IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells

Abstract

Background: Long-term maintenance of neural stem cells in vitro is crucial for their stage specific roles in neurogenesis. To have an in-depth understanding of optimal conditional microenvironmental niche for long-term maintenance of neural stem cells (NSCs), we imposed different combinatorial treatment of growth factors to EGF/FGF-responsive cells. We hypothesized, that IGF-1-treatment can provide an optimal niche for long-term maintenance and proliferation of EGF/FGF-responsive NSCs.

Objective: This study was performed to investigate the cellular morphology and growth of rat embryonic striatal tissue derived-NSCs in long-term culture under the influence of different combinatorial effects of certain growth factors, such as EGF, bFGF, LIF and IGF-1.

Methods: The NSCs were harvested and cultured from striatal tissue of 18 days old rat embryos. We have generated neurospheres from these NSCs and cultured them till passage 7 (28 days in vitro) under four different conditional microenvironments: (A) without growth factor, (B) EGF/bFGF, (C) EGF/bFGF/LIF, (D) EGF/bFGF/IGF-1 and (E) EGF/bFGF/LIF/IGF-1. Isolated NSCs were characterised by Immunoflouroscence for nestin expression. The cell growth and proliferation was evaluated at different time intervals (P1, P3, P5 & P7), assessing the metabolic activity based cell proliferation. Apoptosis was studied in each of these groups by In situ cell death assay.

Results: Our results demonstrated certain important findings relevant to long-term culture and maintenance of striatal NSC-derived neurospheres. This suggested that IGF-1 can induce enhanced cell proliferation during early stages of neurogenesis, impose long-term maintenance (up to passage 7) to cultured NSCs and enhance survival efficiency in vitro, in the presence of EGF and FGF.

Conclusions: Our findings support the hypothesis that the enforcement of IGF-1 treatment to the EGF/FGF-responsive NSCs, can lead to enhanced cell proliferation during early stages of neurogenesis, and an extended life span in vitro. This information will be beneficial for improving future therapeutic implication of NSCs, by addressing improved in vitro production of NSCs.

Keywords: EGF; IGF-1.; LIF; bFGF; neural stem cells; neurospheres; proliferative activity.

Figure 1

Cellular morphological analysis of neurospheres. (A) Neurospheres vary in shape; some are well-formed spheres, whereas others are irregular cell clusters. (B) The P1 of NSCs cultures contained cell debris. (C) By P3, neurosphere cultures contained less debris compared with P1. The neurospheres adopted spherical shapes with bright surfaces. (D) Healthy neurospheres are composed of many individual cells and possess small cilia that are apparent on the outer edge. All images were viewed at 100X magnification using an inverted microscope.

Figure 2

Immunocytochemical staining of embryonic striatal stem cells in culture. Results show that neurospheres were positively stained for nestin (red). (A) Phase-contrast image of a neurosphere. (B) Negative control. (C) Adult rat hippocampal neural stem cells as positive control. (D) Neurosphere at P1. (E) Neurosphere at P3. Neurospheres were viewed at 200X magnification using a Pascal 5 confocal microscope (Carl Zeiss, Germany).

Figure 3

The NSCs at P1 until P7 shows different sizes of neurospheres grown in different groups of growth factors. Group A (without growth factor); Group B (EGF/bFGF); Group C (EGF/bFGF/LIF); Group D (EGF/bFGF/IGF-1) and group E (EGF/bFGF/LIF/IGF-1). Group D had the largest in size of neurospheres. Viewed at 100X magnification using an inverted microscope.

Figure 4

Long-term Proliferation and survival of ESSCs: Embryonic neural stem cells were cultured under different cocktails of growth factors (n=5) and change in proliferation was studied at P1, P3, P5 and P7 days of passage using in vitro cell proliferation assay (A).Two-way anova analysis was performed for all the above cell populations to study the significance changes in the rate of proliferation. The data was represented as optical density (OD) at 540nm in mean ± SD. P-value <0.05 was considered as significant. Survival efficiency was also evaluated for ESSCs from all groups by TUNEL assay, and staining was visualized in fluorescent microscopy at 20X magnification. Panel B demonstrated TUNEL/DAPI ^positive NSCs at P3 from different niche conditions (B). Data only shown for group C and group D derived NSCs. The TUNEL/DAPI ^positive NSCs from control group A cultured without any growth factor.

Figure 5

Pair wise comparative analysis of long-term growth profile of ESSCs at different time intervals: Pair wise comparison was analyzed in detail, among NSCs cultured under the influence of different growth factor conditions at P1, P3, P5 and P7 passages using Tukey's HSD (Tukey's honestly significant difference test). The data was represented as mean ± SD and statistical significance was based on P-value <0.05.