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Expression of MYCN in Multipotent Sympathoadrenal Progenitors Induces Proliferation and Neural Differentiation, but Is Not Sufficient for Tumorigenesis.

Mobley BC, Kwon M, Kraemer BR, Hickman FE, Qiao J, Chung DH, Carter BD - PLoS ONE (2015)

Bottom Line: MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies.MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis.We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology, and Immunology, Division of Neuropathology, Vanderbilt University Medical Center, Nashville, Tennessee, the United States of America.

ABSTRACT
Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Sympathoadrenal Progenitor Sphere Characterization and Differentiation.(A) Spheres grown from postnatal mouse adrenal glands cells express markers of neural crest stem cells (NCSCs), sympathoadrenal progenitors (SAPs), and sympathetic neurons (SNs). mRNA levels were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). Expression data from postnatal mouse superior cervical ganglia (SCG) are included for comparison. Data are the mean values from four experiments run in triplicate. *p<0.05,**p<0.01,***p<0.001,****p < .0001, unpaired Student’s t-test. (B) The majority of sphere cells show nuclear SOX10 reactivity by immunoperoxidase labeling. Cytoplasmic nestin expression with linear morphology was observed by immunofluorescence microscopy in spheres. TH expression was prominent at the periphery of spheres. Representative images from 3 independent experiments. Scale bars equal 20 microns. (C) Sphere differentiation was induced by transfer to poly-d-lysine and fibronectin-coated plates in medium with low CEE content. Immunofluorescent images show the three cell types observed in differentiated spheres: TuJ1/TH-reactive neurons, SMA-reactive myofibroblasts, and S100-reactive Schwann cells. Scale bars 50 μM upper left panel and 25 μM all other panels. (D) A shift in differentiation potential was observed from primary spheres, grown directly from adrenal gland cells by differential plating, to secondary spheres, the products of dissociated primary spheres. Differentiated secondary spheres showed a reduced frequency of bipotent and neuron-containing colonies, and were more likely to show myofibroblast-only colonies (primary spheres n = 3, secondary spheres n = 4). Primary and secondary spheres were grown at clonal density.
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pone.0133897.g001: Sympathoadrenal Progenitor Sphere Characterization and Differentiation.(A) Spheres grown from postnatal mouse adrenal glands cells express markers of neural crest stem cells (NCSCs), sympathoadrenal progenitors (SAPs), and sympathetic neurons (SNs). mRNA levels were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). Expression data from postnatal mouse superior cervical ganglia (SCG) are included for comparison. Data are the mean values from four experiments run in triplicate. *p<0.05,**p<0.01,***p<0.001,****p < .0001, unpaired Student’s t-test. (B) The majority of sphere cells show nuclear SOX10 reactivity by immunoperoxidase labeling. Cytoplasmic nestin expression with linear morphology was observed by immunofluorescence microscopy in spheres. TH expression was prominent at the periphery of spheres. Representative images from 3 independent experiments. Scale bars equal 20 microns. (C) Sphere differentiation was induced by transfer to poly-d-lysine and fibronectin-coated plates in medium with low CEE content. Immunofluorescent images show the three cell types observed in differentiated spheres: TuJ1/TH-reactive neurons, SMA-reactive myofibroblasts, and S100-reactive Schwann cells. Scale bars 50 μM upper left panel and 25 μM all other panels. (D) A shift in differentiation potential was observed from primary spheres, grown directly from adrenal gland cells by differential plating, to secondary spheres, the products of dissociated primary spheres. Differentiated secondary spheres showed a reduced frequency of bipotent and neuron-containing colonies, and were more likely to show myofibroblast-only colonies (primary spheres n = 3, secondary spheres n = 4). Primary and secondary spheres were grown at clonal density.

Mentions: Recent studies have shown that multipotent sympathoadrenal progenitor cells (SAPs) can be isolated from the adrenal gland by differential plating and grown as spheres [18–20]. We employed this strategy to study the impact of Mycn, the murine homolog of MYCN, on neural progenitor cell differentiation and proliferation. Postnatal mouse adrenal progenitors were grown as spheres in chick embryo extract (CEE)-containing self-renewal medium at physiologic oxygen levels [23]. Cells were plated at a density of one cell per μL, a clonal density at which spheres formed would be derived from single cells (S1 Fig). Quantitative PCR was used to examine gene expression in spheres, and expression levels were compared to those found in postnatal superior cervical ganglia (SCG), a sympathoadrenal tissue containing neurons and Schwann cells. Spheres expressed genes characteristic of neural crest stem cells (NCSCs) including Bmi1, Mycn, Snai1, Sox10, and Nestin (Fig 1A). Bmi1 and Snai1 were expressed more highly by spheres than by SCG tissue. Mycn and Nestin showed greater expression on average in spheres, but the differences did not reach statistical significance. Sox10 expression was higher in sympathetic ganglia, where it is expressed by mature glial cells. Expression of the SAP genes Ascl1 and Phox2b was also greater in spheres, while the sympathetic neuron (SN) markers TrkA and Peripherin were more highly expressed by SCG. Tyrosine hydroxylase and dopamine β hydroxylase were expressed highly by the two tissue types, indicating that adrenal-derived spheres express markers common to neural crest progenitors and sympathetic neurons. Quantitative PCR for the adrenal cortical markers Sf-1, Cyp11a1, and Cyp11b2 revealed no significant expression in spheres relative to postnatal adrenal gland control (data not shown). Immunohistochemical studies of primary spheres demonstrated expression of the NCSC proteins SOX10 and nestin (Fig 1B), with expression of tyrosine hydroxylase at the periphery of spheres.


