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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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Generation of motor neurons (MNs) from FT-derived neurospheres.Individual neurospheres were treated with RA and either Shh-N or Hh-Ag1.3 for 4–5 days, plated onto an adhesive substrate, and cultured in serum-containing medium with appropriate growth factors for 7–10 days. Differentiated cells were subsequently evaluated for the expression of MN and MN progenitor markers. a) Differentiated cells from a P7 FT (30 DIV) stained positive for Olig2 (i, red) Pax6 (ii, green). The merged images co-stained for DAPI (blue) are shown in (iii). b) The MN-specific marker MNR2 is expressed by FT-derived neurospheres (green). Donor: P7 FT, 30 DIV. c) Expression of Tuj-1 (i, green) and ChAT (ii, red). The merged image co-stained for DAPI is shown in (iii). Donor: P6 FT, 36 DIV. Scale bars: 100 µm.
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pone-0065974-g007: Generation of motor neurons (MNs) from FT-derived neurospheres.Individual neurospheres were treated with RA and either Shh-N or Hh-Ag1.3 for 4–5 days, plated onto an adhesive substrate, and cultured in serum-containing medium with appropriate growth factors for 7–10 days. Differentiated cells were subsequently evaluated for the expression of MN and MN progenitor markers. a) Differentiated cells from a P7 FT (30 DIV) stained positive for Olig2 (i, red) Pax6 (ii, green). The merged images co-stained for DAPI (blue) are shown in (iii). b) The MN-specific marker MNR2 is expressed by FT-derived neurospheres (green). Donor: P7 FT, 30 DIV. c) Expression of Tuj-1 (i, green) and ChAT (ii, red). The merged image co-stained for DAPI is shown in (iii). Donor: P6 FT, 36 DIV. Scale bars: 100 µm.

Mentions: We next determined whether FT-derived neurospheres were capable of generating spinal cord MNs that could be used in cell replacement strategies in cases of spinal cord trauma or MN degeneration. To produce MN progenitors, single neurospheres were treated for 4–5 days with 2 µM retinoic acid and either 0.5–1 µM sonic hedgehog protein (Shh-N) or 1.5 µM of the hedgehog agonist Hh-Ag1.3. The neurospheres were subsequently plated on an adhesive substrate in the presence of 5% horse serum and 3 neurotrophic factors known to promote MN growth and survival (ciliary-derived neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF); Soundararajan et al., 2006 and Wichterle et al., 2002). In all experiments (n = 25), various proportions of the differentiated cells expressed either MN progenitor or mature MN markers (Figure 7). At first, various proportions of differentiated cells expressed motor neuron progenitor markers including Olig2 and Pax6 (Figure 7a). After 7–10 days, the fraction of MNs produced by each neurosphere was determined using immunocytochemistry for the MN marker Motor Neuron Restricted-2 (MNR-2; Figure 7b). Motor neurons were further characterized by their expression of the neuronal marker Tuj-1 as well as choline acetyl transferase (ChAT), which is the enzyme necessary for the synthesis of acetylcholine, the neurotransmitter used by motor neurons (Figure 7c).


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)

Generation of motor neurons (MNs) from FT-derived neurospheres.Individual neurospheres were treated with RA and either Shh-N or Hh-Ag1.3 for 4–5 days, plated onto an adhesive substrate, and cultured in serum-containing medium with appropriate growth factors for 7–10 days. Differentiated cells were subsequently evaluated for the expression of MN and MN progenitor markers. a) Differentiated cells from a P7 FT (30 DIV) stained positive for Olig2 (i, red) Pax6 (ii, green). The merged images co-stained for DAPI (blue) are shown in (iii). b) The MN-specific marker MNR2 is expressed by FT-derived neurospheres (green). Donor: P7 FT, 30 DIV. c) Expression of Tuj-1 (i, green) and ChAT (ii, red). The merged image co-stained for DAPI is shown in (iii). Donor: P6 FT, 36 DIV. Scale bars: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065974-g007: Generation of motor neurons (MNs) from FT-derived neurospheres.Individual neurospheres were treated with RA and either Shh-N or Hh-Ag1.3 for 4–5 days, plated onto an adhesive substrate, and cultured in serum-containing medium with appropriate growth factors for 7–10 days. Differentiated cells were subsequently evaluated for the expression of MN and MN progenitor markers. a) Differentiated cells from a P7 FT (30 DIV) stained positive for Olig2 (i, red) Pax6 (ii, green). The merged images co-stained for DAPI (blue) are shown in (iii). b) The MN-specific marker MNR2 is expressed by FT-derived neurospheres (green). Donor: P7 FT, 30 DIV. c) Expression of Tuj-1 (i, green) and ChAT (ii, red). The merged image co-stained for DAPI is shown in (iii). Donor: P6 FT, 36 DIV. Scale bars: 100 µm.
Mentions: We next determined whether FT-derived neurospheres were capable of generating spinal cord MNs that could be used in cell replacement strategies in cases of spinal cord trauma or MN degeneration. To produce MN progenitors, single neurospheres were treated for 4–5 days with 2 µM retinoic acid and either 0.5–1 µM sonic hedgehog protein (Shh-N) or 1.5 µM of the hedgehog agonist Hh-Ag1.3. The neurospheres were subsequently plated on an adhesive substrate in the presence of 5% horse serum and 3 neurotrophic factors known to promote MN growth and survival (ciliary-derived neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF); Soundararajan et al., 2006 and Wichterle et al., 2002). In all experiments (n = 25), various proportions of the differentiated cells expressed either MN progenitor or mature MN markers (Figure 7). At first, various proportions of differentiated cells expressed motor neuron progenitor markers including Olig2 and Pax6 (Figure 7a). After 7–10 days, the fraction of MNs produced by each neurosphere was determined using immunocytochemistry for the MN marker Motor Neuron Restricted-2 (MNR-2; Figure 7b). Motor neurons were further characterized by their expression of the neuronal marker Tuj-1 as well as choline acetyl transferase (ChAT), which is the enzyme necessary for the synthesis of acetylcholine, the neurotransmitter used by motor neurons (Figure 7c).

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