Generation of motor neurons from human amygdala-derived neural stem-like cells

Abstract

Objectives: Among several cell sources, adult human neural stem/progenitor cells (hNS/PCs) have been considered outstanding cells for performing mechanistic studies in in vitro and in vivo models of neurological disorders as well as for potential utility in cell-based therapeutic approaches. Previous studies addressed the isolation and culture of hNS/PCs from human neocortical and hippocampal tissues. However, little data are available on hNS/PCs obtained from the adult human amygdala.

Materials and methods: The present study explored the capacity of the amygdala harvested from resected brain tissues of patients with medically refractory epilepsy to generate neurosphere-like bodies and motor neuron-like cells.

Results: Although the proliferation process was slow, a considerable amount of cells was obtained after the 3rd passage. In addition, the cells could generate motor neuron-like cells under appropriate culture conditions.

Conclusion: Isolation and culture of these cells enable us to improve our knowledge of the role of the amygdala in some neurological and psychological disorders and provide a novel source for therapeutic cell transplantation.

Keywords: Brain; Hippocampus; Intractable Epilepsy; Motor neuron; Neural stem cells.

Figure 1

Histological verification of the amygdala tissue. Schematic design of an amygdala section and a represented example of histological confirmation based on cytoarchitecture of the amygdala complex. (morphological aspects of neurons in the amygdala are pyramidal, modified pyramidal, ovoid, and gliaform that have been labeled. types that have been labeled. Magnification of toluidine blue staining photomicrographs is 20X

Figure 2

Formation of neurospheres from the epileptic human amygdala tissue. A: The neurospheres proliferated slowly 4 (A1), 7 (A2), and 14 (A3) days after primary culture of the adult human amygdala (magnification 20X). A4 represents the neurospheres at passage 3. Scale bars for all micrographs are equal to 50 µm. B: Quantification of spheres from the adult human amygdala tissue was obtained from 8 patients with refractory epilepsy during brain surgery (~150 neurospheres/amygdala tissues in passage 3). The mean number of spheres exponentially increased during three passages. C: The mean number of cells obtained after each passage. Calculation of the cell number represented a rising trend during passages

Figure 3

Characterization of the neural stem-like cells isolated from the epileptic human amygdala. Immunocytochemistry analysis showed that almost all of the cells isolated and cultured from the adult human amygdala by the neurosphere culture method expressed nestin (B, green) and Sox2 (C, green). In addition, the expression of GFAP was considerable (D, green). In contrast, only a few MAP-2 (E, green) or CD68 (F) positive cells were seen. A is a phase contrast micrograph of adherent neural stem-like cells. Nuclei are seen in blue

Figure 4

Morphological changes during differentiation of neural stem-like cells. Neural stem-like cells obtained from the epileptic human amygdala differentiated during 14 days exposure to differentiation medium

Figure 5

Generation of motor neurons from neural stem-like cells from human amygdala obtained during epilepsy surgery. The cells were evaluated for motor neuron differentiation. Generated neurons expressed motor neuron-specific markers ChAT, Isl-1, and HB9 but not neural stem cells marker, nestin