Human induced pluripotent stem cell-derived neural stem cells survive, migrate, differentiate, and improve neurologic function in a rat model of middle cerebral artery occlusion
- Ting Yuan†1,
- Wei Liao†3,
- Nian-Hua Feng4,
- Yuan-Lei Lou4,
- Xin Niu1,
- Ai-Jun Zhang2,
- Yang Wang1, 4Email author and
- Zhi-Feng Deng2, 3Email author
© Yuan et al.; licensee BioMed Central Ltd. 2013
Received: 1 April 2013
Accepted: 10 June 2013
Published: 14 June 2013
Stroke is a major cause of permanent neurologic damage, with few effective treatments available to restore lost function. Induced pluripotent stem cells (iPSCs) have the potential to generate all cell types in vitro and can be generated from a stroke patient. Therefore, iPSCs are attractive donor sources of genetically identical “patient-specific” cells to hold promise in therapy for stroke. In the present study, we established a four-stage culture system by using serum-free medium and retinoic acid (RA) to differentiate iPSCs into neural stem cells (NSCs) effectively and stably. Our hypothesis was that iPSC-derived NSCs would survive, migrate, and differentiate in vivo, and improve neurologic function after transplantation into the brains of rats with ischemic stroke.
Human iPSCs (iPS-S-01) and human ESCs (HuES17) were used to differentiate into NSCs by using our four-stage culture system. iPSCs and differentiated NSCs were characterized by immunocytochemistry staining and reverse transcription-polymerase chain reaction (RT-PCR) analysis. After establishment of focal cerebral ischemia with occlusion of the middle cerebral artery (MCA) and cell transplantation, animals were killed at 1 week and 2 weeks to analyze survival, migration, and differentiation of implanted cells in brain tissue. Animal behavior was evaluated via rope grabbing, beam walking, and Morris water maze tests.
iPSCs were efficiently induced into NSCs by using a newly established four-stage induction system in vitro. iPSCs expressed pluripotency-associated genes Oct4, Sox2, and Nanog before NSC differentiation. The iPSC-derived NSCs spontaneously differentiated into neurons and astrocytes, which highly express β-tubulin and glial fibrillary acidic protein (GFAP), respectively. On transplantation into the striatum, CM-DiI labeled iPSC-derived NSCs were found to migrate into the ischemia area at 1 week and 2 weeks, and animal-function recovery was significantly improved in comparison with control groups at 3 weeks.
The four-stage induction system is stable and effective to culture, differentiate, and induce iPSCs to NSCs by using serum-free medium combined with retinoic acid (RA). Implanted iPSC-derived NSCs were able to survive, migrate into the ischemic brain area to differentiate into mature neural cells, and seem to have potential to restore lost neurologic function from damage due to stroke in a rat model.
KeywordsInduced pluripotent stem cell Stoke Neural stem cell Middle cerebral artery occlusion
Stroke is one of the leading causes of death and disability in humans. Currently, no effective treatments can restore lost neurologic function after stroke in the clinic [1, 2]. An increasing number of studies have provided evidence that cell transplantation can lead to the repair of damaged brain tissue and functional recovery in preclinical stroke models [3–8], including transplantation of bone marrow stromal cells (BMSCs) [9, 10], neural stem cells (NSCs) [11, 12], and embryonic stem cells (ESCs) [13, 14]. These stem cells can differentiate into neural lineages to replace lost neurons and partially reconstruct damaged neuronal circuitry. Nevertheless, disadvantages such as immune rejection and less differentiation potential of adult stem cells, as well as ethical considerations of ESCs, now limit their utility in clinical trials.
The establishment of induced pluripotent stem cells (iPSCs) offers new prospects for regenerative medicine in stroke. iPSCs behave in a manner similar to that of ESCs with high reproduction ability and pluripotency to differentiate into various types of cells . Furthermore, iPSCs can be generated from cells of any part of an adult patient, avoiding both ethical problems and immune rejection, and have a potential to provide genetically identical “patient-specific” cells for stroke patients.
iPSCs have been shown to differentiate successfully into neural precursor cells, neurons, hematopoietic cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, and so on, in vitro[16–19]. Some in vivo studies have confirmed that iPSCs transplanted directly into the cerebral tissue of ischemic stroke rats could differentiate into neurons and promote functional recovery [20, 21], but only a very small number of implanted cells exhibited the specific markers of neurons. Furthermore, a portion of grafted iPSCs formed tumors in implanted sites . These results indicate that the differentiation ability of iPSCs into neurons is limited and associated with unacceptable high rates of tumor formation. Therefore, if iPSCs are induced into NSCs before transplantation in vitro, it is possible to improve the effectiveness of cell therapy, while reducing the risk of tumor formation.
