Araya 2009. Localized Donor Cells in Brain of a Hunter Disease Patient After Cord Blood Stem Cell Transplantation

8/11/2019 Araya 2009. Localized Donor Cells in Brain of a Hunter Disease Patient After Cord Blood Stem Cell Transplantation

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Localized donor cells in brain of a Hunter disease patient after cord bloodstem cell transplantation

Ken Araya a , Norio Sakai a , Ikuko Mohri a,b , Kuriko Kagitani-Shimono a , Takeshi Okinaga a , Yoshiko Hashii a ,Hideaki Ohta a , Itsuko Nakamichi c, Katsuyuki Aozasa c, Masako Taniike a,b, * , Keiichi Ozono aa Department of Pediatrics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japanb Department of Molecular Research Center for Childrens Mental Development, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japanc Department of Histopathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

a r t i c l e i n f o

Article history:Received 21 May 2009Accepted 21 May 2009Available online 24 May 2009

Keywords:NeuropathologyImmunohistochemistryHunter diseaseMucopolysaccharidosis IICord blood stem cell transplantationIduronate-2-sulfatase

a b s t r a c t

The efcacy of hematopoietic stem cell transplantation (HSCT) for Hunter disease (deciency of iduronate-2-sulfatase, IDS) remains unclear. We treated a 6-year-old male suffering from a severe typeof Hunter disease with cord blood stem cell transplantation (CBSCT); however, he died at 10 monthspost-therapy due to a laryngeal post-transplantation lymphoproliferative disorder. During the follow-up period after CBSCT, his hyperactivity, estimated mental age, and brain MR ndings had not improved.We assessed the efcacy of CBSCT by biochemical and pathological analyses of the autopsied tissues.There were many distended cells with accumulated substrate in the brain, but not in the liver. IDSenzyme activity in the cerebrum remained very low, although that in the liver reached about 40% of the normal control level. However, a variable number of tandem repeats analyses demonstrated a weakdonor-derived band not only in the liver but also in the cerebrum. Furthermore, IDS-immunoreactivity inthe liver was recognized broadly not only in Kupffer cells but also in hepatocytes. On the other hand, IDS-immunoreactivity was recognized exclusively in CD68-positive microglia/monocytes in the patientsbrain; whereas that in the normal brain was also detected in neurons and oligodendrocytes. These

donor-derived IDS-positive cells were predominantly localized in perivascular spaces and some of themwere evidently present in the brain parenchyma. The efcacy of CBSCT was judged to be insufcient forthe brain at 10 months post-therapy. However, the pathological detection of donor-derived cells in thebrain parenchyma suggests the potential of HSCT for treatment of neurological symptoms in Hunter dis-ease. This is the rst neuropathological report documenting the distribution of donor-derived cells in thebrain after CBSCT into a Hunter disease patient.

2009 Elsevier Inc. All rights reserved.

1. Introduction

Hunter disease [1] , mucopolysaccharidosis (MPS) II (MIM+309900), is an X-linked recessive lysosomal storage disorder(LSD) that arises due to a deciency of iduronate-2-sulfatase(IDS: EC 3.1.6.13), and leads to the accumulation of glycosamino-glycan (GAG) in various organs including the central nervous sys-tem (CNS). In the CNS, distended cells with large clear vacuolesare observed, and dilatation of the perivascular (VirchowRobin)space is conspicuous within the cerebral white matter. The neuro-nal storage materials are positive by periodic acid Schiff (PAS)staining [2] . A considerable degree of neuronal loss and gliosis iscommon [3] . The accumulated substrate with membranous cyto-plasmic bodies or Zebra bodies is observed in electron micrographs

[4,5] . In the liver, hepatocytes and Kupffer cells are also swollenwith a vacuolated cytoplasm, and accumulated substrate in themis positive by colloidal iron staining [6,7] .

The patients present with multiple progressive symptoms suchas coarse facial features, skeletal deformities, hepatosplenomegaly,respiratory tract infection, cardiovascular disorders, and variousdegrees of CNS involvement. There is a wide spectrum from mildto severe types, which are classied on clinical grounds becauseIDS activity appears equally decient in both types. Although themild type is characterized by preservation of intelligence, the se-vere type, with onset usually occurring between 2 and 4 years of age, is characterized by mental retardation and death before adult-hood mainly due to obstructive airway disease and cardiac failure[8] .

There have been no established treatments for MPS II; however,2 therapeutic possibilities have been pursued, that is, hematopoi-etic stem cell transplantation (HSCT) and enzyme replacementtherapy (ERT). Although the enzyme administered intravenouslyby ERT is not expected to cross the bloodbrain barrier (BBB) [9] ,

1096-7192/$ - see front matter 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.ymgme.2009.05.006

* Corresponding author. Address: Department of Pediatrics, Osaka UniversityGraduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

E-mail address: [email protected] (M. Taniike).

Molecular Genetics and Metabolism 98 (2009) 255263

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HSCT is consideredto provide a constant sourceof enzyme replace-ment through the engrafted donor cells, which are not impeded bythe BBB and can thus deliver the enzyme to the CNS [10] . Severalin vitro studies using cultured cells [11] and in vivo ones usingmodel animals [1214] have conrmed the biochemical, patholog-ical or functional improvement; therefore, many LSD patients havebeen treated with HSCT over the last 25 years [15] . However, only afew studies have reported about the pathological evaluation of thehuman CNS after HSCT [16,17] .

