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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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Characterization of FT-derived, undifferentiated neurospheres by the expression of NPC, neuronal, and glial markers.a) Phase microscopy of a single neurosphere derived from a P10 FT culture after 10 days in vitro (DIV) and 1 passage. b) An individual neurosphere derived from a P5 FT culture (5 DIV) stained for the NPC marker Nestin (i, red), DAPI (ii, blue), and merged (iii). The images in (c–f) are co-stained with DAPI (blue). c) The NPC marker Olig2 (red) is expressed in a neurosphere derived from a P7 FT culture (34 DIV). d) Expression of Vimentin (green) in a proportion of cells of an individual neurosphere derived from a P7 FT culture (60 DIV). e) Immunostaining of Sox2 (red) in some cells from a single neurosphere isolated from a P6 FT culture (30 DIV). f) Weak staining of Musashi (red), with some hot-spots, in a neurosphere isolated from a P6 FT culture (30 DIV). g) (i) Sparse expression of β-Tubulin III (Tuj-1) in a proportion of cells of a neurosphere derived from a P7 FT culture (34 DIV). The expression of GFAP in the same neurosphere (g-ii) is restricted to the peripheral cells in vitro. (iii) Merged images of g(i) and (ii). Scale bars: 100 µm.
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pone-0065974-g003: Characterization of FT-derived, undifferentiated neurospheres by the expression of NPC, neuronal, and glial markers.a) Phase microscopy of a single neurosphere derived from a P10 FT culture after 10 days in vitro (DIV) and 1 passage. b) An individual neurosphere derived from a P5 FT culture (5 DIV) stained for the NPC marker Nestin (i, red), DAPI (ii, blue), and merged (iii). The images in (c–f) are co-stained with DAPI (blue). c) The NPC marker Olig2 (red) is expressed in a neurosphere derived from a P7 FT culture (34 DIV). d) Expression of Vimentin (green) in a proportion of cells of an individual neurosphere derived from a P7 FT culture (60 DIV). e) Immunostaining of Sox2 (red) in some cells from a single neurosphere isolated from a P6 FT culture (30 DIV). f) Weak staining of Musashi (red), with some hot-spots, in a neurosphere isolated from a P6 FT culture (30 DIV). g) (i) Sparse expression of β-Tubulin III (Tuj-1) in a proportion of cells of a neurosphere derived from a P7 FT culture (34 DIV). The expression of GFAP in the same neurosphere (g-ii) is restricted to the peripheral cells in vitro. (iii) Merged images of g(i) and (ii). Scale bars: 100 µm.

Mentions: Cells isolated from the FT were dissociated with collagenase and cultured in standard stem cell medium (DMEM/F12+1% N2 supplement) containing bFGF (20 ng/ml) and EGF (20 ng/ml) along with B27 (2%) and N2 (1%) supplements. Previous studies have identified these mitogenic factors as successful stimulants of NPC proliferation [14], [53], [57]. Each culture was derived from a single donor rat (ages P4–P10). After 3–4 days in vitro, neurospheres were observed in 96 out of the 100 primary cultures. These neurospheres were primarily free-floating and were identified by their spherical structure, phase bright appearance, and regular cell membranes (Figure 3a). The neurospheres initially appeared as smaller clusters of 3–4 round cells and eventually grew into larger neurospheres. The size of these larger neurospheres ranged widely from <50 µm to >1 mm. Cell clusters of <30 µm were not counted as neurospheres [58]. The number of neurospheres per primary culture varied from 1 to more than 40. For a subset of the cultures, we examined the effect of donor age upon the ability to produce neurospheres and the number of neurospheres obtained and found no correlation with age (Table 1). To demonstrate their capacity for proliferation and self-renewal, neurospheres were dissociated and passaged up to 19 times. These cultures have been maintained in vitro for up to 7 months. Twelve cultures have been frozen, and two have been tested for viability and were successfully recovered upon thawing.


