1 Introduction
Ischemic stroke is a devastating disease and remains a considerable socioeconomic burden worldwide given its high mortality and long-term morbidity [
1]. Conventional therapeutic strategies, including intravenous thrombolysis and intra-arterial approaches, have a narrow therapeutic time window [
2]. Thus, only a minority of patients can benefit from these therapies. Hence, exploring more promising approaches to treat more patients with ischemic stroke is essential.
Stem cell transplantation is emerging as a favorable therapeutic option for ischemic stroke [
3,
4]. To date, numerous studies have determined the therapeutic effect of various stem cells on ischemic stroke [
5–
7], such as neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). NSCs are distributed in localized brain regions such as the subventricular zone and are expected to proliferate and differentiate into oligodendrocyte progenitors, astrocytes, and neuroblasts in cerebral ischemic insult [
8,
9]. Given the neurogenesis induced by endogenous NSCs, the potential of exogenous transplanted NSCs to replace lost cells and improve neurological function following stroke has been demonstrated [
6,
7,
10]. However, the application of NSCs is limited by ethical issues. Recently, iPSCs have attracted considerable interest as they minimize the risk of immune rejection and avoid ethical issues [
11]. Given their retention of self-renewal and pluripotent characteristics, these cells have been applied in the disease modeling and regenerative therapy of various nervous system diseases [
12], especially ischemic stroke. iPSCs can differentiate into functional neurons, inducing the recovery of brain function after ischemic insult [
5].
At present, growing evidence reveals that the mechanism of stem cell-based therapy involves not only cell differentiation but also the interaction with the ischemic microenvironment [
13]. After cerebral ischemia, the microenvironment in the brain parenchyma changes, including alterations in ionic homeostasis, overproduction of inflammatory cytokines, and abnormal release of proteases [
14,
15]. These changes are related to a series of spatiotemporal pathological events (also known as ischemic cascade), including excitotoxicity, mitochondrial dysfunction, oxidative stress, and apoptosis [
16]. Transplanted stem cells secrete growth or trophic factors to improve the ischemic microenvironment, thus attenuating inflammation and reducing cell death [
4]. In this regard, understanding the ischemic microenvironment provides a novel perspective on the pathology of cerebral ischemia and its therapeutic strategy [
17].
Proteomics can provide detailed information of the microenvironmental changes by obtaining large-scale protein expression profiles in the complex, dynamic, multifactorial pathological process of ischemic stroke [
18]. A number of quantitative proteomic studies on ischemic stroke have been performed. Some were aimed at the pathological changes in the course of stroke [
19–
21], while others were designed to explore the mechanisms of therapeutic strategies [
22,
23], such as stem cell transplantation. He
et al. conducted a proteomic analysis on cerebral ischemic rats after mesenchymal stem cell transplantation, but the research was confined in the short term after transplantation (within 48 h in the acute/subacute phase of stroke) [
22]. Long-term protein changes after stem cell transplantation (in the subacute and chronic phases of stroke) remain unclear, although the long-term adverse effects resulting from ischemic stroke remain widespread.
In this study, we hypothesized that the brain microenvironment after iPSCs transplantation would dramatically change for a long time, and these changes would contribute to neuroprotection in ischemic stroke. To test this hypothesis, 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) scans and neurological tests were performed to determine the metabolic and neurofunctional recovery of cerebral ischemic rats after iPSCs or NSCs transplantation, in which NSCs-transplanted rats were used as a standard for comparison. Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to identify the protein expression profile in the ischemic area of iPSCs-transplanted rats in the subacute and chronic phases.