Expression of MYCN in Multipotent Sympathoadrenal Progenitors Induces Proliferation and Neural Differentiation, but Is Not Sufficient for Tumorigenesis.

Mobley BC, Kwon M, Kraemer BR, Hickman FE, Qiao J, Chung DH, Carter BD - PLoS ONE (2015)

Sympathoadrenal Progenitor Sphere Characterization and Differentiation.(A) Spheres grown from postnatal mouse adrenal glands cells express markers of neural crest stem cells (NCSCs), sympathoadrenal progenitors (SAPs), and sympathetic neurons (SNs). mRNA levels were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). Expression data from postnatal mouse superior cervical ganglia (SCG) are included for comparison. Data are the mean values from four experiments run in triplicate. *p<0.05,**p<0.01,***p<0.001,****p < .0001, unpaired Student’s t-test. (B) The majority of sphere cells show nuclear SOX10 reactivity by immunoperoxidase labeling. Cytoplasmic nestin expression with linear morphology was observed by immunofluorescence microscopy in spheres. TH expression was prominent at the periphery of spheres. Representative images from 3 independent experiments. Scale bars equal 20 microns. (C) Sphere differentiation was induced by transfer to poly-d-lysine and fibronectin-coated plates in medium with low CEE content. Immunofluorescent images show the three cell types observed in differentiated spheres: TuJ1/TH-reactive neurons, SMA-reactive myofibroblasts, and S100-reactive Schwann cells. Scale bars 50 μM upper left panel and 25 μM all other panels. (D) A shift in differentiation potential was observed from primary spheres, grown directly from adrenal gland cells by differential plating, to secondary spheres, the products of dissociated primary spheres. Differentiated secondary spheres showed a reduced frequency of bipotent and neuron-containing colonies, and were more likely to show myofibroblast-only colonies (primary spheres n = 3, secondary spheres n = 4). Primary and secondary spheres were grown at clonal density.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519318&req=5

pone.0133897.g001: Sympathoadrenal Progenitor Sphere Characterization and Differentiation.(A) Spheres grown from postnatal mouse adrenal glands cells express markers of neural crest stem cells (NCSCs), sympathoadrenal progenitors (SAPs), and sympathetic neurons (SNs). mRNA levels were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). Expression data from postnatal mouse superior cervical ganglia (SCG) are included for comparison. Data are the mean values from four experiments run in triplicate. *p<0.05,**p<0.01,***p<0.001,****p < .0001, unpaired Student’s t-test. (B) The majority of sphere cells show nuclear SOX10 reactivity by immunoperoxidase labeling. Cytoplasmic nestin expression with linear morphology was observed by immunofluorescence microscopy in spheres. TH expression was prominent at the periphery of spheres. Representative images from 3 independent experiments. Scale bars equal 20 microns. (C) Sphere differentiation was induced by transfer to poly-d-lysine and fibronectin-coated plates in medium with low CEE content. Immunofluorescent images show the three cell types observed in differentiated spheres: TuJ1/TH-reactive neurons, SMA-reactive myofibroblasts, and S100-reactive Schwann cells. Scale bars 50 μM upper left panel and 25 μM all other panels. (D) A shift in differentiation potential was observed from primary spheres, grown directly from adrenal gland cells by differential plating, to secondary spheres, the products of dissociated primary spheres. Differentiated secondary spheres showed a reduced frequency of bipotent and neuron-containing colonies, and were more likely to show myofibroblast-only colonies (primary spheres n = 3, secondary spheres n = 4). Primary and secondary spheres were grown at clonal density.
Mentions: Recent studies have shown that multipotent sympathoadrenal progenitor cells (SAPs) can be isolated from the adrenal gland by differential plating and grown as spheres [18–20]. We employed this strategy to study the impact of Mycn, the murine homolog of MYCN, on neural progenitor cell differentiation and proliferation. Postnatal mouse adrenal progenitors were grown as spheres in chick embryo extract (CEE)-containing self-renewal medium at physiologic oxygen levels [23]. Cells were plated at a density of one cell per μL, a clonal density at which spheres formed would be derived from single cells (S1 Fig). Quantitative PCR was used to examine gene expression in spheres, and expression levels were compared to those found in postnatal superior cervical ganglia (SCG), a sympathoadrenal tissue containing neurons and Schwann cells. Spheres expressed genes characteristic of neural crest stem cells (NCSCs) including Bmi1, Mycn, Snai1, Sox10, and Nestin (Fig 1A). Bmi1 and Snai1 were expressed more highly by spheres than by SCG tissue. Mycn and Nestin showed greater expression on average in spheres, but the differences did not reach statistical significance. Sox10 expression was higher in sympathetic ganglia, where it is expressed by mature glial cells. Expression of the SAP genes Ascl1 and Phox2b was also greater in spheres, while the sympathetic neuron (SN) markers TrkA and Peripherin were more highly expressed by SCG. Tyrosine hydroxylase and dopamine β hydroxylase were expressed highly by the two tissue types, indicating that adrenal-derived spheres express markers common to neural crest progenitors and sympathetic neurons. Quantitative PCR for the adrenal cortical markers Sf-1, Cyp11a1, and Cyp11b2 revealed no significant expression in spheres relative to postnatal adrenal gland control (data not shown). Immunohistochemical studies of primary spheres demonstrated expression of the NCSC proteins SOX10 and nestin (Fig 1B), with expression of tyrosine hydroxylase at the periphery of spheres.

Bottom Line: MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies.MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis.We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology, and Immunology, Division of Neuropathology, Vanderbilt University Medical Center, Nashville, Tennessee, the United States of America.

ABSTRACT
Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus