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Functional recovery after transplantation of induced pluripotent stem cells in a rat hemorrhagic stroke model
Neuroscience Letters 554 (2013) 70–75
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Functional recovery after transplantation of induced pluripotent stem cells in a rat hemorrhagic stroke model Jie Qin a,1 , Guangming Gong b,1 , Shilei Sun a , Jing Qi a , Huili Zhang a , Yanlin Wang a , Ning Wang c , Qing Mei Wang d , Yan Ji a , Yuan Gao a , Changhe Shi a , Bo Yang e , Yi Zhang f , Bo Song a,∗∗ , Yuming Xu a,∗ a
Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, PR China c Department of Natural Sciences, The University of Virginia’s College at Wise, Wise, VA 24293, United States d Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital, Harvard Medical School, Boston, MA 02135, United States e Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China f Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China b
h i g h l i g h t s • • • • •
We assessed the therapeutic effect of iPSCs in experimental ICH rat. Therapeutic effects of iPSCs in rat ICH model were observed. The grafted iPSCs exhibited neural cell-specific biomarkers in the brain of ICH rats. The levels of BDNF and VEGF were enhanced in the surrounding region of hematoma. Functional improvement is partially due to neuronal replacement and neurophic factors.
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Article history: Received 10 July 2013 Received in revised form 21 August 2013 Accepted 23 August 2013 Keywords: Intracerebral hemorrhage (ICH) Induced pluripotent stem cells (iPSCs) Brain derived neurophic factors (BDNF) Vascular endothelial growth factor (VEGF)
a b s t r a c t Transplantation of induced pluripotent stem cells (iPSCs) has shown promising therapeutic effects for ischemic stroke. However, it is not clear if this treatment would promote recovery after intracerebral hemorrhage (ICH). In this study, we investigated the functional outcome of iPSCs transplantation in experimental ICH in rats. IPSCs were derived from an ICH patient’s fibroblasts and were injected into the ipsilateral side of ICH in rats. IPSCs transplantation significantly improved the neurological functions after ICH as compared to vehicle and fibroblast injection. The grafted iPSCs migrated into brain tissue around the hematoma, survived after 4 weeks of transplantation, and exhibited the neural cell-specific biomarkers nestin, -tubulin, and GFAP. Immunohistochemical staining showed that the densities of brain derived neurophic factors (BDNF)-positive cells and vascular endothelial growth factor (VEGF)positive cells were significantly increased around the hemorrhagic brain tissues of iPSCs-treated rats. In addition, iPSCs treatment increased the protein expression of BDNF and VEGF in the surrounding region of hematoma. These findings demonstrate that the transplantation of ICH patient-derived iPSCs contributes toward the improved neurological function in experimental ICH rats. The mechanisms are possibly due to neuronal replacement and enhanced secretion of neurophic factors. Our data suggest that transplantation of ICH patient-derived iPSCs may be a therapeutic strategy for hemorrhagic stroke. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: 1 Jianshe East Road, Erqi District, Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China. Tel.: +86 371 66862133. ∗∗ Corresponding author at: 1 Jianshe East Road, Erqi District, Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China. Tel.: +86 371 66862132. E-mail addresses: [email protected] (B. Song), [email protected] (Y. Xu). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.08.047
Intracerebral hemorrhage (ICH) has higher mortality and disability rates than that caused by ischemic stroke, although it accounts for only 10–15% of all strokes in the USA and Europe, and 20–30% of strokes in Asia [6]. Following the primary injury of ICH, a series of secondary events such as ischemia, hypoxia, inflammation of brain tissues, degeneration, necrosis, or apoptosis of neurons around the hematoma result in damage to or interrupt the integrity of intracerebral nerve networks, which finally
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causes clinical neurological dysfunction [6]. However, there are no effective treatments to promote functional recovery after ICH [11]. Emerging stem cell therapies have shown potential benefits in treating stroke by targeting the complicated pathological damages caused in multiple pathways [1,5–7,9,12,13,15]. Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have great therapeutic potential and unique advantage due to abundant cell supplies and lack of immunogenic, social, and ethical concerns [13,14,16]. Growing evidence suggests there are therapeutic effects of iPSCs and their derivatives for the treatment of ischemic stroke in rodent stroke models [9,12,13,15]. In a recent study, we showed that neuro-epithelial-like stem cells (NES) derived from iPSCs improve the neurological function after ICH in rats [14]. However, the complex and time-consuming procedure of inducing NES from iPSCs in vitro hinder their application in personalized cell therapy. With more primary and pluripotent capacities than NES, whether iPSCs have therapeutic effect for ICH remains unknown. In this study, we investigated the functional outcome of intracerebral transplantation of an ICH patient’s fibroblasts-derived iPSCs in a rat ICH model and explored the underlying mechanism(s). 2. Materials and methods 2.1. Establishment of rat ICH model Animal feeding and all relevant follow-up experimental procedures were carried out in accordance with the European Council Directive 86/609/European Economic Community for animal experiments and were approved by the Ethics Committee of Zhengzhou University, China. Sprague-Dawley (SD) rats were provided by the animal center of Henan province, Zhengzhou, China. Rat ICH model was generated using intracerebral injection with collagenase VII. After anesthetized with 1% ketamine (30 mg/kg, I.P.; Sigma–Aldrich), male rats (270–300 g) were placed in a rat brain stereotaxic apparatus (Narishige SN-3, Tokyo, Japan) in a prone position. A 1 mm × 1 mm craniectomy was performed, and the needle was inserted perpendicularly into the left corpus striatum (coordinates: 3.5 mm lateral to midline, 0.5 mm anterior to bregma, 6.0 mm depth). Then 2.5 U (0.5 U/2 L) collagenase VIII (Sigma–Aldrich) was injected gently at 1 L/min with a 10 L microsyringe, and the needle was withdrawn slowly. Then the wound surface was cleaned, and the scalp was sutured [14]. In the sham group, stereotactic puncture was performed without collagenase injection. 2.2. IPSCs induction IPSCs were induced from an ICH patient’s skin fibroblasts as described in a previous publication [14]. Briefly, following informed consent and the approval of Ethics Committee of Zhengzhou University, the ICH patient’s skin fibroblasts were taken from the medial upper arm by skin biopsy technique. Fibroblasts were induced by lentiviral vectors carrying the cDNAs of human transcription factor Oct4, Sox2, C-myc, and Klf4. Afterward, embryonic stem cells (ESCs)-like cell clones were gradually formed, and gone through serial identification procedures such as alkaline phosphatase (AP) assay, expressions of hESC-specific biomarkers, and formation of teratoma in severe combined immune deficiency (SCID) mice. Finally, one of these clones was identified as iPSCs successfully [14]. 2.3. Cell therapy Modified neurological severity scoring (mNSS) and modified limb placing test (MLPT) were performed to evaluate neurological
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function at 1 day post operation [2,14]. Thirty-six rats with experimental ICH that scored 8–12 in mNSS and 4–7 in MLPT were randomly and equally divided into 3 groups: phosphate buffered saline (PBS) only group, fibroblast group (total 1 × 106 skin fibroblasts in 10 L PBS), and iPSCs group (total 1 × 106 iPSCs in 10 L PBS). Ten microliters PBS with or without cells was stereotactically injected into all rats at the same coordinates as above to generate ICH, except at a depth of 3.5 mm relative to the bregma. Before being transplanted, all cells were incubated with 20 mol/L of BrdU (Sigma–Aldrich) for 2 days. 2.4. Behavioral tests The functional outcomes (mNSS and MLPT) were evaluated by independent blinded investigators at days 1, 3, 7, 14, and 28 after experimental ICH [14]. 2.5. Extraction of brain tissue Rats were sacrificed in accordance with euthanasia procedures after behavioral testing on day 28 after ICH. Brain tissues of 6 rats in each group were fixed with paraformaldehyde and dehydrated with gradient sucrose. After being frozen, they were cut into coronal slices and used for further immunostaining analysis. For the other 6 rats, brain tissues were quickly extracted, and the brain tissues on the nidus side were ground with liquid nitrogen and degenerated with a protein isolation buffer containing a cocktail of protease inhibitors (Sigma–Aldrich), and then put on ice for 30 min at 4 ◦ C. The supernatant fluids were centrifuged (12,000 × g, 20 min, 4 ◦ C), and the supernatant was stored at −80 ◦ C for western-blotting assay. 2.6. Immunostaining Brain coronal slices were taken from the same position (puncture point) on each rat in all groups. In addition to hematoxylin–eosin staining, immunofluorescence double staining was conducted with antibodies specific for BrdU, nestin, -tubulin, and GFAP (rabbit anti-human were primary antibodies, and goat anti-rabbit, PE/FITC-conjugated goat anti-rabbit antibodies were second antibodies) to identify the transplanted cells, neural stem cells (NSCs), neurons, and astrocytes, respectively. Immunohistochemical staining of brain derived neurophic factors (BDNF) and vascular endothelial growth factor (VEGF) (rabbit anti-rat as primary antibodies, and goat anti-rabbit as second antibodies) was also performed with similar methods. All of the above antibodies were purchased from Santa Cruz Biotech, CA, USA. The slices were fixed with 4% paraformaldehyde for 5 min at room temperature (RT), heated (92–98 ◦ C) in a 0.01 mol/L, pH 6.0 citrate buffer solution for 30 min, and then cooled for 30 min at RT. After blocked with 5% bovine serum albumin (BSA) for 60 min at RT, the slices were incubated with primary antibodies overnight at 4 ◦ C. Afterward, they were incubated with a mixed solution of second antibodies for 1 h at RT. For immunohistochemistry, colors were developed with 3,3 -diaminobenzidine (DBA), and the slices were counterstained with hematoxylin. For immunofluorescence, the slices were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI). In negative control sections for immunostaining, the primary antibodies were omitted, and the remaining procedures were processed in the same manner. Under a fluorescent microscope (Leica), 5 non-overlapping high-power fields in the same magnification (400×) and illumination intensity were randomly selected from the perihematoma area in each slice to determine the gray value of immunohistochemical positive signal, respectively.
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Fig. 1. The improved neural function of ICH rats post iPSCs graftage. The scores of mNSS (A) and MLPT (B) tests at days 1, 3, 7, 14 and 28 after experimental ICH were shown. On days 14 and 28 after ICH, iPSCs-transplanted group showed best performance with lowest scores of mNSS (A) and MLPT (B) among all groups except for sham group without any observed neural dysfunction (n = 12 in each group) (*iPSCs group vs. PBS group and fibroblasts group, P < 0.05 respectively).
