A Comparison of Intracerebral Transplantation of RMNE6 Cells and MSCs on Ischemic Stroke Models
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.273641
Source of Support: None, Conflict of Interest: None
Keywords: Cell transplantation, magnetic resonance imaging, Mesenchymal stem cells, RMNE6 cells, stroke
It is well known that injuries to the central nervous system (CNS) caused by different disorders, including stroke are difficult to reverse. Thus there have been no effective therapies for stroke to date. Using surgical decompression or thrombolytic therapy in the clinic may be of some help, but only for the early stage because the time window for intervention is very short. With respect to the chronic stage of stroke, only supportive and symptomatic treatments are applied, especially for functional rehabilitation of the patient.
Recently, the intracerebral transplantation of stem cells has been thought to be a promising approach for rescuing stroke because stem cells are continuously self-renewing and differentiate into multiple lineages, which may replace the damaged brain areas. Indeed, a number of different stem cells have already been used for the restoration of stroke lesions in animal models, and these stem cells include neural stem cells (NSCs), embryonic stem cells (ESCs), and mesenchymal stem cells (MSCs).
Several problems still exist in using stem cell therapy for repairing brain injuries, including the poor availability of the cells and immune rejection from the host. Although induced pluripotent stem cells (iPSs) and induced neural stem/progenitor cells (iNSCs) have been shown in recent years to overcome these difficulties, the uncertain fate and tumorigenicity in the brain after grafting leads to obstacles in the clinical use of stem cells. Moreover, the bad environment after brain ischemia, including the interruption of the blood supply and the absence of neurotrophic factors impair implanted cell survival and differentiation.
Effective cell replacement therapies for neurologic disease require grafted cells to have the following features. First, C continuously proliferate so that a sufficient quantity can be acquired for transplantation. Second, cells must be neuron-restricted precursors so as not to form tumor-like iPSs after transplantation. Third, cells must be able to secrete some neurotrophic factors to facilitate survival in bad environments. The RMNE6 cell with the three features is a thermally controlled GABAergic neuronal progenitor cell line established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferate rapidly and express a neuron-like phenotype at a permissive temperature (33°C), but eventually stop growing at a nonpermissive temperature (39°C). RMNE6 cells express the neuronal markers, β-tubulin III, and microtubule-associated protein-2 (MAP2). Furthermore, RMNE6 cells exhibit functional γ-aminobutyric acid (GABA) ergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD), as well as the synthesis and release of the neurotransmitter, GABA, in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously express and secrete several neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5, and glial cell-line derived neurotrophic factor (GDNF). Taken together, the results support the notion that RMNE6 cells have some capacity to overcome the deficiencies and obstructions in the practical application of stem cells, and have superiority compared with other stem cells, and are an ideal cell model for transplantation research aimed at the treatment of stroke.
Among the stem cells which have already been tested for stroke, MSCs appear to be a promising candidate. After transplantation into the ischemic brains of animal models, the MSCs survived well, migrated to the injured areas, and improved animal behavior even though differentiation in the brain is very rare.
It is of value, therefore, to compare the effects of RMNE6 cells and MSCs with respect to improvement in behavior and neural repair of rodent stroke models by intracerebral transplantation of the cells. Thus, the experiment entails transplanting the two types of stem cells into the ischemic brains of rats with middle cerebral artery occlusion (MCAO) and observing the fates and therapeutic effects by immunofluorescence staining, behavior testing, and MRI to explore the potentials of RMNE6 cells for stroke.
The animal studies were approved by the guidelines of the Institutional Animal Care and Use Committee of the Capital Medical University. Adult male SD rats weighing 230--250 g were used. The animals were housed under a 12 h light--12 h dark cycle with free access to food and water.
The rats were also divided into three experimental groups (vehicle, MSCs, and RMNE6 [n=8 each]). The transplantation or injection was performed on the 3rd day after the MCAO.
The MSCs marked by GFP and the immortalized GABAergic neuronal progenitor cell line (RMNE6) were created in the Beijing Institute of Neuroscience and were treated according to the following methods. Briefly, MSCs were cultured in α-MEM medium containing 10% FBS at 37°C in a humidified 5% CO2 atmosphere, and passaged every 3--4 days. The medium was changed every other day. The RMNE6 grew in DMEM/F-12 containing 10% FBS and incubated in an incubator at 37°C in 5% CO2, and were passaged every 3 days without a change of medium.
