Intracerebroventricular transplantation of human iPSC-derived neural stem cells (hiPSC-NSCs) into neonatal mice.

Human neural stem cells (hNSCs) hold great promises for the development of cell-based therapies for neurodegenerative diseases, given their capability to provide immunomodulatory and trophic support and to replace, to a limited extent, damaged, or lost cells. Human NSCs are under clinical evaluation for the treatment of several neurodegenerative diseases. Still, issues related to the large-scale production of clinical-grade fetal hNSCs and their allogeneic nature-requiring immunosuppressive regimens-have hampered their full exploitation as therapeutics. NSCs derived from human induced pluripotent stem cells (hiPSCs) provide a valuable alternative to fetal hNSCs since they can be generated from autologous or HLA-matched donors expanded for large-scale clinical-grade production, and are amenable for gene addition/gene editing strategies, thus potentially addressing CNS diseases of genetic origin. The prospective use of hiPSC-derived NSCs (hiPSC-NSCs) for CNS-directed therapies demands a careful evaluation of the efficacy and safety of these cell populations in animal models. Here, we describe a protocol for the transplantation and phenotypical characterization of hiPSC-NSCs in neonatal immunodeficient mice. This protocol is relevant to assessing the safety and the efficacy of hiPSC-NSC transplantation to target early-onset neurodegenerative or demyelinating CNS diseases.

Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis.

Genetic deficiency of ß-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System.

Glial cells (astrocytes, oligodendrocytes, and microglia) are emerging as key players in several physiological and pathological processes of the central nervous system (CNS). Astrocytes and oligodendrocytes are not only supportive cells that release trophic factors or regulate energy metabolism, but they also actively modulate critical neuronal processes and functions in the tripartite synapse. Microglia are defined as CNS-resident cells that provide immune surveillance; however, they also actively contribute to shaping the neuronal microenvironment by scavenging cell debris or regulating synaptogenesis and pruning. Given the many interconnected processes coordinated by glial cells, it is not surprising that both acute and chronic CNS insults not only cause neuronal damage but also trigger complex multifaceted responses, including neuroinflammation, which can critically contribute to the disease progression and worsening of symptoms in several neurodegenerative diseases. Overall, this makes glial cells excellent candidates for targeted therapies to treat CNS disorders. In recent years, the application of gene editing technologies has redefined therapeutic strategies to treat genetic and age-related neurological diseases. In this review, we discuss the advantages and limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing in the treatment of neurodegenerative disorders, focusing on the development of viral- and nanoparticle-based delivery methods for in vivo glial cell targeting.

In vitro Validation of Chimeric ß-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.

Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disease (LSD) caused by inherited defects of the ß-galactosylceramidase (GALC) gene. The infantile forms display a rapid and aggressive central and peripheral nervous system (CNS and PNS) dysfunction. No treatments are available for GLD patients. Effective gene therapy (GT) strategies for GLD require a safe and widespread delivery of the functional GALC enzyme to all affected tissues/organs, and particularly to the CNS. The use of chimeric lysosomal enzymes with increased secretion and enhanced transport across the blood-brain barrier (BBB) that boost the efficacy of GT approaches in pre-clinical models of similar neurodegenerative LSDs may benefit GLD as well. Here, we tested the safety and biological efficacy of chimeric GALC enzymes engineered to express an alternative signal peptide (iduronate-2-sulfatase - IDSsp) and the low-density lipoprotein receptor (LDLr)-binding domain from the Apolipoprotein E II (ApoE II) in GLD murine neural and hematopoietic stem/progenitor cells and progeny, which are relevant cells types in the context of in vivo and ex vivo GT platforms. We show that the lentiviral vector-mediated expression of the chimeric GALC enzymes is safe and leads to supranormal enzymatic activity in both neural and hematopoietic cells. The IDSsp.GALC shows enhanced expression and secretion in comparison to the unmodified GALC. The chimeric GALC enzymes produced by LV-transduced cells reduce intracellular galactosylceramide (GalCer) storage and effectively cross-correct GLD murine neurons and glial cells, indicating that the transgenic enzymes are delivered to lysosomes, efficiently secreted, and functional. Of note, the expression of LDLr and LDLr-related proteins in GLD neurons and glial cells supports the exploitation of this system to enhance the GALC supply in affected CNS cells and tissues. These in vitro studies support the use of chimeric GALC enzymes to develop novel and more effective GT approaches for GLD.

Novel bicistronic lentiviral vectors correct ß-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.

The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or ß-subunits of the lysosomal ß-Hexosaminidase enzyme. In physiological conditions, α- and ß-subunits combine to generate ß-Hexosaminidase A (HexA, αß) and ß-Hexosaminidase B (HexB, ßß). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and ß-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and ß-subunits, leading to supranormal levels of ß-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of ß-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or ß-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.

