In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors

Abstract

In vitro exposure of neural progenitor cell (NPC) populations to reduced O₂ (e.g. 3% versus 20%) can increase their proliferation, survival and neuronal differentiation. Our objective was to determine if an acute (< 1 hr), in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20 × 1 minute; alternating 21% and 10% O₂). Immediately after AIH, SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC, and this was accompanied by increases in a proliferation maker (Ki67), MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally, immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue, and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to “prime” NPC prior to transplantation into the injured central nervous system.

Introduction

The postnatal brain contains neural progenitor cell (NPC) populations within the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone of the hippocampal dentate gyrus. These NPC pools are mitotically active, multipotent (i.e., generate different cell types), and self-renewing (Gage et al., 1995, Reynolds and Weiss, 1992, Temple, 1989). NPC from these regions are candidates for cell-based therapies following neurological insult (Sohur et al., 2006). For example, potential NPC based therapies include targeted recruitment of endogenous populations (Teng et al., 2006), or replacing dead or damaged cells through auto- or allograft transplantation procedures (Eftekharpour et al., 2008). Fundamental to the success of NPC transplant is the ability to increase the overall cell yield, cell robustness (e.g. for subsequent graft survival) and/or generation of neuronal phenotypes [e.g. for the goal of gray matter replacement (Reier, 2004)] in cell cultures. Manipulation of O₂ levels may provide a relatively simple means to achieve these goals. For instance, culture of NPC in a sustained 3% O₂ environment [i.e. approximating physiological brain O₂ levels (Studer et al., 2000)] as compared to standard culture conditions of 20% O₂ yields populations that are both more proliferative and less susceptible to programmed cell death (Chen et al., 2007, Morrison et al., 2000, Pistollato et al., 2007, Studer et al., 2000, Theus et al., 2008). Reducing the O₂ content in cell culture also impacts cell fate choice (Chen et al., 2007) and enhances neuronal yield (Studer et al., 2000) in progeny cells.

The aforementioned in vitro results raise the possibility of using in vivo hypoxia to achieve the same goals. For example, chronic exposure to intermittent hypoxia (CIH) can trigger proliferation in CNS postnatal neurogenic niches including the dentate gyrus of the hippocampus and the SVZ of the lateral ventricle (Zhu et al., 2005). The Zhu et al. study establishes the proof-of-principle that in vivo CIH protocols can affect CNS progenitors. However, that study employed extended periods of IH (i.e., days to weeks), and it is unknown how rapidly NPC can respond to in vivo IH. Further, to our knowledge the impact of in vivo IH on the ex vivo expansion and differentiation of cultured NPC has not been explored. This potential application of AIH is intriguing when considering that pre-harvest in vivo hypoxia could “prime” NPC for increased in vitro expansion prior to transplant into the injured CNS.

In the present study we hypothesized that acute in vivo exposure to AIH can alter the proliferation and neuronal differentiation of NPC isolated and cultured from the SVZ of postnatal mice. We further hypothesized that AIH would activate known hypoxia-driven signaling pathways and neuronal fate choice pathways immediately following hypoxia exposures. We focused on HIF-1α, which has previously been shown to be activated in neurogenic niche sites following hypoxia exposure (Cunningham et al., 2011, Nanduri et al., 2008). In addition, we examined NF-κß since it is associated with NPC proliferation after hypoxia (Widera et al., 2006). Our results demonstrate that in vivo AIH profoundly increases NPC population expansion, and that this effect is mediated through increased proliferation as evidenced by increased Ki67 expression and MTT activity. We further show evidence that HIF-1α and related downstream signaling may be involved with AIH-mediated effects on NPC proliferation and differentiation.