2 Materials and methods
2.1 Experimental design
The experimental procedure is shown in Fig. 1. A total of 64 male Sprague–Dawley rats (weighing 200–250 g) were randomly assigned to one of the following four groups: sham group (n = 8), phosphate-buffered saline (PBS) group (n = 24), iPSCs group (n = 24), and NSCs group (n = 8). On day 0, all rats underwent middle cerebral artery occlusion (MCAO) surgery, except those in the sham group. On day 1, 18F-FDG PET scans and neurological tests were conducted on eight rats from each group to establish a baseline. On day 3, all rats were injected with iPSCs, NSCs, or PBS, except those in the sham group. On days 7 and 14, the rats (eight per group) were subjected to 18F-FDG PET scans followed by neurological tests. The rats (n = 32, eight from the iPSCs and PBS group on day 7 and 14, respectively) were sacrificed for quantitative proteomics and Western blot analysis. In addition, 11C-methionine (MET) PET scans were performed on day 14 to detect tumor formation. Finally, the remaining rats were sacrificed for hematoxylin–eosin (HE) staining and immunofluorescent staining.
This study was approved by the Institutional Animal Care and Use Committee of Zhejiang University School of Medicine (Protocol No. ZJU2015-074-02).
2.2 Establishment of animal model
Cerebral ischemic stroke was established via MCAO using the intraluminal filament technique as previously described [
6]. The rats were anesthetized with 1.5% pentobarbital sodium (50 mg/kg, intraperitoneally). A midline incision was made in the neck to expose the right common carotid artery (CCA), external carotid artery, and internal carotid artery (ICA). A 4-0 rounded tip monofilament nylon suture was inserted from the right CCA into the ICA. Then, the suture was detained with a length of approximately 18 mm from the CCA bifurcation to block the origin of the MCA. Ninety minutes later, reperfusion was performed by withdrawing the suture. The body temperature of rats was monitored continuously and maintained at 37± 0.3 °C during the surgery. The rats were injected with buprenorphine hydrochloride (0.05 mg/kg, subcutaneously) for analgesia at 4, 12, and 24 h after the surgery.
2.3 Stem cell culture and transplantation
Green fluorescent protein (GFP)-labeled mouse iPSCs were cultured on a mouse embryonic fibroblast feeder layer treated with mitomycin C (Roche, Switzerland) in iPSC culture medium. Humanized mulleri GFP-labeled rat NSCs were cultured in Neural Stem Cell Basal Medium (Millipore, USA) supplemented with 20 ng/mL of FGF2 (Millipore, USA) and 1 µg/mL of puromycin (Millipore, USA). Both iPSCs and NSCs were prepared as previously described [
6].
The rats were anesthetized with 1.5% pentobarbital sodium (50 mg/kg, intraperitoneally) and then placed in a stereotaxic frame (RWD Life Science Co., China). A 1-mm-diameter hole was drilled over the right lateral ventricle with a cranial drill (RWD Life Science Co., China) after making a midline incision in the head. Suspended iPSCs or NSCs (2.0×106 cells in 20 µL of culture medium) were stereotactically injected into the right lateral ventricle (AP, –0.9 mm; ML, –1.5 mm; DV, –3.5 mm) within 15 min by using a Hamilton microsyringe (kdScientific, USA). The PBS group was injected with 20 µL of PBS into the same position. After injection, the needle remained in place for 5 min and then was slowly withdrawn over 5 min.
2.4 Neurological tests
Neurological tests were performed weekly by using the Garcia neurological grading criteria [
24]. The neurological behaviors were scored by six tests using an 18-point scale: (1) spontaneous activity, (2) symmetry of movement, (3) symmetry of forelimbs, (4) climbing, (5) body proprioception, and (6) response to vibrissal touch. The total score was correlated closely with the severity of necrotic neurons in a rat model of MCAO. The lower the score, the more severe the injury. The inclusion criteria ranged from 7 to 14 points (except for the sham group). To minimize the influence of initial value and individual difference on the results, the neurological scores before transplantation were defined as the baseline, and the subsequent scores were normalized to the baseline. Investigators experimented with a double-blind method.
2.5 PET imaging and image analysis
The rats were anesthetized with 2% inhalant isoflurane and injected with approximately 18.5 MBq (500 μCi) of
18F-FDG via the tail vein. After 40 min, PET images were acquired using a PET scanner (Siemens Medical Solutions). Semiquantitative assessments of glucose metabolism were expressed by lesion-to-normal (L/N) ratio, which was calculated as follows: L/N ratio= mean counts per pixel of infarcted area/mean counts per pixel of the contralateral normal area [
25]. The L/N ratios before transplantation were defined as the baseline, and the subsequent L/N ratios were normalized to the baseline. To detect whether tumor was formed in iPSCs-transplanted rats,
11C-MET PET imaging was applied followed by injecting approximately 37 MBq (1 mCi) of
11C-MET via the tail vein with 20-min wait.