Multicenter, randomised, open-label, non-comparative phase 2 trial on the efficacy and safety of the combination of bevacizumab and trabectedin with or without carboplatin in women with partially platinum-sensitive recurrent ovarian cancer.

BACKGROUND: Trabectedin, in addition to its antiproliferative effect, can modify the tumour microenvironment and this could be synergistic with bevacizumab. The efficacy and safety of trabectedin and bevacizumab ± carboplatin have never been investigated. METHODS: In this phase 2 study, women progressing between 6 and 12 months since their last platinum-based therapy were randomised to Arm BT: bevacizumab, trabectedin every 21 days, or Arm BT+C: bevacizumab, trabectedin and carboplatin every 28 days, from cycles 1 to 6, then trabectedin and bevacizumab as in Arm BT. Primary endpoints were progression-free survival rate (PFS-6) and severe toxicity rate (ST-6) at 6 months, assuming a PFS-6 ≤35% for BT and ≤40% for BT+C as not of therapeutic interest and, for both arms, a ST-6 ≥ 30% as unacceptable. RESULTS: BT+C (21 patients) did not meet the safety criteria for the second stage (ST-6 45%; 95%CI: 23%-69%) but PFS-6 was 85% (95%CI: 62%-97%). BT (50 patients) had 75% PFS-6 (95%CI: 60%-87%) and 16% ST-6 (95%CI 7%-30%). CONCLUSIONS: BT compared favourably with other platinum- and non-platinum-based regimens. The combination with carboplatin needs to be assessed further in a re-modulated safer schedule to confirm its apparent strong activity. CLINICAL TRIAL REGISTRATION: NCT01735071 (Clinicaltrials.gov).

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

[The ciliopathies--Polycystic Kidney Disease]. / Le Malattie del Ciglio--Rene Policistico.

The primary cilium is an organelle composed of microtubules that emerges from the cell surface. Its inappropriate structure or function leads to the onset of a series of disease conditions collectively defined as ciliopathies, a class of genetic disorders with a variety of manifestations in different organs. In the kidneys the ciliopathies manifest with renal cysts and propotype diseases are autosomal dominant and autosomal recessive polycystic kidney diseases (ADPKD and ARPKD, respectively) . ADPKD is one of the most frequent genetic disorders affecting 1: 500 to 1: 1000 at birth. It manifests primarily with the formation of cysts in both kidneys that increase in number and size during the life of an individual, eventually causing loss of renal function. ADPKD is caused by mutations in the genes PKD1 (85% of cases) or PKD2 (15% of cases), encoding for polycystin-1 (PC-1) and polycystin-2 (PC-2) respectively. The PC-1/PC-2 complex was found on the primary cilium and elsewhere. Here we summarize recent work from our and other labs suggesting that defective planar cell polarity and cellular shape might underly PKD and additional work suggesting that defective glucose metabolism is a feature of this disease. This is the summary of a presentation delivered during a recent meeting on the genetics of renal diseases.

Combined gene/cell therapies provide long-term and pervasive rescue of multiple pathological symptoms in a murine model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by deficient activity of ß-galactocerebrosidase (GALC). The infantile forms manifest with rapid and progressive central and peripheral demyelination, which represent a major hurdle for any treatment approach. We demonstrate here that neonatal lentiviral vector-mediated intracerebral gene therapy (IC GT) or transplantation of GALC-overexpressing neural stem cells (NSC) synergize with bone marrow transplant (BMT) providing dramatic extension of lifespan and global clinical-pathological rescue in a relevant GLD murine model. We show that timely and long-lasting delivery of functional GALC in affected tissues ensured by the exclusive complementary mode of action of the treatments underlies the outstanding benefit. In particular, the contribution of neural stem cell transplantation and IC GT during the early asymptomatic stage of the disease is instrumental to enhance long-term advantage upon BMT. We clarify the input of central nervous system, peripheral nervous system and periphery to the disease, and the relative contribution of treatments to the final therapeutic outcome, with important implications for treatment strategies to be tried in human patients. This study gives proof-of-concept of efficacy, tolerability and clinical relevance of the combined gene/cell therapies proposed here, which may constitute a feasible and effective therapeutic opportunity for children affected by GLD.

Gene delivery targeted to oligodendrocytes using a lentiviral vector.

