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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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In vivo survival and differentiation of FT-derived NPCs transplanted into the developing chick spinal cord.a) Neurospheres were isolated from a P2 transgenic rat that expressed eGFP in every cell. Scale bar: 100 µm. b) GFP+ NPCs from a P2 rat were transplanted into a stage 10 (33 hrs) chick neural tube at the prospective rostral end of the developing spinal cord. This image was taken immediately after injection, and the rostral end is pointing towards the bottom. Scale bar: 200 µm. c) After 3 DIO (∼stage 22), GFP+ cells had survived and begun to take on the morphology of neurons and glia. Scale bar: 50 µm. d–e) Neurospheres from a 6-month-old human were transplanted into a stage 10 (33 hrs) chick spinal cord at the rostral end. After 3 DIO, transplanted cells were labeled with the human-specific antibody HSP27 (green) and can be seen migrating away from the transplantation site. Two different embryos are shown in (d) and (e). The image in (e)i is a magnified view of the migrating cells from the highlighted area in (e). Scale bars: (d) and (e): 200 µm, (e)i: 50 µm. f–h) After 7 DIO, some FT-derived NPCs (green, f–h(i)) expressed the neuronal marker Tuj-1 (red, f(ii)), the astrocyte marker GFAP (red, g(ii)), or the oligodendrocyte marker MBP (red, h(ii)). In all cases (f–h), a merged image of (i) and (ii) along with the nuclear marker DAPI is shown in (iii). Scale bars: 50 µm.
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pone-0065974-g010: In vivo survival and differentiation of FT-derived NPCs transplanted into the developing chick spinal cord.a) Neurospheres were isolated from a P2 transgenic rat that expressed eGFP in every cell. Scale bar: 100 µm. b) GFP+ NPCs from a P2 rat were transplanted into a stage 10 (33 hrs) chick neural tube at the prospective rostral end of the developing spinal cord. This image was taken immediately after injection, and the rostral end is pointing towards the bottom. Scale bar: 200 µm. c) After 3 DIO (∼stage 22), GFP+ cells had survived and begun to take on the morphology of neurons and glia. Scale bar: 50 µm. d–e) Neurospheres from a 6-month-old human were transplanted into a stage 10 (33 hrs) chick spinal cord at the rostral end. After 3 DIO, transplanted cells were labeled with the human-specific antibody HSP27 (green) and can be seen migrating away from the transplantation site. Two different embryos are shown in (d) and (e). The image in (e)i is a magnified view of the migrating cells from the highlighted area in (e). Scale bars: (d) and (e): 200 µm, (e)i: 50 µm. f–h) After 7 DIO, some FT-derived NPCs (green, f–h(i)) expressed the neuronal marker Tuj-1 (red, f(ii)), the astrocyte marker GFAP (red, g(ii)), or the oligodendrocyte marker MBP (red, h(ii)). In all cases (f–h), a merged image of (i) and (ii) along with the nuclear marker DAPI is shown in (iii). Scale bars: 50 µm.

Mentions: Because FT-derived neurospheres could differentiate into both neurons (including motor neurons) and glia in vitro, we wanted to examine the behavior of these cells when they were reintroduced to the spinal cord in vivo. To accomplish this, the FT was isolated from transgenic rats aged P0–P2 in which all cells express GFP under the control of the chicken β-actin promoter and cytomegalovirus enhancer (SD-Tg(Act-EGFP)CZ-004Osb; SLC Japan). Neurospheres were isolated and cultured from this tissue as previously described (Figure 10a). These FT-derived, GFP+ neurospheres were implanted into the neural tube of stage 10–15 chick embryos at the rostral end of the developing spinal cord (Figure 10b; n = 31). In some cases, the neurospheres were dissociated before being implanted. The embryos were then permitted to develop for an additional 3–7 days post-transplantation before their ability to survive, migrate into the host tissue, and differentiate was assessed.