2.7. Western blotting Each sample of the supernatant fluids from rat brain tissue was diluted to equal protein concentrations after being measured with the bicinchoninic acid (BCA) protein assay kit (DingGuo, China). Equal volumes of each sample were loaded onto each lane. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels and transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane. After blocking non-specific binding sites with 5% defatted milk powder in Tris buffered saline with Tween-20 (TBST) for 1 h, the PVDF membrane was incubated with primary antibodies, including rabbit anti-rat BDNF, VEGF, and internal reference (GAPDH) (all from ABcam) overnight at 4 ◦ C. Subsequently, the washed PVDF membrane was incubated with goat anti-rabbit second antibody marked with AP for 2 h at 37 ◦ C. After being washed three more times with TBST, the targeted strips were displayed with AP staining (BoShiDe, China). Optical band densities were analyzed with a gel image analysis system (BIO-RAD). The relative levels of the proteins of interest were shown in density as the ratio of the targeted strips divided by the internal reference strips.
were significant decreases in the scores of both mNSS (Fig. 1A) and MLPT (Fig. 1B) in iPSCs-transplanted rats as compared to those of the PBS group and fibroblasts group (P < 0.05 respectively), while there was no significant difference between the scores of the PBS group and fibroblasts group (Fig. 1A and B). 3.2. Neural differentiation of iPSCs in vivo To examine the survival of transplanted iPSCs in vivo, we measured the BrdU-positive cells in rat brain on day 28 post-ICH. IPSCs were incubated with BrdU for 2 days prior to transplantation; therefore, the BrdU-positive cells in the brain represented the viable transplanted cells. BrdU-positive cells were found in the surrounding area of hematoma on day 28 post-ICH (Fig. 2B, F, and J). In the corresponding region of the contralateral side brain, few BrdUpositive iPSCs were found. Furthermore, we investigated the neural differentiation ability of transplanted iPSCs in vivo by doublefluorescence immunostaining of BrdU and neural markers on day 28 post-ICH. Indeed, survivor iPSCs displayed the biomarkers of neural precursor-like cells (nestin+ /BrdU+ ) (Fig. 2A–D), neuronlike cells (-tubulin+ /BrdU+ ) (Fig. 2E–H), and astrocyte-like cells (GFAP+ /BrdU+ ) (Fig. 2I–L) in ICH brain of rats.
2.8. Statistical analysis SPSS 13.0 statistical software was applied to analyze the data. All measurement data were presented in mean ± standard deviation (X¯ ± S). Repeated measurement analysis was conducted on mNSS and MALP scoring data. One-factor analysis of variance was conducted on the rest of the data with pairwise comparison among groups. Multiple comparisons between the groups were performed using the S–N–K method. Data were considered statistically significant if P-value < 0.05. 3. Results 3.1. Continuous improvement of neurological function after ICH in rats with iPSCs transplantation IPSCs were transplanted into the brain near the hematoma on day 1 post experimental ICH. Neurological function of ICH rats in each group was periodically evaluated after iPSCs transplantation to assess their therapeutic effect. On days 14 and 28 post ICH, there
3.3. Increased expression of BDNF and VEGF in rat ICH brain after iPSCs transplantation On day 28 post-ICH, BDNF-positive cells were found around the hematoma using immunohistochemistry (Fig. 3A). The statistical analysis showed that the gray value of BDNF-positive cells (Fig. 3A) in the iPSCs group was significantly higher than that of the sham group, PBS group, and fibroblasts group (P < 0.05 respectively) (Fig. 3C). Only a few BDNF-positive cells could be seen in the sham group (Fig. 3A), which had no hematoma formation. Consistently, the protein level of BDNF in the iPSCs group detected with western-blot also increased significantly as compared to that of the control groups (P < 0.05 respectively) (Fig. 3B and D). The levels of VEGF in rat brain were also detected using immunocytochemistry and western-blot (Fig. 3). Similarly, both the gray value of VEGF-positive cells (P < 0.05 respectively) (Fig. 3A and C) and the protein level of VEGF (P < 0.05 respectively) (Fig. 3B and D) in rat brain of the iPSCs group were significantly higher than those of the control groups (Fig. 3).
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Fig. 2. Neural differentiation of iPSCs in the perihematomal area at day 28 post experimental ICH. Double-fluorescence immunostaining revealed that transplanted iPSCs were able to survive and displayed different biomarkers of neural cells in ICH brain. Double immunostaining of BrdU and neural marker nestin (A), BrdU (B), DAPI (C) and merged image (D); double immunostaining of BrdU and neural marker -III tubulin (E), BrdU (F), DAPI (G) and merged image (H); double immunostaining of BrdU and neural marker GFAP (I), BrdU (J), DAPI (K) and merged image (L). The graphs of the contralateral side and negative control were not show. Scale bars = 100 m.
