Article Figures & Data
Figures
- Fig. 7.
Regions of the adult CNS examined for the presence of growth factor-responsive stem cells. A–D, Ventral view of the adult mouse brain (A), illustrating the coronal sectioned regions that were used to dissect lateral ventricle (B), third ventricle (C), and fourth ventricle (D). Dark lines illustrate the regions considered ventricular, whereas stippled linesillustrate nonventricular regions of the same thick section.E, Adult mouse spinal cord, illustrating the regions dissected as thoracic (T1–T13) and lumbar/sacral (L6–Co3). As detailed in Results, stem cells were isolated from all ventricular regions examined but not from the adjacent parenchyma. Scale bar: each graduation is 1 mm.
- Fig. 5.
Schematic representation of approaches used to establish adult spinal cord stem cell proliferation, self-renewal and expansion, and production of neurons, astrocytes, and oligodendrocytes. The experimental approaches to demonstrating self-renewal and expansion of stem cells in response to EGF+bFGF are shown. When primary, dissociated adult cells are exposed to EGF+bFGF, spheres of undifferentiated cells are generated. (1) Differentiation of single primary spheres results in the production of neurons, astrocytes, and oligodendrocytes. (2) Dissociation of single primary spheres into single cells, which are plated after serial dilution as 1 cell/well, generates clonally derived secondary spheres. Differentiation of single secondary spheres results in the production of neurons, astrocytes, and oligodendrocytes. (3) Dissociation of single primary spheres into single cells, all of which are plated into one well, results in more than one secondary sphere. Once again, differentiation of these single secondary spheres results in the production of neurons, astrocytes, and oligodendrocytes.
- Fig. 1.
Characteristics of spheres generated from isolated cells of the adult thoracic spinal cord. A, B, An example of a single sphere generated from the adult thoracic cord (A), which was dissociated into single cells that yielded close to 300 spheres 1 week later, some of which are illustrated in B. C, D, Spheres generated from the adult thoracic spinal cord contained no differentiated cells; however, virtually all cells within these spheres (C) expressed nestin (D), an intermediate filament characteristic of undifferentiated neuroepithelial cells. Scale bars:A, 100 μm; B, 50 μm;C, 50 μm; D, 30 μm.
- Fig. 2.
Single spheres derived from the adult thoracic spinal cord yielded neurons, astrocytes, and oligodendrocytes.A, A single, isolated sphere was transferred onto poly-l-ornithine-coated glass coverslips, cultured for 3 weeks in the presence of EGF+bFGF, fixed, and processed for indirect immunocytochemistry. B–E, Triple-label immunocytochemistry of the sphere in A, illustrating the cells within the box (B), shows (C) MAP-2-, (D) GFAP-, and (E) O4-immunoreactive cells, with the morphology of neurons, astrocytes, and oligodendrocytes, respectively. Scale bars: A, 200 μm; B–E (shown in E), 20 μm.
- Fig. 3.
Examples of O4-immunoreactive cells in plated adult thoracic spinal cord spheres, with and without permeabilization.A, An O4-immunoreactive cell in a fixed, but nonpermeabilized, single thoracic sphere culture, demonstrating typical oligodendrocyte morphology. B, C, In a fixed and permeabilized sister culture, a single oligodendrocyte (arrow) is specifically stained in a punctate fashion with the O4 antibody. Scale bar, 20 μm.
- Fig. 4.
Further characterization of the phenotypes of cells derived from stem cells of the thoracic spinal cord.A–D, Triple-label immunocytochemistry of single spheres derived from the thoracic spinal cord, after 3 weeks of plating on poly-l-ornithine. A, Representative field shows (B) neurofilament M (160 kDa;arrows), (C) GFAP, and (D) O4 immunoreactivity, characteristic of neurons, astrocytes, and oligodendrocytes, respectively. E, F, The principal neuronal phenotype detected, after 3 weeks of plating on poly-l-ornithine, was GABA. Indirect immunocytochemistry of a representative field (E) shows cells with neuronal morphology that were GABA-immunoreactive (F). Scale bars: A–D (shown in D), 20 μm;E, F (shown in F), 30 μm.
