RESUMEN
Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células-Madre Neurales , Humanos , Reproducibilidad de los Resultados , Criopreservación , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Control de CalidadRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14-15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. METHODS: By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a "mitogen-independent" phenotype (I-GSCs) from patient's tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs' critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher's exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs' key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett's multiple comparison test with the distribution of survival compared by Kaplan-Meier method. RESULTS: Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs' transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs' tumorigenic and invasive ability, thus increasing survival. CONCLUSION: We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs' lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Mitógenos/metabolismo , Mitógenos/farmacología , Mitógenos/uso terapéutico , Células Madre Neoplásicas/metabolismo , Fenotipo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMEN
BACKGROUND: Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. METHODS AND FINDINGS: We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). INTERPRETATION: Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. FUND: This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.
Asunto(s)
Autorrenovación de las Células , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Biomarcadores , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Técnica del Anticuerpo Fluorescente , Xenoinjertos , Humanos , Inmunohistoquímica , Pérdida de Heterocigocidad , Ratones , Clasificación del Tumor , Estadificación de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Células Madre Neoplásicas/patologíaRESUMEN
Stem cells are emerging as a therapeutic option for incurable diseases, such as Amyotrophic Lateral Sclerosis (ALS). However, critical issues are related to their origin as well as to the need to deepen our knowledge of the therapeutic actions exerted by these cells. Here, we investigate the therapeutic potential of clinical-grade human neural stem cells (hNSCs) that have been successfully used in a recently concluded phase I clinical trial for ALS patients (NCT01640067). The hNSCs were transplanted bilaterally into the anterior horns of the lumbar spinal cord (four grafts each, segments L3-L4) of superoxide dismutase 1 G93A transgenic rats (SOD1 rats) at the symptomatic stage. Controls included untreated SOD1 rats (CTRL) and those treated with HBSS (HBSS). Motor symptoms and histological hallmarks of the disease were evaluated at three progressive time points: 15 and 40 days after transplant (DAT), and end stage. Animals were treated by transient immunosuppression (for 15 days, starting at time of transplantation). Under these conditions, hNSCs integrated extensively within the cord, differentiated into neural phenotypes and migrated rostro-caudally, up to 3.77 ± 0.63 cm from the injection site. The transplanted cells delayed decreases in body weight and deterioration of motor performance in the SOD1 rats. At 40DAT, the anterior horns at L3-L4 revealed a higher density of motoneurons and fewer activated astroglial and microglial cells. Accordingly, the overall survival of transplanted rats was significantly enhanced with no rejection of hNSCs observed. We demonstrated that the beneficial effects observed after stem cell transplantation arises from multiple events that counteract several aspects of the disease, a crucial feature for multifactorial diseases, such as ALS. The combination of therapeutic approaches that target different pathogenic mechanisms of the disorder, including pharmacology, molecular therapy and cell transplantation, will increase the chances of a clinically successful therapy for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Células-Madre Neurales/trasplante , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/terapia , Animales , Diferenciación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Estimación de Kaplan-Meier , Masculino , Microglía/citología , Microglía/metabolismo , Neuronas Motoras/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Médula Espinal/patología , Superóxido Dismutasa/genéticaRESUMEN
Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the iduronate 2-sulfatase (IDS) enzyme, causing progressive neurodegeneration in patients. Neural stem cells (NSCs) derived from the IDS-ko mouse can recapitulate MPSII pathogenesis in vitro. In differentiating IDS-ko NSCs and in the aging IDS-ko mouse brain, glial degeneration precedes neuronal degeneration. Here we show that pure IDS-ko NSC-derived astrocytes are selectively able to drive neuronal degeneration when cocultured with healthy neurons. This phenotype suggests concurrent oxidative damage with metabolic dysfunction. Similar patterns were observed in murine IDS-ko animals and in human MPSII brains. Most importantly, the mutant phenotype of IDS-ko astrocytes was reversed by low oxygen conditions and treatment with vitamin E, which also reversed the toxic effect on cocultured neurons. Moreover, at very early stages of disease we detected in vivo the development of a neuroinflammatory background that precedes astroglial degeneration, thus suggesting a novel model of MPSII pathogenesis, with neuroinflammation preceding glial degeneration, which is finally followed by neuronal death. This hypothesis is also consistent with the progression of white matter abnormalities in MPSII patients. Our study represents a novel breakthrough in the elucidation of MPSII brain pathogenesis and suggests the antioxidant molecules as potential therapeutic tools to delay MPSII onset and progression.
Asunto(s)
Mucopolisacaridosis II/patología , Neuroglía/patología , Estrés Oxidativo , Adolescente , Animales , Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/patología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Técnicas de Cocultivo , Femenino , Humanos , Iduronato Sulfatasa/metabolismo , Lactante , Inflamación/complicaciones , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Mutación/genética , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/patología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuroglía/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxígeno/farmacología , Fenotipo , Ratas , Vitamina E/farmacología , Sustancia Blanca/patología , Adulto JovenRESUMEN
In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.
