Resveratrol promotes the survival and neuronal differentiation of hypoxia-conditioned neuronal progenitor cells in rats with cerebral ischemia

Abstract

Hypoxia conditioning could increase the survival of transplanted neuronal progenitor cells (NPCs) in rats with cerebral ischemia but could also hinder neuronal differentiation partly by suppressing mitochondrial metabolism. In this work, the mitochondrial metabolism of hypoxia-conditioned NPCs (hcNPCs) was upregulated via the additional administration of resveratrol, an herbal compound, to resolve the limitation of hypoxia conditioning on neuronal differentiation. Resveratrol was first applied during the in vitro neuronal differentiation of hcNPCs and concurrently promoted the differentiation, synaptogenesis, and functional development of neurons derived from hcNPCs and restored the mitochondrial metabolism. Furthermore, this herbal compound was used as an adjuvant during hcNPC transplantation in a photothrombotic stroke rat model. Resveratrol promoted neuronal differentiation and increased the long-term survival of transplanted hcNPCs. 18-fluorine fluorodeoxyglucose positron emission tomography and rotarod test showed that resveratrol and hcNPC transplantation synergistically improved the neurological and metabolic recovery of stroke rats. In conclusion, resveratrol promoted the neuronal differentiation and therapeutic efficiency of hcNPCs in stroke rats via restoring mitochondrial metabolism. This work suggested a novel approach to promote the clinical translation of NPC transplantation therapy.

Keywords

neuronal progenitor cells / resveratrol / cerebral ischemia / neuronal differentiation / mitochondrial metabolism / positron emission tomography

1 Introduction

Cerebral ischemia (i.e., stroke) is one of the leading causes of disability worldwide [1] and is characterized as neuronal cell loss and neuronal tissue damage due to the lack of blood flow [2]. Owing to the poor regenerative ability of the adult brain, stroke induced neuronal cell or tissue loss is permanent and results in long-term neurological dysfunction [3]. Therefore, novel therapies must be developed to regenerate lost cells and tissues after stroke.

Neuronal progenitor cell (NPC) transplantation has become a focus of stroke treatment research due to its potential to repopulate lost cells and regenerate neuronal tissues after stroke [3,4]. NPCs derived from induced pluripotent stem cells bypass ethical concerns and show mild immune rejection after transplantation, thereby providing a promising cell resource for transplantation therapy [2,5]. Nevertheless, NPC transplantation is still far from clinical application due to restrictions including high mortality and limited neuronal differentiation [6–8].

Hypoxia conditioning is a widely used approach to increase the tolerance of cells to detrimental elements in transplanted environment through various mechanisms including the downregulated activity of apoptotic factors, reduced reactive oxygen species (ROS) generation, and suppressed mitochondrial metabolism [7,9]. Hypoxia-conditioned NPCs (hcNPCs) show increased in vivo survival and improved therapeutic effect in recipients compared with those in control NPCs [10–12]. Nevertheless, hypoxia conditioning is reported to inhibit the neuronal differentiation of NPCs [13,14].

Suppressed neuronal differentiation under hypoxia condition is partly due to the altered energy metabolism. NPCs adapted to hypoxia condition show reduced mitochondrial metabolism and depend on glycolysis for energy production [9,13]. However, neuronal differentiation involves a metabolic shift from glycolysis to mitochondrial oxidative phosphorylation [15], which depends on peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1)-mediated mitochondrial biogenesis [15,16] and regulates neuronal fate commitment, synaptogenesis, and the development of electrophysiological activities [17–19]. Deficient mitochondrial metabolism would hinder the differentiation and function of neurons, whereas restored mitochondrial function could alleviate neuronal developmental and degeneration disease [20]. A recent stem cell study proved that reducing glycolysis is a viable approach to promote the neuronal differentiation of NPCs [13]. However, to our knowledge, no research attempted to regulate mitochondrial metabolism to promote the neuronal differentiation of NPCs under hypoxia conditions.

Resveratrol is a natural polyphenol present in Chinese herbal medicine white hellebore (Veratrum grandiflorum Loes. fil.) and Polygonum cuspidatum [21]. This compound could easily cross the blood brain barrier and shows neuroprotective effect [22,23]. In particular, resveratrol promotes mitochondrial biogenesis and function by activating PGC-1 [24]. Considering the above characteristics, resveratrol is a promising agent to promote the neuronal differentiation and transplantation efficiency of hcNPCs.