- Fig. 6.
Primary spheres from the adult thoracic spinal cord give rise to clonally derived, multipotent secondary spheres.A–H, Multipotent secondary spheres are derived from a single cell. A single cell (arrow) dissociated from a primary sphere (A) after 24 hr. After 5 d in vitro (B), the cell has begun to proliferate and has formed a large sphere after 14 d in vitro(C). The sphere was transferred to a glass coverslip and cultured in the presence of EGF+bFGF. After 3 weeks (D), the sphere was processed for indirect immunocytochemistry. Thebox designates the field (E) that, through triple-labeling for MAP-2, GFAP, and O4 immunoreactivities, revealed the presence of neurons (F, short arrow), astrocytes (G, long arrow), and oligodendrocytes (H,arrowhead), respectively. Scale bars:A–C (shown in C), 50 μm;D, 140 μm; E–H (shown inH), 30 μm.
- Fig. 8.
The in vitro generation of spheres derived from isolated cells of the adult spinal cord is greatest in theLumbar/Sacral segment. The number of spheres generated in the presence of EGF+bFGF in vitro was determined for the three regions of the spinal cord indicated and was normalized to the length of spinal cord tissue dissected. The data represent the mean ± SEM of duplicate determinations in six independent culture preparations.
Tables
- Table 1.
Multipotency of primary and renewed adult thoracic spinal cord stem cell-derived spheres
Experimental protocol Number of independent primary cultures Frequency of spheres containing neurons, astrocytes, and oligodendrocytes Primary culture 23 106 /106 Single cell culture 6 11 /11 Single sphere dissociation 15 79 /79 The experimental protocols correspond to those outlined schematically in Figure 5. Indirect immunocytochemistry for the three neural antigens is described in Materials and Methods.
- Table 3.
Expansion and multipotency of growth factor-generated spheres derived from various brain regions
Brain region No. 2° spheres (mean ± SEM) No. of cultures No. of spheres No. N+A+O Lateral ventricle 79 ± 20 1° 9 37 37 2° 4 9 9 Third ventricle 84 ± 21 1° 10 33 33 2° 6 33 303-a Fourth ventricle 107 ± 22 1° 12 37 37 2° 9 54 54 Thoracic cord 127 ± 14 1° 23 106 106 2° 15 79 79 Lumbar/sacral cord 110 ± 11 1° 9 34 34 2° 5 11 11 The left-hand column is a comparison of the expandability of primary spheres, generated in the presence of EGF + bFGF, from the indicated regions. No. 2° spheres refers to the numbers of secondary spheres counted in a well where all of the dissociated cells of a primary sphere had been seeded. The data for those single-sphere dissociations are the mean ± SEM of 10–20 primary spheres dissociated within each region. The remaining three columns refer to the multipotential nature of primary spheres and their progeny, the secondary spheres produced through subcloning. The total numbers of individual experiments and spheres examined are given. 1°, Primary culture; 2°, secondary culture.
↵F3-a Three spheres contained only neurons and astrocytes. N+A+O, Neurons + astrocytes + oligodendrocytes.
- Table 2.
Frequency and growth factor dependence of primary multipotent stem cell-derived spheres
Brain region Numbers of self-renewing, expandable spheres/5000 cells plated (mean ± SEM) EGF bFGF EGF + bFGF Lateral ventricle 26.7 ± 3.7 – 26.8 ± 4.5 Third ventricle – – 6.1 ± 1.42-a Fourth ventricle – – 1.0 ± 0.3 Thoracic cord – – 8.6 ± 3.4 Lumbar/sacral cord – – 32.6 ± 3.2 The brain regions indicated are those shown schematically in Figure 7. The data are the mean ± SEM of spheres formed after 8 d in culture in at least four independent culture preparations, each condition performed in duplicate. Basic FGF was tested in the absence of added heparin [see Discussion and Gritti et al. (1996)].
↵F2-a Counted after 14–16 d.
