In Utero Fetal Liver Cell Transplantation without Toxic Irradiation Alleviates Lysosomal Storage in Mice with Mucopolysaccharidosis Type VII

Barker et al.

Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 861– 873 doi:10.1006/bcmd.2001.0453, available online at http://www.idealibrary.com on

In Utero Fetal Liver Cell Transplantation without Toxic Irradiation Alleviates Lysosomal Storage in Mice with Mucopolysaccharidosis Type VII Submitted 08/13/01 (Communicated by H. Ranney, M.D., 08/27/01)

Jane E. Barker,1 Sue Deveau,1 Mark Lessard,1 Nancy Hamblen,1 Carole Vogler,2 and Beth Levy2 ABSTRACT: Lysosomal storage diseases, such as Mucopolysaccharidosis type VII (MPS VII), cause progressive loss of mobility and intellect and result in early death. Treatment of progressive diseases must occur before the blood– brain barrier closes. In MPS VII mice, normal donor hematopoietic cells secrete the missing enzyme ␤-glucuronidase (GUSB) that reverses disease manifestations. Correction of lysosomal storage is limited to the visceral organs unless transplantation is preceded by high-dose irradiation. We hypothesize that irradiation opens the blood– brain barrier allowing passage of corrective cells. Here we transplanted genetically myeloablated MPS VII fetuses to determine whether earlier treatment without toxic irradiation is systemically corrective. Cells with a selective advantage in utero were identified. Donor fetal liver cells (FLC), a substitute for difficult to obtain murine cord blood cells, were increased 10-fold in the host peripheral blood over equivalent numbers of adult marrow cells injected simultaneously and were stable long term in both primary and secondary hosts. GUSB⫺ MPS VII fetuses injected with GUSB⫹ FLC were assessed longitudinally after birth. Donor FLC replaced host stem cell descendants, prolonged life dramatically, and reduced bone dysplasia and lysosomal storage in all tissues long term. GUSB, donor leptomeningeal cells, and microglia were present in the brain at 11 months postinjection. Lysosomal storage in cortical neurons and glia, although not completely corrected, was reduced. We conclude that in utero intervention without toxic pretreatment in this model reduces the storage disease long term and improves the length and quality of life despite exerting only minor effects on the brain. © 2001 Academic Press Key Words: MPS VII; lysosomal storage; fetal liver cells; myeloablation; transplantation.

INTRODUCTION

is feasible but can elicit anaphylactic shock and the enzyme may not cross the blood– brain barrier once interendothelial tight junctions close (5). Hematopoietic stem cell or organ transplantation are therapeutic options for some diseases but require a compatible donor and pre-treatment can be toxic to the already debilitated patients (6). Even with therapy, the clinical manifestations of lysosomal storage disease are not eliminated. Treated patients generally have a slower learning curve that may even decrease posttreatment (4, 7). Sight is adversely affected (8, 9), and coordination is limited even in successfully transplanted patients. It is not clear whether these effects are conse-

Progressive lysosomal storage diseases can affect bone formation, liver, spleen, and cardiac function, hearing, sight, and the central nervous system (CNS) (1, 2). Children diagnosed with any one of the multiple forms of storage diseases affecting the CNS usually have learning disabilities, may acquire behavioral problems, have a poor quality of life, and die within the first one to three decades of life. Early diagnosis and treatment before disease symptoms manifest is imperative but treatment modalities are limited (3, 4). Enzyme replacement for the more prevalent forms

Correspondence and reprint requests to: Jane E. Barker. Fax: (207) 288-6079. E-mail: [email protected]. 1 The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609. 2 Department of Pathology, St. Louis Medical School, St. Louis, Missouri 63110. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved

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TABLE 1 Genotypes of Mutant and Recipient Mice Experiment

Genotype FLC donor

Genotype ABM donor

Primary recipient

Secondary recipient

ID, competitive repopulation

B6-⫹/⫹, Gpi1a/Gpi1a, Hbbs/Hbbs (⫹, a, s)a

B6-⫹/⫹, Gpi1b/Gpi1b, Hbbd/Hbbd (⫹, b, d)