In this study, resveratrol was administered during the in vitro neuronal differentiation of hcNPCs to determine whether it could alleviate the deficient neuronal differentiation of hcNPCs. This herbal compound was also applied as an adjuvant during hcNPC transplantation to investigate whether the combined use of resveratrol and hcNPC transplantation could further improve the neurological and metabolic outcome in a rat model of stroke compared with hcNPC transplantation alone.

2 Materials and methods

2.1 NPC culture and neuronal differentiation

NPCs derived from human induced pluripotent stem cells (Cellapy) were cultured as the neurospheres in NeuroEasy Human Neuronal Progenitor Cell Maintenance Medium (Cellapy) under humanized atmosphere with 5% CO₂ at 37 °C. When their diameter reached approximately 200 mm, the neurospheres were dissociated with NeuroEasy Human Neuronal Progenitor Cell Dissociation Medium (Cellapy), resuspended in NeuroEasy Human Neuronal Progenitor Cell Inoculation Medium (Cellapy), and transferred into a suspension culture flask (Nunc) at a density of 10⁵ cells/mL. After 24 h, the inoculation medium was replaced by maintenance medium, which was changed every 2 days.

NeuroEasy Human Neuron Differentiation Kit (Cellapy) was used to differentiate NPCs into mature neurons. NPCs cultured as neurospheres were dissociated with dissociation medium (Cellapy), resuspended with Neuron Differentiation Inoculation Medium (Cellapy), and planted on a poly-ornithine (Sigma) and laminin (Millipore) coated dish at 5 × 10⁴ cells/cm². After 24 h, the differentiation inoculation medium was aspirated and replaced by Neuron Differentiation Maintenance Medium, which was changed every 3 days.

2.2 Hypoxia conditioning of NPCs

Dimethyloxalyl Glycine (DMOG, MCE), a hypoxia mimicking reagent, was used for the hypoxia conditioning of NPCs [25]. NPCs were incubated in culture medium supplemented with 200 mmol/L DMOG for 24 h and defined as hypoxia conditioned NPCs [25,26].

2.3 Resveratrol administration

Resveratrol (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) at 5 mmol/L as stock solution and then added into neuronal differentiation medium at the final concentration of 5 mmol/L [22,27]. For the treatment of stroke rats, resveratrol was given intraperitoneally at 10 mg/kg (body weight) [22].

2.4 In vitro experimental design

NPCs pretreated with DMOG or equivalent amount of DMSO were termed as the hcNPC and control groups, respectively. After the pretreatment, both groups were tested by Trypan blue assay to analyze cell viability under ROS insult and by 5-ethynyl-2-deoxyuridine (EdU) assay to test their proliferation rate. The mitochondrial membrane potential (Dym), glucose consumption, and energy metabolism of NPCs were also tested by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo-cyanineiodid (JC-1) staining, 18-fluorine fluorodeoxyglucose (¹⁸F-FDG) uptake assay, and Seahorse assay, respectively (Fig. 1A).

The control NPCs and hcNPCs were induced for neuronal differentiation for 4 weeks in vitro. hcNPCs treated with 5 mmol/L resveratrol during neuronal differentiation were termed as the hcNPC+ Res group. The neuronal fate commitment of NPCs was analyzed by bIII-tubulin (TUJ1, neuron marker) immunofluorescence staining. Mitochondrial mass and biogenesis were examined by the translocase of mitochondrial outer membrane 20 (TOMM20) expression and PGC-1 (including PGC-1a and PGC-1b) mRNA level, respectively. After 4 weeks of differentiation, the mitochondrial function and energy metabolism of neurons were tested by tetramethylrhodamine methyl ester (TMRM) fluorescence assay and Seahorse bioenergetics assay, respectively, and their synaptogenesis and calcium homeostasis of neurons were analyzed by postsynaptic density 95 (PSD95) expression and Fluo-4 calcium imaging, respectively (Fig. 1A).

2.5 Measurement of energy metabolism

The glycolysis and mitochondrial metabolism of NPCs or neurons were measured by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, using a Seahorse XF96 analyzer (Seahorse Bioscience) [28] (details are provided in the supplemental material).