Here we document a clinicopathological evaluation of the ef-cacy of umbilical cord blood stem cell transplantation (CBSCT) onthe neuropathology made by detecting the accumulated substrateand donor-derived cells in the autopsied tissue of a 6-year-oldmale with a severe type of MPS II, involving moderate mentalretardation and brain atrophy, 10 months after CBSCT. Since sex-matched transplantation had been performed, the strategy of thedetection of donor cells by the use of uorescence in situ hybridiza-tion for sex chromosomes [18] was not available. Therefore, thedetection of donor cells was performed by immunohistochemicaldemonstration of IDS-positive cells, assays of IDS activity, and var-iable number of tandem repeats (VNTR) analysis.

2. Patient, materials, and methods

2.1. Patient and case history

The patient was born by normal delivery at 40 weeks gestation.His developmental milestones were normal by the age of 18 months; however, a delay in his language and motor skillswas rst recognized at the age of 2 years. He was suspected of suf-fering from MPS II because of the developmental delay, the charac-teristic facial features, and the characteristic X-ray ndings of skeletal deformities. At the age of 3 years 8 months, the diagnosisof MPS II was conrmed based on the low activity of IDS in his lym-phocytes (0.5 nmol/4 h/mg vs. adult control range of 58.4114 nmol/4 h/mg, measured by SRL Inc., Tokyo, Japan). Geneticanalysis revealed that he harbored a nonsense mutation, W267X,in exon 6 of his IDS gene on Xq28.1.

At the age of 5 years 11 months, CBSCT was performed byusing cells from an unrelated male donor who had completelymatched HLA phenotypes but 3 loci mismatched HLA genotypes(donor/recipient: A0201/0206, DR0406/0410, DR1406/1402), afterobtaining informed consent from his parents. The conditioningregimen for CBSCT consisted of busulfan, cyclophosphamide,and udarabine. He received short-term methotrexate (15 mg/m 2 on day 1, 10 mg/m 2 on days 3, 6, and 11) and tacrolimus of which whole blood concentration was maintained at a trough le-vel of 515 ng/ml. The detailed protocol was described by Toki-masa et al. (as Patient 5) [19] . He had not experienced chronic

graft-versus-host-disease (GVHD) but only grade I acute GVHDrequiring no steroid therapy. His bone marrow was conrmedto be completely of the donor type by VNTR analysis 1 month,6 months, and 9 months after CBSCT ( Supplemental Fig. 1 ). How-ever, at the age of 6 years 7 months, he developed severe upper-airway obstruction necessitating intensive care. The biopsy of hisswollen larynx yielded the diagnosis of EpsteinBarr virus-unas-sociated post-transplantation lymphoproliferative disorder, andhis condition deteriorated despite 6 courses of rituximab admin-istration. He eventually died at the age of 6 years 9 months, thatis, 10 months post-CBSCT.

At the age of 5 years 11 months, just before CBSCT, he washyperactive and had an estimated mental age of 28 months. His li-ver was palpable 6 cm below the right costal margin. By the age of

6 years 6 months, 7 months post-CBSCT, his hyperactivity andmental retardation had not changed signicantly (estimated men-

tal age of 30 months), although his hepatomegaly had improved(palpable 3 cm below the right costal margin).

2.2. Materials and post-mortem brain tissue processing

We performed a general autopsy of the patient after havingobtained written informed consent from his parents. The post-

mortem interval (PMI) was 3 h. At the autopsy, small blocks of liverand brain samples were snap-frozen or xed for the electronmicroscopical investigation. After 10 months of formalin-xation,blocks from cerebrum, hippocampus, and cerebellum were cutout and used for the study. For comparative analysis, formalin-xed brain blocks (13 months of xation) from a 6-year-5-month-old boy who had died in our hospital were used as anon-MPS II control, with informed consent. The formalin-xedbrain blocks were embedded in Tissue-Tek Optimal Cutting Tem-perature compound (Sakura Finetek Inc., Torrance, CA, USA) afterimmersion for 6 days in 0.1 M phosphate buffer (PB) in whichthe concentration of saccharose was increased from 5% to 30%,by 5% per day. Thereafter, they were quickly frozen and sectionedat 5 l m by a cryostat ( JUNG CM 1800, Leica Microsystems GmbH,

Wetzlar, Germany) and mounted onto the aminosilane-coatedmicroscope slides (Matsunami Glass Ind. Ltd., Osaka, Japan).In addition, we obtained unxed-frozen cerebrumsamples from

an 11-year-old untreated MPS II patient and a 12-year-old non-MPS II control, whose PMI were 15 h and 18.5 h, respectively, fromthe NICHD Brain and Tissue Bank for Developmental Disorders atthe University of Maryland, Baltimore, MD, USA. The unxed-fro-zen cerebral samples were sectioned at 5 l m, mounted onto ami-nosilane-coated microscope slides, and xed by immersion in 20%formaldehyde solution for 5 min at room temperature. Further-more, we obtained three unxed-frozen liver samples fromnon-MPS II controls (27 years old) from Dr. Masahiro Nakayama,Osaka Medical Center and Research Institute for Maternal andChild Health, Osaka, Japan.

This study was approved by the Institutional Review Boards of Osaka University Graduate School of Medicine.

2.3. Methods

2.3.1. Colloidal iron staining studyAfter having been rinsed in 12% acetic acid, 5 l m-thick unxed-

frozen liver sections were placed in a solution of 0.25% ferric chlo-ride and 12% acetic acid for 1 h. They were then rinsed again in 12%acetic acid, immersed in a mixture of 2.5% potassium ferrocyanideand 2.5% hydrochloric acid, and counterstained with 0.1% nuclearfast red and 5% aluminum sulfate. They were observed under aMicroscope BX40 (Olympus Co., Tokyo, Japan) equipped with ahigh-sensitivity cooled CCD color camera VB-7010 (Keyence Co.,Osaka, Japan).