2.6 HE staining
After deep anesthesia by 1.5% pentobarbital sodium (50 mg/kg, intraperitoneal), the rats were transcardially perfused with 4% chilled paraformaldehyde in PBS. The fixed brains were embedded in paraffin and then cut into 4 µm coronal serial sections with a microtome (Microm HM-340 E microtome, Walldorf, Germany). The sections were dewaxed with xylene, dehydrated with gradient ethanol, and then stained with hematoxylin for 10 min. After rinsing with running water, 1% hydrochloric acid alcohol was added to visualize the blue color followed by rinsing with 95% ethanol. The sections were stained with 0.5% eosin for 1 min, dehydrated with gradient ethanol, cleared with xylene, and mounted with neutral gum. Finally, the histopathological changes were observed under a light microscope (BX60, Olympus, Japan).
2.7 Immunofluorescent staining
After being frozen by powdered dry ice, the brain tissues were cut into 20-µm-thick sections, and then blocked and incubated at 4°C overnight with primary antibodies against neurons (mouse monoclonal antibody against neuronal nuclei (NeuN) (1:200 dilution, Millipore, USA)), astrocytes (rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP) (1:300 dilution, Abcam, USA)), and endothelial cells (rabbit polyclonal antibody against von Willebrand factor (vWF) (1:200 dilution, DAKO, Denmark)). Sections were incubated with fluorescent-conjugated secondary antibodies (1:500 dilution, Invitrogen, USA) for 1 h at room temperature after washing with PBS. Then, sections were counterstained with the nuclear dye 4,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was imaged on a Zeiss confocal microscope (LSM710, Zeiss, Germany).
2.8 Quantitative proteomic analysis
The rats were sacrificed under deep anesthesia, and the samples of infarction were taken from the brain. To minimize the biological variation between individual subjects, the protein extracts from five distinct rats were pooled for LC-MS/MS analysis. Each group contained three biological replicates. Briefly, the samples were first ground by liquid nitrogen and transferred into lysis buffer (8 mol/L urea, 10 mmol/L DTT, and 1% Protease Inhibitor Cocktail). The required protein was obtained by repeated centrifugations, and the concentration was determined with the 2-D Quant kit (GE Healthcare, USA) according to the manufacturer’s instructions. Thereafter, the protein was digested by sequencing grade modified trypsin (Promega, USA) for the following experiments. The resulting peptides were desalted by using the Strata X C18 SPE column (Phenomenex, USA), vacuum-dried, and then processed by using the 8-plex iTRAQ kit (AB Sciex, USA) according to the manufacturer’s protocol. The sample was then separated into fractions by high-pH reversed-phase HPLC using the Agilent 300Extend C18 column.
The separation of peptide was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific, USA). The step linear gradient of solvent B (0.1% formic acid in 98% acetonitrile) started from 7% to 22% over 26 min, 22% to 35% in 8 min, and 35% to 80% in 3 min and then held at 80% for 3 min at a constant flow rate on an EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. The resulting peptides were subjected to NSI source followed by MS/MS in Q ExactiveTM (Thermo Fisher Scientific, USA) coupled online to the UPLC. The MS/MS data were searched by Mascot search engine (version 2.3.0). A strict cutoff of fold-change<0.83 and fold-change>1.2 was used as the qualification criteria, which corresponds to a peptide confidence level of 90%. The ratios were sorted by a P-value cutoff of 0.05 to obtain the list of differentially expressed proteins. For bioinformatic analysis, the UniProt-GOA database was used for Gene Ontology (GO) annotation.