Retinoic acid (RA) has been shown to have the potential to improve the differentiation of ESCs into NSCs [22, 23], but the efficiency is low. In recent years, the alternative serum-free induction protocols have been used widely to differentiate ESCs to NSCs for the advantage of definite components of culture medium, but this induction process needs a long time [24, 25]. We used a modified protocol (a four-stage culture system) combining RA induction and serum-free medium culture. The human iPSCs and ESCs were efficiently induced into neural precursor cells and then differentiated into mature neural cells in this newly established four-stage culture system. After we induced them from iPSCs to NSCs, we implanted iPSC-derived NSCs stereotactically into the striatum of rats with ischemic stroke. To evaluate their therapeutic potential, we observed the differentiation, migration, and distribution of grafted cells after transplantation, and analyzed the neurologic and functional status. To our knowledge, we are the first to study the effects of combining RA and serum-free medium to induce iPSCs into NSCs and observe the direct migration of human iPSC-derived NSCs from implanted sites to ischemic brain areas to restore lost neurologic function in a rat model.
Materials and methods
Human iPSCs culture
Human iPSCs (iPS-S-01) and human ESCs (HuES17) were provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences in agreement with Liao J and Xiao L . The iPS-S-01 cells were induced by six transcription factors (Oct4, Sox2, Nanog, Lin28, C-myc, and Klf4) and resembled the phenotype and pluripotency of undifferentiated human ESCs . Both iPSCs and ESCs were maintained on mouse embryonic fibroblast (MEF) feeder layer, which was inactivated by mitomycin. The medium was changed every day. In general, every 5 to 7 days, when colonies approached approximately 70% confluence, or greater than 700 microns, the cultured cells were then passaged by 1 mg/ml collagenase type IV and mechanical dissociation, and split at a 1:4 to 1:6 ratio. The medium for iPSCs and ESCs culture consisted of Knockout DMEM supplemented with 20% Knockout serum replacement, 1% nonessential amino acid, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml bFGF (Gibco, Grand Island, NY, USA).
Establishment of focal cerebral ischemia and cell transplantation
All animal procedures were approved by the Institutional Animal Care and Use Committee of Jiangxi Province, China. All experiments on animals were carried out in an ethical manner. Adult male Sprague–Dawley rats (250 to 300 g) were used to establish the model of middle cerebral artery occlusion (MCAO), as previously described . Rats were anesthetized, and a 4–0 nylon intraluminal suture was introduced from the minimal incision of the external carotid artery into the internal carotid artery at about a depth of 18 mm to 20 mm until it blocked the origin of the MCA. After 2-hour temporary MCA occlusion, reperfusion was performed by withdrawal of the suture. The rats with significant neurologic deficit of the left forelimb, according to the Longa 5-point scale , were chosen as candidates for cell transplantation. Sham-operated animals underwent the same surgical procedure without suture insertion.
Cells and rat brains were fixed with 4% paraformaldehyde for 15 minutes. Nonspecific sites were blocked with 1% BSA/PBS (for extracellular epitopes) or 1% BSA/PBS/0.5% triton X-100 (for intracellular epitopes) for 30 minutes at 37°C. Then the specimens were incubated with primary antibodies at 4°C overnight. The redundant primary antibodies were removed by washing with PBS for 5 minutes and repeated 3 times the next day. Second antibodies labeled with FITC were used. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 0.5 μg/ml, Invitrogen, Grand Island, NY, USA). The following antibodies were used: Anti-Nestin (monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Anti-Sox2 (monoclonal, Cell Signaling, Danvers, MA, USA), Anti-β-Tubulin (monoclonal, Chemicon, Billerica, MA, USA), Anti-glial fibrillary acidic protein (GFAP, monoclonal, Santa Cruz Biotechnology), Anti-Oct3/4 (monoclonal, Cell Signaling, Danvers, MA, USA). Primary and secondary antibodies were diluted in 1% BSA (Roche, Indianapolis, IN, USA) at 1:100 and 1:200, respectively. Fluorescence was observed under a fluorescence microscope (Olympus, Tokyo, Japan), and photos were taken with a DP70 digital camera system (Olympus). Positive cells in brain tissue were counted blindly in five coronal sections per animal.
