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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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Neuromuscular junction formation.a) A single neurosphere derived from a P6 FT was treated for 24 hours with Shh and RA and then labeled with DiI. b) A cell derived from the DiI-labeled neurosphere that has been co-cultured with rat myocytes for 2 days. c) After 20 days of co-culture, muscle cells were labeled with α-bungarotoxin-AF594 (red) to identify acetylcholine receptors clustered at neuromuscular junctions. Cells derived from individual neurospheres were pre-labeled with CFSE (green) prior to being added to the co-culture.
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pone-0065974-g009: Neuromuscular junction formation.a) A single neurosphere derived from a P6 FT was treated for 24 hours with Shh and RA and then labeled with DiI. b) A cell derived from the DiI-labeled neurosphere that has been co-cultured with rat myocytes for 2 days. c) After 20 days of co-culture, muscle cells were labeled with α-bungarotoxin-AF594 (red) to identify acetylcholine receptors clustered at neuromuscular junctions. Cells derived from individual neurospheres were pre-labeled with CFSE (green) prior to being added to the co-culture.

Mentions: To assess the potential of FT-derived MNs to innervate muscle, we used the same motor neuron/muscle co-culture system that we had previously developed for human FT-derived NPCs [51]. Single neurospheres were treated with RA & Shh and co-cultured with postnatal rat striated muscle fibers under differentiating conditions in the presence of BDNF, CNTF, and GDNF (n = 24). To confirm that the neurons observed in the co-culture were derived from plated neurospheres, 14 neurospheres were pre-incubated with either a lipophilic carbocyanine dye (Di-I or DI-D; Figure 9a,b) or carboxyfluorescein diacetate succinimidyl ester (CFSE; Figure 9c). Treated neurospheres were co-cultured with muscle for 6–21 days and then incubated with fluorescent α-bungarotoxin to mark the nicotinic acetylcholine receptors and identify the presence of neuromuscular junctions. After 6–21 days, clustered α-bungarotoxin staining on muscle fibers was detected in all of the co-cultures (Figure 9c). In contrast, control cultures containing muscle fibers only do not contain neuromuscular junctions as defined by acetylcholine receptor clustering [51]. These findings are highly suggestive that FT-derived neurospheres have the potential to form neuromuscular junctions.


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)

Neuromuscular junction formation.a) A single neurosphere derived from a P6 FT was treated for 24 hours with Shh and RA and then labeled with DiI. b) A cell derived from the DiI-labeled neurosphere that has been co-cultured with rat myocytes for 2 days. c) After 20 days of co-culture, muscle cells were labeled with α-bungarotoxin-AF594 (red) to identify acetylcholine receptors clustered at neuromuscular junctions. Cells derived from individual neurospheres were pre-labeled with CFSE (green) prior to being added to the co-culture.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065974-g009: Neuromuscular junction formation.a) A single neurosphere derived from a P6 FT was treated for 24 hours with Shh and RA and then labeled with DiI. b) A cell derived from the DiI-labeled neurosphere that has been co-cultured with rat myocytes for 2 days. c) After 20 days of co-culture, muscle cells were labeled with α-bungarotoxin-AF594 (red) to identify acetylcholine receptors clustered at neuromuscular junctions. Cells derived from individual neurospheres were pre-labeled with CFSE (green) prior to being added to the co-culture.
Mentions: To assess the potential of FT-derived MNs to innervate muscle, we used the same motor neuron/muscle co-culture system that we had previously developed for human FT-derived NPCs [51]. Single neurospheres were treated with RA & Shh and co-cultured with postnatal rat striated muscle fibers under differentiating conditions in the presence of BDNF, CNTF, and GDNF (n = 24). To confirm that the neurons observed in the co-culture were derived from plated neurospheres, 14 neurospheres were pre-incubated with either a lipophilic carbocyanine dye (Di-I or DI-D; Figure 9a,b) or carboxyfluorescein diacetate succinimidyl ester (CFSE; Figure 9c). Treated neurospheres were co-cultured with muscle for 6–21 days and then incubated with fluorescent α-bungarotoxin to mark the nicotinic acetylcholine receptors and identify the presence of neuromuscular junctions. After 6–21 days, clustered α-bungarotoxin staining on muscle fibers was detected in all of the co-cultures (Figure 9c). In contrast, control cultures containing muscle fibers only do not contain neuromuscular junctions as defined by acetylcholine receptor clustering [51]. These findings are highly suggestive that FT-derived neurospheres have the potential to form neuromuscular junctions.

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