Asunto(s)
Transformación Celular Neoplásica/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Receptor EphA2/genética , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Regulación hacia Abajo/genética , Efrina-A1/genética , Efrina-A1/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Receptor EphA2/metabolismoRESUMEN
Skin stem/progenitor cells (SKPs) derive from the dermis and in culture can generate mesodermal and neural progenies. To investigate their potential for the treatment of brain diseases, we first injected SKPs into the brain of syngeneic mice. Brain histology indicated that most SKPs remained undifferentiated and clustered at the injection site, while, in vitro, 17% of SKPs expressed neural markers, as assessed by flow cytometry. After labeling with magnetodendrimers, murine and human SKPs were detected by magnetic resonance imaging even 5 months after brain injection. To evaluate their therapeutic potential on malignant gliomas, IL-4 SKPs (i.e. SKPs transduced by a lentiviral vector carrying the cDNA of the anti-glioma cytokine interleukin-4) were injected into GL261 experimental gliomas. IL-4-SKPs prolonged significantly the survival of tumor-bearing mice: furthermore, GL261 gliomas attracted SKPs originally injected into the contralateral hemisphere. Thus, prolonged survival, capacity for transgene expression, and lack of uncontrolled proliferation suggest that SKPs warrant further consideration as therapeutic tools for brain tumors and, possibly, other neurological disorders.
Asunto(s)
Neoplasias Encefálicas/terapia , Glioma/terapia , Piel/citología , Trasplante de Células Madre , Animales , Biomarcadores/análisis , Encéfalo/metabolismo , Neoplasias Encefálicas/inmunología , Movimiento Celular , Glioma/inmunología , Humanos , Interleucina-4/genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Twitcher (GALC(twi/twi)) is the murine model of globoid cell leukodystrophy (GLD or Krabbe disease), a disease caused by mutations of the lysosomal enzyme galactocerebrosidase (GALC). To verify the therapeutic potential on twitcher of neural stem/progenitor cells (NSPC), we transduced them with a GALC lentiviral vector. Brain injection of NSPC-GALC increased survival of GALC(twi/twi) from 36.1 +/- 4.1 to 52.2 +/- 5.6 days (P < 0.0001). Detection of GALC activity and flow cytometry showed that NSPC-GALC and NSPC expressing the green fluorescent protein were attracted to the posterior area of twitcher brain, where demyelination occurs first. GALC(twi/twi) microglia, also more abundant in posterior regions of the brain, released significant amounts of the cytotoxic cytokine TNF-alpha when matched with NSPC-GALC. Thus, in murine GLD, and possibly in other demyelinating diseases, NSPC are attracted to regions of active demyelination but have limited survival and therapeutic potential if attacked by activated macrophages/microglia.
Asunto(s)
Leucodistrofia de Células Globoides/terapia , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Microglía/metabolismo , Neuronas/trasplante , Trasplante de Células Madre , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Galactosilceramidasa/genética , Galactosilceramidasa/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Ratones , Neuronas/citología , Transducción GenéticaRESUMEN
Malignant gliomas are associated with risk of thromboembolism, but the molecular link between tumor and peripheral pro-coagulant status has not been elucidated. Vascular Endothelial Growth Factor (VEGF), tissue-type Plasminogen Activator (tPA), Plasminogen Activator Inhibitor-1 (PAI-1) and lipoprotein (lp) (a) influence the pro-coagulant status. To assess whether the presence of the tumor influenced the peripheral levels of VEGF, tPA, PAI-1 and lp(a), we studied the expression and secretion of VEGF, tPA, PAI-1 and lp(a) in glioma specimens, in peripheral blood and in primary glioma-derived cultures. We also measured lp(a), VEGF, tPA and PAI-1 in the peripheral circulation of patients, before and after surgery for glioma. VEGF, tPA and PAI-1 were expressed in glioma specimens. Glioma cells were indeed a major source of tPA and PAI-1; these molecules were significantly more expressed in glioma than in patient's blood cells. Lp(a) was rarely expressed in glioma specimens and not expressed in blood cells. In glioma, VEGF, tPA and PAI-1 were localized mainly in tumor cells; tPA was localized also in the extracellular matrix and PAI-1 in tumor vascular lumen. Glioma cells were indeed able to produce and release VEGF, tPA and PAI-1. After surgery, peripheral levels of VEGF and PAI-1 were increased, while tPA and lp(a) were unchanged. The great amount of VEGF, tPA and PAI-1 produced by glioma could influence peripheral levels of these molecules. The partial resection of the tumor by surgery was not able to decrease plasma levels of these molecules.
Asunto(s)
Neoplasias Encefálicas/química , Glioma/química , Inhibidor 1 de Activador Plasminogénico/análisis , Trombosis/diagnóstico , Activador de Tejido Plasminógeno/análisis , Factor A de Crecimiento Endotelial Vascular/análisis , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/cirugía , Femenino , Glioma/complicaciones , Glioma/cirugía , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/sangre , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/análisis , Trombosis/etiología , Activador de Tejido Plasminógeno/sangre , Activador de Tejido Plasminógeno/genética , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Pediatric brain tumors may originate from cells endowed with neural stem/precursor cell properties, growing in vitro as neurospheres. We have found that these cells can also be present in adult brain tumors and form highly infiltrating gliomas in the brain of immunodeficient mice. Neurospheres were grown from three adult brain tumors and two pediatric gliomas. Differentiation of the neurospheres from one adult glioblastoma decreased nestin expression and increased that of glial and neuronal markers. Loss of heterozygosity of 10q and 9p was present in the original glioblastoma, in the neurospheres and in tumors grown into mice, suggesting that PTEN and CDKN2A alterations are key genetic events in tumor initiating cells with neural precursor properties.