2.6 Animals and in vivo experimental design

Male Sprague Dawley rats (250–300 g, Silaike Experimental Animal Company) were kept under standard laboratory conditions with freely accessed food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University (Protocol No. ZJU20190033).

The rats receiving photothrombosis were randomly assigned to one of the following four groups: Vehicle group (receive vehicle, no treatment), Res group (receive resveratrol treatment), hcNPC group (receive hcNPC transplantation), and hcNPC+ Res group (receive the combined therapy of hcNPCs and resveratrol). The day of photothrombosis treatment was termed as day -7. Ischemia lesion and metabolic deficiency induced by photothrombosis were confirmed by 2,3,5-triphenyltetrazolium chloride (TTC) staining and ¹⁸F-FDG positron emission tomography (PET) scanning at day -6. NPCs or vehicle medium were injected into rat brain after photothrombosis at day 0. Resveratrol was used daily from the day of transplantation to the day the animals were sacrificed. After transplantation, NPCs were traced by bioluminescence image at day 1, week 2, and week 4. Metabolic change of rat brain after photothrombosis was assessed by ¹⁸F-FDG PET at week 4. Neurological function of rats was analyzed by rotarod test prior to photothrombosis (baseline) and at day -6, day 1, week 2, and week 4. After the last time of rotarod test and PET scan, the rats were sacrificed, and the brain was removed for immunofluorescence staining (Fig. 4A).

2.7 Animal model preparation and cell transplantation

Photothrombotic assay was used to establish rat stroke model [29]. The rats were anesthetized through intraperitoneal injection with 1.5% pentobarbital sodium (50 mg/kg, i.p.) and fixed at a stereotactic apparatus (RWD Life Science Corporation). The skull was then exposed and disinfected with 5% iodophor (Minsheng). A unilateral skull hole (1.00 mm in diameter, 2.50 mm posterior to bregma, 3.00 mm lateral to sagittal suture) was drilled using dental drill (RWD Life Science Corporation) to expose the sensory cortex. Rose Bengal (30 mg/mL solved in saline, Sigma) was injected into tail vein according to the body weight (0.25 mL/100 g), and the exposed sensory cortex was illuminated by a customized laser light resource (5 mW, 525 nm) for 5 min to induce cerebral infarction. After saturation, the rats were put on a warm blanket and received penicillin (80 000 units/rat; LKPC).

Seven days after photothrombosis, the stroke rats were anesthetized through intraperitoneal injection with 1.5% pentobarbital sodium (50 mg/kg, i.p.) and fixed in stereotactic apparatus. 1 × 10⁵ human NPCs (transduced with lentivirus carrying a pLV-eGFP-T2A-Luciferase cassette) were suspended in 1 mL of Dulbecco’s Phosphate Buffer Saline (DPBS) (Hyclone, USA) and microinjected into peri-infarct brain area (2.50 mm posterior to bregma, 2.00 mm lateral to sagittal suture, 2.00 mm ventral to brain surface) using a Hamilton microsyringe. Cyclosporine A (1 mg/kg; MCE) injection was given daily to the rats from the day of photothrombosis to the day the animals were sacrificed.

2.8 Neurofunctional test

Rotarod test was used to assess the neurological function of rats to determine whether transplantation or resveratrol treatment could improve the neurological deficiency induced by stroke. A customized rotarod device (diameter= 10 cm) was used. The rats were trained with steady speed mode (10 rpm) for 3 days prior to photothrombosis. Their neurological function was tested with accelerating mode (speed gradually accelerates from 5 to 50 rpm in 100 s), and the time that the rats remained on rotarod was recorded for statistical analysis.

2.9 ¹⁸F-FDG PET imaging and image analysis

¹⁸F-FDG PET was used to evaluate the metabolic recovery of stroke brain after NPC transplantation or resveratrol treatment [30]. The rats were fasted overnight prior to PET imaging. On the day of PET scanning, the rats were intraperitoneally given 18.5 MBq (500 mCi) ¹⁸F-FDG. After 40 min interval, the rats were anesthetized with 2.0% isoflurane and placed on a high-resolution microPET (Siemens Medical Solutions) for 10 min static acquisition. PET images were reconstructed using maximum posteriori 2 (MAP2) algorithms. Reconstructed PET image was displayed and analyzed by IDL VM (Exelis Inc) and ASIPro VM (Siemens Medical Solutions) software. Regions of interest were drawn symmetrically on the infarcted sensory cortex and the contralateral normal sensory cortex. The lesion to normal (L/N) ratio was calculated by the following formula: L/N ratio= mean counts per pixel of lesion region of interest/mean counts per pixel of normal region of interest.