ID, ABM only ID, ABM only In utero cure

NAb NA (⫹, a, s)

(⫹, a, s) (⫹, a, s) NA

Fetus B6-kitW41/kitW41, Gpi1b/Gpi1b, Hbbs/Hbbs (W41, b, s) Fetus (W41, b, s) Adult (W41, b, s) Fetus B6-kitW41/kitW41, gusmps/gusmps Gpi1b/ Gpi1b, Hbbs/Hbbs (W41, MPS VII b, s)

Adult B6-kitW/kitWv, Gpi1b/Gpi1b, Hbbd/Hbbs (W41, b, d/s) NA NA NA

a b

Genotype defined hereafter as ⵹. Not applicable.

quences of the pre-treatment or of the limited numbers of enzyme-producing cells that seed the CNS (10). MPS VII is rare in humans but shares clinical and pathological features with other more common lysosomal storage diseases (11). Murine MPS VII, a model of human MPS, has proved useful for testing therapeutic strategies and for characterizing functional disorders (12). The bone dysplasia, cognitive defects, sight and hearing loss and general ill health that accompany increasing lysosomal storage develop gradually after birth in the MPS VII mice (13). Transplantation of GUSB⫹ donor cells into 10 Gy irradiated adults alleviates many of the defects in vivo and increases levels of GUSB in the brain significantly but intervention occurs too late to correct bone and learning defects (14). Enzyme released from the donor hematopoietic cells is present in the serum and in host cells adjacent to donor macrophages in various tissues. Transplantation in 2 Gy irradiated neonates reduces host cell lysosomal storage and increases catabolism of the lysosomal glycosaminoglycans. The treatment increases life span two- to fourfold, reduces bone dysplasia (15), rescues hearing (16) and sight (17) but has no effect on neurological defects (18). Despite correction of bone and visceral defects, there is no increase in brain GUSB following neonatal transplantation without irradiation (19). Irradiation appears to be required for donor cells to implant in the brain and provide significant levels of GUSB.

Enzyme injected intravenously into MPS VII pups prior to the second postnatal week, reduces storage in neurons long term (20) and normalizes learning capabilities (21). This observation supported prior reports that the mouse blood– brain barrier closes during the first two weeks of life (22). We exploited this information to determine whether donor cells can enter the brain in utero without toxic pre-treatment. We used genetically myeloablated, kitW-41J/kitW-41J, MPS VII fetuses as recipients (23). The kitW-41J/kitW-41J (W41) mice have a stem cell deficit and a mild anemia but normal numbers of leukocytes in the peripheral blood. Our transplantation results show a long-term reduction in lysosomal storage disease in visceral tissues and bone, but regional and incomplete correction in the brain of genetically myeloablated MPS VII mice. METHODS Mice and Identification of Donor Cells Two separate experiments were employed. The first identified cells with a selective advantage in utero, involved both competitive repopulation between FLC and adult bone marrow (ABM) and bone marrow transplantation alone, and is called ID. The second determined whether cells with a selective advantage could alleviate lysosomal storage disease and is called cure. Donors and recipients for all these experiments are listed in Table 1. The ID recipients were 12- to 862

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16-day postcoitum (dpc) C57BL/6J (B6)-W41. Markers to differentiate donors and host include: Gpi1/Gpi1 (glucose phosphate isomerase I electrophoretic variants a and b) and Hbb/Hbb (hemoglobin electrophoretic variants s and d). Competitive repopulation donors provided 12- to 16dpc FLC or ABM. ABM was subsequently tested alone in utero and in adult W41 mice. Differences between the donor red blood cells (RBC) and those from the host were identified by electrophoretic separation of hemoglobin isoforms (24) and of GPI1 isoforms (25). Quantitation was achieved on a computing densitometer (Molecular Dynamics, Sunnyvale, CA). Long-term repopulation in the competitive experiment was confirmed by secondary injection of marrow cells from a primary recipient of donor FLC and ABM into genetically myeloablated WBB6F1⫺ kitW/kitWv (W/Wv) recipients. To generate 14-dpc fetal recipients for the in utero cure experiments, W41, ⫹/gusmps or gusmps/ gusmps females were mated to W41, ⫹/gusmps males. These initial matings produced few W41 MPS VII pups that survived. Subsequent matings were between W41, MPS VII females and males to generate exclusively W41 MPS VII fetuses. The inability of MPS VII males to breed (26) was corrected by ABM transplantation into nonablated neonates (19). Donor FLC for in utero cure were from 13- or 14-dpc B6-⫹/⫹ fetuses. In neither experiment did the mothers care for their young. Albino BALB/cByJ-⫹/⫹ females with 1- to 3-day-old pups were subsequently used successfully as foster mothers. Pups were easily differentiated by coat and eye color.