All animals were anesthetized with 6% chloral hydrate (6 ml/kg, i.p.). Body temperature was maintained at approximately 37°C using a heating bed during the surgical procedures
Focal brain ischemia was induced by the intraluminal filament technique in the rats. A midline skin incision was made in the neck and subsequently the left common carotid artery (CCA), the external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A monofilament nylon thread (40 mm) with a diameter 0.34 mm tip was advanced from the left CCA bifurcation until the origin of the MCA was blocked
The animals were kept warm on an electric blanket until they were aroused.
The cells were dissociated with trypsin and thrice washed in PBS. The cell density was adjusted to 1 × 105/μl to 1 × 106/μl, and put on ice in preparation for transplantation. All animals were anesthetized with 6% chloral hydrate (6 ml/kg, i.p.) and fixed in a stereotaxic instrument on the 3rd day after MCAO. A midline skin incision was made in the skull with subsequent drilling for a burr hole. Then, 1 × 106 cells were stereotaxically injected into the left corpus striatum of the ischemic rats, 3 mm lateral to the bregma, and at a depth of 5.5 mm using a Hamilton syringe.
The injection speed was 1 μl/min under the control of a syringe pump. The needle was retained in place for an additional 10 min before slow retraction 1 mm every 3 min. The vehicle group received 0.01 M PBS using the same method.
The rotarod test was performed to evaluate the degree of hemiparesis and coordinated movements. All of the rats were trained to stay on the accelerating rotarod with a slowly increasing speed from 4 to 40 rpm within 5 min, until they could remain on the rotarod for≥200s before the MCAO. The times the rats remained on the same accelerating rotarod were measured at different time points after the MCAO. The data are presented as a percentage of the average time on the rotarod for three trials compared with the baseline control obtained before the MCAO.
The cell-transplanted rats were perfused with 0.9% physiologic saline to rinse the blood and subsequently perfused with 4% PFA (in PB) to fix the brain tissue on the 28th day after transplantation. The brains were removed and refixed in 4% PFA (in PB) for 4--6 h, then sunk to the bottom in 30% sucrose (in PB)
The coronal sections with a thickness of 20--30 μm from the whole brain were cut in series by a freezing microtome set (Leica, Germany). Sections were mounted on the slides and prepared for staining with an immunohistochemical method.
Establishment of the MCAO model was determined, as well as infarction location and size. TTC staining was performed in the MCAO mice and rats. The additional MCAO mice and rats were anesthetized and sacrificed the day after ischemia. The brains were removed immediately and cut into a series of 1-mm coronal slices. These slices were stained in 2% TTC in PBS at 37°C for 15--30 min while protected from light.
From each brain, a set of 12 slices were washed with 0.3% PBST, then incubated in 3% donkey serum or 10% goat serum in 0.3% PBST at room temperature for 1 h. The slices were then incubated in mouse anti-NeuN antibody-conjugated biotin (1:200; Chemicon Millipore), rabbit anti-GFAP antibody (1:5000; Abcam), mouse anti-SV40 antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-GABA antibody (1:1000; Sigma, St. Louis, MO, USA), and 1% donkey serum or 10% normal goat serum at 4°C overnight. The sections were then thrice washed in PBS with subsequent incubation in Streptavidin-Cy3 (1:400; Jackson), donkey anti-rabbit Cy3 (1:400; Jackson), goat anti-mouse Alexa Fluor 594 (1:500; Invitrogen), and goat anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) for 2--3 h. After the sections had been washed in PBS, the nuclei were stained by Hochest33258 for 10 min. Finally, the slides were coverslipped using mounting medium. The primary antibody was substituted for 3% donkey serum or 10% normal goat serum in PBS in the control studies. For the cells cultured in vitro, the immunofluorescence staining was similar to the slices. The name, origin, and dilution ratio of the primary antibody and the corresponding second antibody were as follows: mouse anti-SV40 antibody (1:50; Santa Cruz Biotechnology, Inc.), rabbit anti-GABA antibody (1:1000; Sigma), rabbit anti-β-Tub-III antibody (1:800; Sigma), mouse anti-nestin antibody (1:1000; Abcam), Alexa Fluor 594 goat anti-rabbit IgG (1:500; Invitrogen), and Alexa Fluor 594 goat anti-mouse IgG (1:500; Invitrogen).
