Effect of leukemia inhibitory factor on long-term propagation of precursor cells derived from rat forebrain subventricular zone and ventral mesencephalon

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Abstract

Tissue blocks containing neural precursor cells were isolated from the rat forebrain subventricular zone (SVZ) and ventral mesencephalon (VM) and propagated as neural tissue-spheres (NTS). In the presence of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF), SVZ-derived NTS were propagated and maintained for more than 6 months with a cell population doubling time of 21.5 days. The replacement of EGF by leukemia inhibitory factor (LIF) resulted in a cell population doubling time of 19.8 days, corresponding to a 10-fold increase in estimated cell numbers over a period of 70 days, at which point these NTS ceased to grow. In the presence of FGF2 and LIF, VM-derived NTS displayed a cell population doubling time of 24.6 days, which was maintained over a period of more than 200 days. However, when LIF was replaced by EGF, the cell numbers only increased 1.2 fold over 50 days. Using different immunohistochemical markers, we observed a distinct compartmentalization of cells within the spheres. In SVZ-derived NTS an outer compartment of proliferating (nestin+/Ki67+), preferentially neurogenic (β-tubulin III+/MAP2+) cells, surrounded by an inner compartment of glial (GFAP+/CNPase+) cells. The inner compartment of long-term propagated VM-derived NTS contained GFAP+ cells as well as cells immunoreactive for the precursor cell marker nestin, even where minimal cell proliferation was observed.

Our results demonstrate that tissues from rat SVZ and VM can be propagated as NTS. However, the cellular organization of the NTS and the need for mitogens to maintain long-term proliferative capacity differ with the origin of the tissue.

Introduction

Neural stem cells have gained considerable interest as a possible source of cells for regenerative therapies for neurological disorders. For such purposes, high numbers of cells are needed and therefore methods for propagation of cells over long periods of time are desirable.

Endogenous neural stem cells appear to be present not only in highly proliferative regions like the subventricular zone (SVZ) and the hippocampal dentate gyrus, but also in several other brain areas (Lie et al., 2002, Weiss et al., 1996). However, stem cells derived from various regions or different species appear to have different growth factor requirements for propagation in vitro (Weiss et al., 1996). These differences may be critical for defining standardized conditions for long-term propagation of cells.

Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) are widely used as mitogens for the in vitro propagation of neural stem cells, either alone, together, or in combination with other factors. EGF and FGF2 often seem interchangeable, although FGF2 appears to have greatest effects on prenatal tissues, whereas EGF mainly acts on early postnatal and adult-derived cultures (Morshead et al., 1994, Zhu et al., 1999). Within the SVZ, neural precursor cells have been reported to respond to either EGF or FGF2 (Tropepe et al., 1999). Effects may also differ between species as exemplified by the continued proliferative effect of EGF and FGF2 on fetal (E15) mouse cells, while similarly exposed fetal (E16) rat cells fail to propagate after 5 passages (Svendsen et al., 1997).

Another factor used for propagation of stem cells is the IL-6 family member leukemia inhibitory factor (LIF), which is necessary and sufficient to sustain self-renewal of mouse embryonic stem cells (Nichols et al., 1990), but reported to be without significant effect on human ES cells (Thomson et al., 1998). LIF has, however, been shown to prolong the proliferative capacity and to increase expansion rates of human forebrain-derived fetal cells when added to EGF-propagated cultures (Carpenter et al., 1999, Tarasenko et al., 2004).

Enzymatic digestion or mechanical trituration is often used for passaging of stem/progenitor cell cultures. However, intercellular contacts may be important for optimal proliferation of cells in vitro even in the presence of exogenously administered mitogens (Ostenfeld and Svendsen, 2003). The growth of neurospheres, containing developing neural cells, have thus been reported to increase if dissociation is avoided during sub-culturing (Svendsen et al., 1998). Enzymatic treatment and trituration may moreover distort the cellular composition, leaving only the most robust cells to survive. Neurospheres are nonetheless composed of cells at several stages of development and differentiation and are not pure stem cell preparations. Cells found in these aggregates tend to arrange themselves into layers with different immunocytochemical properties, possibly either imitating the cellular organization found in vivo or simply reflecting the accessibility to the nutrients or growth factors in vitro (Mokry et al., 1996). By using small tissue blocks for expansion as free-floating explant cultures (Andersen et al., 2007), precursor cells are not initially separated from their niche (more differentiated cells), which might preserve a more in vivo-like behavior of the cells. Employing such a procedure for expansion of neural tissue-spheres, we investigated the effects of FGF2, EGF, and LIF during long-term propagation of precursor cells derived from the developing rat forebrain SVZ and ventral mesencephalon.

Section snippets

Forebrain neural tissue-spheres (NTS)

In accordance with the local animal ethics committee approval, newborn Sprague Dawley rats (M&B, DK) were killed by decapitation, the forebrains isolated and chopped into 400 μm coronal slices using a McIlwain tissue chopper. The tissue slices were subsequently transferred to Gey's Balanced Salt Solution (GBSS; Gibco) with 0.5% d-glucose (Merck), and 30 mM KCL (Merck). The most rostral 4–5 slices were discarded, but from the following 3 slices, the ventricular/subventricular (SVZ) region lining

Propagation of forebrain subventricular zone neural tissue-spheres (SVZ NTS)

When SVZ NTS were propagated in the presence of EGF (20 ng/ml) and FGF2 (20 ng/ml) (E/F propagation) (Fig. 1A), cell numbers increased 200-fold during an observation period of more than 6 months with an estimated cell population doubling time of 21.5 days, increasing from 17.4 × 106 to more than 3.2 × 109 cells. Replacing EGF with LIF (10 ng/ml) (L/F propagation) did not significantly affect the expansion rate of SVZ NTS, with a cell population doubling time of 19.8 days observed over a period of

Discussion

The subventricular zone (SVZ) gives rise to forebrain neurons and glia during development and persists throughout adult life, harbouring a heterogeneous population of undifferentiated cells of which up to one-third are mitotically active (Morshead and van der Kooy, 1992). It is still debated whether the adult ventral mesencephalon (VM) contains stem cells (Frielingsdorf et al., 2004, Lie et al., 2002, Shan et al., 2006, Zhao et al., 2003), but propagation of rat and human VM cells has been

Acknowledgments

The technical help from Dorte Lyholmer, Maibritt V. Damm, Inge Nielsen, and Randi Godskesen is gratefully acknowledged. This research was supported by the Danish Parkinson Association, the Danish Medical Research Council (J. No. 22-02-0150), the Danish Centre for Stem Cell Research (www.dasc.dk), and as part of the European Science Foundation EUROCORES Programme EuroSTELLS, by funds from the European Commision Sixth Framework Programme (contract no. ERAS-CT-2003-980409).

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