2.9 Western blot analysis
To confirm the results from the iTRAQ-LC-MS/MS experiments, Western blot analysis was used to quantify the proteins of interest. Proteins (20 µg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The transblotted membranes were blocked with 0.5% defatted milk in TBST for 1 h and incubated with primary antibodies against GALE (1:30 000 dilution, Abcam, USA), MEK1 (1:2000 dilution, Abcam, USA), and GAPDH (1:20 000 dilution, Abcam, USA) at 4 °C overnight. Then, the membranes were washed and incubated with horseradish peroxidase-coupled secondary antibodies (1:5000 dilution, Thermo Pierce, USA) for 1 h. The protein bands detected by the antibodies were visualized by the enhanced chemiluminescence method and exposed to autoradiography film. Then, the Western bolt signals were densitometrically quantified with Quantity One 4.4 (Bio-Rad, Hercules, USA).
2.10 Statistical analysis
Data were presented as mean±SEM. Two-way ANOVA was used to assess the L/N ratios and neurological scores, while a two-tailed Student’s t-test was used to analyze the Western blot results. All statistical analyses were performed with SPSS software (version 15.0, SPSS Inc.). The result with a corrected P-value<0.05 was considered significant.
3 Results
3.1 Effect of iPSCs and NSCs on metabolic and functional recovery
To assess the metabolic alterations after stem cell transplantation, 18F-FDG PET scans were performed to visualize glucose uptake in the brain tissues of ischemic rats. Visual assessment of the cerebral 18F-FDG PET images demonstrated that the iPSCs group exhibited better performance in enhancing glucose uptake compared with the NSCs and PBS groups (Fig. 2A). Semiquantitative assessments of glucose uptake (L/N ratios, expressed as values relative to the baseline) showed no significant increase in the iPSCs and NSCs groups compared with the PBS group on day 7 (Fig. 2B). On day 14, significantly increased glucose uptake was observed in the iPSCs group compared with the PBS group (P = 0.010) without a significant difference between the iPSCs group and the NSCs group. This result indicated that the iPSCs transplantation promoted the restoration of glucose metabolism, whereas the effect of NSCs on glucose metabolism was not as significant as that of iPSCs.
The Garcia neurological grading system was used to evaluate the neurofunctional behaviors of cerebral ischemic rats (Fig. 2C). On day 7, the neurological scores (expressed as values relative to the baseline) in the iPSCs group were significantly higher than those in the NSCs group (P = 0.024) and PBS group (P<0.001); however, the NSCs group had higher scores than the PBS group (P = 0.029). On day 14, the scores in the iPSCs group were significantly increased compared with those in the PBS group (P = 0.047), but no significant difference was observed between the iPSCs group and the NSCs group. These results showed that iPSCs and NSCs transplantation could improve neurological function on day 7. However, on day 14, significant improvement was only observed in the iPSCs group, suggesting that iPSCs transplantation had a more obvious facilitating effect on neurological function restoration to a certain extent.
3.2 Tumor formation detection and cell morphology observation
No tumor formation was detected according to 11C-MET PET imaging (Fig. 2D). The distribution of radioactivity in the cerebral cortex was uniform, and no significant increase in radioactive uptake was observed. HE staining showed that the nucleus (blue) and cytoplasm (red) in the sham group were dyed clearly, and the distribution of nerve cells was evenly organized with complete structure and clear arrangements (Fig. 2E). However, in the PBS group, the nerve cells showed degenerative changes, such as the formation of vacuoles and necrosis, and the neuron arrangements were irregular with pyknotic nuclei. The above results demonstrated massive cell death in the peri-infarct area after MCAO. The damage to the cells was milder with fewer vacuoles and a more regular arrangement in the iPSCs and NSCs groups than in the PBS group. Moreover, alternations in cell morphology and structure were less notable in the iPSCs group than in the NSCs group, demonstrating that iPSCs transplantation had a better performance in alleviating cell damage in the peri-infarct area.
3.3 Differentiation of iPSCs and NSCs in the peri-infarct area
The green fluorescence of GFP-labeled iPSCs (Fig. 3A) and NSCs (Fig. 3B) showed the survival and distribution of transplanted cells. In merged images, a part of GFP-labeled stem cells was positive for NeuN, GFAP, or vWF, which are markers for neurons, astrocytes, and endothelial cells, respectively. The immunofluorescence results indicated that the transplanted iPSCs and NSCs could differentiate into nerve cells and angiogenic cells.