On day 1 after MCAO and 1, 2, 3, and 4w after PBS injection or cell transplantation, all of the animals had MRI scans at 1.5T (TR=3,000; TE=85) to demonstrate changes in the infarct areas. To avoid interference of brain edema or atrophy, the corrected infarct area (CIA) was calculated as follows: CIA=RT−(LT − LI), where LT and RT are the areas of the left and right hemispheres, respectively, and LI is the high-intensity area of T2W1 in mm 2. The infarct volumes were calculated using CIA and the slice thickness (1 mm) on MRI examinations.
All data are expressed as the mean ± SEM, and the behavioral data and infarct volume were analyzed using two-way (time and treatment) ANOVA for repeated measures. A P < 0.05 was considered statistically significant.
1. Establishment of the MCAO model
1.1 TTC staining
TTC staining showed that the white ischemic area was located in the cerebral cortex and the corpus striatum of the MCAO rat [Figure 1].
The rotarod test results showed that the MCAO rats lacked motor coordination because the percentage of duration in all groups of MCAO rats was<100% [Figure 2].
2. The identification of RMNE6 cells in vitro and the cultured in vitro MSCs transfected by Enhanced Green Fluorescent Protein (EGFP)
Immunofluorescence showed that the RMNE6 cells expressing SV40 in vitro were mainly concentrated in the cell nucleus and purple color of SV40 colocalized with Hochest33258. Thus, SV40 can serve as a tracer for RMNE6 cells in vivo [Figure 3]. Moreover, RMNE6 cells also expressed nestin, a marker of NSCs [Figure 4], and β-tub-III, a marker of NPCs distributed in the cytoplasm [Figure 5]. The neurotransmitter, GABA, was positive in PMNE6 cells [Figure 6], which indicates that RMNE6 cells will differentiate into GABAergic neurons. [Figure 7] shows cultured in vitro MSCs transfected by EGFP, and [Figure 8] is the image of [Figure 7] under a fluorescence microscope.
3. Fates of transplanted cells in the brains
3.1 Survival and migration
The MSCs and RMNE6 cells (SV40/GABA double-positive cells) survived in the ischemic brains 4 weeks after implantation in the boundary zone adjacent to the ischemic core [Figure 9] and [Figure 10]. The MSCs are round, elliptical, and fusiform; the developed processes have not been demonstrated. The RMNE6 cells are mainly fusiform with slim processes at the end of the cell. Based on survival, the MSCs migrated to the ischemic area [Figure 9]. To our surprise, the MSCs were found in the necrotic cavity and the necrosis area [Figure 9].
For the green MSCs in the necrotic cavity and area, differentiation into neurons and astrocytes was not observed [Figure 9]
No immunoreactivity was observed in the controls.
Neoplasms appeared in the MCAO rats in which RMNE6 cells were transplanted [Figure 11]; however, no tumors were noted in MSC-transplanted rats.
Statistical analysis of the behavioral tests indicated that the difference had no statistical significance between the vehicle, MSC, and RMNE6 groups in the rotarod test (P > 0.05; [Figure 6]C). This finding showed that cell transplantation had no effects on the behavior of MCAO rats.
5. MRI and the infarct volume
Two-way (time and treatment) ANOVA of repeated measures showed that the infarct volume had no statistically significant changes (P < 0.05) between the different experimental groups and the different time points after cell transplantation [Table 1]. [Figure 12], [Figure 13], [Figure 14], [Figure 15] show the MRI images of the different time points after cell transplantation in the different experimental groups, which are only a part of the completed data due to limited space.
The RMNE6 cells and MSCs were transplanted into the ischemic brains of the model animals. The fates of the cells in the ischemic brains were observed by immunofluorescence, and the effects on the behavior of model animals were evaluated using the behavioral method. The effects on the size of the infarct volume were shown by MRI in the current study to explore the efficacy and to compare the advantages and disadvantages in cerebral ischemia. It was found that the two kinds of stem cells could survive, did not differentiate and had no effects on the behavior of the model animals. Furthermore, the size of the infarct volume and had no change after transplantation. RMNE6 cells formed the tumors. The results from the present study, however, did not show curative effects, indicating that the approach of stem cell therapy for stroke is still in need of improvement.