2.3.2 PAS staining studiesThe brain sections were oxidized with 0.5% orthoperiodic acid,

and placed in Lillies Cold Schiffs Reagent (0.5% basic fuchsin,100 mM hydrochloric acid, and 0.5% acid sodium sulte). Theywere then rinsed in 0.5% sodium bisulte and 50 mM hydrochloricacid, counterstained with Mayers hematoxylin (Wako Pure Chem-ical Industries Ltd., Osaka, Japan), and observed under the Micro-scope BX40.

2.3.3. Electron microscope (EM) studiesThecerebral and cerebellar samples were xed with 1% parafor-

maldehyde and 3% glutaraldehyde in 0.1 M PB, postxed in 1%

osmium tetroxide, stained with 1% uranyl acetate, and embeddedin Epon 812(TAABLaboratoriesEquipmentLtd., Berkshire,UK). After

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observation of semi-thin sections, ultra-thin sections were stainedwithleadcitrate andobservedunder a Transmission ElectronMicro-scope H-7650 (Hitachi High-Technologies Co., Tokyo, Japan).

2.3.4. Enzyme assaysEach unxed tissue sample was homogenized mechanically in

ice-cold deionized water. After having been frozen and thawed,the homogenate was centrifuged at 1000

g for 10 min. After the

determination of protein concentration by the Lowry method[20] , each supernatant was used for enzyme assays. The enzymeactivity of IDS was measured by the method described by Voznyiet al. [21] , and those activities of b-galactosidase (EC 3.2.1.23)and b-hexosaminidase (EC 3.2.1.52) were measured as describedby Hindman and Cotlier [22] .

2.3.5. VNTR analysisDeoxyribonucleic acid (DNA) samples (0.2 l g), extracted from

each blood or tissue sample, were amplied with 35 cycles of poly-merase chain reaction in a reaction mixture containing 50 l l of 200 l M deoxynucleotide triphosphate mixture, 2.5 U of AmpliTaqGold DNA Polymerase (Applied Biosystems Inc., Foster City, CA,USA), 10 mM Trishydrochloric acid (pH 8.3), 50 mM potassiumchloride, 1.5 mM magnesium chloride, and an 1 l M concentrationof each primer for amplication of a polymorphic DNA sequence(pMCT118) on chromosome 1p (D1S80) [23,24] . Each cycle con-sisted of 1 min at 95 C for denaturation, 1 min at 65 C for anneal-ing, and 2 min at 70 C for extension. After amplication,polymorphic bands were detected by agarose gel electrophoresis.

2.3.6. Immunoblot analysisAfter solubilization in lysis buffer (50 mM Tris, 150 mM NaCl,

1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 2 l g/ml aprotinin,2 l g/ml leupeptin, 2 l g/ml pepstatin A, 0.5 mM phenylmethylsul-fonyl uoride, and 1 mM dithiothreitol), 40 ng and 0.4 ng recombi-nant human IDS ( Elaprase , Shire Human Genetic Therapies Inc.,Cambridge, MA, USA) and 40 ng recombinant human a -glucosidase(Myozyme , Genzyme Co., Cambridge, MA, USA) as a negative con-trol were separated by sodium dodecyl sulfatepolyacrylamidegel electrophoresis and transferred electrophoretically to a nitro-cellulose membrane ( Trans-Blot Transfer Medium, Bio-Rad Labora-tories Inc., Hercules, CA, USA). The membrane was incubatedovernight at 4 C with goat antibody against IDS (0.1 l g/ml,AF2449, R&D Systems Inc., Minneapolis, MN, USA) after treatmentwith 5% skim milk/Tris-buffered saline for blocking nonspecicreactions. The immune complexes were detected with peroxi-dase-labeled horse anti-goat IgG (1:10,000, Vector LaboratoriesInc., Burlingame, CA, USA) and SuperSignal West Dura ExtendedDuration Substrate (Pierce Chemical Co., Rockford, IL, USA).

2.3.7. Immunohistochemical studies

Brain sections were incubated for 30 min in 0.3% hydrogen per-oxide/methanol for quenching endogenous peroxidase activity,and then pretreated with 1% bovine serum albumin (BSA)/phos-phate buffered saline (PBS) for blocking nonspecic reactions. Theywere next incubated overnight at 4 C with the goat antibodyagainst IDS (10 l g/ml) in 1% BSA/PBS, followed by biotinylated rab-bit anti-goat IgG (1:100, Vector Laboratories Inc.) for 2 h at roomtemperature. The reaction products were visualized by using a Vec-stain ABC Peroxidase Kit (Vector Laboratories Inc.) and diam-inobenzidine, and counterstained with Mayers hematoxylin.They were observed under the Microscope BX40.