2.10 Statistical analysis

Statistical analysis was performed using SPSS 22 (IBM Corp) and GraphPad Prism 6.0 (GraphPad Software). Statistical significance was determined by one-way ANOVA, followed by post hoc multiple-comparisons tests (Turkey’s Correction) to analyze differences among three or more groups in Fig. 2, Fig. 3, and Fig. 5. Unpaired Student’s t-test was used to analyze statistical differences between two groups in Fig. 1 and Fig. 4. P≤0.05 was considered significant.

3 Results

3.1 hcNPCs exhibited increased ROS tolerance, reduced proliferation, and suppressed mitochondrial metabolism

Trypan blue assay was performed on NPCs incubated in 50 mmol/L H₂O₂ for 24 h to validate the benefit of hypoxia conditioning on NPC survival. The result showed that the hcNPC group showed increased cell survival than the control group (P<0.05, Fig. 1B), indicating the increased ROS tolerance of hcNPCs. Furthermore, EdU immunostaining showed that nucleus with EdU incorporation was decreased in the hcNPC group compared with that in the control (P<0.05, Fig. S1 and Fig. 1C), implying the reduced proliferation of hcNPCs. Mitochondrial membrane potential was first examined by JC-1 staining to analyze the energy metabolism of NPCs. The red/green JC-1 fluorescence ratio was significantly reduced in the hcNPC group compared with that in the control group (P<0.05, Fig. 1D and 1E), suggesting the reduced Dym in hcNPCs. Seahorse assay and ¹⁸F-FDG uptake assay were performed to further analyze the mitochondrial oxidative phosphorylation and glycolysis in NPCs. The results showed that hcNPCs had reduced OCR (P<0.05, Fig. 1F), increased ECAR (P<0.001, Fig. 1G), and increased ¹⁸F-FDG uptake compared with the control counterparts (P<0.05, Fig. 1H), indicating a metabolic shift from mitochondrial metabolism to glycolysis.

3.2 Resveratrol promoted the mitochondrial biogenesis and neuronal differentiation of hcNPCs

The neuronal differentiation of NPCs involves upregulated mitochondrial metabolism, which depends on mitochondrial biogenesis. TOMM20 expression was first assessed by Western blot to analyze mitochondrial biogenesis during the neuronal differentiation of NPCs. The result showed that TOMM20 expression was downregulated in the hcNPC group compared with that in the control group from week 1 to week 4, and resveratrol administration remarkably upregulated TOMM20 expression during the neuronal differentiation of hcNPCs (Fig. 2A). The expression of PGC-1, a core regulator for mitochondrial biogenesis, was also examined at week 2 after neuronal differentiation. The results showed that PGC-1a mRNA showed a decreasing trend in the hcNPC group compared with that in the control group (P = 0.05) and was significantly increased in the hcNPC+ Res group compared with that in the hcNPC (P<0.01) and the control group (P<0.05, Fig. 2B). PGC-1b mRNA level was significantly decreased in the hcNPC group compared with that in the control (P<0.001) and the hcNPC+ Res group (P<0.01, Fig. 2C) and showed no statistical difference between the control group and hcNPC+ Res group. These results revealed the reduced mitochondrial biogenesis during the neuronal differentiation of hcNPCs, which was relieved by resveratrol. In addition, RT-PCR results indicated that PGC-1b may play a more important role than PGC-1a in suppressing the mitochondrial biogenesis of hcNPCs.

The immunostaining of TUJ1, a neuronal marker, was used to detect neurons derived from NPCs (Fig. 2D). The percentage of TUJ1 positive neurons was significantly reduced in the hcNPC group compared with that in the control group (P<0.05) and hcNPC+ Res group (P<0.01) at week 1 but showed no statistical difference between the control and hcNPC+ Res groups. From week 2 to week 4, TUJ1 positive neurons showed no statistical difference among the three experimental groups (Fig. 2E). These results revealed the delayed neuronal fate commitment of hcNPCs at the early time of neuronal differentiation, which was relieved by resveratrol administration.

