Growth hormone promotes proliferation of adult neurosphere cultures

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Abstract

Objectives

Growth hormone (GH) and its receptor (GHR) are widely expressed in the CNS. During development, GH signaling regulates both proliferation of neural progenitor cells as well as their differentiation into neurons and glia. Here we have examined the effect of GH signaling on adult subventricular zone derived neural progenitor cells cultured as neurospheres.

Design

GH was added to adult wild-type (WT) neurosphere cultures and neurosphere growth measured using the MTT cell proliferation assay. To examine the influence of endogenous GH production on neural progenitors, neurospheres derived from GH receptor knockout (GHRKO) mice were examined by measuring neurosphere sizes and Ki67 and TUNEL immunoreactivity. In addition, neurosphere growth curves were compared following long term culture. Finally, the differentiation of WT vs. GHRKO neurospheres was compared using immunocytochemistry for βIII-tubulin and GFAP.

Results

While GH alone was insufficient to support neurosphere formation, it enhanced neurosphere growth by 20% in the presence of epidermal growth factor and fibroblast growth factor-2. Compared to wildtype neurospheres, GHRKO neurospheres were smaller, contained fewer proliferating cells and exhibited reduced self-renewal in long term culture. Addition of GH increased STAT5 phosphorylation levels in neurosphere cells. Upon differentiation, GHRKO neurospheres showed accelerated neurogenesis, although over time similar numbers of βIII-tubulin positive neurons were generated by cells of both genotypes.

Conclusions

GH functions as an autocrine mitogen in adult neurosphere cultures and promotes proliferation of neural progenitor cells as well as self-renewal of neurosphere cultures. In addition, signaling through the GHR appeared to delay neuronal differentiation in adult neurospheres.

Introduction

Growth hormone (GH) and its receptor (GHR) are expressed by many cell types throughout the body. Low levels of circulating GH are present in the blood at birth and increase steadily until reaching peak levels at puberty. Produced by the pituitary gland, systemic GH signal is an important driver of postnatal growth and metabolism.

In addition to its prominent role in postnatal development, local production of GH in the embryonic central nervous system (CNS) [1], [2] has been implicated in progenitor cell proliferation and the histogenesis of the brain [3], [4]. GH is synthesized by neurons [5] and astrocytes [6], as well as neural progenitor cells [7], and the neural GH expression follows a similar temporal pattern to systemic GH, rising into adulthood [8], [9].

Expression of the GHR is widespread throughout the CNS and is found in neurons and glia as well as endothelial cells, ventricular lining and microglia [10], [11]. In the hypothalamus, levels of GHR mRNA peak in the early postnatal period and are maintained at maximal levels until adulthood, declining thereafter [12], [13]. GHR immunoreactivity in the hypothalamus as well as other areas of the CNS was found to follow a similar time course, with peak expression observed in juvenile animals [10].

Addition of GH to rat embryonic neural progenitor cultures stimulates proliferation [3], [14], suggesting a mitogenic role for GH during brain development. This role appears to continue into post-natal development, with GH deficient mice exhibiting reduced glial proliferation 1 week after birth resulting in hypomyelination and deficient synaptogenesis in the adult [3], [14], [15].

In addition to promoting proliferation of embryonic neural progenitors and later expanding glial populations, GH may play a role in the differentiation of these cells. Addition of GH to embryonic neural cultures enhanced both neurogenesis and gliogenesis [3], however GH was shown to inhibit neurogenesis in neural stem cell cultures from neonatal mice [3]. Moreover, knockout mice lacking the GHR exhibited increased neuronal densities in the cortex [4] supporting the conclusion that GH signaling inhibits neurogenesis during early postnatal development. GH may promote proliferation of progenitor cells at the expense of their differentiation.

Less is known about the role of GH in the adult CNS. Unlike the embryonic and early postnatal brain, the adult brain is largely post-mitotic, with pools of multipotent neural stem cells (NSCs) retreating to niches proximal to the ventricles [16]. NSCs divide very slowly, with cell cycle times of 15–20 days [17], [18], either symmetrically, to produce two new NSCs or asymmetrically, producing an NSC and a neural progenitor cell. Neural progenitors are highly mitotic and migrate long distances to the olfactory bulb [19], and the hippocampus [20], where they differentiate into neurons and glia. The continuing supply of new neurons into parts of the adult brain plays an important role in regulating CNS function and adult neurogenesis has been implicated in learning and memory processes. Moreover, the prospect that the stem cell pools in the adult brain can be directed towards repair of neural injuries has received strong interest. If such therapies are to be realized, factors that activate adult NSCs to enhance neurogenesis must be identified.

The adult subventricular zone (SVZ) of the lateral ventricles is known to contain NSCs, which exhibit an intermediate phenotype between radial glia and astrocytes [21]. SVZ NSCs can be isolated and propagated in vitro as neurospheres in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) [22], [23], [24]. In this study, we have investigated the role of GH in adult neural progenitor cells, using neurosphere cultures derived from wild-type mice and knockout mice lacking the GHR (GHRKO).

Section snippets

Mouse strains

C57BL/6 wild-type and GHRKO mice were obtained from stocks maintained at the University of Melbourne. The use of experimental animals was approved by the Pathology, Anatomy and Cell Biology, Microbiology and Immunology and Dental Science Animal Ethics Committee of the University of Melbourne. All procedures were conducted in strict accordance with the guidelines of the National Health and Medical Research Council of Australia.

Neurosphere culture and differentiation

Adult mice were culled by cervical dislocation and brains removed

Establishment of GHRKO neurosphere cultures

We derived primary neurospheres from 3 adult (3–4 months) GHR knockout (GHRKO) mice alongside 3 age- and sex-matched littermate controls. Upon passaging, both WT and GHRKO neurospheres gave rise to new neurospheres, demonstrating self-renewal over an extended culture period (8–20 passages) (Fig. 1a). Furthermore, GHRKO neurospheres generated neurons, oligodendrocytes and astrocytes upon differentiation, even after extended cell culture (Fig. 1b). These results demonstrate that neurosphere

Discussion

In this study we have employed the neurosphere culture system to examine the effects of GH on adult neural progenitor cells. We have shown that GH is neither necessary nor sufficient for the propagation of neural stem cells in vitro (Fig. 1). However, in the presence of the neurosphere mitogens EGF and FGF-2, GH increased neurosphere growth suggesting a synergistic, mitogenic influence. Both GH and the GHR were previously shown to be expressed by neurosphere cells [7] and further validated at

Acknowledgements

We thank Alisa Turbic and Reuben Klein for technical assistance. This work was supported by a project grant from the National Health and Medical Research Council of Australia (#350227). AMT is supported by a Senior Research Fellowship from the NH&MRC (#350226). We thank Dr. J. Kopchick and Dr. K. Coschigano for the generous donation of GHR−/− mice.

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