For the double immunouorescence study, after the endoge-nous peroxidase activity had been quenched, brain sections wereincubated for 20 min in 0.1% Sudan black B/70% ethanol for block-

ing autouorescence. After pretreatment with TNB blocking buffer(PerkinElmer Inc., Waltham, MA, USA), they were incubated

overnight at 4 C with the goat antibody against IDS (2 l g/ml)and primary antibody against Lamp2 (1:500; made in mouse,Developmental Studies Hybridoma Bank at the Univ. of Iowa, Iowa,IA, USA), NSE (1:50; made in rabbit, AB951, Chemicon InternationalInc., Temecula, CA, USA), transferrin (1:50; made in rabbit, A0061,Dako A/S, Glostrup, Denmark), S-100 (1:50; made in rabbit, Z0311,Dako A/S) or CD68 (1:50; made in mouse, M0876, Dako A/S) in 1%BSA/PBS, followed by the peroxidase-labeled horse anti-goat IgG(1:100) and biotinylated horse anti-mouse/rabbit IgG (1:100, Vec-tor Laboratories Inc.) for 2 h at room temperature. They were sub-sequently incubated in TSA Plus uorescence reagent workingsolution (1:50, PerkinElmer Inc.) for 5 min, followed by Texas Redstreptavidin (1:100, Vector Laboratories Inc.) for 2 h at room tem-perature, and mounted by using Vectashield mounting mediumwith DAPI (Vector Laboratories Inc.). Fluorescence was observedunder a laser scanning confocal microscope LSM 510 META (CarlZeiss MicroImaging GmbH, Jena, Germany).

Frozen liver sections were xed by immersion in 4% paraformal-dehyde solution for 10 min at room temperature. After quenchingendogenous peroxidase activity, they were incubated for 30 min in0.1% Triton X-100/the TNB blocking buffer with Avidin D solution(in Avidin/Biotin Blocking Kit, Vector Laboratories Inc.). They werenext incubated overnight at 4 C with the goat antibody against IDS(2 l g/ml) in 1% BSA/PBS with Biotin solution (in the Avidin/BiotinBlocking Kit) and subsequently with the peroxidase-labeled horseanti-goat IgG (1:100) for 30 min at room temperature. As a nega-tive control, the sections reacted with IDS antibody which hadbeen preabsorbed overnight at 4 C with an excess amount(20 l g/ml) of the recombinant human IDS. They were subse-quently incubated in Biotinyl TSA reagent working solution (1:50,PerkinElmer Inc.) for 10 min, followed by Streptavidin horseradishperoxidase (1:100, provided in the TSA kit) for 30 min at roomtem-perature. After having been visualized by use of diaminobenzidine,the reaction products were counterstained and observed as de-scribed above.

A double immunouorescence study using the antibody againstIDS (2 l g/ml) and CD68 (1:50) was also performed as describedabove.

3. Results

3.1. Brain MR imaging before and after CBSCT

The patients brain MR images just before CBSCT showed evi-dence of brain atrophy including widening of the cortical sulciand ventricles ( Fig. 1 A and B). The uid-attenuated inversionrecovery image also revealed enlarged VirchowRobin spaces(Fig. 1 B, arrowheads) and an abnormally high intensity of thewhite matter ( Fig. 1 B, arrows). At 7 months post-CBSCT, calculation

using the software for structural image evaluation of atrophy ( SIE-NA [25] ; a part of Functional MRI of the Brain Software Library, FSL[26] ) estimated the rate of brain volume loss to be 9.9% for7 months, indicating the progression of brain atrophy ( Fig. 1 C).This reduced volume may have been due to not only the procedureof HSCT [27] but also the natural course of MPS II. These ndingsindicate that the patient had the CNS abnormalities of MPS II be-fore CBSCT and that the brain atrophy was progressive after CBSCT.

3.2. Detection of accumulated substrate

At autopsy, the crown-heel length was 116 cm and the bodyweight was 25.4 kg. The brain was 935 g in weight and showedmarked dilatation of the lateral ventricles and widening of the

cortical sulci. Histologically, the neurons in the cerebral cortexseemed to be reduced in number with reactive gliosis and

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showed moderate cytoplasmic ballooning ( Fig. 2 A, arrows). Onthe other hand, the liver weight (1645 g) had increased (standardliver weight calculated with reference to the body surface areawas 650 g [28] ); and the edge of the organ was dull. However,hepatocytes and Kupffer cells did not appear to be swollen withvacuolar changes ( Fig. 2 B), and most of them were not stainablewith colloidal iron ( Fig. 2 C). The CNS showed many PAS-positivecells in the MPS II post-CBSCT ( Fig. 2 D and E, arrows), whereas noPAS-positive neurons were observed in the non-MPS II control(Fig. 2 F and G). Toluidine blue-stained cerebellar Purkinje cellswere also ballooned ( Fig. 3 A, asterisks), and membranous cyto-plasmic bodies were observed in these cells on electron micro-graphs ( Fig. 3 B). In addition, many phagocytic cells withenlarged intracytoplasmic vacuoles in the VirchowRobin spaceswere observed in the toluidine blue-stained sections ( Fig. 3 C, ar-rows) and on electron micrographs ( Fig. 3 D).

These results indicate that the characteristic abnormal ndingsof MPS II and the accumulated substrate had persisted in the CNSof the patient at 10 months post-CBSCT, although the characteristic

pathology was not observed in the liver.

3.3. IDS enzyme activity and VNTR analysis

After conrming that the IDS enzyme activity of normal bro-blasts (88.7 nmol/4 h/mg) was in the normal range (31110 nmol/4 h/mg [21] ) by our assay, we measured IDS activity incerebrum and liver of the normal control, the patient (MPS IIpost-CBSCT), and the untreated MPS II ( Table 1 ). IDS enzyme activ-ity in the cerebrum of the patient, as well as in that of the un-treated MPS II, was only about 1% of that of the normal control.On the other hand, in the liver of the patient, it was about 40% of that of the normal control.