4. Discussion In this study, we have demonstrated that ICH patient-derived iPSCs treatment can promote functional recovery in ICH rats. In a previous study, we showed that NES derived from iPSCs improved neurological function in a rat ICH model [14]. In addition, the therapeutic effects of iPSCs were demonstrated in an experimental ischemic stroke model [12,13]. Therefore, transplantation of iPSCs and their derivatives may provide a novel therapeutic approach for stroke [9,12–14]. The mechanisms by which transplanted iPSCs repair strokeinduced damage are largely unknown. Multiple mechanisms have been proposed for the actions of various transplanted stem cells, including neuronal cell replacement, secretion of neurotrophic factors, promotion of angiogenesis, modulation of inflammatory response, and neuroprotection [1,5,7,9,12–15]. The iPSCs with the capability of multi-lineage differentiation are ideal candidates for cell replacement therapy against stroke [6,13,14]. However, the low survival rate and incapacitation of the grafted cells in vivo are the main obstacles in the clinical application of stem cell transplantation. The abilities of transplanted iPSCs to survive and differentiate into neuronal cells in damaged brain are critical to achieve maximum therapeutic effect. In this research, we found that transplanted iPSCs survived at the transplantation site and migrated to the proximity of the damaged area in ICH brain for at least 27 days post transplantation. Furthermore, transplanted iPSCs display the biomarkers of neural-like cells such as NSCs, neurons, and astrocytes around the cerebral hematoma. This suggests that transplanted iPSCs are multipotent and able to differentiate into neural-like cells in ICH-damaged brain; however, it is not clear if these cells really integrate into the host brain tissue to exert neurorestorative function persistently, which need to be verified by further work [6,13,14]. Besides neuronal cell replacement, enhanced release of neurotrophic factors is necessary for the therapeutic effect of stem cell grafts in stroke [5,8,15]. Transplanted stem cells not only
secret neurotrophic factors and growth factors, but also provide an enriched environment to enhance the secretions from host neural tissues [10]. Elevated neurotrophic factors, in return, promote the differentiation of grafted stem cells into neural-like cells to form regenerative feedback loops to support the survival and regeneration of damaged neural cells in a feedback way [15]. Since transplanted iPSCs in rat ICH brain can survive and differentiate into neural cells, we hypothesized that neurotrophic factors in a local microenvironment of rat ICH brain may contribute to the beneficial effects of iPSCs, which may jointly be involved in the recovery of neural dysfunction in ICH rats after iPSCs transplantation. Among neurotrophic factors, BDNF can modulate the survival, growth, differentiation, and regeneration of various neurocytes [1,3,15]. The latest report confirmed that transplanted bone marrow mesenchymal stem cells (BMSCs) promote BDNF secretion in the microenvironments of injured brain after ischemic stroke [15]. In this study, by means of immunohistochemical staining and western blotting, we detected significant increases in the gray values of BDNF-positive cells and BDNF protein respectively, in rat ICH brains after iPSCs transplantation. These results disclose that grafted iPSCs may exert trophic effects on host brain by enhancing the expression and secretion of BDNF in rat ICH brains [15]. The angiogenic factor VEGF also possesses neurotrophic and neuroprotective activities in the central nervous system (CNS). VEGF exerts a direct action on NSCs, neurons, astrocytes, and microglia by promoting neurogenesis and angiogenesis, as well as their possible functional links, to enhance endogenous plasticity in CNS [4]. Driven by its versatile protective effects in CNS, VEGF and its signaling pathways are becoming potential targets for the effective treatment of stroke [4]. Accumulating evidence has indicated that VEGF is involved in the therapeutic effects of various stem cell transplantations in experimental stroke [4,15]. Another recent study [12] showed that transplantation of iPSCsderived NES enhanced VEGF expression in the brain of ischemic stroke models. In this study, we found that the expression of VEGF was also elevated in the local microenvironment of rat ICH brains
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Fig. 3. Increased expression of BDNF and VEGF in brain surrounding area of iPSCs-transplanted rats at day 28 post experimental ICH. Immunohistochemical staining of BDNF in sham group, PBS group, fibroblasts group and iPSCs group (A, top panels). Immunohistochemical staining of VEGF in sham group, PBS group, fibroblasts group and iPSCs group (A, bottom panels) (400×). Gray value analysis of immunohistochemical stainings of BDNF and VEGF showed that their gray values in the brain perihematoma of iPSCstransplanted rats were highest in all groups (n = 6 in each group) (*iPSCs group vs. sham group, PBS group and fibroblasts group, P < 0.05 respectively) (C). Western-blotting images of BDNF, VEGF and internal standard GAPDH in each group (B). Graphs showed that the relative expressing levels of BDNF and VEGF proteins in the same area of iPSCs-transplanted rats were obviously highest in all groups (n = 6 in each group) (*iPSCs group vs. sham group, PBS group and fibroblasts group, P < 0.05 respectively) (D).
after iPSCs transplantation [15]. Increased VEGF not only facilitates the survival and differentiation of iPSCs, but also promotes neurocyte regeneration and neurovascular remodeling, which may jointly contribute to the reconstruction of damaged nerve networks post-hemorrhagic stroke in iPSCs-transplanted rats [13,15]. Collectively, these results suggest that iPSCs therapy has a continuous neurotrophic function for ICH brain by promoting BDNF and VEGF that together may contribute to the functional recovery of ICH rats. In this experiment, we have not observed severe side effects of iPSCs transplantation, such as tumors or seizures, during the entire observation periods. However, tumorigenicity and other safety concerns associated with iPSCs transplantation should be further evaluated during extended observation periods, with a view
to eventual cell replacement therapy for stroke patients. Furthermore, multiple therapeutic mechanisms on iPSCs transplantation, which are not limited to the above-mentioned, need to be further explored in future investigations [12,14,15]. In conclusion, the present study provides the first evidence that transplantation of ICH patient-derived iPSCs significantly promotes neurological function after ICH in rats. The enhanced functional recovery after iPSCs transplantation might be partly due to the replacement of damaged brain tissue and enhanced secretion of neurotrophic factors. These findings are expected to facilitate the development of ICH patient-derived iPSCs autografting in regenerative therapies, which might be a potential strategy for the treatment of refractory hemorrhagic stroke.