Equal numbers of donor nucleated ABM and FLC were mixed together for the ID competitive repopulations. Total cell concentration ranged from 0.29 to 8.1 ⫻ 108 nucleated cells/ml and was dependent on the total number of nucleated FLC retrieved and the estimated number of fetuses to be injected on any one day. The number of cells injected did not affect results since FLC and marrow cells were always present in equivalent numbers in the inoculum. The concentration of ABM cells injected alone was in the same range as ABM in the competitive repopulation assays (0.17 to 4.1 ⫻ 108 nucleated cells/ml). Total FLC concentration in the cure of W41, MPS VII fetuses ranged from 0.24 to 1.2 ⫻ 108 nucleated cells/ml. In both ID and cure, fetuses were injected immediately after cells were collected. The pregnant females were anesthetized with avertin and a midline incision in the skin extending approximately 1 cm anteriorly was made at the level of the 4th mammary glands (30). The peritoneum was opened and the uterine horns were lifted out one at a time. Fetuses were counted and each live fetus was injected via a 33-gauge beveled needle (Hamilton, Reno, NV) attached to a hand-held 25-␮l Hamilton syringe. Dose was 5 ␮l at 12–13 dpc and 10 ␮l at 14 –16 dpc. All injections were made directly into the fetal liver (31). After injection, each uterine horn was replaced and the peritoneum and skin were closed separately. The pregnant females were checked daily thereafter. BALB/cByJ foster mothers were added near birth. Newborns were counted at birth and at weekly intervals and weaned at 4 – 6 weeks of age.

In Utero Injections Cells for injection were minimally manipulated to avoid damage to the hematopoietic stem cells (HSC) and enrichment was avoided since it caused HSC functional loss (27). Marrow was prepared as described previously (28) in 1⫻ PBS (Life Technologies, No. 114200-075, Gaithersburg, MD). FLC were dissociated in 1⫻ PBS (29). For all timed pregnancies, the parents were caged together and females were checked daily for a vaginal plug, signifying day 0 of pregnancy.

Assays Prenecropsy One hematocrit tube of blood was removed from the retro-orbital sinus at 1 month after birth, at 1- to 3-month intervals thereafter, and on the day of necropsy. Levels of host and donor GPI1 (25) and, in the ID experimental mice, HBB (24), were quantified to monitor percent repopulation of the peripheral blood (PBL). 863

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TABLE 2 Characteristics of W41 Recipients Injected in Utero

Experimental protocol

Total no. injected

Cells injected

No. (%) born

No. (%) born that survive

No. (%) donor positive

(a) ID ⫽ competitive repopulation (b) ID ⫽ ABM alone

347 247

FLC ⫽ to ABM ABM

150 (43.2) 90 (36.4)

89 (59.3) 54 (60.0)

47 (52.8)a 16 (29.7)b,*

a

Four mice were transiently repopulated; one incompletely repopulated. Two mice were transiently repopulated; eight incompletely repopulated. * Significant difference from (a) (P ⫽ 0.018). b

Assays Postnecropsy

and beta-hexosaminidase were determined from lysates of tissues saved for biochemistry (14). The fluorescent substrates were 4-methylumbelliferyl␤-D-glucuronide and 4-methylumbelliferyl-␤-Dglucosaminide, respectively (Sigma Chemical Co., St. Louis, MO). Values for enzyme activity were expressed as percent W41 ⫹/⫹ specific activity. Femurs stored in 95% ethanol were dissected free of connective and muscle tissue and analyzed by pQCT on the platform of a Stratec XCT 960M densitometer (Norland Medical Systemic, Ft. Atkinson, WI) as described (32). Sequential crosssectional scans taken at 2-mm intervals down the length of each bone were assigned numerical values for total mineral and density; cortical mineral, density, and thickness; periosteal circumference; and bone length. Values were compared statistically by Student’s t test or by the Mann–Whitney rank-sum test using SigmaStat software (Jandel Scientific, San Rafael, CA).