The analysis of the VNTR locus D1S80 revealed that the recipi-

ent blood (pre-CBSCT) showed a single band (about 600 bp;Fig. 4 , black arrow) and the donor cord blood had two bands due

Fig. 1. MR images of our MPS II patientjustbefore CBSCT (Aand B) and at 7 monthspost-CBSCT (C). A and C, T1-weighted image; B, uid-attenuated inversion recovery(FLAIR) image. The lower gure in B is an enlarged view of the square outlined inthe upper gure in B. Just before CBSCT, the whole brain is atrophic, and thecortical sulci and ventricles are enlarged (A and B). The enlargement of Virchow-Robin spaces (B, arrowheads) and abnormally high intensity of the white matter (B,arrows) are evident. At 7 months post-CBSCT, evidence of the progression of brainatrophy is observed (C).

Fig. 2. (AC) Hematoxylin and eosin (HE) staining (A and B) and colloidal iron (CI)staining (C) of MPS II post-CBSCT. A, cerebrum; B and C, liver. Neurons (A, arrows),but not hepatocytes (B), show cytoplasmic ballooning. Hepatocytes are notstainable with colloidal iron (C). DG, PAS staining of MPS II post-CBSCT (D andE) and non-MPS II control (F and G). D and F, cerebrum and E and G, cerebellum.Many PAS-positive cells (D and E; arrows) are observed in the MPS II post-CBSCT,

whereas no PAS-positive neurons are observed in the non-MPS II control. P,cerebellar Purkinje cells.

Fig. 3. Toluidine blue-stained semi-thin sections (A and C) and correspondingelectron micrographs (B and D) of MPS II post-CBSCT. In the cerebellum, Purkinjecells are ballooned (A, asterisks) with membranous cytoplasmic bodies (B). In the

VirchowRobin space, many enlarged cells lled with vacuoles are observed (C,arrows; D). V, blood vessel; N, nucleus of a vacuolated cell.



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to its heterozygosity. Therefore, the presence of donor-derivedcells was able to be conrmed by their lower band (about450 bp; Fig. 4 , white arrow). In addition to the concrete recipi-ent-derived band which was detected in both liver and cerebrumof the patient after CBSCT, the weak donor-derived bands were de-tected in the liver and the cerebrum ( Fig. 4 , white arrowhead) of

the patient at 10 months post-CBSCT, indicating that some donorcells had migrated into the liver and the cerebrum. The lowerintensity of the donor-derived band in the cerebrum than in the li-ver suggested fewer donor-derived cells residing in the cerebrum.

3.4. Immunohistochemical study on IDS

Before performing immunohistochemical studies on IDS, weinvestigated the validity of the antibody. According to immunoblotanalysis of IDS, the antibody reacted with recombinant human IDShaving the predicted size of 76 kDa but not with recombinant hu-man a -glucosidase as a negative control ( Fig. 5 A, arrow). Althoughby IDS immunoblot analysis the antibody did not react with nor-mal human broblasts, probably because of low level of IDS in

them (data not shown), immunocytochemical study revealedIDS-immunoreactivity in normal control broblasts ( Supplementalmethod and Supplemental Fig. 2). According to immunohisto-chemical studies on unxed-frozen cerebrum samples, IDS-immu-noreactivity was positive in the non-MPS II control ( Fig. 5 B,arrows) but negative in the untreated MPS II ( Fig. 5 C). Furthermore,immunoreactivity of IDS ( Fig. 5 D, green arrows) and that of Lamp2(Fig. 5 E, red arrows), a lysosomal marker, were co-localized in thecerebrum of the non-MPS II control ( Fig. 5 F, yellow arrows). Theseresults conrmed the validity of the antibody against IDS used inthis study.

3.4.1. Distribution of IDS-positive cells in the liver The routine avidinbiotin complex procedure used in the

immunohistochemistry for the CNS did not work in the normalliver (data not shown), perhaps because the IDS enzyme activity

there was only about one-tenth of that in the normal cerebrum(Table 1 ). Therefore, the Tyramide Signal Amplication systemwas applied for the study of the liver.

IDS-immunoreactivity was detected in many cells of the MPS IIpost-CBSCT ( Fig. 6 A) as well as in the non-MPS II control (data notshown). Prior incubation of IDS antibody with excess antigen com-pletely abolished the immunoreactivity, conrming the validity of the staining ( Fig. 6 B). In the double immunouorescence stainingfor IDS and CD68 ( Fig. 6 CE), IDS-immunoreactivity was detected

in many cells ( Fig. 6 C, green arrows), not only in CD68-positiveKupffer cells ( Fig. 6 D, red arrows; and 6E, yellow arrows) but alsoin hepatocytes ( Fig. 6 E, green arrows) in the liver of the MPS II at10 months post-CBSCT. These results indicate that there weremany IDS-positive cells were widely distributed not only amongKupffer cells but also among hepatocytes in the liver at 10 monthsafter CBSCT.

Table 1

Enzyme activities in cerebrum and liver.

IDSa b-Hex b b -Gal b

CerebrumContro lc 131.5 1141 71Patient c 1.4 2135 44Untreated MPS II c 0.5 1931 19

Liver Contro l (n = 3) 14.019.7 11441197 214270Patient c 6.3 1303 233

Fibroblast Control 88.7 Not done Not done

a nmol/4 h/mg.b nmol/h/mg.c Average of duplicate measurements from different pieces of the tissue.

