Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis - PubMed (original) (raw)

Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis

S Weiss et al. J Neurosci. 1996.

Abstract

Neural stem cells in the lateral ventricles of the adult mouse CNS participate in repopulation of forebrain structures in vivo and are amenable to in vitro expansion by epidermal growth factor (EGF). There have been no reports of stem cells in more caudal brain regions or in the spinal cord of adult mammals. In this study we found that although ineffective alone, EGF and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cells isolated from the adult mouse thoracic spinal cord. The proliferating stem cells, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Neural stem cells, whose proliferation was dependent on EGF+bFGF, were also isolated from the lumbar/sacral segment of the spinal cord as well as the third and fourth ventricles (but not adjacent brain parenchyma). Although all of the stem cells examined were similarly multipotent and expandable, quantitative analyses demonstrated that the lateral ventricles (EGF-dependent) and lumbar/sacral spinal cord (EGF+bFGF-dependent) yielded the greatest number of these cells. Thus, the spinal cord and the entire ventricular neuroaxis of the adult mammalian CNS contain multipotent stem cells, present at variable frequency and with unique in vitro activation requirements.

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Figures

Fig. 7.

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 lines_illustrate 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.

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.

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.

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.

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.

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.

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. The_box_ 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 in_H_), 30 μm.

Fig. 8.

Fig. 8.

The in vitro generation of spheres derived from isolated cells of the adult spinal cord is greatest in the_Lumbar/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.

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