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Conflict of interest The authors declare no competing financial interests. Acknowledgments This work was supported by the Natural Science Foundation of China (Grant no. 81070920 to Y.M. Xu; Grant no. 81301007 to J. Qin) and performed in the Key Disciplines Laboratory of Clinical Medicine of Henan Province, China.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Functional recovery after transplantation of induced pluripotent stem cells in a rat hemorrhagic stroke model Jie Qin a,1 , Guangming Gong b,1 , Shilei Sun a , Jing Qi a , Huili Zhang a , Yanlin Wang a , Ning Wang c , Qing Mei Wang d , Yan Ji a , Yuan Gao a , Changhe Shi a , Bo Yang e , Yi Zhang f , Bo Song a,∗∗ , Yuming Xu a,∗ a
Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, PR China c Department of Natural Sciences, The University of Virginia’s College at Wise, Wise, VA 24293, United States d Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital, Harvard Medical School, Boston, MA 02135, United States e Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China f Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China b
h i g h l i g h t s • • • • •
We assessed the therapeutic effect of iPSCs in experimental ICH rat. Therapeutic effects of iPSCs in rat ICH model were observed. The grafted iPSCs exhibited neural cell-specific biomarkers in the brain of ICH rats. The levels of BDNF and VEGF were enhanced in the surrounding region of hematoma. Functional improvement is partially due to neuronal replacement and neurophic factors.
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 21 August 2013 Accepted 23 August 2013 Keywords: Intracerebral hemorrhage (ICH) Induced pluripotent stem cells (iPSCs) Brain derived neurophic factors (BDNF) Vascular endothelial growth factor (VEGF)
a b s t r a c t Transplantation of induced pluripotent stem cells (iPSCs) has shown promising therapeutic effects for ischemic stroke. However, it is not clear if this treatment would promote recovery after intracerebral hemorrhage (ICH). In this study, we investigated the functional outcome of iPSCs transplantation in experimental ICH in rats. IPSCs were derived from an ICH patient’s fibroblasts and were injected into the ipsilateral side of ICH in rats. IPSCs transplantation significantly improved the neurological functions after ICH as compared to vehicle and fibroblast injection. The grafted iPSCs migrated into brain tissue around the hematoma, survived after 4 weeks of transplantation, and exhibited the neural cell-specific biomarkers nestin, -tubulin, and GFAP. Immunohistochemical staining showed that the densities of brain derived neurophic factors (BDNF)-positive cells and vascular endothelial growth factor (VEGF)positive cells were significantly increased around the hemorrhagic brain tissues of iPSCs-treated rats. In addition, iPSCs treatment increased the protein expression of BDNF and VEGF in the surrounding region of hematoma. These findings demonstrate that the transplantation of ICH patient-derived iPSCs contributes toward the improved neurological function in experimental ICH rats. The mechanisms are possibly due to neuronal replacement and enhanced secretion of neurophic factors. Our data suggest that transplantation of ICH patient-derived iPSCs may be a therapeutic strategy for hemorrhagic stroke. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: 1 Jianshe East Road, Erqi District, Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China. Tel.: +86 371 66862133. ∗∗ Corresponding author at: 1 Jianshe East Road, Erqi District, Third Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China. Tel.: +86 371 66862132. E-mail addresses: [email protected] (B. Song), [email protected] (Y. Xu). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.08.047
Intracerebral hemorrhage (ICH) has higher mortality and disability rates than that caused by ischemic stroke, although it accounts for only 10–15% of all strokes in the USA and Europe, and 20–30% of strokes in Asia [6]. Following the primary injury of ICH, a series of secondary events such as ischemia, hypoxia, inflammation of brain tissues, degeneration, necrosis, or apoptosis of neurons around the hematoma result in damage to or interrupt the integrity of intracerebral nerve networks, which finally
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causes clinical neurological dysfunction [6]. However, there are no effective treatments to promote functional recovery after ICH [11]. Emerging stem cell therapies have shown potential benefits in treating stroke by targeting the complicated pathological damages caused in multiple pathways [1,5–7,9,12,13,15]. Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have great therapeutic potential and unique advantage due to abundant cell supplies and lack of immunogenic, social, and ethical concerns [13,14,16]. Growing evidence suggests there are therapeutic effects of iPSCs and their derivatives for the treatment of ischemic stroke in rodent stroke models [9,12,13,15]. In a recent study, we showed that neuro-epithelial-like stem cells (NES) derived from iPSCs improve the neurological function after ICH in rats [14]. However, the complex and time-consuming procedure of inducing NES from iPSCs in vitro hinder their application in personalized cell therapy. With more primary and pluripotent capacities than NES, whether iPSCs have therapeutic effect for ICH remains unknown. In this study, we investigated the functional outcome of intracerebral transplantation of an ICH patient’s fibroblasts-derived iPSCs in a rat ICH model and explored the underlying mechanism(s). 