1. Differentiation of transplanted cells
The previous experiments showed that the RMNE6 cells expressed nestin, β-Tub-III, MAP-2, and GABA in vitro, and did not differentiate into GFAP-positive astrocytes in vivo. Thus, RMNE6 cells can be considered to be neural precursor cells differentiating into mature neurons. It is difficult to envision RMNE6 cells differentiating into more mature neurons because the RMNE6 cells proliferated and formed a neoplasm after grafting in the ischemic brains in the experiments herein. Combined with the results that the cells did not form tumors in the normal rat brain, it can be concluded that the phenomenon is a comprehensive effect caused by the tsA58 SV40 large tumor antigen existing in RMNE6 cells and the ischemic microenvironment of the brain, which is different from the normal state of the brain. The local brain environment underlies the neural differentiation of stem cells involving MSCs in stroke patients. The boundary zone (penumbra) adjacent to the ischemic core is threatened tissue by ischemia. Penumbral tissue robustly expresses developmental proteins and genes, and thereby simulates embryonic tissue., Environmental signals can apparently elicit the expression of pluripotentiality that extends well beyond the accepted fate restrictions of cells originating in classical embryonic germ layers. Thus, progenitor-like cells, such as MSCs, when placed in this microenvironment, may be prompted to proliferate and differentiate into neural-like cells
Chen et al. reported that 14 days after transplantation, the majority of BrdU-labeled MSCs were located in the ischemic boundary zone. Double staining revealed that some BrdU-positive cells in the graft core and in the adjacent host striatum and cortex were double labeled for neuronal markers (NeuN and MAP2) and the astrocytic marker, GFAP. The data of Li et al. also demonstrated that the BrdU-reactive cells were highly concentrated in the ischemic boundary zone after MCAO. Molecules expressed in the ischemic boundary zone might play a role in the subsequent survival and differentiation or migration or both of transplanted bone marrow nonhematopoietic cells. Unlike the above two reports, in this study MSCs marked with GFP were distant from the injection, mainly concentrating in the necrotic cavity and area. Having nothing to do with the ischemic boundary zone explained why MSCs do not differentiate; however, a new question was raised regarding why MSCs are able to survive in areas of ischemic necrosis. Moreover, it is controversial whether or not the MSCs differentiate into the neuron. In the above-mentioned experiments, MSCs differentiated into neurons and astrocytes using BrdU as a trace marker; however, the BrdU integrated into the donor cells may be absorbed by the host cells because of the death of the donor cells to be transferred into the host cells. Thus, it was discounted that the grafted cells marked by the thymidine analogue, such as BrdU, differentiate nerve cells in vivo.
Currently, transgenic tracers (GFP or EGFP) marking the donor cells are more ideal than thymidine analogues; however, it is rarely reported that the GFP-marked MSCs grafted into the ischemic brain differentiate into mature neurons and integrate with the host brain. The results of this experiment in which the EGFP-marked MSCs have not differentiated into nerves is not in contradiction with previous findings.
From the above analysis involving the two types of stem cells, it can be inferred that the fate of the grafted cells also appears to be determined by the local brain environment, and not only from the intrinsic properties of these cells. Thus, exploring the intrinsic reasons for the differentiation of the stem cells and intensive study about the ischemic microenviroment in which the stem cells are located should be the focus of research involving stem cell differentiation in the future.
Because of the weakness and uncertainty of the differentiation after the stem cells were grafted, it was impossible that the improvements in deficient behaviors in the model animals depended on the replacement of lost neurons with functional neurons derived from the grafted stem cells.
However, instead of integrating into the host brain and replacing the lost neurons, RMNE6 cells should have played a neuroprotective role in cerebral injury because RMNE6 cells could spontaneously express and release a lot of neuroprotective factors, such a NGF, BDNF, NT-3, NT-4/5, and GDNF in vitro, although there were no reports about treatment of ischemic brain injury with RMNE6 cells.
In the case of the MSCs, there are a number of theories about improvement in behavior of the animal model after cell transplantation in addition to the neural replacement already mentioned. The MSCs may play an important role in neural protection and regeneration by releasing neuroprotective or neurotrophic factors, inhibiting inflammation,, decreasing apoptosis, promoting and strengthening the endogenous neurogenesis of the SVZ and SGZ, and vascular regeneration. Furthermore, the mechanisms depend on the timing, route of administration, and cell dose.
