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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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Characterization and grafting of serum-stimulated NS/PCs grown as adherent cultures.A, Growth curves (left) and immunophenotype (right) of NS/PCs (P6) cultured in medium containing mitogens and 5% serum (serum-stimulated, SS-NS/PCs). B, Growth curves (left), absorbance test (right), and immunophenotype (right) of SS-NS/PCs cultured in 1% serum. C, Grafting of SS-NS/PCs onto the spinal cord of ciclosporine treated rats. Intramedullary tumor developed four weeks after grafting of SS-OB2a cells (left). Low (a) and high (b) magnification sections show a neuroesthesioblastoma-like tumor (T) (H&E) expressing both neurofilament (c) and GFAP (d). Tumor cells labeled with GFP (e; green) stain with the oligodendrocyte cell marker NG2 (red). Homing and differentiation of SS-NS/PCs after grafting onto the spinal cord (left). GFP-labeled SS-OB1a cells showing neuronal cytology (a). GFP-labeled SS-OB4a cells expressing the neuronal marker neurofilament (GFP green, neurofilament red, merged signal yellow) (b and c). D, Intracerebral grafts of SS-NS/PCs in SCID mice. Brain tumor developed by two weeks after grafting of GFP-labeled SS-OB2a cells (a–c; a–b, H&E c, fluorescence microscopy). Grafted SS-OB1a cells do not form tumor by eight weeks after implantation (d–f; d–e, H&E f, fluorescence microscopy). Left. a, Scale bar 300 µm; b–e, Scale bar 50 µm. Center. a–c, Scale bar 30 µm. Right. a and d, Scale bar 250 µm; b–c and e–f, Scale bar 70 µm.
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pone-0004434-g003: Characterization and grafting of serum-stimulated NS/PCs grown as adherent cultures.A, Growth curves (left) and immunophenotype (right) of NS/PCs (P6) cultured in medium containing mitogens and 5% serum (serum-stimulated, SS-NS/PCs). B, Growth curves (left), absorbance test (right), and immunophenotype (right) of SS-NS/PCs cultured in 1% serum. C, Grafting of SS-NS/PCs onto the spinal cord of ciclosporine treated rats. Intramedullary tumor developed four weeks after grafting of SS-OB2a cells (left). Low (a) and high (b) magnification sections show a neuroesthesioblastoma-like tumor (T) (H&E) expressing both neurofilament (c) and GFAP (d). Tumor cells labeled with GFP (e; green) stain with the oligodendrocyte cell marker NG2 (red). Homing and differentiation of SS-NS/PCs after grafting onto the spinal cord (left). GFP-labeled SS-OB1a cells showing neuronal cytology (a). GFP-labeled SS-OB4a cells expressing the neuronal marker neurofilament (GFP green, neurofilament red, merged signal yellow) (b and c). D, Intracerebral grafts of SS-NS/PCs in SCID mice. Brain tumor developed by two weeks after grafting of GFP-labeled SS-OB2a cells (a–c; a–b, H&E c, fluorescence microscopy). Grafted SS-OB1a cells do not form tumor by eight weeks after implantation (d–f; d–e, H&E f, fluorescence microscopy). Left. a, Scale bar 300 µm; b–e, Scale bar 50 µm. Center. a–c, Scale bar 30 µm. Right. a and d, Scale bar 250 µm; b–c and e–f, Scale bar 70 µm.

Mentions: Transplantation technologies of adult human NS/PCs imply strategies where minimal donor material is highly expanded in vitro to the adequate cell number before implantation. In general, NSCs can be expanded either as floating neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing both mitogens and serum [17]. Neuronal and oligodendroglial differentiation of adherently growing NSCs can be enhanced by growth factor withdrawal and exposure to triiodothyronine (T3) and ascorbic acid [18]. Then, we propagated GFP-positive OB1, OB2, OB4, and OB5 NS/PCs between P7 and P10 either under mitogens or under mitogens and 5% serum (Fig. 1). In mitogens and serum, the NS/PCs became adherent, continued to proliferate, and either maintained an undifferentiated phenotype or differentiated, mainly as astrocytes (Fig. 3A). When such adherent serum-stimulated (SS) NS/PCs were returned to serum-free medium with mitogens, they formed floating neurospheres within one week maintaining their clonal efficiency. Upon removal of mitogens and exposure to 1% serum supplemented with T3 and retinoic acid, the SS-NS/PCs slowered down their growth and further differentiated towards the neuronal, astrocytic, and oligodendrocytic lineages (Fig. 3B). Aberrant coexpression of neuronal and glial markers by the SS-NS/PCs was not seen.