3.3 Resveratrol promoted the mitochondrial metabolism and maturation of neurons derived from hcNPCs

Given the crucial role of mitochondrial metabolism during neuron maturation, the energy metabolism of neurons at week 4 was analyzed by TMRM staining, Seahorse extracellular flux analysis, and ¹⁸F-FDG uptake assay. TMRM fluorescence images showed reduced Dym in the hcNPC group compared with that in the control and hcNPC+ Res group (P<0.05, Fig. 3A and 3B). Seahorse extracellular flux analysis indicated that the hcNPC group showed reduced OCR and increased ECAR compared with the control (P<0.001) and hcNPC+ Res groups (P<0.05, Fig. 3C and 3D). Furthermore, increased ¹⁸F-FDG uptake was observed in the hcNPC group compared with that in the control (P<0.01) and hcNPC+ Res groups (P<0.05, Fig. 3E). No statistical difference was observed between the control and hcNPC+ Res groups during above experiments. These results indicated the deficient mitochondrial metabolism in neurons derived from hcNPCs, which was restored by resveratrol administration.

Mature neurons are characterized by synaptogenesis and calcium homeostasis. The synaptogenesis of neurons was analyzed by the expression of postsynaptic density protein 95 (PSD95). Western blot analysis showed that the PSD95 protein level was reduced in the hcNPC group compared with that in the control and hcNPC+ Res groups (P<0.05, Fig. 3F and 3G). Furthermore, immunofluorescence staining showed that PSD95 protein clusters on neurites were downregulated in the hcNPC group (Fig. 3H). No statistical difference of PSD95 expression was observed between the control and hcNPC+ Res groups. PSD95 expression change indicated the reduced synaptogenesis in neurons derived from hcNPCs, which could be restored by resveratrol administration. The calcium homeostasis of neurons was analyzed by Fluo-4 calcium imaging. Fluo-4 fluorescence change was detected in all three experimental groups after KCL-induced depolarization stimuli (Fig. 3I). However, compared with the control and hcNPC+ Res groups, hcNPC group showed significantly reduced oscillation amplitude (P<0.05, Fig. 3K). The time from KCL stimuli to peak Ca²⁺ level was also significantly longer in the hcNPC group than that in the control and hcNPC+ Res groups (P<0.05, Fig. 3L). At the end of Fluo-4 imaging, the hcNPC group showed higher intracellular Ca²⁺ level compared with the control group and hcNPC+ Res group (P<0.05, Fig. 3M). No statistical difference in calcium homeostasis was observed between the control and hcNPC+ Res groups. Fluo-4 fluorescence imaging suggested the disturbed calcium homeostasis in neurons derived from hcNPCs, which was significantly relieved by resveratrol administration.

3.4 Resveratrol improved the efficiency of hcNPCs transplanted in stroke rats

In vivo survival and neuronal differentiation are main criteria to assess NPC transplantation efficiency. Given that resveratrol promoted the neuronal differentiation and maturation of hcNPCs in vitro, its possible promotion for the transplantation efficiency of hcNPCs in rats with cerebral ischemia was also examined. Infarcted area in the sensory cortex of rats was induced via photothrombosis assay (Fig. S2). NPCs were transplanted into rat brain after photothrombosis at day 0 (seven days post photothrombosis). At day 1, week 2, and week 4, the transplanted NPCs were traced by bioluminescence imaging (Fig. 4B). The luminescence signaling from the transplanted NPCs was significantly higher in the hcNPC+ Res group than in the hcNPC group at week 4 (P<0.05, Fig. 4C), indicating that resveratrol promoted the long-term survival of transplanted NPCs. At week 4, postmortem TUJ1 immunostaining was performed on coronal brain sections from two positions (100 and 500 mm adjacent to the transplantation site). The colocalization of TUJ1 and eGFP indicated that the neurons were derived from transplanted NPCs (TUJ1⁺/eGFP⁺ neurons) (Fig. 4D). The number of TUJ1⁺/eGFP⁺ neurons per section was significantly increased in the hcNPC+ Res group than in the hcNPC group at both positions (P<0.05, Fig. 4E), implying that resveratrol promoted the neuronal differentiation of hcNPCs. In addition, the number of TUJ1⁺/eGFP⁺ neurons per section was significantly reduced with the increasing distance to the transplantation site in the hcNPC group (P<0.05, Fig. 4E) but showed no difference between two positions in the hcNPC+ Res group. This finding indicated that resveratrol promoted the neuronal migration of transplanted hcNPCs.