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)

In vivo survival and differentiation of FT-derived NPCs transplanted into the developing chick spinal cord.a) Neurospheres were isolated from a P2 transgenic rat that expressed eGFP in every cell. Scale bar: 100 µm. b) GFP+ NPCs from a P2 rat were transplanted into a stage 10 (33 hrs) chick neural tube at the prospective rostral end of the developing spinal cord. This image was taken immediately after injection, and the rostral end is pointing towards the bottom. Scale bar: 200 µm. c) After 3 DIO (∼stage 22), GFP+ cells had survived and begun to take on the morphology of neurons and glia. Scale bar: 50 µm. d–e) Neurospheres from a 6-month-old human were transplanted into a stage 10 (33 hrs) chick spinal cord at the rostral end. After 3 DIO, transplanted cells were labeled with the human-specific antibody HSP27 (green) and can be seen migrating away from the transplantation site. Two different embryos are shown in (d) and (e). The image in (e)i is a magnified view of the migrating cells from the highlighted area in (e). Scale bars: (d) and (e): 200 µm, (e)i: 50 µm. f–h) After 7 DIO, some FT-derived NPCs (green, f–h(i)) expressed the neuronal marker Tuj-1 (red, f(ii)), the astrocyte marker GFAP (red, g(ii)), or the oligodendrocyte marker MBP (red, h(ii)). In all cases (f–h), a merged image of (i) and (ii) along with the nuclear marker DAPI is shown in (iii). Scale bars: 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675200&req=5

pone-0065974-g010: In vivo survival and differentiation of FT-derived NPCs transplanted into the developing chick spinal cord.a) Neurospheres were isolated from a P2 transgenic rat that expressed eGFP in every cell. Scale bar: 100 µm. b) GFP+ NPCs from a P2 rat were transplanted into a stage 10 (33 hrs) chick neural tube at the prospective rostral end of the developing spinal cord. This image was taken immediately after injection, and the rostral end is pointing towards the bottom. Scale bar: 200 µm. c) After 3 DIO (∼stage 22), GFP+ cells had survived and begun to take on the morphology of neurons and glia. Scale bar: 50 µm. d–e) Neurospheres from a 6-month-old human were transplanted into a stage 10 (33 hrs) chick spinal cord at the rostral end. After 3 DIO, transplanted cells were labeled with the human-specific antibody HSP27 (green) and can be seen migrating away from the transplantation site. Two different embryos are shown in (d) and (e). The image in (e)i is a magnified view of the migrating cells from the highlighted area in (e). Scale bars: (d) and (e): 200 µm, (e)i: 50 µm. f–h) After 7 DIO, some FT-derived NPCs (green, f–h(i)) expressed the neuronal marker Tuj-1 (red, f(ii)), the astrocyte marker GFAP (red, g(ii)), or the oligodendrocyte marker MBP (red, h(ii)). In all cases (f–h), a merged image of (i) and (ii) along with the nuclear marker DAPI is shown in (iii). Scale bars: 50 µm.
Mentions: Because FT-derived neurospheres could differentiate into both neurons (including motor neurons) and glia in vitro, we wanted to examine the behavior of these cells when they were reintroduced to the spinal cord in vivo. To accomplish this, the FT was isolated from transgenic rats aged P0–P2 in which all cells express GFP under the control of the chicken β-actin promoter and cytomegalovirus enhancer (SD-Tg(Act-EGFP)CZ-004Osb; SLC Japan). Neurospheres were isolated and cultured from this tissue as previously described (Figure 10a). These FT-derived, GFP+ neurospheres were implanted into the neural tube of stage 10–15 chick embryos at the rostral end of the developing spinal cord (Figure 10b; n = 31). In some cases, the neurospheres were dissociated before being implanted. The embryos were then permitted to develop for an additional 3–7 days post-transplantation before their ability to survive, migrate into the host tissue, and differentiate was assessed.

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