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Differentiation and Neurological Benefit of the Mesenchymal Stem Cells Transplanted into the Rat Brain Following Intracerebral Hemorrhage

Posted on: Tuesday, 14 February 2006, 03:03 CST

By Zhang, Huabiao; Huang, Zhiyong; Xu, Yuming; Zhang, Suming

Spontaneous intracerebral hemorrhage (ICH) is often a fatal event. In a patient who survives the initial ictus, the resulting hematoma within brain parenchyma can trigger a series of events that lead to secondary insults and severe neurological deficits. Great efforts have been focused on searching for new approaches to help patients recover neurological function after ICH. Previous studies indicate that mesenchymal stem cells (MSCs) grafted into the ischemic rat brain can improve neurological function. However, there is no report regarding whether MSCs can be used in the same way to improve the neurological function after ICH. We generated the ICH model by injecting collagenase VII into rat brain. Subsequently, 5- bromo-2-deoxyuridine (BrdU)-labeled mesenchymal stem cells were delivered into the brain through carotid artery, cervical vein or lateral ventricle. The distribution and differentiation of MSCs were investigated by methods of immunohistochemistry. We found that MSCs were able to differentiate into neural cells in vitro as well as in the rat brain after ICH. The injected MSCs were able to migrate into hippocampus, blooding foci and ipsilateral cortex. In the hippocampus, MSCs differentiated into neurons; but in surrounding bleeding foci, they differentiated into neurons and astrocytes. In the ipsilateral cortex, MSCs differentiated into neurons, astrocytes and oligodendrocytes. Notably, the motor function of the rats in the carotid artery (CA) group and the lateral ventricle (L V) group improved significantly. Collectively, our study indicates that MSCs are able to differentiate into neural cells in the rat brain after ICH and can significantly improve motor function. [Neurol Res 2006; 28: 104-112]

Keywords: Mesenchymal stem cells; differentiation; delivery; intracerebral hemorrhage

INTRODUCTION

Spontaneous intracerebral hemorrhage (ICH) is often fatal and can initiate a series of potentially fatal events. Even if the patient survives the initial ictus, the resulting hematoma within the brain parenchyma can still lead to secondary insults and severe neurological deficits1. Currently, there is no effective treatment for ICH and subsequent neurological damage.

Stem cells are characterized by their ability to generate progenitors capable of differentiating into various and distinct cell lineages2-4. It is this ability of stem cells that makes them potentially ideal candidates as treatments for central nervous system disease. Previous research indicates that rodent bone marrow cells grafted into the ischemic rat brain differentiated into neural cells and improved the neurological function5. In addition, neural stem cells (NSCs) and embryonic stem cells (ESCs) have been transplanted into adult rat brain to treat neurodegenerative disorder such as Parkinson's disease6. It is commonly held that stem cells derived from early embryos can differentiate into all somatic cell types7-9, whereas those derived from adult tissues can only differentiate into the cell types of areas from which they are derived. However, recently this notion has been challenged. Bjornson and his colleagues10 reported that when NSCs were transplanted into the hematopoitic system of irradiated hosts, they differentiated into myeloid and lymphoid cell lineages. Furthermore, hematopoitic stem cells (HSCs) were able to differentiate into the epithelia of various organs in irradiated mice11, and also into microgliaand macroglia in the brains of adult mice12. Mesenchymal stem cells (MSCs) can not only give rise to mesoderm-derived cells such as osteoblasts, chondrocytes, adipocytes and muscular cells, but also adopt neuroectodermal cell fate13-16. For example, the donorderived neurons and astrocytes have been found in the host rodent brain following transplantation of rodent bone marrow12,17-19. However, even if NSCs and ESCs were capable of differentiating into neural cells, several problems remain when developing their use for treatment of neurological diseases. For example, NSCs are difficult to be obtained and often trigger immune rejection when used for xenotransplantation. Additionally, the ethics of ESCs use remains highly debated. Therefore, the potential for rapidly developing NSCs and ESCs as a treatment for neurological diseases is somewhat limited.

MSCs are easily harvested form a patient's own bone marrow, and can be stimulated to differentiate into neurons, astrocytes and oligodendrocytes in vitro20-22. Work from several groups suggests that subpopulations of MSCs derived from the bone marrow are able to differentiate into neural cells if given the appropriate environmental cues17-19,23. lntracerebral transplantation of MSCs into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their lifespan24. Furthermore, transplanting MSCs into focal brain regions following cerebral infarction significantly improves neurological function25. However, to our knowledge, there are no reports concerning the use of MSCs to improve neurological function following ICH. In the current study, we have investigated whether rat MSCs will differentiate into neural cells in vitro. Furthermore, we examine whether MSCs, when delivered to the brain through various routes, can differentiate into neural cells in the rat brain and give a functional benefit to animal following ICH.

MATERIALS AND METHODS

Isolation and purification of MSCs

Rats were killed by decapitation and immersed in 75% ethanol for 10 minutes. Subsequently, their limbs were cut off for MSCs isolation. Rat bone marrow was flushed with the culture medium containing 1 M α-MEM (Eagle's minimal essential medium) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 25 ng/ml amphotericin B. The culture medium was harvested and put onto Percoll separation medium in a 10 ml test tube that was centrifuged at 2000 rev/min for 30 minutes. The layer of mononuclear cells was obtained from the third layer in the separation medium. And the cells were then resuspensed with culture medium. Approximately 1 10^sup 7^ cells were placed in a 75 cm^sup 2^ flask and cultured with culture medium plus 10 ng/ml basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The culture medium was replaced every 3 to 4 days. After the cells became confluent, they were split. The cells were passaged to the tenth generation.

MSCs identification

The stem cells obtained from bone marrow include HSCs and MSCs. CD45 is a specific cell surface marker of HSCs, while CD90 and CD106 are negative for HSCs but positive for MSCs. Therefore, fluorescence- labeled CY-chrome(TM) mouse anti-rat CD45 monoclonal antibody, mouse anti-rat CD90-fluorescein isothiocyanate (FITC) antibody and R- phycoerythrin conjugated mouse anti-rat CD106 monoclonal antibody were employed to identify the purity of MSCs. The cultured cells were harvested by incubation with 0.25% trypsin for 5 minutes at room temperature and then fixed with 4% paraformaldehyde for 30 minutes. One separate group of the cells were incubated with anti- CD45, CD90 or CDI06 antibody respectively, or with three antibodies together at 4C for 1 hour, and then incubated with three fluorescence-labeled secondary antibodies: anti-mouse CY- chrome(TM), CD90-FITC and R-phycoerythrin antibodies. The other separate group of cells were not incubated with any antibody. The cells were washed with phosphate-buffered saline (PBS), centrifuged three times and subsequently analysed by flow cytometry. The levels of staining for the single antibody as well as staining for all the three antibodies were calculated.

Cell induction and differentiation in vitro

The induction method employed was described previously by Woodbury et al.20 Briefly, subconfluent cultures of rat MSCs were maintained in culture medium. Twenty-four hours before the induction, the culture medium was replaced by the pre-induction medium containing 1 mM β-mercaptoethanol (BME) in culture medium. To induce neuron differentiation, the preinduction medium was removed, then the cells were washed with PBS and they grew for 5 hours in induction medium consisting of 1 mol/l α-MEM and 2% dimethylsulfoxide (DMSO). Finally, the cellular differentiation was induced with 1 M α-MEM and 200 mol/l butylated hydroxyanisole (BHA) for additional 5 hours. The differentiation of neuron and NSCs at three time points including 24 hours before induction, growing for 5 hours in culture medium of DMSO and another 5 hours in culture medium of BHA was identified by immunocytochemistry. The cellular markers used were neuronal nuclei (NeuN), a neuron-specific marker; glial fibrillary acidic protein (GFAP), a marker for astrocytes; and 2',3'-cyclic nucleotide 3'-phosphorylase (CNP), a marker for oligodendrocytes. The mouse anti-rat NeuN (1:100; Chemicon), anti- rat GFAP Ab-6 and CNP Ab-1 (1 : 100; NeoMarker) were used to identify the cell differentiation following induction. The induced cells were fixed with 4% paraformaldehyde, and incubated with the primary antibodies overnight at 4C. The cells were then washed three times in PBS and incubated with alkaline phosphatase-anti-mouse secondary antibody for 1 hour. Subsequently, the cells were washed three times in PBS, and incubatedwith nitroblue tetrazolium/ bromochloroindolylphosphate (NBT/BCIP) complex for 1 hour at room temperature, followed by three additional washes in PBS. For each of the three induction steps, anti-rat nestin (1 :500; Pharmingen) was used for NSCs identification.

Rat ICH model

The ICH model was created using a collagenase VII injection as described by Rosenberg etal.26. Briefly, the rats were anesthetized, and a 15 mm scalp incision was made along with the midline. Two microliters of saline containing 0.5 unit of bacterial collagenase were injected into the left caudate nucleus of the adult rat brain in 5 minutes. The stereotactic coordinate is 3.2 mm left to the sagittal suture, 1.4 mm behind the interior fontanelle, and 5.6 mm deep to meninges by cutting off 1.4 mm wide and 3.2 mm long skull in this region. After injection, the scalp of operation was sutured and sterilized by iodine. A separate group of rats received a sham operation consisting of a two-microliter injection of saline into the left caudate nucleus.

MSCs labeling and delivery

One hundred and five adult Sprague-Dawley rats weighing 270 to 300 g were randomly divided into six groups including three treatment groups, a sham operation (SO) group, an ICH-only (IO) group and notreatment (NT) control group. The three treatment groups: the carotid artery (CA) group, the cervical vein (CV) group and the lateral ventricle (LV) group, received injections of 5- bromo-2-deoxyuridine (BrdU)-labeled MSCs through the carotid artery, cervical vein or lateral ventricle respectively, on days 1, 3, 5 and 7 after ICH. The SO group was given BrdU-labeled MSCs into the lateral ventricle on days 1, 3, 5 and 7 after the sham operation. Five rats were used for each time point. The IO group included 20 rats that did not receive any treatment after ICH. The NT group included five rats that received neither an ICH or sham treatment nor a cell injection.

The cultured MSCs, at a density of 2 10^sup 6^ cells/ml, were labeled with 10 M BrdU in cell culture medium27, after which the cells were incubated for 5 hours, rinsed twice with PBS, and then incubated in culture medium for 16 hours. The BrdU-labeled MSCs were harvested, washed twice and re-suspended in PBS. Approximately 2 10^sup 6^ cells in 20 l saline were slowly injected into the carotid artery, cervical vein or lateral ventricle. On days 1, 3, 5 and 7 post-injection, rats were killed and perfused with 4% paraformaldehyde. The brain was cut into 10 m-thick sections and processed for immunohistochemistry18.

Behavioral evaluations

The behavior evaluation included a test of coordinated motor control, and was able to detect difference between young and aged rats. The cage was placed at one end of a wooden beam (2.5 cm wide, 2 cm long) elevated 45 cm from the table surface. At the beginning of the training session, the rat was placed at the end of the beam opposite to the home cage. Initially, the rat was placed on the beam near the cage and naturally returned to it without the need of negative reinforcement. Gradually, the rat was placed further from the cage until it returned to the cage by walking the full length of the beam. The test was conducted after training had been finished. After training, rats without impairments always maintained all four paws on the upper surface (2.5 cm wide) of the beam. When a motor deficit appeared, this pattern changed dramatically. For example, on the first day after ICH, the rats were unable to run on the beam, and often would roll over and lay on the side of their body contralateral to the ICH. This behavior rated the maximum deficit score 6. An animal received a '5' if it traversed the beam while dragging a h'mdlimb, a '4' if it fell or traversed the beam slipping off on more than half of its steps, a '3' if it traversed the beam without slipping but with the contralateral hind paw touching the lateral aspect (edge) of the beam, a '2' if the animal limped with one hindlimb (hypotonous) and a '1' if the animal widened its base with four toes off the beam bilaterally.

The behavior was rated on each quarter section of the beam, and the final score was obtained by adding together the scores on each section of the beam. For instance, an animal which dragged its affected hindlimb (motor deficit=5) over the first two quarters of the beam and made repetitive hindlimb slips (motor deficit=4) over the final two section of the beam, obtained a final motor score of 5 + 5 + 4 + 4=18. Initially, rats were trained to climb the beam (2.5 cm wide) and lay at a 45 angle.

Identification of MSCs distribution and differentiation in rat brain

Brain sections were treated with 0.3%H^sub 2^O^sub 2^ for 30 minutes to remove endogenous peroxidase activity, followed by incubation in antigen retrieval solution at 89C for 10 minutes. The sections were blocked with FBS, and then incubated with anti-BrdU Ab- 2 (1 :1000; Sigma) at 4C overnight. The distribution of BrdU- labeled MSCs were observed after the cells were incubated with alkaline phosphatase-labeled secondary antibody and stained with NBT/ BCIP solution.

The MSCs differentiation in the rat brain was identified with double fluorescence staining for BrdU as well as one of specific neural cell markers. The primary antibodies: anti-NeuN (1:100; Chemicon), anti-GFAP Ab-6 and CNP Ab-1 (1 :100; NeoMarker) were applied at 4C overnight. After the sections were rinsed in tris buffered saline (TBS) three times, a goat anti-mouse IgG1-FITC secondary antibody (1:50; Serotec) was used for BrdU staining, whereas streptavidin-Cy3 (1 :500; Sigma) was used for anti-NeuN, anti-GFAP Ab-6 or CNP Ab-1 staining. The sections were observed under a confocal (Tcs-st) microscope at 200 magnification.

Statistical analysis

Data are presented as mean SE. The behavioral evaluation was analysed by χ^sub 2^ test, and processed with repeated measures one-way analysis of variance (ANOVA) software with groups as the between-subjects factor used at a 99.9% significant level.

RESULTS

Identification of MSCs

The cultured cells were incubated with the fluorescence-labeled CD45, CD106 and CD90 antibodies, and then analysed by flow cytometry. The majority of cells were positive for CD90 and CD106, and negative for CD45. The proportion of labeling was 95.92, 98.47 and 0.17% respectively (Figure 1A-C). When the cells were labeled with the three antibodies together, 95.04% of cells were positive for both CD90 and CD106 (Figure 1E). However, 93.79% of the cells positive for CD90 were negative for CD45. (Figure 1G)

Differentiation of MSCs

Within 3 hours induction with α-MEM/20% FBS/ 1 mM BME, morphologic changes in MSCs appeared. The cytoplasm of the flat MSCs retracted towards the nucleus, forming a contracted multipolar cell body and peripheral process-like extensions. The process continued to elaborate, displaying primary and secondary branches (Figure 2B). These process-like structures were similar to axons and dendrites of neurons and astrocytes in appearance (Figure 2C,D).

Figure 1: The cellular phenoltypes of undifferentiated MSCs were identified by flow cytometry. When the cells were labeled with an individual antibody, 95.92% of the cells were positive for CD90 (A), 98.47% were positive for CD106 (B) and only 0.17% were positive for CD45 (C). When the cells were labeled with combined antibodies, 95.04% of the cells were positive for both CD90 and CD106 (E), whereas 93.79% of the cells were positive for CD45 and CD90 (G). (D) and (F) show control staining where the cells were labeled with the three fluorescence-labeled secondary antibodies: anti-mouse CY- chrome(TM), CD90-FITC and R-phycoerythrin antibodies

Nestin was expressed for 24 hours after induced with 1 M α- MEM/20% FBS/1 mM BME, after which the expression gradually decreased when the induction medium was changed into 1 M α-MEM/2% DMSO for 5 hours, and into α-MEM/200 mol/l BHA for additional 5 hours. At the end of induction, immunocytochemistry staining revealed that the induced cells were positive for NeuN, CFAP and CNP. The expression rates of the proteins were 75, 20 and 2%. There were also a few cells that remained undifferentiated.

The distribution and differentiation of MSCs in the rat brain

The percentage of MSCs labeled with BrdU was about 40-50% in vitro (Figure 3A). The BrdU-labeled MSCs were injected into rat brain via carotid artery, cervical vein or lateral ventricle. The BrdU immunostaining allowed us to determine whether MSCs could migrate into rat brain, and if so, where the cells became established. At all time points post-injection, BrdU-labeled MSCs were able to migrate into the brain when injected through the carotid artery and lateral ventricle, but not when injected through the cervical vein. The majority of labeled MSCs were found in ipsilateral cortex, bleeding foci and hippocampus of the injection side (Figure 3B-D). In the side contralateral to the injection, few labeled MSCs were found in the corresponding brain regions, suggesting that the injected MSCs were capable of migrating into the relevant areas throughout the entire rat brain.

Double labeling for BrdU and NeuN, CFAP or CNP revealed that MSCs were able to differentiate into neurons, astrocytes and oligodendrocytes in the brain. In the hippocampus, MSCs primarily differentiated into neurons, whereas around bleeding foci they differentiated into neurons and astrocytes. In the ispilateral cortex, MSCs differentiated into neurons (Figure 4A-C), astrocytes (Figure 4-F) and oligodendrocytes.

Figure 2: MSCs differentiate in vitro. (A) The morphology of the in vitro cultured MSCs ( 100). (B) When MSCs were induced, they differentiated into neuron-like cells that protruded axons and dendrites ( 200). (C) At the end of induction, the neuron-like cells were positive for NeuN (200). (D) The astrocyte-like cells were positive for GFAP ( 400).

Behavioral improvements following delivery of \MSCs into rat brain

The rats receiving the intracerebral injection of collagenase VII showed significant limb-placing deficits as well as overall asymmetry. There was no apparent motor dysfunction in the NT group. The limb motor function of the rats in the CA group and the LV group were significantly better than those in the IO group and the CV group. The behavioral scores of all groups were culminated on day 3 after ICH and began to descend on day 5. The behavioral scores of the CA group and LV group were significantly lower (better) than those of the IO and CV groups, and there was no difference in the behavior scores between the IO group and the CV group (p>0.05). The behavioral scores of the LV group and the CA group were significantly lower than those of the IO group on day 1 to day 7 (p< 0.001) respectively. And there was no significant statistical difference between the LV group and the CA group after the third day (p>0.05) (Table 1).

Table 1: The rat behavioral scores of different groups

Figure 3: MSCs distributed in the brain. (A) The in vitro cultured MSCs were stained with a BrdU antibody ( 100). The BrdU- labeled MSCs distributed around bleeding focus (B), in ispilateral cortex (C) and around the hippocampus(D). (B1), (C1) and (D1) are negative controls without MSCs transplantation for (B), (C) and (D), respectively ( 100)

DISCUSSION

MSCs can not only differentiate into mosedermal cells, such as bone, cartilage, muscle, ligament, tendon, adipose and marrow stroma, but also transdifferentiate into ectodermal and endodermal cells, particularly neural cells14. In this regard, MSCs are similar to ESCs that are able to differentiate into ectoderm, mesoderm and endoderm during embryogenesis28,29. Because of these qualities, MSCs can be developed into effective treatments for a wide variety of neurological disease, including cerebrovascular diseases, neurodegenerative diseases and genetic diseases. In addition, MSCs are easily available from patients' own bone marrow and can rapidly multiply over a million-fold in culture30,31, which would overcome the immune rejection and ethical issues caused by xenon-delivery of ESCs. In the past several years, MSCs have been extensively studied as a cell and gene therapy method and great progress has been made in both laboratory and clinic32-36.

Figure 4: The labeled MSCs differentiated into neurons and astrocytes in the rat brain, lmmunofluorescence demonstrated that the BrdU-positive MSCs expressed NeuN: (A) BrdU was stained on MSCs; (B) NeuN was stained on MSCs; (C) brain sections were double- stained for MSCs and NeuN. At the same time immuneofluorescence revealed that BrdU-positive MSCs expressed GFAP: (D) BrdU was stained on MSCs; (E) GFAP was stained on MSCs; (F) brain sections were double-stained for MSCs and GFAP

Stroke is the third leading cause of death and serious, long- term disability37. ICH accounts for 10-20% of all strokes all over the world38,39. The incidence of ICH is between 13.5 and 32 per 100,000 (Refs. 40 and 41). Spontaneous ICH is a relatively common fatal disease. Even if the patient survives the initial ictus, the resulting hematoma within the brain parenchyma can still trigger a series of events leading to secondary insults and severe neurological deficits1. Previous studies indicate that MSCs can greatly proliferate in vitro and subsequently differentiate into neural cells around the foci of the cerebral ischemia, including the hippocampus and cerebellum, when delivered into the ischemic rat brain through the focus injection24, carotid artery, lateral ventricle17or cervical vein injection42. When MSCs were injected into the lateral ventricle of neonatal mice, they differentiated into neural cells and migrated throughout the forebrain and cerebellum in 12 days post-injection17. Somatosensory asymmetries and limb placement impairments resulting from unilateral brain ischemia were significantly improved in 2 to 6 weeks after MSCs grafting21. Gene-modified MSCs are useful as a therapeutic tool for brain tissue damage (e.g. brain infarction) and MSCs transplantation protects the brain tissue from acute ischemie damage in the midcerebral artery occlusion animal model . However, to our knowledge, there is no report in literature regarding whether MSCs are able to differentiate into neural cells and improve the neurological function of the ischemie brain. The present study investigated the potential of MSCs as an effective treatment for ICH.

The stem cells derived from bone marrow include HSCs and MSCs. MSCs express CD29, CD44, CD54, CD58, CD90 and CD106, but are negative for CD45, CD34, CD50, CD14, CD68 and CDl 1 75'42'44. CD45 is a specific cell surface marker of HSCs, whereas, CD90 and CD106 labeling is present in MSCs but absent in HSCs. Our flow cytometry data indicated that over 95% of cells cultured in vitro were negative for CD45, and positive for CD90 and CD106, demonstrating that those cells were likely to be MSCs rather than HSCs.

In addition, our data indicated that the MSCs cultured in vitro were negative for nestin, NeuN, GFAP and CNP, indicating that there were no NSCs and neural cells in the cultured cells prior to differentiation induction. After the cells were induced with a-MEM plus BME for 24 hours, nestin was significantly expressed; furthermore, its expression gradually decreased when the induction medium was changed into a-MEM plus DMSO for 5 hours and then into a- MEM plus BHA for additional 5 hours. By the end of the last additional 5 hours, all induced cells were negative for nestin; however, 75, 20 and 2% of the induced cells expressed NeuN, GFAP and CNP, respectively. These data indicated that MSCs initially differentiated into NSCs and then differentiated into neural cells in vitro. Recently there were demonstrations that when MSCs were co- cultured with cerebellar granule neurons, MSCs could express neuronal markers45, and when MSCs were cultured with EGF and bFGF, RNA expression of neuronal specific markers nestin, microtubule- associated protein 2 and tyrosine hydroxylase were observed22. Although some studies showed that molecular pathways involved in neural in vitro differentiation of MSCs, such as cAMP, the classic protein kinase A pathway, etc.46, the mechanism was not clear.

