebral vessel occlusion is rarely permanent because of spontaneous or thrombolytic therapy-mediated reperfusion. These results have clinical implications indicating a much extended therapeutic window for transplantation of human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs; 24 hours after stroke as opposed to the 5-hour window with tissue plasminogen activator [tPA]). In addition, there is potential for a synergistic effect by combining hiPSC-NSC transplantation with tPA to attenuate stroke's adverse effects.

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Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.