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

Casalbore P, Budoni M, Ricci-Vitiani L, Cenciarelli C, Petrucci G, Milazzo L, Montano N, Tabolacci E, Maira G, Larocca LM, Pallini R - PLoS ONE (2009)

Bottom Line: However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy.

ABSTRACT

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.

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Summary of experimental design.The OB was obtained from adult patients who underwent neurosurgical operations. On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133. The nestin-expressing cells colocalize glial fibrillary acid protein (GFAP). These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells. In the external plexiform layer, a few proliferating cells (n, 200–300) are detected by Ki67 labeling. Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Primary neurospheres were dissociated into single cells and plated one cell per mini-well. Clonal cell cultures were established by dissociation of secondary neurospheres. Clonal cultures from each OB were passaged up to P30 in mitogens. NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo. At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP. The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents.
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pone-0004434-g001: Summary of experimental design.The OB was obtained from adult patients who underwent neurosurgical operations. On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133. The nestin-expressing cells colocalize glial fibrillary acid protein (GFAP). These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells. In the external plexiform layer, a few proliferating cells (n, 200–300) are detected by Ki67 labeling. Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Primary neurospheres were dissociated into single cells and plated one cell per mini-well. Clonal cell cultures were established by dissociation of secondary neurospheres. Clonal cultures from each OB were passaged up to P30 in mitogens. NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo. At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP. The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents.

Mentions: The OB was harvested from five adult patients who had undergone surgery for extracerebral benign lesions (Table S1). Unilateral division of the OB, which is often necessary for surgical exposure, is well tolerated by the patients because the olfactory function is preserved. Immunohistochemistry showed that the human adult OB contains a few hundreds of putative NS/PCs (Fig. 1). Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Under these conditions, the OB cells generated primary neurospheres with latencies that ranged from 6 to 8 weeks (Table S2). An exception was Case OB3 where primary neurosphere formation was observed as early as 3 weeks of culturing. Primary neurospheres were dissociated into single cells and plated one cell per mini-well (Fig. 1). Clonal cell cultures were established by dissociation of secondary neurospheres and passaged up to P30 in mitogens. The ability to form spheres after serial passaging, the number and diameter of spheres produced during each passage, and the cloning efficiency were similar among different cultures (Table S2). Upon removal of mitogens and serum exposure, the NS/PC cultures obtained from four of the OB specimens arrested their growth and gave rise to adherent cells that expressed neuronal, astrocytic, and oligodendrocytic markers (Figs. 2A–2B). In contrast, OB3 NS/PC cultures lost both growth factor dependence and potential to differentiate as neurons between P4 and P6. Notably, the OB3 patient harbored a meningioma adjacent to the OB. Losing growth factor dependence and capacity to differentiate by NSCs may indicate transformation. On soft agar assay, an in vitro correlate of transformation, the OB3 NS/PCs developed colonies (Fig. S2). Then, we assessed tumorigenicity in vivo using hetero- and orthotopic xenografts in immunodeficient mice. Two to 3 weeks after grafting, NS/PCs from all OB3 cultures developed subcutaneous tumors with a 88.6 percent take (Fig. 2C and Table 1). Histologically, these tumors showed glioblastoma features, like perinecrotic pseudo-palisading and vascular proliferation. Tumorigenicity of OB3 NS/PCs was demonstrated both at early (P6) and at late passages (P30). Subcutaneous injection of OB1, OB2, OB4, and OB5 NS/PCs resulted in amorphous tissue grafts with embedded scarce cells showing heterogenous morphology and occasional GFAP staining without neoplastic features (not shown). Intracerebral injection of OB3 NS/PCs also produced tumors which developed at 63.1 percent of injection sites by 4 to 6 weeks after grafting (Fig. 2D and Table 1). Histologically, these tumors featured anaplastic astrocytoma with predilection for growing into the ventricles. Intracerebral injection of OB1, OB2, OB4, and OB5-derived NS/PCs did not result in tumor formation (Table 1).