3.5 Resveratrol and hcNPCs synergistically benefited the metabolic and neurological recovery of stroke rats

¹⁸F-FDG PET image was performed at week 4 to illustrate the metabolic recovery of stroke rats (Fig. 5A). The lesion to normal (L/N) ratio of ¹⁸F-FDG radioactivity in ROI was significantly increased in Res, hcNPC, and hcNPC+ Res groups compared with that in the Vehicle group (P<0.0001, Fig. 5B). This result indicated that resveratrol and hcNPC transplantation or their combination could promote the metabolic recovery of rat brain after photothrombosis. In addition, the L/N ratio in the hcNPC+ Res group was increased compared with that in the Res and hcNPC groups (P<0.01, Fig. 5B), implying that the combined use of resveratrol and hcNPC transplantation lead to better metabolic recovery of stroke rats compared with individual treatment.

Rotarod test was performed to analyze the change of neurological function of stroke rats in Vehicle, Res, hcNPC, and hcNPC+ Res groups. At week 2, the hcNPC+ Res group showed improved neurological function compared with the Vehicle group (P<0.05, Fig. 5C), but no significant neurological recovery was observed in the hcNPC and Res groups. At week 4, the hcNPC and hcNPC+ Res groups showed increased neurological function compared with the Vehicle group (P<0.05 and P<0.001, respectively, Fig. 5C). Furthermore, the neurological function in hcNPC+ Res group was significantly increased compared with that in the Res group (P<0.001) and showed a tendency to increase compared with that in the hcNPC group (P = 0.10, Fig. 5C). These results indicated that the combination of resveratrol and hcNPC transplantation significantly promoted the neurological recovery of stroke rats compared with individual treatment.

4 Discussion

In vitro and in vivo experiments proved that resveratrol promoted the differentiation and maturation of neurons derived from hcNPCs partly via restoring mitochondrial metabolism and improved the therapeutic effect of transplanted hcNPCs on stroke rats.

Hypoxia conditioning mediates a metabolic shift from mitochondrial metabolism to glycolysis, maintains ATP production under hypoxia circumstance, and reduces ROS-induced cell death [9]. In this study, hcNPCs showed a metabolic shift to glycolysis and increased tolerance to ROS insults, thus further validating the benefit of hypoxia conditioning on cell survival. Hypoxia also influences the proliferation of cells in an oxygen-tension dependent manner. Moderate hypoxia promotes the proliferation of NSCs, whereas severe hypoxia inhibits NSC proliferation [26]. In this study, hcNPCs showed reduced proliferation compared with the control group. The reduced proliferation under severe hypoxia was an adaptive change, which maintains hypometabolism states and increases the tolerance of cells to severe detriments in ischemia brain [31]. Therefore, the reduced proliferation may also contribute to the increased resistance of hcNPCs to ROS stress.

Neuronal differentiation is concurrent with a metabolic shift from glycolysis to mitochondrial metabolism, which is pivotal for the neuronal fate commitment and maturation of neurons [17–19]. In this study, the neuronal fate commitment of hcNPCs was hindered at week 1. Furthermore, deficient neurons maturation and reduced mitochondrial metabolism were observed in the hcNPC group at week 4. These results were in accordance with previous studies, indicating that low oxygen tension maintains undifferentiated state and prevents the neuronal differentiation of NPCs [14,32], and further proved the essential role of mitochondrial metabolism on neuronal differentiation. The activation of Wnt, Notch, or Shh signaling pathways contribute to the hindered neuronal differentiation of hcNPCs [32–34]. However, in this study, mitochondrial metabolism was selected as a main therapeutic target because its restoration was proven as a viable approach to treat neuronal developmental or degenerative disease [20,35].

Activated mitochondrial metabolism depends on PGC-1-mediated mitochondrial biogenesis [15], which lasts through out the 4 weeks of neuronal differentiation and peaks during 2–3 weeks [36,37]. In this study, PGC-1b expression was reduced during the neuronal differentiation of hcNPCs. Concurrently, TOMM20 expression decreased mitochondrial mass in the hcNPC group. These results indicated that hypoxia conditioning suppressed mitochondrial biogenesis through transcriptional regulation [38]. Considering the core regulatory role of PGC-1 during increased mitochondrial function [39], increasing its expression and activity may be a promising approach to restore mitochondrial metabolism.