In the cure experiment, untreated (Utr) W41 MPS VII, FLC-injected (Tr) W41 ⫹/⫹, and Tr W41 MPS VII mice were necropsied as adults. Tissue pieces were frozen in Tissue-Tek for cryosectioning and histochemistry, stored in 2% paraformaldehyde for pathology, and frozen in liquid nitrogen for biochemistry. A femur from one of the Utr W41 MPS VII; one of the Tr W41 ⫹/⫹; and four of the Tr W41 MPS VII mice was flushed with 1⫻ PBS to collect cells for flow cytometry and the other femur was dissected and stored in 95% ethanol for peripheral quantitative computer tomography (pQCT) (32). As described previously (23), following ACK lysis of RBCs, repopulation percentages of the various GUSB⫹ cell types in marrow were quantified by flow cytometry. Cells exposed to the GUS substrate Imagine Green C12FdGlcU (lipophilic analog of fluorescein-di-␤-D-glucuronic acid, Molecular Probes, Inc., Eugene, OR) were also labeled with individual fluorescent phycoerythrin cell surface markers: Mac-1 (CD11b), Gr-1 (RB6-8C5), CD45R (RA3-6B2), or TcR (H57-597) from Pharmingen (San Diego, CA). Cytometric comparative analysis was with similarly treated cells from Utr W41 MPS VII and W41 ⫹/⫹ mice. The localization of donor cells was by microscopic evaluation of 10-␮m cryostat sections histochemically stained for GUSB and counterstained with methyl green (33). For pathology, tissues were embedded in Spurr’s resin and examined by light microscopy for reduction of lysosomal storage (34). The lysosomal enzyme activities of GUSB

RESULTS FLC Have an Advantage in Utero Losses in the in utero experiments were predictably high. In the ID experiment, a total of 347 W41 fetuses were injected in utero between 12 and 16 dpc with equivalent numbers of FLC and ABM cells that were genetically differentiable from one another and from the host. Of the 150 mice (43% of injected) born, 89 survived beyond weaning and 47 of these were positive for donor cells (Table 2, a). Age matching of fetal donors and recipients did not provide a birth advantage, 864

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ment is not propitious for the ABM cells, normal ABM at a dose of 1.7 ⫻ 105 to 41 ⫻ 105 cells was injected into 12- to 16-dpc W41 fetuses (Table 2, b). The percentage of mice born and of those surviving was not significantly different in recipients of marrow alone and of ABM plus FLC (␹2, P ⫽ 0.31 and 0.95, respectively) (Table 2, a/b) indicating that neither donor cell number nor source affects these two parameters. There were, however, significantly (␹2, P ⫽ 0.018) fewer donor positive mice among the recipients injected with ABM alone suggesting ABM is at a disadvantage in the fetal environment. This is supported by the fact that 8 of 14 (57.1%) long term (9 months) repopulated ABM recipients were incompletely repopulated. This difference is significant (P ⫽ 0.005) when compared to the percentage of mice completely repopulated with mixtures of FLC and ABM. The lowest successful donor ABM dose of 1.7 ⫻ 105 cells was similar to that in the successful competitive repopulations. The in utero recipients in both experiments were repopulated long-term as evidenced by the complete replacement of primary host with donor cells for at least 9 months. In addition, secondary W/Wv recipients of primary W41 hosts repopulated with FLC and ABM cells retained donor FLC and ABM until necropsy at 16 months posttransplantation (data not shown).