3.4 Identification of differentially expressed proteins
In this work, iTRAQ-LC-MS/MS was applied to obtain the difference in protein expression between the iPSCs group and the PBS group in the subacute and chronic phases of ischemic stroke. A total of 2294 and 2303 proteins were identified and quantified on days 7 and 14, respectively. The volcano plots visualized all the identified proteins and highlighted the differentially expressed proteins (Fig. 4A and 4B). The clustering of 39 differentially expressed proteins between the iPSCs group and the PBS group is summarized in Fig. 4C. Compared with the PBS group, the expression levels of 21 proteins (15 upregulated and 6 downregulated) on day 7 and 18 proteins (4 upregulated and 14 downregulated) on day 14 were significantly altered in the iPSCs group (P<0.05). These differentially expressed proteins can be functionally classified into four categories related to neuronal survival, axonal remodeling, mitochondrial function, and oxidative stress. The categories and detailed quantitative information are displayed in Table 1.
3.5 Characterization of differentially expressed proteins
GO annotations of proteins were classified into three categories: biological process, cellular component, and molecular function. In the cellular component category, the differentially expressed proteins between the iPSCs group and the PBS group on day 7 (Fig. 5A) were mainly distributed in “extracellular” (42.86%), “nuclear” (23.81%), and “cytosol” (14.29%) categories. The representative molecular functions of these proteins were “binding” (33.33%), “catalytic activity” (19.05%), and “transporter activity” (14.29%). Their biological processes were mainly associated with “cellular process” (38.10%), “single-organism process” (33.33%), and “biological regulation” (33.33%).
On day 14 (Fig. 5B), the differentially expressed proteins were mostly distributed in “nuclear” (27.78%), “cytosol” (22.22%), and “plasma membrane” (16.67%) categories. In terms of molecular function, “binding” (27.78%), “catalytic activity” (27.78%), and “molecular function regulator” (11.11%) were the main items. Regarding biological process, the differentially expressed proteins were related to “single-organism process” (33.33%), “cellular process” (27.78%), and “biological regulation” (22.22%).
3.6 Validation of differentially expressed proteins
Western blot analysis was used to validate the differentially expressed proteins determined by iTRAQ-LC-MS/MS, and one protein was randomly selected from the differentially expressed proteins on days 7 and 14 (Fig. 6). Compared with the PBS group, GALE was upregulated continuously in the iPSCs group on days 7 and 14 (P = 0.048, P = 0.041, respectively). The expression of MEK1 also showed an increase in the iPSCs group on days 7 and 14 (P = 0.060, P = 0.006, respectively).
4 Discussion
In this study, 18F-FDG PET imaging combined with neurological tests and immunofluorescent staining was used to evaluate the therapeutic response of iPSCs and NSCs in ischemic stroke. Glucose metabolism and neurological function were improved after iPSCs and NSCs transplantation. Immunofluorescence showed that iPSCs and NSCs transplanted in the lateral ventricle could migrate to the peri-infarct area and induce neurogenesis and angiogenesis. More importantly, using quantitative proteomic analysis, significant differences in the protein expression profile between the iPSCs group and the PBS group were observed in the subacute and chronic phases of ischemic stroke.
During cerebral ischemia, the reduction of cerebral blood flow and subsequent decrease of glucose supply to the brain initiate a series of pathophysiological changes, leading to impaired glucose metabolism [
26]. As a noninvasive and dynamic imaging technique,
18F-FDG PET visualizes the glucose metabolism
in vivo at the molecular level [
27]. In this study, the results from
18F-FDG PET imaging showed that the tendency of increased glucose uptake in the iPSCs group was greater than in the NSCs group on days 7 and 14, which coincided with our previous findings [
6]. These results indicated that transplanted iPSCs had a more active and stable effect on glucose metabolic restoration than NSCs, ameliorating the disruption of glucose metabolism in the ischemic area. In addition, in terms of neurological behaviors, iPSCs demonstrated a better capability to attenuate neurofunctional deficits than NSCs.
