Identification and early characterization of genetically modified NGF-producing neural stem cells grafted into the injured adult rat brain.

OBJECTIVE: To investigate whether genetically modified mouse neural stem cells (NSC) expressing recombinant human nerve growth factor (rhNGF) and transplanted in chemically injured rat brain, can survive and eventually acquire phenotypic characteristics of early nerve cells. METHODS: Stably high expression of rhNGF in NSC was obtained by a new lentivirus-mediated expression system. To test the effectiveness of hNGF secreted by rhNGF-NSC, hereby we performed either a bioassay for neurite outgrowth in PC12 rat cells or immunoblot analysis for TrkA, the high-affinity NGF receptor, from engineered NSC. rhNGF and mock-NSC were grafted into adult injured rats striatum and 3 days later, animals were killed, and brains were removed and examined by immunohistochemical analysis. RESULTS: The results showed that rhNGF-producing NSC cultured for extended period of time release bioactive hNGF in the culture media which promotes PC12 neuronal differentiation and correlates with the up-regulation of TrkA. rhNGF-NSC transplanted into the injured brain can survive, produce hNGF and induce the expression of NGF receptors, p75(NTR) and TrkA. DISCUSSION: In vitro and in vivo experiments confirmed the ability of rhNGF-NSC to secrete bioactive hNGF. Our data provide by means of genetically modified rhNGF-producing NSC, a useful experimental tool to test the potential clinical effectiveness of trophic factors relevant to central nervous system (CNS).

In vitro analysis of mouse neural stem cells genetically modified to stably express human NGF by a novel multigenic viral expression system.

OBJECTIVES: The purpose of this study was to characterize mouse neural stem cells (NSC) transduced by a multigenic lentiviral vector (LV) and stably express recombinant human nerve growth factor (rhNGF). We obtained NSC-derived cell lines which express human NGF in relevant amount to exploit their ability for therapeutic applications. METHODS: We constructed advanced multigenic LV vectors which contain a tricistronic cassette to express simultaneously up to three independent genes: (1) rhNGF (beta subunit); (2) EGFP (enhanced green fluorescent protein) and (3) Neo(R) (neomycin antibiotic resistance gene). Lentiviruses were obtained by transfecting LV constructs plus helper plasmids in human embryonic kidney (HEK)-293T packaging cells. Lentiviral virions were released in culture media and subsequent used to infect mouse NSC. Genetycin 418-resistant NSC were obtained after 1 month of selection in the presence of antibiotic (G418). Levels of human NGF secreted by rhNGF-NSC were determined by ELISA (enzyme-linked immunosorbent assay). Features of multipotentiality of engineered NSC-derived cell lines versus naive cells (control-NSC) were assessed by immunocytochemical analysis in differentiation conditions. Self-renewal of NSC was tested by neurospheres assay (NSA). RESULTS: Levels of secreted human NGF, from conditioned media obtained by rhNGF-NSC cultures, were found to be elevated in either proliferation or differentiation conditions as compared with control cells. Moreover, released hNGF demonstrated biologic activity on PC12 cells by a functional test of neurite outgrowth. Immunocytochemical analysis revealed that engineered NSC showed to be all positives for EGFP. After thirty passages in vitro in the presence of G418, engineered cells versus naive NSC cultures maintained their multipotentiality to differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, it was found that rhNGF-NSC-derived neurons expressed choline acetyltransferase (ChAT) and displayed an enhanced axonal growth. NSA showed an altered sphere forming frequency either in rhNGF-NSC or both group of control NSC. DISCUSSION: Lentivirus-mediated rhNGF gene transfer into NSC was achieved using a new version of LV vectors. We obtained rhNGF-NSC-derived cell lines which released hNGF to high levels in the culture medium. The expression of neural differentiation markers, like microtubule associated protein 2 (MAP2) (a/b), glial fibrillary acidic protein (GFAP) and chondroitin sulphate proteoglycan (NG2), was not enhanced in rhNGF-NSC compared with control cells. Secreted hNGF increased axonal sprouting by rhNGF-NSC-derived neurons which was associated with ChAT expression. rhNGF-NSC may prospectively be good candidates for the treatment of either neurodegenerative diseases such as Alzheimer disease or central nervous system injuries.

The immunosuppressive effect of mesenchymal stromal cells on B lymphocytes is mediated by membrane vesicles.

The immunomodulatory properties of mesenchymal stromal cells are the subject of increasing interest and of widening clinical applications, but the reproducibility of their effects is controversial and the underlying mechanisms have not been fully clarified. We investigated the transfer of membrane vesicles, a recently recognized pathway of intercellular communication, as possible mediator of the interaction between mesenchymal stromal cells and B lymphocytes. Mesenchymal stromal cells exhibited a strong dose-dependent inhibition of B-cell proliferation and differentiation in a CpG-stimulated peripheral blood mononuclear cell coculture system. We observed that these effects could be fully reproduced by membrane vesicles isolated from mesenchymal stromal cell culture supernatants in a dose-dependent fashion. Next, we evaluated the localization of fluorescently labeled membrane vesicles within specific cell subtypes both by flow cytometry and by confocal microscopy analysis. Membrane vesicles were found to be associated with stimulated B lymphocytes, but not with other cell phenotypes (T lymphocytes, dendritic cells, natural killer cells), in peripheral blood mononuclear cell culture. These results suggest that membrane vesicles derived from mesenchymal stromal cells are the conveyors of the immunosuppressive effect on B lymphocytes. These particles should be further evaluated as immunosuppressive agents in place of the parent cells, with possible advantages in term of standardization, safety, and feasibility.

Tumorigenic potential of olfactory bulb-derived human adult neural stem cells associates with activation of TERT and NOTCH1.

BACKGROUND: Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation. METHODOLOGY/PRINCIPAL FINDINGS: Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with maintenance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity. CONCLUSIONS/SIGNIFICANCE: Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.

Transplantation of foetal neural stem cells into the rat hippocampus during trimethyltin-induced neurodegeneration.

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair.

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation.

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.