An avian model for the reversal of neurobehavioral teratogenicity with neural stem cells

Abstract

A fast and simple model which uses lower animals on the evolutionary scale is beneficial for developing procedures for the reversal of neurobehavioral teratogenicity with neural stem cells. Here, we established a procedure for the derivation of chick neural stem cells, establishing embryonic day (E) 10 as optimal for progression to neuronal phenotypes. Cells were obtained from the embryonic cerebral hemispheres and incubated for 5-7 days in enriched medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) according to a procedure originally developed for mice. A small percentage of the cells survived, proliferated and formed nestin-positive neurospheres. After removal of the growth factors to allow differentiation (5 days), 74% of the cells differentiated into all major lineages of the nervous system, including neurons (Beta III tubulin-positive, 54% of the total number of differentiated cells), astrocytes (GFAP-positive, 26%), and oligodendrocytes (O4-positive, 20%). These findings demonstrate that the cells were indeed neural stem cells. Next, the cells were transplanted in two allograft chick models; (1) direct cerebral transplantation to 24-h-old chicks, followed by post-transplantation cell tracking at 24 h, 6 days and 14 days, and (2) intravenous transplantation to chick embryos on E13, followed by cell tracking on E19. With both methods, transplanted cells were found in the brain. The chick embryo provides a convenient, precisely-timed and unlimited supply of neural progenitors for therapy by transplantation, as well as constituting a fast and simple model in which to evaluate the ability of neural stem cell transplantation to repair neural damage, steps that are critical for progress toward therapeutic applications.

Figure 1. Undifferentiated E10 neurospheres are nestin+

Immunofluorescent staining for nestin as a neural stem cell marker. E10 cells formed neurospheres that were attached to Petri dishes on day 5.

Figure 2. E10 cells gradually form neurospheres

Phase-bright micrographs showing progressive growth of cells into clusters during the first days in culture with EGF and FGF2. (A) E10 cells at plating day (day 1) are separated into single cells. (B) E10 cells on day 2 have formed clusters of 5–15 cells. (C) E10 cells at day 3 show the formation of bigger, non-rounded clusters. (D) E10 cells at day 4, after being re-plated on day 3, form bigger and rounder clusters. (E) E10 cells at day 5 have grown into round neurospheres. Scale bar 50µm; applies A–E.

Figure 3. Cultures derived from different embryonic days display diverse differentiation patterns

E10, E11, E12 and E13 cells were grown in the presence of EGF and FGF2, dissociated into single cells, attached to Petri dishes and allowed to differentiate for 5 days without growth factors. Each plate was stained for a different cell marker; β III tubulin for neurons, GFAP for astrocytes and O₄ for oligodendrocytes. The number of differentiated cells on each plate was blind counted as percentage out of the total number of cells counted on that plate, marked by DAPI nuclear stain. The sum of the percentages from each plate was used to calculate the total differentiation rate, and the presented data are percentage out of the differentiated cells. Data are mean of 3–5 plates. As derivation day progressed, fewer neurons were formed and more astrocytes emerged. Beta3T, β III tubulin; GFAP, glial fibrillary acidic protein. Every embryonic day (E) represents one derivation, 3–5 Petri dishes per derivation.

Figure 4. E10 cells grow best in the presence of both EGF and FGF2

Daily counts of viable cells using trypan blue in the presence of either EGF or FGF2, both or none. 10⁷ E10 cells were plated on day 1. Exposure of cells to EGF, FGF2 or none resulted in lower number of spheres compared with the group exposed to both growth factors. Data are presented as percent of living spheres compared to both EGF and FGF2 (the 100% line). EGF, epidermal growth factor; FGF2, basic fibroblast growth factor. The line representing EGF+FGF is an average of three derivations; all other lines represent percentage survival of only one derivation (not an average).

Figure 5. E10 neurospheres differentiate into neurons, astrocytes and oligodendrocytes

E10 neurospheres were grown for 5 days with EGF and FGF2, then attached to Petri dishes and allowed to differentiate for 5 days without growth factors. Cells differentiated into (A,B) neurons (β III tubulin, green), (C) astrocytes (GFAP, red) and (D) oligodendrocytes (O₄, red). Nuclei were stained using DAPI (blue). (E,F) Triple labeling of β III tubulin, GFAP and DAPI. (G) Triple labeling of β III tubulin, O₄ and DAPI. GFAP, glial fibrillary acidic protein; DAPI, 4',6-diamidino-2-phenylindole.

Figure 6. Intra-cerebrally transplanted Neural stem cells survive and migrate in the posthatched chick brain for at least 14 days

CM-Dil labeled single cells were transplanted intra-cerebrally into 24h-old chicks and the cells were tracked 24h (A–C), 6 days (D,E) and 14 days (F) post transplantation. (A) Transplanted cells migrated and seen along the left cerebral cortex 24h post transplantation. (B) Transplanted cells migrated and seen around the left ventricle 24h post transplantation. (C) Transplanted cells migrated and seen inside the left ventricle 24h post transplantation. (D,E) Transplanted cells are seen around the left ventricle 6 days post transplantation. (F) Transplanted cells are seen along the left cerebral cortex 14 days post transplantation. (G) Quantification of in vivo cell survival 24h, 6 days and 14 days post transplantation. Transplanted cells – red (CM-Dil). Nuclei – blue (DAPI). h, hours Every bar represents two brains (total of 6). Error bars indicate SEM.

Figure 7. IV transplanted neural stem cells in the chick embryo migrated to its brain and survived for at least 6 days

CM-Dil labeled single cells were IV transplanted into E13 chick embryos and the cells were tracked in E19 chick embryos’ brains. Transplanted cells have migrated along the blood vessels and reached the third ventricle of the chick embryos’ brains.