Our immunofluorescence staining showed that some surviving transplanted cells could migrate to the peri-infarct area and induce neurogenesis and angiogenesis. In a previous study, Kosi
et al. found that in stroke-affected animals, a majority of NSCs transplanted via the lateral ventricle remained in the ventricles, while only 15% of the cells migrated outside of the cerebrospinal fluid into the surrounding brain parenchyma [
28]. Consistent with this study, we observed that only a small number of transplanted cells survived and reached the peri-infarct area on day 14. Furthermore, significantly improved glucose metabolism and neurological function were still observed in cerebral ischemic rats. A reasonable explanation is that changes in the microenvironment may contribute to tissue protection and repair or that the interaction of transplanted stem cells with the host brain may lead to the production of a wide range of adaptive factors, such as VEGF, BDNF, EPO, and SDF-1α [
13,
25].
For detailed information on the microenvironmental changes after iPSCs transplantation, quantitative proteomics was used to analyze the protein expression profiles in the ischemic area on days 7 and 14. As a classic animal model of cerebral ischemia, the MCAO model can simulate the course of human ischemic stroke. The period within 24 h after MCAO is generally considered as the acute phase of ischemic stroke, 3–7 days is considered the subacute phase, and 14 days later is considered the chronic phase [
29]. Therefore, we obtained the brain protein expression profiles of iPSCs-transplanted rats in the subacute and chronic phases. We identified a total of 39 differentially expressed proteins between the iPSCs group and the PBS group. GO annotation of these proteins showed that the main biological processes involved were cellular process, biological regulation, and metabolic process, and their molecular functions included binding, catalytic activity, transporter activity, and enzyme regulation. According to their role in the molecular events of cerebral ischemic stroke pathophysiology, the differentially expressed proteins could be subdivided into enhancement of neuronal survival, promotion of axonal remodeling, regulation of mitochondrial function, and suppression of oxidative stress. Two proteins were selected from each category for discussion on the basis of expression levels and bioinformatics.
Due to the microenvironmental changes after cerebral ischemia, multiple signaling pathways affecting neuronal growth and metabolism are activated, such as MAPK [
30], mTOR, and Wnt [
31] signaling pathways. MEK1 is an indispensable component of the classical MAPK/ERK signaling pathway and is involved in cell differentiation, proliferation, and survival [
32]. Increasing evidence has shown that the activation of the MAPK/ERK pathway mediates neuroprotection against cerebral ischemic injury [
30,
33]. In addition to its role in ischemic stroke, the MAPK/ERK signaling pathway functions as a pro-survival pathway in ischemia/reperfusion injury of the intestines and heart [
34,
35]. In our study, the expression of MEK1 showed an increased trend on days 7 and 14, indicating that the function of the MAPK/ERK signaling pathway was extended in the long term after iPSCs transplantation. However, due to the complexity of the pathways involved in MEK1, more detailed research is needed. In addition, ensuring neuron survival in the penumbra is of importance for the treatment of ischemic stroke with effectively diminishing infarct size and accelerating metabolic restoration, on the grounds that neurons in the ischemic core die rapidly after the onset of focal ischemia, whereas neurons located in the ischemic penumbra are potentially salvageable [
36,
37]. Our results showed that the expression of several proteins related to neuronal survival, such as CHMP5 and MEK1, was significantly altered compared with the PBS group. As a constantly upregulated protein, CHMP5 has been identified with a new antiapoptotic function [
38], which is expected to salvage neurons in the ischemic penumbra even in the subacute and chronic phases.