2. Materials and methods 2.1. Establishment of rat ICH model Animal feeding and all relevant follow-up experimental procedures were carried out in accordance with the European Council Directive 86/609/European Economic Community for animal experiments and were approved by the Ethics Committee of Zhengzhou University, China. Sprague-Dawley (SD) rats were provided by the animal center of Henan province, Zhengzhou, China. Rat ICH model was generated using intracerebral injection with collagenase VII. After anesthetized with 1% ketamine (30 mg/kg, I.P.; Sigma–Aldrich), male rats (270–300 g) were placed in a rat brain stereotaxic apparatus (Narishige SN-3, Tokyo, Japan) in a prone position. A 1 mm × 1 mm craniectomy was performed, and the needle was inserted perpendicularly into the left corpus striatum (coordinates: 3.5 mm lateral to midline, 0.5 mm anterior to bregma, 6.0 mm depth). Then 2.5 U (0.5 U/2 L) collagenase VIII (Sigma–Aldrich) was injected gently at 1 L/min with a 10 L microsyringe, and the needle was withdrawn slowly. Then the wound surface was cleaned, and the scalp was sutured [14]. In the sham group, stereotactic puncture was performed without collagenase injection. 2.2. IPSCs induction IPSCs were induced from an ICH patient’s skin fibroblasts as described in a previous publication [14]. Briefly, following informed consent and the approval of Ethics Committee of Zhengzhou University, the ICH patient’s skin fibroblasts were taken from the medial upper arm by skin biopsy technique. Fibroblasts were induced by lentiviral vectors carrying the cDNAs of human transcription factor Oct4, Sox2, C-myc, and Klf4. Afterward, embryonic stem cells (ESCs)-like cell clones were gradually formed, and gone through serial identification procedures such as alkaline phosphatase (AP) assay, expressions of hESC-specific biomarkers, and formation of teratoma in severe combined immune deficiency (SCID) mice. Finally, one of these clones was identified as iPSCs successfully [14]. 2.3. Cell therapy Modified neurological severity scoring (mNSS) and modified limb placing test (MLPT) were performed to evaluate neurological
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function at 1 day post operation [2,14]. Thirty-six rats with experimental ICH that scored 8–12 in mNSS and 4–7 in MLPT were randomly and equally divided into 3 groups: phosphate buffered saline (PBS) only group, fibroblast group (total 1 × 106 skin fibroblasts in 10 L PBS), and iPSCs group (total 1 × 106 iPSCs in 10 L PBS). Ten microliters PBS with or without cells was stereotactically injected into all rats at the same coordinates as above to generate ICH, except at a depth of 3.5 mm relative to the bregma. Before being transplanted, all cells were incubated with 20 mol/L of BrdU (Sigma–Aldrich) for 2 days. 2.4. Behavioral tests The functional outcomes (mNSS and MLPT) were evaluated by independent blinded investigators at days 1, 3, 7, 14, and 28 after experimental ICH [14]. 2.5. Extraction of brain tissue Rats were sacrificed in accordance with euthanasia procedures after behavioral testing on day 28 after ICH. Brain tissues of 6 rats in each group were fixed with paraformaldehyde and dehydrated with gradient sucrose. After being frozen, they were cut into coronal slices and used for further immunostaining analysis. For the other 6 rats, brain tissues were quickly extracted, and the brain tissues on the nidus side were ground with liquid nitrogen and degenerated with a protein isolation buffer containing a cocktail of protease inhibitors (Sigma–Aldrich), and then put on ice for 30 min at 4 ◦ C. The supernatant fluids were centrifuged (12,000 × g, 20 min, 4 ◦ C), and the supernatant was stored at −80 ◦ C for western-blotting assay. 2.6. Immunostaining Brain coronal slices were taken from the same position (puncture point) on each rat in all groups. In addition to hematoxylin–eosin staining, immunofluorescence double staining was conducted with antibodies specific for BrdU, nestin, -tubulin, and GFAP (rabbit anti-human were primary antibodies, and goat anti-rabbit, PE/FITC-conjugated goat anti-rabbit antibodies were second antibodies) to identify the transplanted cells, neural stem cells (NSCs), neurons, and astrocytes, respectively. Immunohistochemical staining of brain derived neurophic factors (BDNF) and vascular endothelial growth factor (VEGF) (rabbit anti-rat as primary antibodies, and goat anti-rabbit as second antibodies) was also performed with similar methods. All of the above antibodies were purchased from Santa Cruz Biotech, CA, USA. The slices were fixed with 4% paraformaldehyde for 5 min at room temperature (RT), heated (92–98 ◦ C) in a 0.01 mol/L, pH 6.0 citrate buffer solution for 30 min, and then cooled for 30 min at RT. After blocked with 5% bovine serum albumin (BSA) for 60 min at RT, the slices were incubated with primary antibodies overnight at 4 ◦ C. Afterward, they were incubated with a mixed solution of second antibodies for 1 h at RT. For immunohistochemistry, colors were developed with 3,3 -diaminobenzidine (DBA), and the slices were counterstained with hematoxylin. For immunofluorescence, the slices were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI). In negative control sections for immunostaining, the primary antibodies were omitted, and the remaining procedures were processed in the same manner. Under a fluorescent microscope (Leica), 5 non-overlapping high-power fields in the same magnification (400×) and illumination intensity were randomly selected from the perihematoma area in each slice to determine the gray value of immunohistochemical positive signal, respectively.
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Fig. 1. The improved neural function of ICH rats post iPSCs graftage. The scores of mNSS (A) and MLPT (B) tests at days 1, 3, 7, 14 and 28 after experimental ICH were shown. On days 14 and 28 after ICH, iPSCs-transplanted group showed best performance with lowest scores of mNSS (A) and MLPT (B) among all groups except for sham group without any observed neural dysfunction (n = 12 in each group) (*iPSCs group vs. PBS group and fibroblasts group, P < 0.05 respectively).