Fig. 4. VNTR analysis of MPS II post-CBSCT. The donor cord blood-derived band(about 450 bp, white arrow) can be distinguished from that of the recipient (pre-CBSCT). The brain of the patient after CBSCT, as well as the liver, seems to have thedonor-derived band (white arrowhead) in addition to the recipient-derived band(about 600 bp, black arrow).

Fig. 5. (A) Immunoblot analysis of IDS. The antibody against IDS reacts specicallywith recombinant human IDS having the predicted size of 76 kDa (arrow). GAAindicates recombinant human a -glucosidase, used as a negative control. (B and C)Immunohistochemical study on IDS in the cerebrum of non-MPS II control (B) anduntreated MPS II (C). IDS-immunoreactivity is positive in the non-MPS II control (B,

arrows) butnegative in theuntreated MPS II (C). (DF) Doubleimmunouorescencefor IDS and Lamp2, a lysosomal marker, in the cerebrum of the non-MPS II control.Immunoreactivity of IDS (D,green arrows) andthat of Lamp2 (E, red arrows) are co-localized (F, yellow arrows). Blue uorescence in F is from DAPI.

Fig. 6. Immunohistochemical studies on IDS in the liver of MPS II post-CBSCT. (Aand B) IDS-immunoreactivity is extensively detected in many cells (A); however, itdisappears completely when the antibody is preabsorbed with the excessrecombinant human IDS (B). (CE) Double immunouorescence for IDS and CD68.IDS-immunoreactivity is detected in many cells (C, green arrows), notonly in CD68-

positive Kupffer cells (D, red arrows; and E, yellow arrows) but also hepatocytes (E,green arrows). The blue uorescence is from DAPI.

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3.4.2. Distribution of IDS-positive cells in the CNS IDS-immunoreactivity of the antibody was more evident in the

formalin-xed frozen sections of the non-MPSII control ( Fig. 7 A)than in the unxed-frozen sections ( Fig. 5 B), but the antibodywas not reactive with the parafn sections (data not shown).Therefore, we used the formalin-xed frozen CNS samples in thefollowing studies.

In the immunohistochemical study on IDS of the non-MPS IIcontrol and MPS II post-CBSCT, many IDS-immunoreactive largecells, presumably neurons, were found in the non-MPS II control(Fig. 7 A and B); whereas only a few IDS-immunoreactive smallcells, reminiscent of microglia/monocytes, were found in the MPSII post-CBSCT ( Fig. 7 CF). Most IDS-positive small cells were local-ized in perivascular spaces in the MPS II post-CBSCT ( Fig. 7 CE).Although many of them seemed to be in the distended Virchow-Robin spaces ( Fig. 7 C, area between red dashed lines), some of them were evidently found in the brain parenchyma ( Fig. 7 E, ar-rows). Also, a very small minority of them were present in theparenchyma where no blood vessels were found in theneighborhood ( Fig. 7 F). On the other hand, no IDS-immunoreactiveneurons were found in the MPS II post-CBSCT.

We next performed double immunouorescence staining forIDS and Lamp2, NSE, transferrin, S-100 or CD68. IDS-immunoreac-tivity in the cerebrum of the non-MPS II control was observed inNSE-positive neurons ( Fig. 8 AC) and transferrin-positive oligo-dendrocytes ( Fig. 8 DF) but not in S-100-positive astrocytes(Fig. 8 GI). Although CD68-positive microglia/monocytes wereonly few in number, they were also immunoreactive for IDS(Fig. 8 JL). On the other hand, IDS-immunoreactivity in thecerebrum of the MPS II post-CBSCT was co-localized with that of Lamp2 ( Fig. 8 MO) and found exclusively in CD68-positive cells(Fig. 8 PR) but neither in NSE-, transferrin-nor S-100-positive cells(data not shown). A cell count showed that about 5.7% (17/297) of CD68-positive microglia/monocytes had IDS-immunoreactivity.

Fig. 7. Immunohistochemical studies on IDS in the CNS of non-MPS II control (Aand B) and MPS II post-CBSCT (CF). A, C, and E, cerebrum; B and D, cerebellum; F,hippocampus. Insets represent higher magnication of the rectangle outlined in thecorresponding gures, focusing on IDS-positive cells. The non-MPS II control hasmany IDS-positive neurons (A and B), whereas theMPS II post-CBSCT has only a fewIDS-positive small cells predominantly localized in the perivascular space (CE).Many of IDS-positive cells in the MPS II post-CBSCT seemed to be in the distendedVirchowRobin space (C, area between red dashed lines); however, some of themhave migrated into the brain parenchyma nearby the blood vessel (E, arrows). Avery small number of them are present in the parenchyma where no blood vesselsare found in the neighborhood (F). V, blood vessel; VR, VirchowRobin space, and

arrowhead: an enlarged cell with many vacuoles in the VirchowRobin space (seealso Fig. 3 C).

Fig. 8. Double immunouorescence in the cerebrum for IDS and NSE (AC),transferrin (DF), S-100 (GI) or CD68 (JL) of the non-MPS II control; and for IDSand Lamp2 (MO) or CD68 (PR) of MPS II post-CBSCT. Green arrows indicate IDS-positive cells; and red ones, NSE-, transferrin-, S-100-, CD68- or Lamp2-positivecells. Yellowones indicate double-positive cells. Theblue uorescence is from DAPI.IDS-immunoreactivity in the non-MPS II control is observed in NSE-, transferrin-,and CD68-positive cells but not in S-100-positive cells. On the other hand, IDS-

immunoreactivity in the MPS II post-CBSCT is co-localized with that of Lamp2, andobserved exclusively in CD68-positive cells.