Axon remodeling is regarded as an important reparative method for the improvement of neurological deficits after stroke [
39]. The elongation and regeneration capacity of damaged axons in the nervous system are partly determined by the environment in which they are located [
40]. In our results, several representative proteins were associated with axon remodeling. PTGDS, which is also known as L-PGDS, is a key mediator of myelination in the peripheral nervous system and is involved in myelin formation and maintenance [
41]. In addition, studies have demonstrated that PTGDS could protect neuronal cells against apoptosis
in vitro and
in vivo [
42,
43]. PTGDS showed continuing increased expression during the experimental period. Consistent with this result, an increase in DARPP-32 expression was also observed. DARPP-32 is a specific marker of mature medium-sized spiny neurons (MSNs) given its typical high expression [
44]. A previous study has shown that human pluripotent stem cells could be induced to differentiate into DARPP-32
+ MSNs in Huntington’s disease [
45]. The high expression of these regeneration-related proteins combined with the immunofluorescence results indicated that iPSCs retained the role in cell differentiation in the subacute and chronic phases (within two weeks after transplantation) and that cell differentiation is not limited to the acute phase.
The brain mainly uses glucose as fuel for energy generation, and mitochondria are the powerhouses of neurons. Therefore, the improvement of mitochondrial function is essential for the restoration of energy metabolism. Some upregulated differentially expressed proteins provide relevant information regarding our results. TOMM20, a mitochondrial outer membrane receptor, binds with TOMM22 to ensure the import of proteins required for mitochondrial biogenesis [
46], which reflects cellular function at the organelle level. Loss of TOMM20 is associated with mitochondrial dysfunction in Parkinson’s disease [
47]. In our study, the expression of TOMM20 was upregulated on days 7 and 14, demonstrating a continuing effect on mitochondrial function. Increased expression of GALE was also observed. GALE is a key enzyme in the last step of the Leloir pathway and reversibly converts UDP-galactose into UDP-glucose [
48], both of which are immediate glucose donors in the synthesis of various glycoproteins and glycolipids. Given the critical role in glucose homeostasis and galactose homeostasis, the deletion or mutation of GALE can lead to severe galactosemia [
49].
Oxidative stress is a critical contributor to cerebrovascular diseases, especially in ischemic stroke [
50,
51]. It damages brain tissues via the overproduction of reactive oxygen species (ROS) and aggravation of the inflammatory response. Hence, the suppression or elimination of oxidative stress offers a potential therapeutic approach for stroke. In this study, the expression of some oxidative stress-related proteins, such as EIF6 and NDUFS6, was altered. Recently, a novel connection between EIF6 expression and ROS production was unveiled. In
eIF6+/− mice, the transcription of mitochondrial respiratory chain complex genes was impaired, leading to defects in ROS synthesis [
52]. Its decreased expression level was observed on day 14. NDUFS6, an accessory subunit of mitochondrial complex I, is essential for the biogenesis of mitochondrial complex I and participates in the regulation of activity [
53]. Of note, complex I is a key enzyme of energy metabolism in cells, providing large amounts of proton-motive force for ATP production [
54]. However, it is also one of the major sources of ROS in mitochondria. Mitochondrial complex I deactivates during cerebral ischemia, but reactivates upon reperfusion of ischemic tissue, which is accompanied by transient overwhelming production of ROS [
54]. The downregulation of NDUFS6 on day 7 and the upregulation of NDUFS6 on day 14 may be ascribed to the dynamic change in the dual function of complex I [
53]. However, future studies are needed to confirm these hypotheses.
The present study has several limitations. The functional classification of differentially expressed proteins is based on published data, and some proteins with undefined functions need to be further explored. In addition, despite a risk of tumorigenicity, no tumor formation was detected within two weeks after iPSCs transplantation in our study. To the best of our knowledge, increasing evidence has indicated that tumor formation from iPSCs can be prevented by eliminating the remaining undifferentiated stem cells with small-molecules inhibitors [
55,
56], which should increase the safety of iPSCs-based therapy.
In summary, our study identified multiple differentially expressed proteins in the ischemic microenvironment after iPSCs transplantation. In the subacute and chronic phases of ischemic stroke, the extensive protein expression changes after iPSCs transplantation were mainly related to neuronal survival, axonal remodeling, oxidative stress, and mitochondrial function. The findings of these spatiotemporal responses in the ischemic microenvironment help to obtain a deeper understanding on the pathology of ischemic stroke and its therapeutic strategies.
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