FIG. 1. Donor FLC outcompete donor adult bone marrow after injection in utero. Equivalent numbers of nucleated cells from ⫹, a, s fetal liver and ⫹, b, d adult marrow were mixed and injected into fetal W41, b, s recipients. The percentage of donor marked fetal liver-derived RBC (hatched bars) and marrow-derived RBC (open bars) shows a 10-fold increment in liver-derived RBC that persists long term. The levels stabilized by 6 months (not shown). Bars indicate standard deviations from the mean.

postnatal survival advantage, or repopulation advantage (data not shown). Among the 47 donor-positive survivors, FLCderived RBCs gained a 10-fold advantage over marrow following in utero injection (Fig. 1). The FLC progeny increased rapidly postnatally to comprise 85% of the PBL RBCs by three months of age. By contrast, the marrow-derived RBCs did not increase, stabilizing at an average of 15%. Cells from both donors implanted successfully since 45 of the 47 recipients had both FLC- and marrow-derived RBCs. To test the possibility that the concentration of donor ABM cells in the in utero ID experiment was limiting, the number of ABM cells needed to repopulate substantially larger W41 adults was titrated. A dose of 1.1 ⫻ 105 cells replaced 57.8% of the host RBCs and replacement was complete with 11.0 ⫻ 105 donor cells within 3.5 months (data not shown). The lowest donor marrow cell dose in successful competitive ID in utero transfers was 1.44 ⫻ 105. To test the possibility that the fetal environ-

FLC Replace Host W41 MPS VII Cells Long Term Following in Utero Injection The larger, more clearly defined hematopoietic liver of 14 dpc fetuses and lack of survival advantages noted above prompted injection into 14 dpc liver in the cure experiment. Preweaning losses were more extensive than in the ID experiment (Table 3). W41 MPS VII loss was the highest. Expectations based on parental genotypes for the first two matings were 32 W41 MPS VII:43 ⫹/gusmps:10 W41 ⫹/⫹. Surviving to weaning were 7 W41 MPS VII:43 W41 ⫹/gusmps:13 W41, ⫹/⫹. In matings between W41 MPS VII mice rescued by postnatal bone marrow transplantation, 121 fetuses were injected and 12 doubly homozygous pups (9.9%) were born, sug865

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TABLE 3 Observed and Expected Numbers of Pups Based on Parental Genotype Number of mice at weaning Mating/treatment results Parent genotype (all W41/W41)

Total injected

Total born (%)

W41 ⫹/⫹ observed

W41 ⫹/⫹ expected

W41 ⫹/ gusmps observed

⫹/gusmps ⫻ ⫹/gusmps gusmps/gusmps ⫻ ⫹/gusmps gusmps/gusmps ⫻ gusmps/gusmps

140 109 121

40 (36.6) 45 (32.7) 12 (9.9)

13 0 0

10 0 0

18 25 0

a

W41 ⫹/ gusmps expected

W41 gusmps/ gusmps observed

W41 gusmps/ gusmp expected

20 22.5 0

1a 6a 12

10 22.5 12

The MPS VII young are often lost before weaning.

gesting extensive in utero loss of manipulated W41 MPS VII fetuses. A total of 19 W41 MPS VII pups survived to weaning. Ten of these were positive for donor FLC. Results were similar among the 43 W41 ⫹/gusmps pups born where 21 were donor positive. Replacement of donor RBC (Fig. 2) and, more importantly for the cure of the MPS VII phenotype, of the GUSB⫹ white blood cells (WBC) was rapid. RBC replacement increased from 40% at 1 month to 100% by 5 months. Donor WBCs in successfully transplanted W41

MPS VII mice increased from 20% at 1 month postnatally to plateau at 75% by 6 months. The donor GPI1A RBC increment in unaffected, W41 ⫹/gusmps and W41 ⫹/⫹ pups was similar to that in the doubly homozygous mutants, indicating that there was no selective advantage in the W41 MPS VII hosts (data not shown). To corroborate the replacement levels, marrow cells retrieved at necropsy (271–330 days of age) were assessed by flow cytometry for GUSB (FdGlcU) and cell type. More than 50% of the total cells were macrophages and neutrophils and ⬎97% of these were GUSB⫹ (Fig. 3). B and T cells comprised on average 6.8 and 1.3%, respectively, of the total cells with ⬎98% donor-derived. Results indicated donor FLC progeny persisted throughout life. FLCs Are Therapeutic When Injected into W41 MPS VII Fetuses The in utero treatment was successful in increasing the life span from an average of 6 months (Utr) to over 9 months at necropsy. At necropsy, GUSB levels that were therapeutic (35) were present in heart, kidney, liver, lung, spleen, and thymus (Fig. 4a). Beta hexosaminidase levels, predictive of lysosomal storage, were decreased significantly in all tissues except the brain (Fig. 4b). The highest levels of beta hexosaminidase (2500% of normal) were observed in the heart of Utr W41 MPS VII mice. Levels decreased to 400% of normal in the Tr mice and lysosomal storage was reduced in myocardium and heart valves (Figs. 5a and 5b). Lysosomal storage was also markedly reduced in the hematopoietically

FIG. 2. Normal donor FLC-derived RBC and WBC replace MPS VII host cells postnatally. GPI1A FLC were injected into GPI1B W41 MPS VII fetuses. The donor RBCs (open bars) completely replaced the host RBCs by 5 months after birth. The donor WBCs (hatched bars) increased from 20% at the earliest time point to 75% at 6 months of age. No increase was seen thereafter (not shown). Bars represent standard errors of the mean. 866

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FIG. 4. GUSB is increased and BHEX decreased in GUSB⫺ recipients of GUSB⫹ FLC. Levels of GUSB (a) and BHEX (b) were quantified in tissue samples obtained at necropsy with respect to the levels in the same tissues of normal mice. The values are averages of six Tr W41 MPS VII (hatched bars) mice and of three Utr W41 MPS VII (open bars) mice. The results show increments in GUSB sufficient to correct disease pathology (37) in all tissues except brain. HEXB, elevated in Utr MPS VII mice, decreases as the GUSB levels increase.

derived liver Kupffer cells (Fig. 5c), bone marrow lining cells (Fig. 5d) and osteoblasts, kidney mesangial cells, spleen, and neutrophils (not shown). Lysosomal storage in neighboring hepatocytes (Fig. 5c), and kidney tubules (Fig. 5e) was reduced as well. Retinal pigmented epithelium (Fig. 5f) and corneal (Fig. 5g) lysosomal storage was also dramatically decreased. While the bone of Tr MPS VII mice showed morphological alterations, the vacuolization of osteocytes was less than that seen in Utr MPS VII mice in the cancellous bones and the middle ear ossicles. Histochemistry confirmed distribution of GUSB⫹ cells and of enzyme (Figs. 6a– 6d) was more extensive than that seen after transplantation of lethally irradiated (15) or nonablated (19) neonates. All the GUSB⫹ mice had a more normal

FIG. 3. Flow cytometric quantitation of FLC-derived GUSB⫹ myeloid and lymphoid cells in the GUSB⫺ host at necropsy. Fluorescent substrate, FdGlcU, for identification of GUSB⫹ cells and antibody markers for granulocytes (Gran), macrophages (Mac), and T (T)- and B (B)-lymphocytes were reacted with cells retrieved from the marrow at necropsy. Individual cells were quantified by flow cytometry. Comparisons of typical profiles from one Tr W41 MPS VII mouse (left) and one Utr W41 MPS VII mouse (right) show an increase in the GUSB⫹ cells (right half of each square) and in the individual myeloid and lymphoid compartments (top right of each plot). Percentages are of total GUSB⫹ cells. 867

FIG. 5. Comparative histopathology between Utr and Tr W41 MPS VII mice. Tissues fixed at necropsy were stained with toluidine blue and evaluated for storage disease. Tr (left) and Utr (right) samples. Note loss of storage in (a and b) myocardium and heart valve (arrow); (c) liver Kupffer cells (arrow) and hepatic cells; (d) osteoclasts (arrow) and sinus lining cells (arrowhead); (e) mesangial cells (arrowhead) and kidney tubules (arrow); (f) retinal pigmented epithelium (arrow); and (g) cornea of the Tr tissues. The neurons (arrow) and glia (arrowhead) in the neocortex (h) and leptomeninges (arrow) (i) of Tr mice also show reduced storage. Bars are 50 ␮m in length. 868