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)

Characterization and grafting of serum-stimulated NS/PCs grown as adherent cultures.A, Growth curves (left) and immunophenotype (right) of NS/PCs (P6) cultured in medium containing mitogens and 5% serum (serum-stimulated, SS-NS/PCs). B, Growth curves (left), absorbance test (right), and immunophenotype (right) of SS-NS/PCs cultured in 1% serum. C, Grafting of SS-NS/PCs onto the spinal cord of ciclosporine treated rats. Intramedullary tumor developed four weeks after grafting of SS-OB2a cells (left). Low (a) and high (b) magnification sections show a neuroesthesioblastoma-like tumor (T) (H&E) expressing both neurofilament (c) and GFAP (d). Tumor cells labeled with GFP (e; green) stain with the oligodendrocyte cell marker NG2 (red). Homing and differentiation of SS-NS/PCs after grafting onto the spinal cord (left). GFP-labeled SS-OB1a cells showing neuronal cytology (a). GFP-labeled SS-OB4a cells expressing the neuronal marker neurofilament (GFP green, neurofilament red, merged signal yellow) (b and c). D, Intracerebral grafts of SS-NS/PCs in SCID mice. Brain tumor developed by two weeks after grafting of GFP-labeled SS-OB2a cells (a–c; a–b, H&E c, fluorescence microscopy). Grafted SS-OB1a cells do not form tumor by eight weeks after implantation (d–f; d–e, H&E f, fluorescence microscopy). Left. a, Scale bar 300 µm; b–e, Scale bar 50 µm. Center. a–c, Scale bar 30 µm. Right. a and d, Scale bar 250 µm; b–c and e–f, Scale bar 70 µm.
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Related In: Results  -  Collection

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pone-0004434-g003: Characterization and grafting of serum-stimulated NS/PCs grown as adherent cultures.A, Growth curves (left) and immunophenotype (right) of NS/PCs (P6) cultured in medium containing mitogens and 5% serum (serum-stimulated, SS-NS/PCs). B, Growth curves (left), absorbance test (right), and immunophenotype (right) of SS-NS/PCs cultured in 1% serum. C, Grafting of SS-NS/PCs onto the spinal cord of ciclosporine treated rats. Intramedullary tumor developed four weeks after grafting of SS-OB2a cells (left). Low (a) and high (b) magnification sections show a neuroesthesioblastoma-like tumor (T) (H&E) expressing both neurofilament (c) and GFAP (d). Tumor cells labeled with GFP (e; green) stain with the oligodendrocyte cell marker NG2 (red). Homing and differentiation of SS-NS/PCs after grafting onto the spinal cord (left). GFP-labeled SS-OB1a cells showing neuronal cytology (a). GFP-labeled SS-OB4a cells expressing the neuronal marker neurofilament (GFP green, neurofilament red, merged signal yellow) (b and c). D, Intracerebral grafts of SS-NS/PCs in SCID mice. Brain tumor developed by two weeks after grafting of GFP-labeled SS-OB2a cells (a–c; a–b, H&E c, fluorescence microscopy). Grafted SS-OB1a cells do not form tumor by eight weeks after implantation (d–f; d–e, H&E f, fluorescence microscopy). Left. a, Scale bar 300 µm; b–e, Scale bar 50 µm. Center. a–c, Scale bar 30 µm. Right. a and d, Scale bar 250 µm; b–c and e–f, Scale bar 70 µm.
Mentions: Transplantation technologies of adult human NS/PCs imply strategies where minimal donor material is highly expanded in vitro to the adequate cell number before implantation. In general, NSCs can be expanded either as floating neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing both mitogens and serum [17]. Neuronal and oligodendroglial differentiation of adherently growing NSCs can be enhanced by growth factor withdrawal and exposure to triiodothyronine (T3) and ascorbic acid [18]. Then, we propagated GFP-positive OB1, OB2, OB4, and OB5 NS/PCs between P7 and P10 either under mitogens or under mitogens and 5% serum (Fig. 1). In mitogens and serum, the NS/PCs became adherent, continued to proliferate, and either maintained an undifferentiated phenotype or differentiated, mainly as astrocytes (Fig. 3A). When such adherent serum-stimulated (SS) NS/PCs were returned to serum-free medium with mitogens, they formed floating neurospheres within one week maintaining their clonal efficiency. Upon removal of mitogens and exposure to 1% serum supplemented with T3 and retinoic acid, the SS-NS/PCs slowered down their growth and further differentiated towards the neuronal, astrocytic, and oligodendrocytic lineages (Fig. 3B). Aberrant coexpression of neuronal and glial markers by the SS-NS/PCs was not seen.

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