The current experiments showed that the behavior of stroke animals was not improved after transplantation of MSCs and RMNE6 cells. In the case of MSCs, the results of Chen  are consistent with our results; specifically no difference was found between the MCAO rats with intracranial injection of the MSCs and the control group who underwent the rotarod test. In contrast, the transplantation of MSCs could prolong the retaining time of MCAO mice on the rotarod in the study of Li. The difference may be attributed to the animal species, ischemia model, and purity of transplanted cells. Compared with other experiments in which MSC transplantation contributed to the functional recovery of model animals, the difference in route of administration may be the reason for the different effects on behavior. For the RMNE6 cells, the negative effect of tumor formation may overcome the neuroprotective effect of cells to result in the absence of improvement of behavior; the specific reason warrants further study.
Therefore, the MSCs are more reliable in improving the behavior of cerebral ischemia animals than RMNE6 cells; however, the optimum protection for the CNS may be related to many parameters, such as timing, administration route, cell dose, state and treatment of cells before transplantation, and immunosuppressants.
RMNE6 cells are derived from an immortalized GABAergic neuronal progenitor cell line transduced with the temperature-sensitive A58 allele of the SV40 large T antigen (SV40 T tsA58) gene. In contrast to the current experimental results, there were no tumors appearing in the brains of the rats after the same amount of cells had been injected in the corpora striata of the normal rats. Tumors were not found when transgenic RMN cells were injected into the corpora striata of the PD rats explore the therapeutic effects on the PD because the intrinsic growth rule of the cells has been changed and the temperature-controlled growth characteristic determined by the SV40 disappeared with the increasing passages in vitro. Definitive evidence is still needed to be confirmed by more experiments in vitro. Furthermore, compared with the above two experiments, it was possible that it was the ischemic microenvironment that induced the formation of tumors, although the possibility was very small.
In contrast, there were no tumors appearing in the MSCs-transplanted animals, which may be related to the origin of the MSCs without any genetic modifications.
Therefore, the MSCs are more secure in cell transplantation than the RMNE6 cells from the perspective of tumorigenicity.
5. MRI and infarct volume
The infarct volume is a common index used to evaluate the effects of cell transplantation on stroke. The changes in pathology of the brain can be observed in the same living animal for some time by MRI. With respect to ischemic brain injuries, MRI can identify the injury, the size of the injury, and the change in the injury over time in the same animal. Compared with the previous observation by histology, this method can reduce the number of experimental animals and acquire data about the successive changes of the infarct area in the same animal, which is beneficial for statistical analysis.
The current experiments showed that the infarct volume had no changes before and after transplantation. The results are the same as the results reported by others,, who measured the images of the HE-stained slices using a Global Lab Image analysis system. Whether or not the MSCs were grafted has no effect on the infarct volume; however, there were experiments that showed that the infarct volume of the model animals was reduced after MSC transplantation., The different routes of administration may lead to the difference in the two results. The intravenous injection has better effects on stroke than intracranial injection because the intravenous injection imitated the passage of the MSCs from the blood into the brain in vivo. The intracranial injection, including the current experiments, may be injury to the brain following ischemia.
Although the results were not ideal, our study compared the effects of MSC and RMNE6 cell transplantation on stroke for the first time. From the point of view of security, the MSCs are more suitable for cell transplantation than RMNE6 cells. Based on the current data, it is likely that stem cell transplantation may rescue dying neural cells to reduce the degree of brain injury after cerebral ischemia through a variety of mechanisms. As mentioned before, however, how to optimize the effect still needs comparative studies based on a unified standard because of many factors involved.
Currently, there are no promising measures for stroke other than stem cell transplantation; however, it seemed unlikely that new brain tissue created by stem cells substitutes for the lost brain tissue to function in the stroke model. Thus, while the stem cells will need to be remolded continuously, the mechanism of the development and maturation of the brain should be explored to reconstruct the tissue and function of the brain.
Thanks to Professor Xu Qun-Yuan for the design and guidance, and Teacher Lei for the magnetic resonance scanning of the experiments at Capital Medical University.
Financial support and sponsorship
This subject was supported by the National Natural Science Foundation of China (No.: 81070977), the Scientific Research Fund of the Doctoral Young Scholars of Shanxi University of Traditional Chinese Medicine (No.: 2016BK01), the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No.: 201804005), and the Scientific Research Project (No.: 201601113 and No.: 201601106) of the Health and Family Planning Commission of Shanxi Province.
Conflicts of interest
There are no conflicts of interest.
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