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

Casalbore P, Budoni M, Ricci-Vitiani L, Cenciarelli C, Petrucci G, Milazzo L, Montano N, Tabolacci E, Maira G, Larocca LM, Pallini R - PLoS ONE (2009)

Summary of experimental design.The OB was obtained from adult patients who underwent neurosurgical operations. On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133. The nestin-expressing cells colocalize glial fibrillary acid protein (GFAP). These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells. In the external plexiform layer, a few proliferating cells (n, 200–300) are detected by Ki67 labeling. Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Primary neurospheres were dissociated into single cells and plated one cell per mini-well. Clonal cell cultures were established by dissociation of secondary neurospheres. Clonal cultures from each OB were passaged up to P30 in mitogens. NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo. At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP. The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637538&req=5

pone-0004434-g001: Summary of experimental design.The OB was obtained from adult patients who underwent neurosurgical operations. On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133. The nestin-expressing cells colocalize glial fibrillary acid protein (GFAP). These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells. In the external plexiform layer, a few proliferating cells (n, 200–300) are detected by Ki67 labeling. Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Primary neurospheres were dissociated into single cells and plated one cell per mini-well. Clonal cell cultures were established by dissociation of secondary neurospheres. Clonal cultures from each OB were passaged up to P30 in mitogens. NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo. At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP. The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents.
Mentions: The OB was harvested from five adult patients who had undergone surgery for extracerebral benign lesions (Table S1). Unilateral division of the OB, which is often necessary for surgical exposure, is well tolerated by the patients because the olfactory function is preserved. Immunohistochemistry showed that the human adult OB contains a few hundreds of putative NS/PCs (Fig. 1). Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Under these conditions, the OB cells generated primary neurospheres with latencies that ranged from 6 to 8 weeks (Table S2). An exception was Case OB3 where primary neurosphere formation was observed as early as 3 weeks of culturing. Primary neurospheres were dissociated into single cells and plated one cell per mini-well (Fig. 1). Clonal cell cultures were established by dissociation of secondary neurospheres and passaged up to P30 in mitogens. The ability to form spheres after serial passaging, the number and diameter of spheres produced during each passage, and the cloning efficiency were similar among different cultures (Table S2). Upon removal of mitogens and serum exposure, the NS/PC cultures obtained from four of the OB specimens arrested their growth and gave rise to adherent cells that expressed neuronal, astrocytic, and oligodendrocytic markers (Figs. 2A–2B). In contrast, OB3 NS/PC cultures lost both growth factor dependence and potential to differentiate as neurons between P4 and P6. Notably, the OB3 patient harbored a meningioma adjacent to the OB. Losing growth factor dependence and capacity to differentiate by NSCs may indicate transformation. On soft agar assay, an in vitro correlate of transformation, the OB3 NS/PCs developed colonies (Fig. S2). Then, we assessed tumorigenicity in vivo using hetero- and orthotopic xenografts in immunodeficient mice. Two to 3 weeks after grafting, NS/PCs from all OB3 cultures developed subcutaneous tumors with a 88.6 percent take (Fig. 2C and Table 1). Histologically, these tumors showed glioblastoma features, like perinecrotic pseudo-palisading and vascular proliferation. Tumorigenicity of OB3 NS/PCs was demonstrated both at early (P6) and at late passages (P30). Subcutaneous injection of OB1, OB2, OB4, and OB5 NS/PCs resulted in amorphous tissue grafts with embedded scarce cells showing heterogenous morphology and occasional GFAP staining without neoplastic features (not shown). Intracerebral injection of OB3 NS/PCs also produced tumors which developed at 63.1 percent of injection sites by 4 to 6 weeks after grafting (Fig. 2D and Table 1). Histologically, these tumors featured anaplastic astrocytoma with predilection for growing into the ventricles. Intracerebral injection of OB1, OB2, OB4, and OB5-derived NS/PCs did not result in tumor formation (Table 1).

Bottom Line: However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus