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Journal of Clinical Neuroscience 15 (2008) 907–912

Laboratory Study

Intracerebral transplantation of human adipose tissue stromalcells after middle cerebral artery occlusion in rats

Tae-Hoon Lee a,*, Jung-Gyu Yoon b

a Department of Health Administration, Namseoul University, 21 Maeju-ri, Seonghwan-eup, Cheonan-city, Choongnam 330-707, Koreab Department of Physical Theraphy, Namseoul University, 21 Maeju-ri, Seonghwan-eup, Cheonan-city, Choongnam 330-707, Korea

Received 22 January 2007; accepted 20 March 2007

Abstract

The transplantation of cells capable of neuronal differentiation has great potential for the treatment of neurological conditions. Iexamined whether human adipose tissue stromal cells (hATSCs) can be induced to undergo neuronal differentiation. I isolated hATSCsfrom human liposuction tissue and induced neuronal differentiation using azacytidine. After neuronal induction, the hATSCs adopted amore neuronal morphology. These hATSCs were injected into the lateral ventricle of the rat brain, after which they migrated to variousparts of the brain. After ischemic brain injury induced by middle cerebral artery occlusion (MCAO), a large number of cells migrated tothe injured cortex. Intracerebral grafting of hATSCs significantly enhanced the recovery of functional motor deficits in MCAO rats.These data indicate that transplanted hATSCs survive, migrate and differentiate in the ischemic microenvironment and improve neuro-logical recovery after stroke in rats.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Human adipose tissue stromal cell; Brain ischemia; Middle cerebral artery occlusion; Transplantation

1. Introduction

Neurotransplantation has been to investigate develop-ment, plasticity and regeneration of the central nervoussystem.1 Transplanted adipose tissue stromal cells mayaid in the restoration of lost function by contributing tro-phic support or integrating into functional synaptic net-works with host tissues. Experiments involving animalmodels and humans have produced promising results inthis regard.2–4

Stem cells have the capacity for self-renewal and differ-entiation into diverse cell types. During embryogenesis,totipotent stem cells give rise to ectoderm, mesoderm,and endoderm.5 Transplantation of embryonic stem cellsinto the brain ameliorates neurological deficits in animalmodels of Parkinson’s disease6 and spinal cord injury.7

0967-5868/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jocn.2007.03.016

* Corresponding author. Tel.: +82 41 580 2338; fax: +82 41 580 2330.E-mail address: thlee@nsu.ac.kr (T.-H. Lee).

Transplanted embryonic stem cells have been shown to sur-vive and differentiate into oligodendrocytes, astrocytes anddopaminergic neurons.6,7 However, immune rejectionneeds to be overcome to allow the clinical use of embryonicstem cell transplantation. Recent research has suggestedthat mesenchymal stem cells (MSCs) from bone marroware capable of differentiating into various brain cells. Bonemarrow stromal cells (BMSCs) have also been reported todifferentiate into neural cells in vitro.8–10 When BMSCs aretransplanted into the lateral ventricles of neonatal mice,they migrate to various brain regions and differentiate intocells with astrocytic and neuronal phenotypes.11 Similarly,when human MSCs are directly infused into rat striatum,engraftment and differentiation into astrocytes occurs.12

Transplanted BMSCs have also been shown to migrateextensively throughout adult animals. Following intrave-nous bone marrow transplantation in rodents, BMSCshave been detected in many non-hematopoietic tissues,13,14

including the brain.15–17 Interestingly, bone marrow trans-plantation has been shown to effectively prevent the

908 T.-H. Lee, J.-G. Yoon / Journal of Clinical Neuroscience 15 (2008) 907–912

progression of neurological signs and symptoms in someclinical trials, if performed at a sufficiently early stage ofParkinson’s disease. Recently, rodent bone marrow cellsgrafted into the ischemic rat brain were found to yield afunctional improvement.18 BMSCs have also been usedas vehicles for gene delivery to various tissues, includingthe brain.19–22 These findings suggest that BMSCs are a po-tential source of brain progenitor cells. However, they canonly be obtained by bone marrow biopsy, a potentiallypainful procedure. Thus, it would be advantageous to iden-tify similar multipotent stromal cells in tissue sites outsidethe bone marrow microenvironment.

Adipose tissue, like bone marrow, is derived from theembryonic mesoderm and contains a heterogenous stromalcell population.23,24 These similarities between adipose andbone marrow tissue, together with the fact that MSCs havebeen identified in several tissues, make it seem likely that astem cell population could be isolated from human adiposetissue. In fact, MSCs isolated from adipose tissue havebeen shown to differentiate into multiple mesodermal tis-sues, including bone, fat and muscle.25–27 Differentiationinto neuron-like cells expressing neuronal markers has beenreported.28

Therefore, adipose tissue has been identified as an alter-native source of pluripotent stromal cells.25,26,29 These cellshave been termed adipose tissue stromal cells (ATSCs), asthey are self-renewing and can be induced to differentiateinto various mesenchymal tissues, including chondrocytes,adipocytes, osteoblasts and myocytes.25–27 ATSCs havebeen shown to display both epithelial cell and hepatocyticmorphological features, neither of which is a mesenchymallineage.28 However, the fate of human ATSCs (hATSCs)and the functional outcome after in vivo transplantationhas not been determined. In the present study, I investi-gated whether hATSCs could integrate into various partsof the brain, and ameliorate neurological deficits in ratswith ischemic brain injury.

2. Materials and methods

2.1. Adipose tissue preparation

The subcutaneous adipose tissue was acquired from pa-tients undergoing elective surgery. Patients consented tothe procedure, which was approved by the institution re-view board. The adipose tissue was transported to the lab-oratory in saline solution within 2 h of removal. The tissuewas washed at least three times with two volumes ofHank’s balanced salt solution (HBSS) buffer to removeany blood. The tissue was then digested with one volumeof type I collagenase (1 g/L in HBSS buffer with 1% bovineserum albumen) for 60 min at 37 �C with intermittent shak-ing. The floating adipocytes were separated from the stro-mal-vascular fraction by centrifugation (300 g) for 5 min.The preadipocytes in the stromal-vascular fraction wereplated onto tissue culture dishes at 3500 cells/cm2 in a-modified Eagle’s medium (MEM) supplemented with 10%

fetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin (preadipocyte medium). The primarycells were cultured for 4–5 days until they reached conflu-ence; these cells were defined as passage 0. The cells werethen harvested by digestion with 0.5 mmol/L edetic acid/0.05% trypsin, centrifuged at 1200 rpm for 5 min, resus-pended in preadipocyte medium and plated at a densityof approximately 10 000 cells/cm2. The cells were passagedand then used for in vitro differentiation and in vivo trans-plantation experiments. The cells were cryopreserved in li-quid nitrogen in media supplemented with 10% dimethylsulfoxide (DMSO) prior to subsequent experimentation.

For transplantation, after 2 days of culture, the cell cul-ture medium was changed to a half volume of the LacZinfection medium (a-MEM supplemented with 2% FBS,penicillin and streptomycin) containing the appropriate ti-ter of adenoviruses (Adv, CMV, LacZ+). The cells wereincubated for 8 hr and were gently rocked twice, and thenan equal volume of medium containing 20% FBS withoutthe adenoviruses was added. The cells were cultured for12 h before in vivo injection.

2.2. In vitro differentiation of ATSC to neural cell

Subconfluent cultures of ATSCs were incubated for 24 hwith a-MEM containing 1 mmol b-mercaptoethanol, thenthe medium was removed, and cells were washed withHBSS (pH 7.4) and placed in neural induction media con-sisting of a-MEM, 2% DMSO and 200 lmol/L butylatedhydroxyanisole (Sigma, St Louis, MO, USA) for 60 min.Cell were washed with HBSS and transferred to neurobasalmedium supplemented with B27, 50 ng nerve growth factor(NGF), 10 ng brain-derived growth factor (BDNF) and 5ng basic fibroblast growth factor (bFGF) (Sigma). Theywere then incubated for 10 days.

The other condition used for induction of differentiationwas 96 h culture in 10 lg/L 5-azacytidine medium (inDMEM, with 10% FBS) supplemented with NGF (50 ng/L)/BDNF (10 ng/L)/bFGF (5 ng/L). To induce further dif-ferentiation, the medium was replaced with neurobasalmedium containing B27 and the medium was changedevery 4 days.

2.3. Middle cerebral artery occlusion model

All experimental protocols of this study were approvedby the Animal Care Committee of Namseoul Universityin accordance with the policies established in the Guideto the Care and Use of Experimental Animals of the Cana-dian Council on Animal Care. Adult male Wistar rats(n = 37) weighing 270–300 g were used in our experiments.Briefly, anesthesia was induced with 5% enflurane andmaintained with 2% enflurane in O2 delivered via a face-mask. Rectal temperature was maintained at 37 �Cthroughout the surgical procedure using a feedback regu-lated heating system. I induced transient middle cerebralartery occlusion (MCAO) using the intraluminal vascular

T.-H. Lee, J.-G. Yoon / Journal of Clinical Neuroscience 15 (2008) 907–912 909

occlusion method.30 The right common carotid artery,external carotid artery (ECA) and internal carotid artery(ICA) were exposed. A length of 4–0 monofilament nylonsuture (18.5–19.5 mm), with the length determined accord-ing to animal weight, with its tip rounded by heating neara flame, was advanced from the ECA into the lumen ofthe ICA until it blocked the origin of the MCA. Twohours after MCAO, reperfusion was permitted by with-drawal of the suture until the tip cleared the lumen of theECA.

2.4. Neurotransplantation

The experimental groups established were as follows:group 1 (control), MCAO without cell transplantation(n = 10); group 2, MCAO and injection of phosphate buf-fered saline (PBS; 10 lL, n = 5); group 3, MCAO andtransplantation of hATSC (1 � 105/10 lL, n = 22). Ratsthat had undergone MCAO (270–300 g) were anesthetizedin a sealed chamber using 5% enflurane in oxygen. Anes-thesia was maintained using 2% enflurane delivered via aface mask. The animals were transferred to a stereotaxicapparatus in a clean field. A 2–5 mm incision was madein the scalp 1.5 mm lateral to the bregma. A burr holewas made in the bone 3 mm lateral to bregma with a dentaldrill, and about 10 lL of the adenovirus-infected cell sus-pension (1 � 105 cells) was slowly injected over 30 min intothe lateral ventricle at a depth 3.5 mm from the surface ofthe brain using a 10 lL Hamilton microsyringe (Hamilton,Reno, NV, USA). After injection, the syringe was left inplace for an additional 5 min before retraction. Trans-planted hATSC were labeled with Cell Tracker CM-Dil(Molecular Probes, Eugene, OR, USA). The wound wasclosed with interrupted surgical sutures.

2.5. Histological and immunological assessments

Animals were killed 14 days after MCAO by deep anes-thesia with xylozine (13 mg/kg) and ketamine (44 mg/kg).Their brains were fixed by transcardial perfusion with sal-ine, followed by perfusion and immersion in 4% parafor-maldehyde, and brain tissue blocks were embedded inparaffin. The brain was sectioned into seven equally spaced2 mm coronal sections. A series of adjacent 6-lm thick sec-tions were cut from each 2-mm section in the coronal planefor immunohistochemistry. The sections were air-dried andincubated in PBS with 2% bovine serum albumin (Sigma)for 1 h at room temperature to block non-specific binding.Then the sections were washed three times in PBS and incu-bated in fluorescein isothiocyanate-conjugated F(ab0)2

fragment donkey anti-rabbito IgG (1:100; Jackson Immu-noResearch Laboratories, West Grove, PA, USA) in 2%BSA for 3 h at room temperature. The sections weremounted on glass slides with mounting medium (GVAmount; Zymed, CA, USA) and hATSCs in the sectionswere traced using confocal microscopy.

2.6. Behavioral testing

In all animals behavioral tests were performed beforeMCAO and at 7 and 14 days after MACO. All rats wereevaluated using a modified neurological severity score(mNSS)18,31,32 and rotarod test.33 The mNSS is a compos-ite of motor (muscle status, abnormal movement), sensory(visual, tactile, proprioceptive), reflex and balance tests.Neurological function was graded on a scale of 0 to 18(normal score, 0; maximal deficit score, 18). In the assess-ment of injury, 1 point was awarded for the inability to per-form a test or for the lack of a tested reflex. In the rotarodmotor test, the rats were placed on a rotarod cylinder, andthe time the animals remained on the rotarod was mea-sured. A trial ended if the animal fell off the rungs orgripped the device and spun around for two consecutiverevolutions without attempting to walk on the rungs.The animals were trained 3 days before MCAO. Themean duration (in seconds) on the device was recordedfor three measurements sessions 1 day before surgery. Dataare presented as mean duration on the rotarod as a per-centage relative to the internal baseline control (beforesurgery).

2.7. Statistical analysis

The neurological deficit scores (the rotarod test and themNSS test) were subjected to paired t-tests to evaluate dif-ferences between the control (MCAO) and treatment(hATSC) groups. Data are presented as mean ± standarderror of the mean (SEM). A probability value less than0.05 was considered significant.

3. Results

3.1. ATSC characterization

Within 2–3 passages after the initial plating of the pri-mary culture, the hATSCs appeared as a monolayer oflarge, flat cells. As the cells approached confluence, theyshowed a more spindle-shaped, fibroblastic morphology(Fig. 1). The hATSCs became relatively homogeneous inappearance as the cells were passaged. However, two dis-tinct populations were seen, large flattened cells and rela-tively elongated or spindle-shaped cells (Fig. 1).

3.2. Detection of ischemic brain region

To demonstrate the extent of infarction, 2-mm coronalbrain sections were immersed in 2% 2,3,5-triphenyltetrazo-lium hydrochloride (TTC) in saline for 20 min at 37�C andfixed for 30 min in 4% paraformaldehyde. Ischemic braininjury was prominent in areas such as the cerebral cortexand striatum, which are supplied by the MCA and under-went infarction (Fig. 2). White matter tracts including thecorpus callusum also appeared as white infarct areas.

Fig. 2. Sections of brain stained with 2,3,5-triphenyltetrazolium hydrochloride (TTC) to reveal brain ischemia. Rats underwent middle cerbral arteryocclusion for 2 h, then reperfusion; sections were obtained 14 days after ischemia. TTC-stained brain slices used to examine the cerebral cortex, striatumand corpus callosum are shown. Red is viable and white is nonviable tissue. White matter tracts including the corpus callosum also appear white with thismethod. This figure is available in colour at www.sciencedirect.com.

Fig. 1. Phase-contrast photomicrographs of cultured human adipose tissue stromal cells (hATSCs). hATSCs were grown under control conditions in a-modified Eagle’s medium/10% fetal bovine serum. Stromal cells grew as monolayer of either large and flat or spindle-shaped cells (�100).

Motor Test

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Fig. 4. Behavioral functional testscores before and after middle cerebralartery occlusion (MCAO). Groups were as follows: group 1, MCAO alone(n = 10); group 2, MCAO with phosphate-buffered saline (PBS) injection(sham control; n = 5); group 3, intracerebral infusion of adipose tissuestromal cells (ATSC) (1 � 105; n = 22) at 24 h after MCAO. Rats werekilled at 14 days after MCAO.

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3.3. Migration of cells

To examine the migration pathways of hATSCs trans-planted in the brain, hATSCs were stained with Cell Track-er CM-Dil. Labeled hATSCs were injected into the lateralventricles of rat brains and CM-Dil-positive cells werereadily detected in the resulting brain sections. ImplantedhATSCs integrated and migrated to multiple areas of thebrain, including the cortex and the striatum (Fig. 3). Theheaviest concentration of cells was found in the striatumand along the corpus callosum. hATSCs in the infarct re-gion were mostly located at the border between the intactbrain tissue and the area of the infarction and in other sec-tions within the infarct cavity.

3.4. Functional recovery after MCAO

Injection of hATSCs had a significant effect on func-tional recovery as assessed using the rotarod and mNSStests. Similar results were obtained for the group that

Fig. 3. Immunohistochemically stained sections of brain from rats 14 days after middle cerebral artery occlusion. Rats were injected with human adiposetissue stromal cells (hATSCs). Implanted hATSCs integrated into and migrated to multiple areas of the brain including the cortex and striatum. A, �400;B, �200; C, �40.

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underwent MCAO alone, and the group that underwentMCAO and received an injection of PBS. Treatment at 1day after MCAO with hATSCs significantly improvedfunctional recovery, as evidenced by improved rotarod testand mNSS scores during 15 days (p < 0.05) (Fig. 4). Slightdifferences were observed between the treatment and con-trol groups before hATSC treatment. However, the differ-ences were not significant. The treatment effects over timewere significant for the rotarod and mNSS tests. Both therotarod and mNSS tests demonstrated that motor and sen-sory behaviors were impaired by the MCAO ischemic in-sult. Significant recovery of motor and neurologicalsensory behavior was found in animals that received hAT-SCs compared with control ischemic animals or animalsthat received PBS instead of hATSCs.

4. Discussion

The main findings of the present study are that thetransplanted hATSCs survived and migrated in the rodentbrain, with no evidence of immune destruction, and thatthe rats showed improved neurological function aftertransplantation following ischemia. Similar effects of bonemarrow stem cells on the functional deficits induced byischemic brain injury have been reported elsewhere.18,34

The surface phenotype of ATSCs is similar to that of bonemarrow-derived stromal cells.35–37 ATSCs and marrowstromal cells share many of the same adhesion and receptormolecules,36–39 and hATSCs and hBMSCs have similarexpression profiles at the protein and mRNA levels. MorehATSCs were found in the cortex in MCAO rats than inrats that did not undergo MCAO, which suggests thatischemia-induced chemotactic factors facilitate hATSCmigration. These data suggest that hATSCs may have sim-ilar potential to BMSCs with respect to tissue engineeringand regenerative medicine.28

The mechanisms by which transplanted hATSCs inducefunctional benefit after stroke are not clear. In this study,the morphology of transplanted cells was still primitiveand the infarct size in hATSC-treated rats was not signifi-cantly different from that in control ischemic rats at thetime that functional recovery was observed and at trans-plantation. Therefore, it is highly unlike that transplantedcells integrate into the cerebral tissue and make appropri-ate connections within days after transplantation. Neuro-trophic factors could participate in hATSC-mediatedfunctional improvement. Neurotrophic factors are survivaland/or differentiation factors for neuronal progenitorcells,40 and they may play an important role in the prolifer-ation or differentiation of neural tissue. A recent reportshowed that hMSCs from bone marrow express severalneurotrophic factors.41,42

Growth factors are the molecular signals by which thebody regulates cell survival, proliferation and differentia-tion. Exogenously administered neurotrophic growth fac-tors may limit the extent of acute ischemic neural injuryand enhance functional recovery after stroke.43 The intra-

cerebral administration of hATSCs and the migration ofthese cells into the injured tissue may provide a trophic fac-tor production source that bypasses the blood-brain bar-rier. hATSC treatment at 1 day after MCAO significantlyimproved functional recovery (according to motor rotarodtest and mNSS scores) after stroke. A significant increase inhATSC migration activity was detected in ischemic cere-bral tissue harvested at 1 day after stroke. Early treatmentwith hATSCs after stroke may promote hATSC migrationinto ischemic brain and facilitate functional recovery afterMCAO.

In conclusion, I have shown that intracerebrally im-planted hATSCs survive, migrate, and improve functionalrecovery after stoke. Since hATSCs are widely availableand have been used clinically, they may be an excellentsource of cells for treatment of early stroke. Furthermore,hATSCs appear to seed the brain globally but may prefer-entially migrate to the site of brain pathology. Potentially,adipose tissue may provide a powerful autoplastic therapyfor human neurological degenerative disorders and notonly stroke. The mechanisms underlying the functionalrecovery after transplantation of hATSCs remain to be fur-ther investigated.

Acknowledgement

This paper was supported by Namseoul Universityfunding.

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