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4. Discussion

In this study, we evaluated the CNS pathology of a 6-year-oldMPS II male who died at 10 months post-CBSCT. There were manydistended cells with accumulated substrate in the CNS, like in un-treated cases previously reported [35] ; and IDS enzyme activitythere remained very low. However, IDS-immunoreactivity was

found in a few microglia/monocytes predominantly localized inthe perivascular spaces. Furthermore, as far as we know, this studyalso identied the IDS-positive cells in the normal brain for the rsttime. The fact that oligodendrocytes expressed IDS in the non-MPSII control, but not in the MPS II patient, may partly be the reason forthe white matter lesions seen in MPS II [4,29] , since impairment of oligodendrocytes due to IDS deciency may lead to the secondarydysmyelination.

The antibody we used was produced in goats immunizedwith recombinant human IDS (2449-SU, R&D Systems Inc.),which contains whole mature protein (Ser 26Pro 550); there-fore, there might be some residual recipient IDS detected bythe antibody in vitro . However, this was not the case in situ be-cause nonsense-mediated mRNA decay generally degradesmRNAs that terminate translation more than 5055 nucleotidesupstream of a splicing-generated exonexon junction such asin our patient [30] . In addition, Chang et al. reported in anexpression study in vitro of the W267X mutation of the IDS genethat the truncated IDS proteins produced seemed to be trappedin the endoplasmic reticulum, not in the lysosomes, of transfec-ted COS-7 [31] ; therefore, the trapped proteins were probablydegraded by proteasomes [32] . Furthermore, even if there werethe residual recipient IDS proteins despite of the conditions de-scribed above, it is very unlikely that they would exist exclu-sively in microglia/monocytes predominantly localized in theperivascular spaces. These lines of evidence indicate that theIDS-immunoreactive cells in our MPS II post-CBSCT patient are judged to be donor-derived microglia/monocytes that had pene-trated the blood vessels.

The precise physiological events after HSCT still remain to bedetermined. Especially, the efciency of microglia replenishmentby hematopoietic stem cells has remained controversial [33] . In astudy on B6/129 F2 mice after bone marrow transplantation(BMT), Kennedy and Abkowitz found that donor microglia repre-sented only 30% of the total microglia at 12 months and that thedonor cells were predominantly seen at perivascular and leptome-ningeal, but not parenchymal, sites; although 89% of the splenicmonocytes/macrophages were of donor origin by 1 month [34] .Furthermore, in the same report they also showed that the engraft-ment rate of Kupffer cells in the liver was higher than that of microglia in the CNS at 6 months after BMT (36% vs. 23%) and thatthe engraftment rate increased at 12 months after BMT (52% vs.30%). Cogle et al. demonstrated that transgender microglia, con-

taining a Y chromosome, made up 12% of all microglia and thattransgender neurons and astrocytes were present in the hippocam-pus of female patients up to 6 years after HSCT [18] . On the otherhand, microglia progenitor recruitment from the circulation wasnot found in denervation or CNS neurodegenerative disease of chi-meric animals obtained by parabiosis without brain conditioningprocedures such as irradiation [35] . The discrepancy about themicroglia progenitor recruitment in the CNS might reect the dif-ference in neurological disorders, the age, and the conditioningregimen for HSCT. In our study, IDS-immunoreactivity was foundin about 5.7% of the microglia/monocytes predominantly localizedin perivascular spaces of the patient at 10 months post-CBSCT. Ourresult is largely compatible with the above-mentioned study byKennedy et al. The engraftment rate might have been higher if

the patient had lived longer. On the other hand, we detected no

IDS-immunoreactivity in neurons, oligodendrocytes or astrocytesof the patient.

HSCT has been reported to be much more effective in the liverthanintheCNS [16,34,36] . Resnicket al.foundthatliverbiopsyspec-imens fromMPSpatientswhohad achievedmetaboliccorrection byBMTwerenot stainablewith colloidal iron [37] , althoughthose fromuntreated cases hadhepatocellulardilatation withrarefactionof thecytoplasm, which gave positive staining with colloidal iron [38] . Inaddition, in a studyperforming BMTon modelmice ofHurlerdisease(MPS I: MIM +607014), Zheng et al. reported the disappearance of the storage vacuoles in both Kupffer cells and hepatocytes, and thebroad distribution of a -L -iduronidase (EC 3.2.1.76), a decient en-zyme of MPSI, withsparse distribution of the bone-marrow derivedcells in the liver [14] . In our case, the hepatomegaly was clinicallyimproved at 7 months post-CBSCT, and hepatocytes and Kupffercells did not appear to be swollen with apparent intracytoplasmiccolloidal iron-positive substrate at 10 months post-CBSCT. Also, ahigher intensityof thedonor-derived band was detectedin the liverthan in the cerebrum by VNTR analysis. Furthermore, the livershowed about 40% of the normal IDS enzyme activity, and manyIDS-immunoreactiveKupffercells and hepatocytes. Althoughevalu-ation of the hepatic pathology of the patient before CBSCT had beennot performed,it is very unlikely that residualIDS activity was pres-ent in a tissue-specic way such that the residual IDS activity waspreserved only in the liver. Therefore, IDS-immunoreactivity in theliver after CBSCT was judged to be donor-derived. Our result andthose ofpreviousstudiessuggestthat donor-derivedmigratingcells,such as Kupffer cells, secreted IDS enzyme that was taken up byneighboring recipient hepatocytes and Kupffer cells sufciently tocorrect GAG metabolism in the liver of the MPS II patient at10 months post-CBSCT.