BACKGROUND: Most leukodystrophies result from mutations in genes expressed in oligodendrocytes that may cause autonomous loss of function of cell structural proteins. Therefore, effective gene delivery to oligodendrocytes is necessary to develop future treatments. MATERIALS: To achieve this, we cloned a lentiviral vector in which the enhanced green fluorescent protein (EGFP) expression was driven by the oligodendrocyte specific 2,3-cyclic nucleotide 3-phosphodiesterase promoter. The vector was inserted into C57BL/6 neonatal mouse brain by combined intraventricular and parenchymal injections. RESULTS: Assessment of EGFP expression revealed a widespread distribution, specifically in cells of the oligodendrocyte linage, starting from postnatal day 6 (P6) in the subventricular zone and spreading through migrating oligodendrocyte precursors. By P30, it was detectable throughout the brain and persisted for at least 3 months, showing an increase both in the number of expressing cells and in intensity over time. EGFP expression was restricted to oligodendrocyte linage cells. On average, 20.3 ± 2.56% of all oligodendrocytes in different central nervous system areas were EGFP-positive, with regional variations. CONCLUSIONS: Lentiviral gene delivery using an oligodendrocyte-specific promoter may achieve widespread and long-lasting expression selectively in oligodendrocytes, offering a possibility for gene therapy in certain leukodystrophies, although the relatively low rates of oligodendrocyte transduction are a limitation that remains to be overcome.

The galactocerebrosidase enzyme contributes to maintain a functional neurogenic niche during early post-natal CNS development.

We report a novel role for the lysosomal galactosylceramidase (GALC), which is defective in globoid cell leukodystrophy (GLD), in maintaining a functional post-natal subventricular zone (SVZ) neurogenic niche. We show that proliferation/self-renewal of neural stem cells (NSCs) and survival of their neuronal and oligodendroglial progeny are impaired in GALC-deficient mice. Using drugs to modulate inflammation and gene transfer to rescue GALC expression and activity, we show that lipid accumulation resulting from GALC deficiency acts as a cell-autonomous pathogenic stimulus in enzyme-deficient NSCs and progeny before upregulation of inflammatory markers, which later sustain a non-cell-autonomous dysfunction. Importantly, we provide evidence that supply of functional GALC provided by neonatal intracerebral transplantation of NSCs ameliorates the functional impairment in endogenous SVZ cells. Insights into the mechanism/s underlying GALC-mediated regulation of early post-natal neurogenic niches improve our understanding of the multi-component pathology of GLD. The occurrence of a restricted period of SVZ neurogenesis in infancy supports the implications of our study for the development of therapeutic strategies to treat this severe pediatric neurodegenerative disorder.

Site-specific integration and tailoring of cassette design for sustainable gene transfer.

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

Neural stem cell gene therapy ameliorates pathology and function in a mouse model of globoid cell leukodystrophy.

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of ß-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit.

Gene therapy for lysosomal storage disorders.

INTRODUCTION: Lysosomal storage disorders (LSDs) encompass more than 50 distinct diseases, caused by defects in various aspects of lysosomal function. Neurodegeneration and/or dysmyelination are the hallmark of roughly 70% of LSDs. Gene therapy represents a promising approach for the treatment of CNS manifestations in LSDs, as it has the potential to provide a permanent source of the deficient enzyme, either by direct injection of vectors or by transplantation of gene-corrected cells. In this latter approach, the biology of neural stem/progenitor cells and hematopoietic cells might be exploited. AREAS COVERED: Based on an extensive literature search up until March 2011, the author reviews and discusses the progress, the crucial aspects and the major challenges towards the development of novel gene therapy strategies aimed to target the CNS, with particular attention to direct intracerebral gene delivery and transplantation of neural stem/progenitor cells. EXPERT OPINION: The implementation of viral vector delivery systems with specific tropism, regulated transgene expression, low immunogenicity and low genotoxic risk and the improvement in isolation and manipulation of relevant cell types to be transplanted, are fundamental challenges to the field. Also, combinatorial strategies might be required to achieve full correction in LSDs with neurological involvement.

Coordinated involvement of cathepsins S, D and cystatin C in the commitment of hematopoietic stem cells to dendritic cells.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

Robust generation of oligodendrocyte progenitors from human neural stem cells and engraftment in experimental demyelination models in mice.