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volume, density, and thickness; in periosteal circumference; and in femur length (Table 4). Data comparisons were performed between each set of control mice and five Tr W41 MPS VII mice with significant levels of donor PBL cells during postnatal life when bone growth occurs. Dramatic improvement was shown by the assumption of bone characteristics typical of the ⫹/⫹ but not of the Utr MPS VII mice. Femur length and periostal circumference did not improve significantly (not shown). GUSB levels in the brain (Fig. 4a) of six mice ⬎270 days old at necropsy averaged 1.4% of normal. Neurons in the neocortex (Fig 5h), but not in the hippocampus nor in the cerebellum, had slightly reduced storage. Glia throughout the CNS had a mild decrease in storage as did the ependymal cells. Storage in the leptomeninges and perivascular cells of the parenchyma was reduced as well. Donor cells in the cortex appeared to be primarily perivascular (Fig. 6d). Donor replaced host cells in the meninges and GUSB enzyme, in some cases, extended from the meninges into adjacent spinal nerve tracts (Fig. 6e). Control W41 ⫹/⫹ mice had more diffuse GUSB (Fig. 6g) while Utr W41 MPS VII mice had none (Fig. 6h). The W41 MPS VII mouse necropsied at 65 days after birth, while it had 100% donor RBCs and 76% WBCs, had no GUSB activity in the brain nor donor cells visible by histochemical analyses. Other tissues had enzyme levels equivalent to those in the ⬎270-day-old W41 MPS VII recipients. A complete deficiency of GUSB in the brain of the younger mouse but not of the older recipients suggests donor cells accumulate over time.

FIG. 6. Identification of donor GUSB⫹ cells in various tissues by histochemistry. Histochemical staining of tissues for GUSB⫹ (red) cells among GUSB⫺ (green) cells show intensely positive donor cells: (a) heart interstitial cells, (b) liver Kupffer cells (arrow) and hepatic cells (arrowhead), (c) bone osteoclasts (arrow) and fibroblasts (arrowhead), (d) neocortex, and (e) meninges (arrow). GUSB has cross-corrected (b) hepatic cells and (e) spinal nerves (arrowhead) adjacent to the leptomeninges. (f, g, and h) are brains from Tr W41 MPS VII, W41 ⫹/⫹, and Utr W41 MPS VII mice, respectively, stained simultaneously for comparison. Bars are 50 ␮m in length.

DISCUSSION The rationale for these studies is that BMT for the mucopolysaccharidoses must be preceded in mice and possibly also in human patients by toxic myeloablation to obtain significant levels of donor enzyme in the brain. Our objective was to test the theory that toxic myeloablation is necessary for systemic enzyme accumulation and storage correction in MPS VII mice injected in utero. This was, in part, borne out by experimentation. Genetically myeloablated MPS VII mouse fetuses

appearance. Bone structure, when evaluated by pQCT, had improved. GUSB was provided by donor fibroblasts that paralleled the bone surface and by donor osteoclasts within the chondyles (Fig. 6c). Control Utr MPS VII and Utr ⫹/⫹ mice had statistically significant differences in total bone mineral and density; in cortical mineral, 869

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TABLE 4 Improvement in Bone Characteristics Determined by pQCT Totala

Corticala

Mouse genotype

Number of mice

Mineral

Density

Mineral

Volume

Thickness

Tr W41 MPS VII Utr ⫹/⫹ Utr MPS VII

5 10 8

9.79 ⫾ 0.32* 10.48 ⫾ 0.42 12.51 ⫾ 0.45

0.47 ⫾ 0.03* 0.50 ⫾ 0.01 0.56 ⫾ 0.01

8.36 ⫾ 0.40* 10.48 ⫾ 0.30 11.52 ⫾ 0.42

13.21 ⫾ 0.43* 14.63 ⫾ 0.55 16.93 ⫾ 0.54

0.36 ⫾ 0.03* 0.33 ⫾ 0.00 0.43 ⫾ 0.01

a Values are means ⫾ standard error of the mean. * Tr W41 MPS VII values are significantly different (P ⱖ 0.022) than Utr MPS VII values but not Utr ⫹/⫹ values. This indicates correction of the defect.

