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Nava S.,Cell Therapy Production Unit | Lisini D.,Cell Therapy Production Unit | Pogliani S.,Cell Therapy Production Unit | Dossena M.,Laboratory of Cellular Neurobiology | And 7 more authors.
Stem Cells Translational Medicine | Year: 2015

Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysateloaded DCs as treatment for GB are ongoing. From2010 to 2014, 54 patients were enrolled in the studiesand 54 batches of DCs were prepared.Weretrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. ThenumberofmDCsobtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%–98%), and all batches showed high viability after thawing (mean ± SD: 94.6%±2.9%). Phenotype evaluation of mDCsshowed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches. © AlphaMed Press.

Cristini S.,Laboratory of Cellular Neurobiology | Alessandri G.,Laboratory of Cellular Neurobiology | Acerbi F.,Fondazione Istituto Neurologico Carlo Besta | Acerbi F.,University of Milan | And 8 more authors.
Tissue Engineering - Part C: Methods | Year: 2011

Complex microenvironmental stimuli influence neural cell properties. To study this, we developed a three-dimensional (3-D) neural culture system, composed of different populations including neurons, astrocytes, and neural stem cells (NSCs). In particular, these last-mentioned cells represent a source potentially exploitable to test drugs, to study neurodevelopment and cell-therapies for neuroregenerations. On seeding on matrigel in a medium supplemented with serum and mitogens, cells obtained from human fetal brain tissue formed 3-D self-organizing neural architectures. Immunocytochemical analysis demonstrated the presence of undifferentiated nestin+ and CD133+ cells, surrounded by β-tub-III+ and GFAP+ cells, suggesting the formation of niches containing potential human NSCs (hNSCs). The presence of hNSCs was confirmed by both neurosphere assay and RT-PCR, and their multipotentiality was demonstrated by both immunofluorescent staining and RT-PCR. Flow cytometry analysis revealed that neurosphere forming cells originating from at least two different subsets expressing, respectively, CD133 and CD146 markers were endowed with different proliferative and differentiation potential. Our data implicate that the complexity of environment within niches and aggregates of heterogeneous neural cell subsets may represent an innovative platform for neurobiological and neurodevelopmental investigations and a reservoir for a rapid expansion of hNSCs. © 2011, Mary Ann Liebert, Inc.

Cristini S.,Laboratory of Cellular Neurobiology | Navone S.,Laboratory of Cellular Neurobiology | Canzi L.,Laboratory of Cellular Neurobiology | Acerbi F.,Fondazione Istituto Neurologico Carlo Besta | And 8 more authors.
Human Molecular Genetics | Year: 2010

The study of Lesch-Nyhan-diseased (LND) human brain is crucial for understanding how mutant hypoxanthine-phosphoribosyltransferase (HPRT) might lead to neuronal dysfunction. Since LND is a rare, inherited disorder caused by a deficiency of the enzyme HPRT, human neural stem cells (hNSCs) that carry this mutation are a precious source for delineating the consequences of HPRT deficiency and for developing new treatments. In our study we have examined the effect of HPRT deficiency on the differentiation of neurons in hNSCs isolated from human LND fetal brain. We have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in dopamine (DA) biosynthetic pathway. LND hNSCs demonstrate aberrant expression of several transcription factors and DA markers. HPRT-deficient dopaminergic neurons also demonstrate a striking deficit in neurite outgrowth. These results represent direct experimental evidence for aberrant neurogenesis in LND hNSCs and suggest developmental roles for other housekeeping genes in neurodevelopmental disease. Moreover, exposure of the LND hNSCs to retinoic acid medium elicited the generation of dopaminergic neurons. The lack of precise understanding of the neurological dysfunction in LND has precluded development of useful therapies. These results evidence aberrant neurogenesis in LND hNSCs and suggest a role for HPRT gene in neurodevelopment. These cells combine the peculiarity of a neurodevelopmental model and a human, neural origin to provide an important tool to investigate the pathophysiology of HPRT deficiency and more broadly demonstrate the utility of human neural stem cells for studying the disease and identifying potential therapeutics. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

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