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The isolation, differentiation, and survival in vivo of multipotent cells from the postnatal rat filum terminale.

Jha RM, Chrenek R, Magnotti LM, Cardozo DL - PLoS ONE (2013)

Bottom Line: Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors.Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro.In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

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Inducing differentiation of FT-derived neurospheres.a) An individual neurosphere from a P5 FT (42 DIV) was plated at T = 0 on a laminin-coated coverslip and cultured in media that contained 10% serum. b) Morphological properties of differentiation were evident after 18 hours of exposure to these differentiating conditions. Scale bar: 100 µm.
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pone-0065974-g004: Inducing differentiation of FT-derived neurospheres.a) An individual neurosphere from a P5 FT (42 DIV) was plated at T = 0 on a laminin-coated coverslip and cultured in media that contained 10% serum. b) Morphological properties of differentiation were evident after 18 hours of exposure to these differentiating conditions. Scale bar: 100 µm.

Mentions: Some neurospheres adhered to the cultureware and spontaneously differentiated into cells having the morphological characteristics of neurons and glia without the addition or removal of factors from the medium. We sought to determine the conditions required to differentiate FT-derived neurospheres into neurons and glia. After withdrawing bFGF and EGF, single neurospheres were plated onto coverslips that had been treated with 7 different combinations of adhesive substrates ± exposure to 5–10% fetal bovine serum (Table 2). For each condition, 5 experiments were performed. After 7 days, the cultures were stained for Tuj-1, neurofilament, O1, GFAP, and Nestin. Although the use of either an adhesive substrate alone or serum alone was sufficient to initiate morphological differentiation, the addition of serum resulted in a more rapid differentiation. For example, an individual neurosphere plated on a poly-L-lysine- and laminin-coated coverslip began to exhibit morphological properties of differentiation after 42 hours (data not shown). In contrast, a neurosphere plated on a laminin-coated coverslip and cultured in 10% serum-containing medium began to differentiate after only 18 hours (Figures 4a,b). In all cases, we detected cells derived from the neurospheres that expressed either neuronal or glial markers including Tuj-1, neurofilament, O1, and GFAP (data not shown).


The isolation, differentiation, and survival in vivo of multipotent cells from the postnatal rat filum terminale.

Jha RM, Chrenek R, Magnotti LM, Cardozo DL - PLoS ONE (2013)

Inducing differentiation of FT-derived neurospheres.a) An individual neurosphere from a P5 FT (42 DIV) was plated at T = 0 on a laminin-coated coverslip and cultured in media that contained 10% serum. b) Morphological properties of differentiation were evident after 18 hours of exposure to these differentiating conditions. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675200&req=5

pone-0065974-g004: Inducing differentiation of FT-derived neurospheres.a) An individual neurosphere from a P5 FT (42 DIV) was plated at T = 0 on a laminin-coated coverslip and cultured in media that contained 10% serum. b) Morphological properties of differentiation were evident after 18 hours of exposure to these differentiating conditions. Scale bar: 100 µm.
Mentions: Some neurospheres adhered to the cultureware and spontaneously differentiated into cells having the morphological characteristics of neurons and glia without the addition or removal of factors from the medium. We sought to determine the conditions required to differentiate FT-derived neurospheres into neurons and glia. After withdrawing bFGF and EGF, single neurospheres were plated onto coverslips that had been treated with 7 different combinations of adhesive substrates ± exposure to 5–10% fetal bovine serum (Table 2). For each condition, 5 experiments were performed. After 7 days, the cultures were stained for Tuj-1, neurofilament, O1, GFAP, and Nestin. Although the use of either an adhesive substrate alone or serum alone was sufficient to initiate morphological differentiation, the addition of serum resulted in a more rapid differentiation. For example, an individual neurosphere plated on a poly-L-lysine- and laminin-coated coverslip began to exhibit morphological properties of differentiation after 42 hours (data not shown). In contrast, a neurosphere plated on a laminin-coated coverslip and cultured in 10% serum-containing medium began to differentiate after only 18 hours (Figures 4a,b). In all cases, we detected cells derived from the neurospheres that expressed either neuronal or glial markers including Tuj-1, neurofilament, O1, and GFAP (data not shown).

Bottom Line: Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors.Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro.In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

Show MeSH
Related in: MedlinePlus