There are some studies documenting that accumulated sub-strate persists in the peripheral nerves of MPS II patients for upto 2 years after HSCT [36,39] . With regard to other inherited met-abolic diseases, Will et al. reported that pronounced cytoplasmicvacuolation was seen in the brain of a 7-year-old boy with a -man-nosidosis (MIM #248500) at 18 weeks post-BMT [16] . Althoughthe precise evaluation of peripheral nerves was impossible, ourdata seem to be compatible with their study showing that theaccumulated substrate in neural tissues had not diminished lessthan 2 years after HSCT.

Thereare many clinical studies and reviews that have concludedthat HSCT does not signicantly alter the natural history of severecases ofMPSII [4044] . Guffon et al. reported that 4 MPS II children,whose development or intelligence quotient at BMT had been from65 to 70, had deteriorated after BMT [44] . On the other hand, in astudy of 54 MPS I children, many patients could achieve a favorablelong-term outcome after successful BMT [45] . The reason for theneurologically poorer outcome in MPS II than in MPS I is not clear;however, there may be two explanations for the different efcacy.

First of all, it may be because thediagnosis andtreatmentare gener-ally delayedmore inchildren withMPSII due tothe slower onsetandprogressionthanin those withMPSI. Indeed,Escolar et al.concludedthat CBSCT in newborns with infantile Krabbes disease (MIM#245200),butnot symptomaticbabies, favorably alteredthe naturalhistoryof thedisease.Secondly,it maybe becauseHSCTitself maybeless effective in the CNS of MPS II than in that of MPS I even at theearly stage of disease. Zheng et al. reported a reduction in the num-ber of vacuolated neurons and migration of hematopoietic donorcells to the brain of MPS I mice with retrovirally transduced bonemarrow [14] , and Ellinwood et al. reported a signicant decreasein the brain GAG levels of MPS I felines after BMT [13] . In addi-tion, Bikenmeier et al. reported a dramatic reduction in storageof accumulated substrate in perivascular and meningeal cells,

but not in neurons and scattered glial cells, in the brain of MPS VII mice after BMT [46] . In our study, there were many dis-



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tended cells with accumulated substrate and only a few IDS-po-sitive microglia/monocytes in the CNS of our patient. The ab-sence of IDS-immunoreactivity in oligodendrocytes or neuronscan be due to either the true absence of IDS in these cells (theabsence of effective transfer of enzyme) or the limitation of the immunohistochemical method to detect IDS present at lowconcentrations in these cells. Our ndings might explain the rea-son why HSCT fails to arrest neurological progression in MPS II.However, despite our results and those of the previous studiesdescribed above, evidence for the reason for this issue has beeninsufcient yet; because there have been no studies about thebrain pathology of any type of MPS patients, not model animals,after HSCT. Therefore, the issue may need more neuropathologi-cal studies on human MPS patients after HSCT.

Our study is unique and valuable in evaluating the efcacy of HSCT by detecting the donor-derived cells; since there has beenonly one report documenting the fate of donor-derived cells afterHCST in patients with inherited metabolic diseases. Schonbergeret al. reported that immunohistochemical staining for adrenoleu-kodystrophy (ALD, MIM #300100) protein revealed no differ-ences between the brain of a 15-year-old ALD patient at76 days post-HSCT and that of the control [17] . Their resultsare in sharp contrast with the study by Yamada et al., who doc-umented that little ALD protein was detected in the brain of anALD model mouse at 6 months post-HSCT [47] . After evaluationof the accumulated substrate and IDS enzyme activity, we con-clude that only a few donor-derived cells had penetrated intothe CNS of our MPS II patient at 10 months post-HSCT and thatthe number of donor-derived cells was insufcient for metabolicimprovement. However, our ndings showing the existence of donor cells in the brain parenchyma at 10 months post-CBSCTsuggest the potential of HSCT for treatment of MPS II.

The issue of HSCT for LSD patients requires detailed discussionregarding the indication, timing, and conditioning regimen. There-fore, the accumulation of neuropathological evidence, such as ourstudy, is very valuable for establishment of evidence-based proto-cols of HSCT for these patients.

Acknowledgments

We thank Dr. Kinuko Suzuki, Tokyo Metropolitan Institute of Gerontology, for critical reading of the manuscript; Dr. AkemiTanaka, Department of Pediatrics, Osaka City University Gradu-ate School of Medicine, for the gift of reagents and advice onthe IDS enzyme assay; and Dr. Kazuhiko Bessho and Dr. Hidet-oshi Taniguchi, Department of Pediatrics, Osaka UniversityGraduate School of Medicine, for their advice and technicalsupervision.

This research was supported by a grant from Research on Mea-sures for Intractable Diseases, Japanese Ministry of Health, Welfare

and Labor (to N.S.); and Grants-in-Aid for Scientic Research Cfrom the Ministry of Education, Culture, Sports, Science, and Tech-nology of Japan (No. 19591205 to I.M. and No. 18790715 to K.K.S.).

We obtained unxed-frozen cerebrum samples from an 11-year-old untreated MPS II patient and a 12-year-old non-MPS IIcontrol from the NICHD Brain and Tissue Bank for DevelopmentalDisorders at the University of Maryland, Baltimore, MD, USA. Therole of the NICHD Brain and Tissue Bank is to distribute tissue;and, therefore, it cannot endorse the studies performed or theinterpretations of results.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.ymgme.2009.05.006 .

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Araya 2009. Localized Donor Cells in Brain of a Hunter Disease Patient After Cord Blood Stem Cell Transplantation