Distribution and characterization of progenitor cells within the human filum terminale

Abstract

Background: Filum terminale (FT) is a structure that is intimately associated with conus medullaris, the most caudal part of the spinal cord. It is well documented that certain regions of the adult human central nervous system contains undifferentiated, progenitor cells or multipotent precursors. The primary objective of this study was to describe the distribution and progenitor features of this cell population in humans, and to confirm their ability to differentiate within the neuroectodermal lineage.

Methodology/principal findings: We demonstrate that neural stem/progenitor cells are present in FT obtained from patients treated for tethered cord. When human or rat FT-derived cells were cultured in defined medium, they proliferated and formed neurospheres in 13 out of 21 individuals. Cells expressing Sox2 and Musashi-1 were found to outline the central canal, and also to be distributed in islets throughout the whole FT. Following plating, the cells developed antigen profiles characteristic of astrocytes (GFAP) and neurons (β-III-tubulin). Addition of PDGF-BB directed the cells towards a neuronal fate. Moreover, the cells obtained from young donors shows higher capacity for proliferation and are easier to expand than cells derived from older donors.

Conclusion/significance: The identification of bona fide neural progenitor cells in FT suggests a possible role for progenitor cells in this extension of conus medullaris and may provide an additional source of such cells for possible therapeutic purposes. Filum terminale, human, progenitor cells, neuron, astrocytes, spinal cord.

Figure 1. Distribution of Sox2-positive cells within filum terminale.

(A) Longitudinal section represents an overview of the distribution and localization of the neuronal progenitor cells in human FT revealed by Sox2-positive cells (red) and the nuclear staining DAPI (blue). (B–E) Represent higher magnifications demonstrating Sox2-positive cells localized subependymally and outlining the central canal. (F and G) represent coronal adjacent sections, (F) stained with florescence staining. The central canal is indicated with an asterix (G) Coronar section stained with Haematoxylin–eosin staining. Arrows (in F and G) indicates corresponding areas. A–F; confocal, G; light microscopy. Scale bars: A = 100 µm; B = 50 µm; C−G = 40 µm.

Figure 2. Presence of additional progenitor markers in the filum terminale.

(A) Longitudinal section and (B) coronal section shows clusters of Musashi-positive cells within the FT (red) and nucleus staining DAPI (blue). (A) A large cluster of Musashi-positive cells at the surface of the FT. Insert represents higher magnification of the boxed area. (B) Small clusters of Musashi-positive cells within the FT. (C–F) Double-immunolabeled sections from human FT show that Sox2 immunoreactivity (green) was colocalized with Musashi immunoreactivity (red) (C). The Sox2 (red) cells were also expressing GFAP (green) (D–F). Scale bars: A,D,E,F = 40 μm, B,C and insert box = 20 μm.

Figure 3. Quantitative determination of cell expansion from young donors (1–7years) and old donors (42–60years).

The number of obtainable cell passages in young donors was significantly higher than old donors. *P <0.05.

Figure 4. Human FT cells in culture. (A) Neurosphere after second passage.

BrdU-positive (green) and Sox2-positive (red) cells in neurosphere from human FT (Insert in A). (B) Nestin-positive cells (green) after 28 days with nuclear marker Hoechst (blue). (C) GFAP-positive cells with astroglial shape (red) and (D) One neuronal (β-III-tubulin) cell as it appears after 11 days of differentiation (blue = DAPI). (E) Expansion of cells without PDGF-BB. (F) Cells expanded in culture medium exposed to PDGF-BB for 8 days, GFAP (red), β-III tubulin (green) and nuclear marker Hoechst (blue). Bars: A and B = 10 μm, Insert = 25 μm, C and D = 5 μm and E−F = 20 μm.

Figure 5. Anatomical comparison between the human and rat filum terminale.

(A) Longitudinal section of human FT stained with haematoxylin-eosin. The human FT mostly consists of astrocytes and ependymal cells (see arrows) (B–D). Transverse sections of rat FT stepwise from conus (B) and further caudally (C and D) the central canal is prominent in the middle of the rat film. Rounded brown structures in the perperiphery represent nerve roots. (E and F). Longitudinal sections of the rat FT labelled with a nestin antibody (red) and a nuclear marker DAPI (blue). (F) Higher magnification of boxed area in (E). Bars: A and E = 40 µm, B and C = 100 µm, F = 5 µm. Asterix =  central canal.