2.7. Western blotting Each sample of the supernatant fluids from rat brain tissue was diluted to equal protein concentrations after being measured with the bicinchoninic acid (BCA) protein assay kit (DingGuo, China). Equal volumes of each sample were loaded onto each lane. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels and transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane. After blocking non-specific binding sites with 5% defatted milk powder in Tris buffered saline with Tween-20 (TBST) for 1 h, the PVDF membrane was incubated with primary antibodies, including rabbit anti-rat BDNF, VEGF, and internal reference (GAPDH) (all from ABcam) overnight at 4 ◦ C. Subsequently, the washed PVDF membrane was incubated with goat anti-rabbit second antibody marked with AP for 2 h at 37 ◦ C. After being washed three more times with TBST, the targeted strips were displayed with AP staining (BoShiDe, China). Optical band densities were analyzed with a gel image analysis system (BIO-RAD). The relative levels of the proteins of interest were shown in density as the ratio of the targeted strips divided by the internal reference strips.
were significant decreases in the scores of both mNSS (Fig. 1A) and MLPT (Fig. 1B) in iPSCs-transplanted rats as compared to those of the PBS group and fibroblasts group (P < 0.05 respectively), while there was no significant difference between the scores of the PBS group and fibroblasts group (Fig. 1A and B). 3.2. Neural differentiation of iPSCs in vivo To examine the survival of transplanted iPSCs in vivo, we measured the BrdU-positive cells in rat brain on day 28 post-ICH. IPSCs were incubated with BrdU for 2 days prior to transplantation; therefore, the BrdU-positive cells in the brain represented the viable transplanted cells. BrdU-positive cells were found in the surrounding area of hematoma on day 28 post-ICH (Fig. 2B, F, and J). In the corresponding region of the contralateral side brain, few BrdUpositive iPSCs were found. Furthermore, we investigated the neural differentiation ability of transplanted iPSCs in vivo by doublefluorescence immunostaining of BrdU and neural markers on day 28 post-ICH. Indeed, survivor iPSCs displayed the biomarkers of neural precursor-like cells (nestin+ /BrdU+ ) (Fig. 2A–D), neuronlike cells (-tubulin+ /BrdU+ ) (Fig. 2E–H), and astrocyte-like cells (GFAP+ /BrdU+ ) (Fig. 2I–L) in ICH brain of rats.
2.8. Statistical analysis SPSS 13.0 statistical software was applied to analyze the data. All measurement data were presented in mean ± standard deviation (X¯ ± S). Repeated measurement analysis was conducted on mNSS and MALP scoring data. One-factor analysis of variance was conducted on the rest of the data with pairwise comparison among groups. Multiple comparisons between the groups were performed using the S–N–K method. Data were considered statistically significant if P-value < 0.05. 3. Results 3.1. Continuous improvement of neurological function after ICH in rats with iPSCs transplantation IPSCs were transplanted into the brain near the hematoma on day 1 post experimental ICH. Neurological function of ICH rats in each group was periodically evaluated after iPSCs transplantation to assess their therapeutic effect. On days 14 and 28 post ICH, there
3.3. Increased expression of BDNF and VEGF in rat ICH brain after iPSCs transplantation On day 28 post-ICH, BDNF-positive cells were found around the hematoma using immunohistochemistry (Fig. 3A). The statistical analysis showed that the gray value of BDNF-positive cells (Fig. 3A) in the iPSCs group was significantly higher than that of the sham group, PBS group, and fibroblasts group (P < 0.05 respectively) (Fig. 3C). Only a few BDNF-positive cells could be seen in the sham group (Fig. 3A), which had no hematoma formation. Consistently, the protein level of BDNF in the iPSCs group detected with western-blot also increased significantly as compared to that of the control groups (P < 0.05 respectively) (Fig. 3B and D). The levels of VEGF in rat brain were also detected using immunocytochemistry and western-blot (Fig. 3). Similarly, both the gray value of VEGF-positive cells (P < 0.05 respectively) (Fig. 3A and C) and the protein level of VEGF (P < 0.05 respectively) (Fig. 3B and D) in rat brain of the iPSCs group were significantly higher than those of the control groups (Fig. 3).
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Fig. 2. Neural differentiation of iPSCs in the perihematomal area at day 28 post experimental ICH. Double-fluorescence immunostaining revealed that transplanted iPSCs were able to survive and displayed different biomarkers of neural cells in ICH brain. Double immunostaining of BrdU and neural marker nestin (A), BrdU (B), DAPI (C) and merged image (D); double immunostaining of BrdU and neural marker -III tubulin (E), BrdU (F), DAPI (G) and merged image (H); double immunostaining of BrdU and neural marker GFAP (I), BrdU (J), DAPI (K) and merged image (L). The graphs of the contralateral side and negative control were not show. Scale bars = 100 m.