It was reported that MSCs treated with BrdU developed the ability to differentiate into neural and retinal cells when provided with the appropriate lineage specific differentiation signals in vitro and in adult animal47. So we treated and labeled MSCs with BrdU in this research. The results further indicated that delivery of cultured MSCs through the carotid artery or lateral ventricle, not the cervical vein, resulted in BrdU-labeled MSCs in the rat brain. The BrdU-labeled MSCs delivered through the carotid artery were primarily distributed through the cortex, hippocampus and bleeding foci, whereas MSCs delivered through the lateral ventricle were found predominantly in areas surrounding the lateral ventricle and bleeding foci, which was in agreement with previous reports48. MSCs were shown to be able to migrate into injured sites of the brain when transplanted systemically or locally, suggesting that MSCs possess migratory capacity, however, the mechanisms underlying the migration of these cells remain unclear49. In addition, BrdU- positive cells integrated in the hippocampus exclusively expressed NeuN, whereas in the areas surrounding bleeding foci, the BrdU- positive cells expressed both NeuN and GFAP. In the cortex of ipsilateral brain, the BrdU-positive cells expressed NeuN, GFAP or CNP. The behavioral scores for the CA group and the LV group were similar at all time points after MSCs delivery. The scores for the CV group, however, were similar to those of the IO group, indicating that there was no effect of cervical vein delivery of the MSCs on ICH-induced damage, most probably owing to the inability of delivered MSCs to enter the brain, as no BrdU-positive cells were observed in cervical veininjected brains. This observation does not agree with previous reports in which MSCs were successfully delivered to the ischemie rat brain though the cervical vein, and improved neurological function in 35 days after the delivery48,50,51. One possible explanation for the different findings could be that the rat infarction model generated by middle cerebral artery occlusion caused extensive damage in the blood- brain barrier of the rat brain, allowing the MSCs to effectively migrate into the rat brain. Also considering that injection into the lateral ventricle compromises the blood-brain barrier, the carotid artery injection may be the best choice for the MSCs delivery. The mechanisms underlying how the delivered MSCs differentiate into various neural cells in distinct brain regions, and furthermore, how they are able to improve the neurological function remain unknown, but it is likely that the delivered MSCs, which differentiated into neural cells expressing NeuN, GFAP and CNP, were responsible for the improvement of the motor function seen in rats with ICH. It was previously reported that the neural cells differentiated from the transplanted MSCs secreted cytokines, such as collagen I and fibronectin, 6 weeks after grafting52,53. These cytokines were involved in modulating peripheral nerve regeneration54 and basic cellular functions, such as cell adhesion, migration, cell growth, differentiation, programmed cell death55,56 and produced specific neurotransmitters57,58. It is possible that these cytokines or other secreted molecules contribute to the improved motor skills.

Taken together, our data demonstrate that MSCs can differentiate into neural cells in vitro. When MSCs are delivered to the ischemie rat brain through the carotid artery or lateral ventricle, they differentiate into neurons, astrocytes and oligodendrocytes, and significantly improve the motor function. There are neither deaths nor significanthealth complications observed in the rats given MSCs, demonstrating that this approach is safe for research in rats. MSCs are readily available from patients' own bone marrow and are therefore safe for transplantation. Finally, MSCs transplantation can be developed into an effective therapeutic approach for ICH in the future.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Hongcan Zhu, Dr Kang Xu, Dr Hongliang Wu, Dr Xinqiao Fu and Dr Baiquan Zhang for their help and advice. We also appreciate Dr Yongdong Feng and Dr Jianhong Wu for their assistance in processing the flow cytometry data. This research was supported by a grant from the Chinese Scientific Fund for Postdoctoral Training (No. 2002032215) and the National Natural Science Foundation of China (No. 30400140).

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Huabiao Zhang*, Zhiyong Huang[dagger], Yuming Xu[double dagger] and Suming Zhang

* Department of Neurology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China, 21002 9

[dagger] Department of Surgery and Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

[double dagger] Department of Neurology, the First Affiliated Hospital, Zhengznou University, Zhengzhou 450052, China

Correspondence and reprint requests to: Huabiao Zhang, Department of Neurology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210009, China, [zhb1969@njmu.edu.cn] Accepted for publication November 2005.

Copyright Maney Publishing Jan 2006

Source: Neurological Research

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