BACKGROUND: Cell-based therapy holds great promises for demyelinating diseases. Human-derived fetal and adult oligodendrocyte progenitors (OPC) gave encouraging results in experimental models of dysmyelination but their limited proliferation in vitro and their potential immunogenicity might restrict their use in clinical applications. Virtually unlimited numbers of oligodendroglial cells could be generated from long-term self-renewing human (h)-derived neural stem cells (hNSC). However, robust oligodendrocyte production from hNSC has not been reported so far, indicating the need for improved understanding of the molecular and environmental signals controlling hNSC progression through the oligodendroglial lineage. The aim of this work was to obtain enriched and renewable cultures of hNSC-derived oligodendroglial cells by means of epigenetic manipulation. METHODOLOGY/PRINCIPAL FINDINGS: We report here the generation of large numbers of hNSC-derived oligodendroglial cells by concurrent/sequential in vitro exposure to combinations of growth factors (FGF2, PDGF-AA), neurotrophins (NT3) and hormones (T3). In particular, the combination FGF2+NT3+PDGF-AA resulted in the maintenance and enrichment of an oligodendroglial cell population displaying immature phenotype (i.e., proliferation capacity and expression of PDGFRalpha, Olig1 and Sox10), limited self-renewal and increased migratory activity in vitro. These cells generate large numbers of oligodendroglial progeny at the early stages of maturation, both in vitro and after transplantation in models of CNS demyelination. CONCLUSIONS/SIGNIFICANCE: We describe a reliable method to generate large numbers of oligodendrocytes from a renewable source of somatic, non-immortalized NSC from the human foetal brain. We also provide insights on the mechanisms underlying the pro-oligodendrogenic effect of the treatments in vitro and discuss potential issues responsible for the limited myelinating capacity shown by hNSC-derived oligodendrocytes in vivo.

Human neural stem cells ameliorate autoimmune encephalomyelitis in non-human primates.

OBJECTIVE: Transplanted neural stem/precursor cells (NPCs) display peculiar therapeutic plasticity in vivo. Although the replacement of cells was first expected as the prime therapeutic mechanism of stem cells in regenerative medicine, it is now clear that transplanted NPCs simultaneously instruct several therapeutic mechanisms, among which replacement of cells might not necessarily prevail. A comprehensive understanding of the mechanism(s) by which NPCs exert their therapeutic plasticity is lacking. This study was designed as a preclinical approach to test the feasibility of human NPC transplantation in an outbreed nonhuman primate experimental autoimmune encephalomyelitis (EAE) model approximating the clinical and complex neuropathological situation of human multiple sclerosis (MS) more closely than EAE in the standard laboratory rodent. METHODS: We examined the safety and efficacy of the intravenous (IV) and intrathecal (IT) administration of human NPCs in common marmosets affected by human myelin oligodendrocyte glycoprotein 1-125-induced EAE. Treatment commenced upon the occurrence of detectable brain lesions on a 4.7T spectrometer. RESULTS: EAE marmosets injected IV or IT with NPCs accumulated lower disability and displayed increased survival, as compared with sham-treated controls. Transplanted NPCs persisted within the host central nervous system (CNS), but were also found in draining lymph nodes, for up to 3 months after transplantation and exhibited remarkable immune regulatory capacity in vitro. INTERPRETATION: Herein, we provide the first evidence that human CNS stem cells ameliorate EAE in nonhuman primates without overt side effects. Immune regulation (rather than neural differentiation) is suggested as the major putative mechanism by which NPCs ameliorate EAE in vivo. Our findings represent a critical step toward the clinical use of human NPCs in MS.

Monitoring disease evolution and treatment response in lysosomal disorders by the peripheral benzodiazepine receptor ligand PK11195.

Microglia activation and neuroinflammation play a pivotal role in the pathogenesis of lysosomal storage disorders (LSD) affecting the central nervous system (CNS), which are amenable to treatment by hematopoietic stem cell transplantation (HSCT). HSCT efficacy relies on replacing the intra- and extra-vascular hematopoietic cell compartments, including CNS microglia, with a cell population expressing the functional enzyme. Non-invasive and quantitative assessment of microglia activation and of its reduction upon HSCT might allow for evaluation of disease evolution and response to treatment in LSD. We here demonstrate that microglia activation can be quantified ex vivo and in vivo by PET using the peripheral benzodiazepine receptor ligand PK11195 in two models of LSD. Furthermore, we show a differential PBR binding following microglia replacement by donor cells in mice undergoing HSCT. Our data indicates that PBR ligands constitute valuable tools for monitoring the evolution and the response to treatment of LSD with CNS involvement, and enable us to evaluate whether the turnover between endogenous and donor microglia following HSCT could be adequate enough to delay disease progression.

Specific determination of beta-galactocerebrosidase activity via competitive inhibition of beta-galactosidase.

BACKGROUND: The determination of cellular beta-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for beta-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of beta-galactocerebrosidase activity can be performed following complete inhibition of beta-galactosidase activity. METHODS: We performed the assay using 2-7.5 microg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-beta-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO(3). Reactions were incubated for 30 min at 37 degrees C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (lambda(ex) 360 nm, lambda(em) 446 nm). RESULTS: AgNO(3) was a competitive inhibitor of beta-galactosidase [inhibition constant (K(i)) = 0.12 micromol/L] and completely inhibited beta-galactosidase activity when used at a concentration of 11 micromol/L. Under this condition, the beta-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect beta-galactocerebrosidase activity in as little as 2 microg cell protein extract or 7.5 microg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC(-/-) hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. CONCLUSIONS: The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.