4. Discussion In this study, we have demonstrated that ICH patient-derived iPSCs treatment can promote functional recovery in ICH rats. In a previous study, we showed that NES derived from iPSCs improved neurological function in a rat ICH model [14]. In addition, the therapeutic effects of iPSCs were demonstrated in an experimental ischemic stroke model [12,13]. Therefore, transplantation of iPSCs and their derivatives may provide a novel therapeutic approach for stroke [9,12–14]. The mechanisms by which transplanted iPSCs repair strokeinduced damage are largely unknown. Multiple mechanisms have been proposed for the actions of various transplanted stem cells, including neuronal cell replacement, secretion of neurotrophic factors, promotion of angiogenesis, modulation of inflammatory response, and neuroprotection [1,5,7,9,12–15]. The iPSCs with the capability of multi-lineage differentiation are ideal candidates for cell replacement therapy against stroke [6,13,14]. However, the low survival rate and incapacitation of the grafted cells in vivo are the main obstacles in the clinical application of stem cell transplantation. The abilities of transplanted iPSCs to survive and differentiate into neuronal cells in damaged brain are critical to achieve maximum therapeutic effect. In this research, we found that transplanted iPSCs survived at the transplantation site and migrated to the proximity of the damaged area in ICH brain for at least 27 days post transplantation. Furthermore, transplanted iPSCs display the biomarkers of neural-like cells such as NSCs, neurons, and astrocytes around the cerebral hematoma. This suggests that transplanted iPSCs are multipotent and able to differentiate into neural-like cells in ICH-damaged brain; however, it is not clear if these cells really integrate into the host brain tissue to exert neurorestorative function persistently, which need to be verified by further work [6,13,14]. Besides neuronal cell replacement, enhanced release of neurotrophic factors is necessary for the therapeutic effect of stem cell grafts in stroke [5,8,15]. Transplanted stem cells not only
secret neurotrophic factors and growth factors, but also provide an enriched environment to enhance the secretions from host neural tissues [10]. Elevated neurotrophic factors, in return, promote the differentiation of grafted stem cells into neural-like cells to form regenerative feedback loops to support the survival and regeneration of damaged neural cells in a feedback way [15]. Since transplanted iPSCs in rat ICH brain can survive and differentiate into neural cells, we hypothesized that neurotrophic factors in a local microenvironment of rat ICH brain may contribute to the beneficial effects of iPSCs, which may jointly be involved in the recovery of neural dysfunction in ICH rats after iPSCs transplantation. Among neurotrophic factors, BDNF can modulate the survival, growth, differentiation, and regeneration of various neurocytes [1,3,15]. The latest report confirmed that transplanted bone marrow mesenchymal stem cells (BMSCs) promote BDNF secretion in the microenvironments of injured brain after ischemic stroke [15]. In this study, by means of immunohistochemical staining and western blotting, we detected significant increases in the gray values of BDNF-positive cells and BDNF protein respectively, in rat ICH brains after iPSCs transplantation. These results disclose that grafted iPSCs may exert trophic effects on host brain by enhancing the expression and secretion of BDNF in rat ICH brains [15]. The angiogenic factor VEGF also possesses neurotrophic and neuroprotective activities in the central nervous system (CNS). VEGF exerts a direct action on NSCs, neurons, astrocytes, and microglia by promoting neurogenesis and angiogenesis, as well as their possible functional links, to enhance endogenous plasticity in CNS [4]. Driven by its versatile protective effects in CNS, VEGF and its signaling pathways are becoming potential targets for the effective treatment of stroke [4]. Accumulating evidence has indicated that VEGF is involved in the therapeutic effects of various stem cell transplantations in experimental stroke [4,15]. Another recent study [12] showed that transplantation of iPSCsderived NES enhanced VEGF expression in the brain of ischemic stroke models. In this study, we found that the expression of VEGF was also elevated in the local microenvironment of rat ICH brains
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Fig. 3. Increased expression of BDNF and VEGF in brain surrounding area of iPSCs-transplanted rats at day 28 post experimental ICH. Immunohistochemical staining of BDNF in sham group, PBS group, fibroblasts group and iPSCs group (A, top panels). Immunohistochemical staining of VEGF in sham group, PBS group, fibroblasts group and iPSCs group (A, bottom panels) (400×). Gray value analysis of immunohistochemical stainings of BDNF and VEGF showed that their gray values in the brain perihematoma of iPSCstransplanted rats were highest in all groups (n = 6 in each group) (*iPSCs group vs. sham group, PBS group and fibroblasts group, P < 0.05 respectively) (C). Western-blotting images of BDNF, VEGF and internal standard GAPDH in each group (B). Graphs showed that the relative expressing levels of BDNF and VEGF proteins in the same area of iPSCs-transplanted rats were obviously highest in all groups (n = 6 in each group) (*iPSCs group vs. sham group, PBS group and fibroblasts group, P < 0.05 respectively) (D).
after iPSCs transplantation [15]. Increased VEGF not only facilitates the survival and differentiation of iPSCs, but also promotes neurocyte regeneration and neurovascular remodeling, which may jointly contribute to the reconstruction of damaged nerve networks post-hemorrhagic stroke in iPSCs-transplanted rats [13,15]. Collectively, these results suggest that iPSCs therapy has a continuous neurotrophic function for ICH brain by promoting BDNF and VEGF that together may contribute to the functional recovery of ICH rats. In this experiment, we have not observed severe side effects of iPSCs transplantation, such as tumors or seizures, during the entire observation periods. However, tumorigenicity and other safety concerns associated with iPSCs transplantation should be further evaluated during extended observation periods, with a view
to eventual cell replacement therapy for stroke patients. Furthermore, multiple therapeutic mechanisms on iPSCs transplantation, which are not limited to the above-mentioned, need to be further explored in future investigations [12,14,15]. In conclusion, the present study provides the first evidence that transplantation of ICH patient-derived iPSCs significantly promotes neurological function after ICH in rats. The enhanced functional recovery after iPSCs transplantation might be partly due to the replacement of damaged brain tissue and enhanced secretion of neurotrophic factors. These findings are expected to facilitate the development of ICH patient-derived iPSCs autografting in regenerative therapies, which might be a potential strategy for the treatment of refractory hemorrhagic stroke.
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Conflict of interest The authors declare no competing financial interests. Acknowledgments This work was supported by the Natural Science Foundation of China (Grant no. 81070920 to Y.M. Xu; Grant no. 81301007 to J. Qin) and performed in the Key Disciplines Laboratory of Clinical Medicine of Henan Province, China.
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