Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus

Abstract

The adult rat spinal cord contains cells that can proliferate and differentiate into astrocytes and oligodendroglia in situ. Using clonal and subclonal analyses we demonstrate that, in contrast to progenitors isolated from the adult mouse spinal cord with a combination of growth factors, progenitors isolated from the adult rat spinal cord using basic fibroblast growth factor alone display stem cell properties as defined by their multipotentiality and self-renewal. Clonal cultures derived from single founder cells generate neurons, astrocytes, and oligodendrocytes, confirming the multipotent nature of the parent cell. Subcloning analysis showed that after serial passaging, recloning, and expansion, these cells retained multipotentiality, indicating that they are self-renewing. Transplantation of an in vitro-expanded clonal population of cells into the adult rat spinal cord resulted in their differentiation into glial cells only. However, after heterotopic transplantation into the hippocampus, transplanted cells that integrated in the granular cell layer differentiated into cells characteristic of this region, whereas engraftment into other hippocampal regions resulted in the differentiation of cells with astroglial and oligodendroglial phenotypes. The data indicate that clonally expanded, multipotent adult progenitor cells from a non-neurogenic region are not lineage-restricted to their developmental origin but can generate region-specific neurons in vivo when exposed to the appropriate environmental cues.

Fig. 1.

Schematic representation of the method used to assess multipotency and self-renewal of FGF-2-responsive, adult-derived spinal cord cells. For cloning, progenitor cells in vitro were labeled with a retroviral marker, and Southern blot analysis was used to show that a cluster of proliferating cells (primary clone) originating from a single cell can generate multiple cell types, including neurons, astrocytes, and oligodendrocytes. For subcloning, primary clones were dissociated and replated as single cells under clonal conditions in FGF-2-containing medium; a subset of cells proliferated to give rise to secondary clones. Secondary clones were also able to generate neurons, astrocytes, and oligodendrocytes. Subcloning analysis demonstrates the capacity of a cell to generate progeny similar to itself (i.e., self-renewal). These experiments demonstrate the presence of multipotent progenitor cells in adult rat spinal cord that proliferate in the presence of FGF-2 and are capable of self-renewal. β-GAL, β-Galactosidase;LTR, long terminal repeat; RSV, Rous sarcoma virus.

Fig. 2.

Expression of lineage-specific markers by a secondary clone derived from cervical spinal cord. A,Retroviral construct map of the vector used for cloning. Thehorizontalarrow beneath the retroviral construct indicates the region detected by the neomycin PCR-generated probe used to indicate the integration site of the retroviral genome.B, Southern blot analysis of BamHI- andPstI-digested genomic DNA from a cervical secondary clone. C, Phase-contrast image of proliferating daughter cells. D–G, Fluorescent confocal micrographs showing that the majority of cells expressed nestin (D). Micrographs also show examples of cell expressing either NF-200 (E), Rip (F), or GFAP (G). Scale bars: C, 50 μm;E–G, 25 μm; D, 15 μm.

Fig. 3.

Distribution of neurons and glia in cervical clonal cultures. A, Quantitation of cells differentiating down neuronal (Tubulin) or glial (GFAP or Rip) lineages in high cell density grown in N2 + FGF (FGF), N2 + 0.5% serum (FBS), or N2 + 0.5% serum + 0.5 μmretinoic acid (FBS+RA) for 6 d. Values represent the mean ± SEM from three separate differentiation experiments.B–G, Representative immunofluorescent staining of β-tubulin-immunoreactive cells (green inB–D), GFAP-immunoreactive cells (red inC, D), and Rip-immunoreactive cells (green in E–G) generated in a secondary clone in response to FGF (B, E), FBS (C, F), orFBS+RA (D, G). Scale bar:B–G, 25 μm.

Fig. 4.

Distribution and differentiation of clonally expanded adult spinal cord stem-like cells (cervical clone) 6 weeks after transplantation into the adult rat spinal cord. A,Horizontal section of the thoracic spinal cord showing the dispersion of transplanted cells. Arrowheads outline the borders of gray matter. B, Glial progenitor phenotype of transplant-derived cells. Arrowhead indicates a NG2 (green) and BrdU (red)-immunoreactive transplanted cell in the white matter of the spinal cord.C, D, Oligodendroglial phenotype of transplant-derived cells at 6 weeks. C, BrdU-immunoreactive transplanted cell expressing APC (red) and not GFAP (blue). D, Merged images of a transplanted cell expressing Rip (red) and BrdU (green). Insets, Single-channel images of the above cell expressing Rip in the cytoplasm and BrdU in the nucleus. E, Colocalization of BrdU-labeled transplanted cells (green) with GFAP (blue) immunoreactivity. F, Expression of the neomycin gene in spinal cord transplant sites of three animals as detected by reverse transcriptase-nested PCR. Scale bars:A, 400 μm; B, 10 μm; C, D, 10 μm; E, 10 μm.

