Bone marrow-derived mesenchymal stem cells prevent the apoptosis of neuron-like PC12 cells via erythropoietin expression
Highlights
► The PC12 cells apoptosis was increased under hypoxia condition mimicked by CoCl2. ► Coculture of BM-MSCs with PC12 cells significantly decreased cell apoptosis. ► CoCl2 induced upregulation of EPO expression in BM-MSCs. ► EPOsiRNA inhibited the expression of EPO and cytoprotective effect of BM-MSCs. ► The expression of EPO is required for BM-MSCs-mediated cytoprotective effect.
Introduction
It is well known that central nervous system (CNS) injury is a leading cause of disability and death, with no effective treatment at present. The pathogenesis of CNS injury includes ischemia, edema, free radical formation, and the manifold loss of neurons, resulting in severe damage such as hemiplegia or paraplegia [4]. Many strategies have been employed to mitigate neuronal loss in CNS injury. One of the most attractive strategies for treatment is bone marrow-derived mesenchymal stem cells (BM-MSCs). It has been suggested that BM-MSCs promote neuronal survival, axonal growth and nerve regeneration through secretion of cytokines and trophic factors in multiple models of CNS injury [14], [16], [27].
Cobalt chloride (CoCl2) is a well-known hypoxia mimetic agent, able to induce mitochondrial apoptotic pathways, and has been widely used in models of CNS injury in vitro [10]. Recent data has shown that CoCl2 induces apoptosis of PC12 cells with an increase in the intracellular levels of reactive oxygen species (ROS) [2]. The hypoxia-sensitive rat pheochromocytoma cell line, PC12, is a useful model for studying CoCl2 neurotoxicity [7]. Much evidence has suggested CoCl2 induced the apoptosis of PC12 cells through the regulation of Bcl-2, Bax and caspase-9 expression [6].
Erythropoietin (EPO) is a hematopoietic and potent survival cytokine involved in post-transcriptional induction associated with hypoxia [5], [26]. Recent research has shown that EPO directly exerts protective properties on PC12 cells [25]. Previous studies have suggested a possible protective mechanism of EPO on neurons and PC12 cells by upregulating Bcl-2 and Bcl-XL expression [11], [23].
The protective mechanisms of BM-MSCs have not been fully elucidated. A recent study demonstrated that hypoxic preconditioning stimulated EPO expression of BM-MSCs and therefore enhanced its ability to promote functional recovery of infarcted myocardium [8]. Our previous study suggested BM-MSCs promoted functional recovery in the brain after stroke by secreting trophic factors [3]. To investigate the potential mechanism of BM-MSCs-mediated cytoprotection, we cultured BM-MSCs with PC12 cells using an indirect co-culture system and investigated the protective effect and mechanisms of BM-MSCs on PC12 cells. Our study showed that BM-MSCs are able to promote survival of PC12 cells and reduce cell death. The cytoprotective effect might be dependent on expression of EPO at least in part via the regulation of Bcl-2 family members and caspases.
Section snippets
Isolation and culture of BM-MSCs
BM-MSCs were isolated from the bone marrow of adolescent, male Sprague-Dawley rats by dissecting tibias and femurs as previously described [15]. The detailed culture method was provided in Supplemental information.
Isolation and culture of PC12 cells
PC12 cells, a rat cell line derived from pheochromocytoma cells, can be induced into neuronal-like cells with nerve growth factor (NGF) [18] and were provided as a kind gift from Professor Jianqiang Feng [20]. PC12 cells were cultured in DMEM supplemented with 10% FBS and maintained
Results and discussion
We observed that CoCl2 (0.6 and 0.8 mM) resulted in significant loss of PC12 cells viability (Supplemental Fig. 1a). We next demonstrated that BM-MSCs co-culture for 24 and 48 h promoted PC12 cells proliferation (Supplemental Fig. 1b).
We investigated whether BM-MSCs exerted protective effects on CoCl2-induced apoptosis in PC12 cells. Compared with the control group, the cell viability of PC12 cells was 49.4 ± 6, 63.5 ± 6, 77.9 ± 3, 69.2 ± 4 and 65.1 ± 7% after 24 h when BM-MSCs were co-cultured at ratios of
Acknowledgments
This work was financially supported by the Natural Science Foundation of China (Nos. 30973093, 801171711), Ministry of Education Fund for the Doctoral (No. 01711100480) and the Natural Science Foundation of Guangdong Province in China (No. 2009B050200010).
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These authors contributed equally.