transplanted with normal FLC had a sustained reduction in lysosomal storage in viscera and bone but limited and localized accumulation of enzyme in the brain. In the brain of all mammals, the interendothelial tight junctions are a barrier to cells and therapeutic chemicals (22). Chemical, irradiation, or inflammatory interventions are considered essential for the disruption of the human blood– brain barrier (3, 5, 36). The murine blood– brain barrier forms between the first and second postnatal week of life (20, 22), significantly later than in the human. We hypothesized that in utero therapeutic modalities would permit entry of cells and enzyme in the brain. In the older (8.5 to 11 months old), but not in the younger (65 days old), W41 MPS VII mice, microglia are donor-derived, GUSB enzyme is present, and lysosomal storage is reduced in cortical neurons, glia, and retinal pigmented epithelium. Results strongly suggest donor cells are delayed in their migration into the brain, even when donor cells are provided at 14 dpc. Repopulation latency periods of a year have been noted in the brain of patients as well (8, 37). Data indicate that enzyme might not accumulate sufficiently early in the brain to deter learning defects. One theory to explain delayed implantation is that severe damage to the brain must occur before donor cell seeding occurs. Irradiation accomplishes this as can the storage disease itself. However, as MPS VII mice show little tissue damage at birth (13), disease may not cause sufficiently severe neuronal defects until later in life. The hypothesis that disease severity is required for cell replacement in the brain is being tested by trans-

plantation into successively older nonablated MPS VII mice. Recently, we have shown that high-dose donor cell injections of non-ablated MPS VII neonates are phenotypically and functionally corrective in all tissues but brain (19). Long-term evaluations of myeloablated human pediatric patients with lysosomal storage diseases also support improvements in prognosis with higher cell doses (6). To the contrary, non-ablated MPS VII fetuses have minimal repopulation in spleen, bone marrow, liver, and brain at lower but not higher cell doses (38). The long-term reduction in bone storage posttransplantation described here is unusual for MPS diseases. In children, bone structure, other than facies and skull, does not show prolonged therapeutic effect following bone marrow transplantation (4, 36, 39, 40). In sheep fetuses with ceroid lipofuscinosis, no improvement is noted following injection of FLC (41). In MPS VII mice treated neonatally, bone structure is improved but not normalized following low dose irradiation but is worsened by higher doses (15). Here there is significant improvement of long bone structure, suggesting toxic myeloablation is unnecessary to effect improvement. The current studies have been abetted by selection of HSC that are highly proliferative and competitive in a fetal environment. Our results indicate that fetal HSC gain and maintain a 10fold advantage over adult marrow HSC following implantation in utero. This is at least five times higher than that detected by Rebel et al. (42) and is two times higher than the best result of Harrison et al. (43) who competed FLC and adult marrow cells in an adult. Fetal HSC are known to be more 870

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abundant and mitotically active than adult marrow HSC (44 – 47) and to differ in cell surface phenotype (42, 46), relative concentration, replicative activity in situ (29, 42– 44) and response to various stimulatory factors (48 –52). There is direct evidence that a developmental match between environment and HSC is essential for maximum responsiveness (53). The seeding and amplification of embryonic yolk sac HSC in a fetus (30) and in a newborn (54) but not in an adult (55) provide evidence that the environment enhances site specific homing and expansion of fetal cells. Our results are consistent with this interpretation. In summary, toxic myeloablation appears essential for donor cells and enzyme to cross the blood– brain barrier at therapeutic levels (14). Unfortunately, irradiation disrupts brain architecture in young animals when enzyme would have maximal effect.

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ACKNOWLEDGMENTS The authors thank their colleagues Brian Soper, Luanne Peters, and Babette Gwynn who critically reviewed the manuscript presubmission; Wes Beamer who performed the pQCT; Greg Martin, Priscilla Jewett, et al. in the Biological Imaging Service; and Jennifer Smith in Graphics Services (funded by Cancer Core CA34196). Grant support was from NIH DK 49525 and 27726 to J.E.B. and subcontracts to C.V. The manuscript was submitted in remembrance of Dr. Edward Birkenmeier who identified, described, and developed treatments for MPS VII mice before his untimely death.

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