Fig. 5.

Distribution and differentiation of clonally expanded, adult spinal cord stem-like cells (cervical clone) 6 weeks after transplantation in the adult rat hippocampus. A,Coronal view of the adult rat hippocampus shows the broad dispersion of transplanted BrdU-immunoreactive cells (green).Arrowheads indicate the needle tract. B,BrdU-immunoreactive cell (green) colocalizes with Rip immunoreactivity (red) in the molecular layer of the hippocampus (cell indicated by arrow).Insets, The shape of the BrdU-labeled nucleus (right) fits the shape of the nucleus of the Rip-expressing cell (left). The arrowhead inB indicates a Rip-expressing endogenous oligodendrocyte.C, D, Merged images of a cell expressing Rip (C) colocalized with BrdU (D) shown. E, F, BrdU-immunoreactive cells (green) expressing GFAP (blue) in the ventral leaf of the GCL (E) and lining the cerebral ventricle (F) are shown. G, Expression of the neomycin gene in hippocampal transplant sites of three animals is shown. H–J,Yellow indicates transplanted cells within the GCL double-labeled for NeuN (red) and BrdU (green). Thearrowhead in H indicates an endogenous astrocyte expressing GFAP. Asterisks inH, M, and P indicate the location of the hilus. The boxed area inI is shown at higher magnification in J.K, L, Merged images of cells (J) immunostained for NeuN (K) and BrdU (L) are shown. M, P,BrdU-immunoreactive cells (green) express calbindin (red) in the GCL. Arrowheads inM indicate transplanted cells that did not differentiate. The arrow in Nindicates the apical process of a transplanted cell extending toward the molecular layer (boxed area in M). N, O, Q, R, Unmerged images of transplanted cells (M, P) immunostained for calbindin (N, Q) and BrdU (O, R) in the dorsal and ventral leaves of the GCL, respectively, are shown. S, BrdU-immunoreactive cells (red) colocalize with NG2 immunoreactivity (green) in the molecular layer of the hippocampus (cells indicated by arrows). Scale bars:A, 500 μm; M, I, 50 μm; (shown inR) B–F, H, J–L, N–R, 15 μm; S, 15 μm.

Fig. 6.

Association of clonally expanded, adult spinal cord stem-like cells (cervical clone) 6 weeks after transplantation in the adult rat hippocampus with the synaptic marker synaptophysin. A, Coronal view of the granule cell layer showing a BrdU (red) and calbindin (blue)-colabeled nucleus that is closely associated with synaptophysin (green)-immunoreactive synaptic processes. Computer-generated XZ and YZ views of the of the Z-series stack are positioned below and to theright, respectively. Views in the XZ and YZ plane are taken from the point indicated by the arrowhead. Note the intimate association of synaptophysin-immunoreactive profiles with the BrdU-labeled cell membrane in all three planes of view.B, Confocal image of the BrdU-labeled cell fromA separated into individual blue,green, and red channels.C–E, Calbindin labeling in the dentate gyrus and CA fields of the hippocampus. The left (CA3) andright (dentate) boxed areas inC are shown at higher magnification in Dand E, respectively. Note that calbindin is rarely expressed by neurons in the CA3 region (D), whereas it is ubiquitously expressed by neurons in the dentate (E). Scale bars:C, 100 μm; A, 50 μm; D, E, 50 μm; B, 30 μm.

Fig. 7.

Proliferation of clonally expanded, adult spinal cord stem-like cells after transplantation. Immunofluorescence of BrdU (red) is combined with labeling for the proliferation marker Ki-67 (green). A, Coronal view of the adult rat hippocampus showing the presence of some proliferating cells in the subgranular zone, GCL, and hilus. Theboxed area is shown at a higher magnification inB. B, A cluster of proliferating cells in the subgranular zone (arrowhead) adjacent to a BrdU-labeled cell (arrow) in the GCL.

Fig. 8.

Distribution and differentiation of control transplants at 6 weeks after transplantation in the adult rat hippocampus. Immunofluorescence for BrdU (green) is combined with labeling for NeuN (red) and GFAP (blue). A, Freeze-thawed, BrdU-labeled, clonally expanded adult spinal cord stem-like cells show minimal dispersion from the injection tract (indicated byarrowheads). Intense GFAP staining is observed surrounding the transplant. The boxed area is shown at higher magnification in B. B,Freeze-thawed, BrdU-labeled nuclei in the GCL do not express NeuN (arrowhead) or GFAP (arrows).C, BrdU-labeled fibroblast transplant into the hippocampus is shown. Minimal dispersion of the transplanted fibroblasts from the injection tract is seen. Arrowheadsindicate the needle tract. D, BrdU-labeled cells in the GCL (arrowhead) do not colocalize with NeuN or with GFAP (arrows). Scale bars: A, C, 100 μm;B, D, 25 μm.