Resveratrol could activate PGC-1-mediated mitochondrial biogenesis [24] and acts as a widely admitted agent to upregulate mitochondrial metabolism. This compound also promotes the neuronal differentiation of mesenchymal stem cells or neurogenesis of elder Wister rats through the activation of Sirt1 [40], a deacetylase of PGC-1 [39]. In this study, resveratrol was used during the neuronal differentiation of hcNPCs. PGC-1 expression, TOMM20 protein level, and mitochondrial metabolism were significantly promoted, and neuronal differentiation and maturation were concurrently improved. These results validated the beneficial effect of resveratrol on the neuronal differentiation of NPCs and indicated that the activation of mitochondrial metabolism is a main underlying mechanism.

The survival and neuronal differentiation of transplanted NPCs directly contribute to the therapeutic outcome of stroke [3,7]. Although resveratrol promotes the endogenous neurogenesis of adult rats [41], its influence on transplanted NPCs remains unreported. In this study, bioluminescence results showed that the bioluminescent signal from transplanted NPCs had no significant difference between the hcNPC and hcNPC+ Res groups at day 1 and week 2 but was significantly increased in the hcNPC+ Res group compared with that in the hcNPC group at week 4. These results indicated that resveratrol had minimal influence on the in vivo population of hcNPCs during the first 2 weeks but significantly promoted the long-term survival of transplants in vivo. The population of transplanted NPCs is seriously influenced by their proliferation rate and the incidence of apoptotic death during the early stage [42–44]. Immunorejection in host brain becomes a main influence on the long-term survival of transplants [42]. The neuroprotection and anti-apoptotic function of resveratrol sharing the similar mechanism with the hypoxia preconditioning assay [22,45] might be the main reason that the combination of resveratrol and hypoxia conditioning did not further improve the in vivo survival of NPCs during the first 2 weeks. Furthermore, resveratrol ameliorates the leukocyte-endothelial cell adhesive interaction and prevents the immunorejection of transplants [46], which may account for the increased long-term survival of NPCs in hcNPC+ Res group.

Transplanted NPCs need at least 4 weeks to differentiate into mature neurons [42]. Therefore, the 4th week post-transplantation is the most widely used time point to analyze the in vivo differentiation of transplanted NPCs and the neurological recovery of stroke rats [8]. In this study, postmortem immunofluorescence staining indicated that the co-administration of resveratrol improved the neuronal differentiation of transplanted NPCs. This result was in accordance with our in vitro experiments and further validated the benefit of resveratrol on neuronal differentiation. ¹⁸F-FDG PET image could non-invasively monitor the metabolic recovery of stroke brain and evaluate the metabolic and neurological recovery of stroke rats receiving NPC transplantation therapy [47]. In this study, ¹⁸F-FDG PET imaging results indicated a better metabolic recovery in stroke rats receiving the combined therapy of resveratrol and hcNPCs compared with those given with single therapy. These findings were highly consistent with the result of rotarod test and proved that the combination of resveratrol and hcNPCs led to the good neurological and metabolic recovery of stroke rats, which was attributed to the increased survival and neuronal differentiation of hcNPCs. Resveratrol used alone promoted the metabolic recovery of stroke rats but did not recover the neurological function. This result was predictable because the chronic administration of resveratrol could prevent the recurrence of stroke [48,49] but showed limited function to promote neurological recovery. In summary, these results suggested that the combined use of resveratrol could improve hcNPC transplantation efficiency and the functional recovery of stroke rats.

This study has some limitations. First, the therapeutic effect of the control NPCs and the hcNPCs on stroke rats was not compared because many studies proved the beneficial effect of hypoxia preconditioning on the transplantation efficiency of NPCs [10–12]. Second, in vitro experiments were mainly performed to investigate the mechanism of resveratrol in promoting the neuronal differentiation of hcNPCs due to the limitations in experimental techniques. Further in vivo investigations should be conducted to prove the influence of resveratrol on the mitochondrial metabolism of transplanted NPCs.

5 Conclusions

This study presented the function of resveratrol to promote neuronal differentiation and improve the transplantation efficiency of hcNPCs partly via restoring mitochondrial biogenesis. The results suggested a novel approach to facilitate the clinical translation of NPC transplantation therapy.

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