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)

Characterization of FT-derived, undifferentiated neurospheres by the expression of NPC, neuronal, and glial markers.a) Phase microscopy of a single neurosphere derived from a P10 FT culture after 10 days in vitro (DIV) and 1 passage. b) An individual neurosphere derived from a P5 FT culture (5 DIV) stained for the NPC marker Nestin (i, red), DAPI (ii, blue), and merged (iii). The images in (c–f) are co-stained with DAPI (blue). c) The NPC marker Olig2 (red) is expressed in a neurosphere derived from a P7 FT culture (34 DIV). d) Expression of Vimentin (green) in a proportion of cells of an individual neurosphere derived from a P7 FT culture (60 DIV). e) Immunostaining of Sox2 (red) in some cells from a single neurosphere isolated from a P6 FT culture (30 DIV). f) Weak staining of Musashi (red), with some hot-spots, in a neurosphere isolated from a P6 FT culture (30 DIV). g) (i) Sparse expression of β-Tubulin III (Tuj-1) in a proportion of cells of a neurosphere derived from a P7 FT culture (34 DIV). The expression of GFAP in the same neurosphere (g-ii) is restricted to the peripheral cells in vitro. (iii) Merged images of g(i) and (ii). Scale bars: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675200&req=5

pone-0065974-g003: Characterization of FT-derived, undifferentiated neurospheres by the expression of NPC, neuronal, and glial markers.a) Phase microscopy of a single neurosphere derived from a P10 FT culture after 10 days in vitro (DIV) and 1 passage. b) An individual neurosphere derived from a P5 FT culture (5 DIV) stained for the NPC marker Nestin (i, red), DAPI (ii, blue), and merged (iii). The images in (c–f) are co-stained with DAPI (blue). c) The NPC marker Olig2 (red) is expressed in a neurosphere derived from a P7 FT culture (34 DIV). d) Expression of Vimentin (green) in a proportion of cells of an individual neurosphere derived from a P7 FT culture (60 DIV). e) Immunostaining of Sox2 (red) in some cells from a single neurosphere isolated from a P6 FT culture (30 DIV). f) Weak staining of Musashi (red), with some hot-spots, in a neurosphere isolated from a P6 FT culture (30 DIV). g) (i) Sparse expression of β-Tubulin III (Tuj-1) in a proportion of cells of a neurosphere derived from a P7 FT culture (34 DIV). The expression of GFAP in the same neurosphere (g-ii) is restricted to the peripheral cells in vitro. (iii) Merged images of g(i) and (ii). Scale bars: 100 µm.
Mentions: Cells isolated from the FT were dissociated with collagenase and cultured in standard stem cell medium (DMEM/F12+1% N2 supplement) containing bFGF (20 ng/ml) and EGF (20 ng/ml) along with B27 (2%) and N2 (1%) supplements. Previous studies have identified these mitogenic factors as successful stimulants of NPC proliferation [14], [53], [57]. Each culture was derived from a single donor rat (ages P4–P10). After 3–4 days in vitro, neurospheres were observed in 96 out of the 100 primary cultures. These neurospheres were primarily free-floating and were identified by their spherical structure, phase bright appearance, and regular cell membranes (Figure 3a). The neurospheres initially appeared as smaller clusters of 3–4 round cells and eventually grew into larger neurospheres. The size of these larger neurospheres ranged widely from <50 µm to >1 mm. Cell clusters of <30 µm were not counted as neurospheres [58]. The number of neurospheres per primary culture varied from 1 to more than 40. For a subset of the cultures, we examined the effect of donor age upon the ability to produce neurospheres and the number of neurospheres obtained and found no correlation with age (Table 1). To demonstrate their capacity for proliferation and self-renewal, neurospheres were dissociated and passaged up to 19 times. These cultures have been maintained in vitro for up to 7 months. Twelve cultures have been frozen, and two have been tested for viability and were successfully recovered upon thawing.

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