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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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Heterogeneous differentiation potential of FT-derived neurospheres.This scatter graph illustrates the variability in the expression of Tuj-1 (neuronal marker) and GFAP (astrocytic marker) in 14 neurosphere differentiation experiments. Individual neurospheres were differentiated by both attachment to an adhesive substrate (poly-L-lysine- and laminin-coated coverslips) and exposure to 5% serum. Differentiated cells were evaluated by immunocytochemistry after either 24 hours or 7–10 days. In both cases, a variable proportion of neurons and astrocytes was observed. The approximate proportion of differentiated cells from a neurosphere that co-stained for both markers decreased after 7–10 days of exposure to the differentiating conditions relative to the proportion of overlap noted after 24 hours.
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pone-0065974-g006: Heterogeneous differentiation potential of FT-derived neurospheres.This scatter graph illustrates the variability in the expression of Tuj-1 (neuronal marker) and GFAP (astrocytic marker) in 14 neurosphere differentiation experiments. Individual neurospheres were differentiated by both attachment to an adhesive substrate (poly-L-lysine- and laminin-coated coverslips) and exposure to 5% serum. Differentiated cells were evaluated by immunocytochemistry after either 24 hours or 7–10 days. In both cases, a variable proportion of neurons and astrocytes was observed. The approximate proportion of differentiated cells from a neurosphere that co-stained for both markers decreased after 7–10 days of exposure to the differentiating conditions relative to the proportion of overlap noted after 24 hours.

Mentions: Figure 6 illustrates the variable expression of Tuj-1 and GFAP in 14 experiments comparing differentiation after 24 hours to differentiation after 7–10 days. Neurospheres differentiated over 24 hours (n = 9) resulted in a high proportion of cells that double stained for both neuronal and glial markers (Figure 6). In the case of Tuj-1 and GFAP staining, 69±14% (n = 5) of the cells were double stained for the two markers. After 7–10 days, the proportion of cells that double stained for both neuronal and glial markers decreased significantly (Figure 6). In the case of Tuj-1 and GFAP staining, only 13±10% (n = 9) of the cells were double stained for the two markers (Figures 5b, 6). On rare occasions, after 7 days of differentiation, >85% of cells derived from a single neurosphere expressed either a neuronal or glial marker (n = 2). However, in most cases, no obvious predominance was observed, and varying proportions of both neuronal and glial cells were noted from the differentiation of a single neurosphere (Figures 5,6). These varying proportions of Tuj-1+ and GFAP+ may reflect the heterogeneous differentiation potential of each neurosphere. This variation persisted in neurospheres either from the same source or different sources regardless of the age of the rat.


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)

Heterogeneous differentiation potential of FT-derived neurospheres.This scatter graph illustrates the variability in the expression of Tuj-1 (neuronal marker) and GFAP (astrocytic marker) in 14 neurosphere differentiation experiments. Individual neurospheres were differentiated by both attachment to an adhesive substrate (poly-L-lysine- and laminin-coated coverslips) and exposure to 5% serum. Differentiated cells were evaluated by immunocytochemistry after either 24 hours or 7–10 days. In both cases, a variable proportion of neurons and astrocytes was observed. The approximate proportion of differentiated cells from a neurosphere that co-stained for both markers decreased after 7–10 days of exposure to the differentiating conditions relative to the proportion of overlap noted after 24 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065974-g006: Heterogeneous differentiation potential of FT-derived neurospheres.This scatter graph illustrates the variability in the expression of Tuj-1 (neuronal marker) and GFAP (astrocytic marker) in 14 neurosphere differentiation experiments. Individual neurospheres were differentiated by both attachment to an adhesive substrate (poly-L-lysine- and laminin-coated coverslips) and exposure to 5% serum. Differentiated cells were evaluated by immunocytochemistry after either 24 hours or 7–10 days. In both cases, a variable proportion of neurons and astrocytes was observed. The approximate proportion of differentiated cells from a neurosphere that co-stained for both markers decreased after 7–10 days of exposure to the differentiating conditions relative to the proportion of overlap noted after 24 hours.
Mentions: Figure 6 illustrates the variable expression of Tuj-1 and GFAP in 14 experiments comparing differentiation after 24 hours to differentiation after 7–10 days. Neurospheres differentiated over 24 hours (n = 9) resulted in a high proportion of cells that double stained for both neuronal and glial markers (Figure 6). In the case of Tuj-1 and GFAP staining, 69±14% (n = 5) of the cells were double stained for the two markers. After 7–10 days, the proportion of cells that double stained for both neuronal and glial markers decreased significantly (Figure 6). In the case of Tuj-1 and GFAP staining, only 13±10% (n = 9) of the cells were double stained for the two markers (Figures 5b, 6). On rare occasions, after 7 days of differentiation, >85% of cells derived from a single neurosphere expressed either a neuronal or glial marker (n = 2). However, in most cases, no obvious predominance was observed, and varying proportions of both neuronal and glial cells were noted from the differentiation of a single neurosphere (Figures 5,6). These varying proportions of Tuj-1+ and GFAP+ may reflect the heterogeneous differentiation potential of each neurosphere. This variation persisted in neurospheres either from the same source or different sources regardless of the age of the rat.

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