Reverse transcription-polymerase chain reaction analysis
Primers used for reverse transcription-polymerase chain reaction (RT-PCR) analysis
CGA AGAGAAAGCGAACCA GTATC
CCCC CGG CGGCAATAGCA
Neurobehavioral evaluation was performed according to the previously published studies [30–32]. The rats were subjected to behavioral testing 1, 2, and 3 weeks after MCAO modeling, by using the rope-grabbing experiment for assaying the myodynamics of the model, beam walking and Morris water maze for testing the spatial, working memory of the models.
Outcome measurement for each experiment was reported as mean ± SEM. All data were analyzed by using SPSS 17.0. Significance of intergroup differences and nerve-assessment data were analyzed with repeated measures of analysis of variance (ANOVA), and the unpaired Student t test. A P value of less than 0.05 was considered to be statistically significant.
IPSCs efficiently induced into NSCs with RA and serum-free medium
iPS-S-01 and HuES17 tended to form packed clones with a high nucleus/cytoplasm ratio (Figure 1B) and displayed a high level of alkaline phosphatase (Figure 1C). To make sure of the undifferentiated state of the cells, pluripotency-associated genes Oct4, Sox2, and Nanog were detected with qRT-PCR before the NSC differentiation. The results showed that those cells expressed high levels of pluripotency-associated genes (Figure 1D).
Transplanted cells survived and migrated into the stroke-damaged host tissue
Transplanted cells improved the behavioral and sensorimotor function of the rats
Animal behavior was evaluated with the rope-grabbing, beam-walking, and Morris water-maze tests 0 days, 1, 2, and 3 weeks after MCAO models were prepared. No obvious differences among the groups were found before the MCAO or transplantation. It was found that no obvious sensorimotor and functional differences among the three groups at 0 days and the first and second weeks (P > 0.05); but at the third week, the transplantation group showed greater function recovery than did those of the other groups (PBS and sham groups, P < 0.05) (Figure 2B through D). These results demonstrate that the NSCs derived from iPSCs improved the behavioral and sensorimotor functions in a rat model of MCAO through transplanting cells in the striatum.
In recent years, with the development of regenerative medicine, stem cell transplantation for the treatment of central nervous system disease is considered to be a way of great potential. NSCs derived from iPSCs seem to be the ideal seed cells for transplantation therapy for neurologic diseases. Because iPSCs share similar characteristics and potential with ESCs, we may study iPSCs by applying the same protocols that had been previously developed in ES cells.
Various culture protocols for the generation of NSCs from iPSCs have been reported in many studies, and the main methods are the following: RA-mediated induction , stromal cell coculture, conditioned medium induction, and serum-free-medium induction . RA has been demonstrated to induce significant neural differentiation of ESCs [33–35]. In 1995, Bain  used RA to induce mouse ESCs into nerve cells successfully, and these cells had the electrophysiological properties and functions of nerve cells. Later, Zhou and Bahrvand [36, 37] reported that the use of RA could promote human ESCs differentiation into neural stem cells, but efficiency was low, and the survival capacity of RA-induced neuronal cells was limited . Stromal cell coculture and conditioned-medium induction are also able to induce ESCs to differentiate into neural cells, but the two methods have a common shortcoming, in that the medium composition is complex, and this complicates the study of its mechanism.
In recent years, induction protocols using cytokines and serum-free medium have been established. Koch  successfully used serum-free medium to induce human ESCs into NSCs. The serum-free medium contains N2, insulin, FGF, and B27, and the data showed that FGF was able to stimulate proliferation and differentiation of a variety of cell types originated from multigerminal layers. A study  showed that neuroepithelial precursor cells differentiated from ESCs proliferate in the presence of basic fibroblast growth factor (bFGF), and differentiate into both neurons and glia after withdrawal of bFGF. Daadi  successfully induced ESCs into NSCs by using serum-free medium containing bFGF, leukemia inhibitory growth factor (LIF), and EGF. These factors are known to stimulate the proliferation of NSCs, and LIF has the potential to maintain the pluripotency of NSCs and to prevent NSCs from differentiating into neurons. Definite components of serum-free medium are beneficial to study the relation between cell growth and medium components.
In the present study, we established a modified four-stage culture system to induce iPSCs to NSCs by taking the advantages of previously developed protocols, by using RA combined with serum-free medium containing bFGF, EGF, LIF, B27, and heparin sodium to induce iPSCs to NSCs in adherent culture. The results showed that this system was stable and effective to induce neural differentiation of iPSCs. Induced cells expressed high levels of NSC markers, Nestin and Sox2, and have been amplified and passaged in vitro for more than 35 cell generations.
In addition, NSCs can be expanded and differentiated in both suspension and adherent cultures. However, it is difficult to control the quality and quantity of cells in the process of suspension culture, because ESC-derived neurosphere cells will lose the potential of self-renewal and differentiation gradually during the long suspension culture, and the types of neurosphere cells are very diverse [40–42]. The adherent culture may avoid these shortcomings.
After implantation into the brain in a rat model of stroke, iPSC-derived NSCs were able to survive and express NSC makers. One week after implantation, implanted cells expressed mainly β-tubulin in addition to Nestin; after 2 weeks, besides continuing to express Nestin and β-tubulin, a few implanted cells expressed GFAP (maker of astrocytes). These results show that grafted NSCs have the potential to differentiate into neurons and glia, and create the possibility of repairing brain injury or other neurologic defects through a neural cell-replacement strategy.
Tumorigenesis is a big challenge in stem cell-replacement therapy. Some animal studies demonstrated that direct implantation of ESCs or iPSCs led to tumor formation [21, 43]. In this study, two rats survived for more than 3 months, and no abnormal proliferation of cells was observed (data not shown). It implied that the implanted cells were not susceptible to tumorigenesis. Although we did not observe tumor formation in this study, it could be that a short period of observation lasting for only 2 weeks was not long enough for tumor development and formation. Therefore, the long-term safety of iPSC-derived NSCs implantation is still a serious problem.
Grafted stem cells have the potential to migrate in vivo. A number of studies [44, 45] observed migration of various implanted stem cells into lesions of the brain, and indicated that possibly stem cells are targeted by inflammatory chemotactic factors and cytokines extracted from ischemic brain tissue. This feature of chemotaxis is the pathophysiologic basis of repairing brain injury by cell-implantation therapy. In this study, implanted NSCs were observed migrating from the striatum toward the ischemic boundary at 2 weeks after implantation.
In our study, we observed a significant increase of neurologic scores 3 weeks after transplantation compared with the control groups, demonstrating a positive effect of repairing ischemic brain with iPSC-derived NSCs. Although 3 weeks is long enough for differentiation of implanted NSCs into neurons, it is not enough for implanted cells to replace dead neurons and function. As a result, some scholars [46, 47] believe that early recovery of function with stem cell transplantation is due to neurologic protection of growth factor release, not new neural cells replacement. Therefore, restoration of neurologic function in this study after cell implantation may be more relevant to a neuroprotective effect.
Note that PBS injection also improved the behavior of rats at 2 weeks (Figure 2B through D). One reason is that collateral circulation has been formed in brain tissue, and the function of impaired cells may be recovered in 2 weeks, which contributes to the improved behavior of rats. Moreover, the improvement of behavior in this rat model may be partly owing to functional compensation after brain injury.
This study established a stable and effective culture system for cell expansion, differentiation, and induction of iPSCs to NSCs in vitro by using serum-free medium combined with RA. After stereotactic implantation of iPSC-derived NSCs into the brain in a rat-stroke model, implanted cells were able to survive, migrate into ischemic brain areas, and differentiate into mature neural cells. Furthermore, this cell-implantation therapy seemed to restore lost neurologic function to a certain extent in a rat stroke model.
Basic fibroblast growth factor
Bone marrow stromal cell
Bovine serum albumin
Dulbecco modified Eagle medium
Epidermal growth factor
Embryonic stem cell
Glial fibrillary acidic protein
Induced pluripotent stem cell
Leukemia inhibitory factor
Middle cerebral artery occlusion
Mouse embryonic fibroblast
Neural stem cell
Reverse transcription-polymerase chain reaction
This work was supported by the National S&T Major Special Project on Major New Drug Innovation (2011ZX09102-010-01), National Nature Science Foundation Grants (81160154,81271270), Nature Science Foundation of Jiangxi Province (2010GZY0256), and the innovation team construction plan of Jiangxi Province (20113BCB24018).
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