help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2131
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cahill, R. A.
Right arrow Articles by Whyte, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cahill, R. A.
Right arrow Articles by Whyte, M. P.
Related Collections
Right arrow Calcium and Bone Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 2923-2930
Copyright © 2007 by The Endocrine Society

Infantile Hypophosphatasia: Transplantation Therapy Trial Using Bone Fragments and Cultured Osteoblasts

Richard A. Cahill, Deborah Wenkert, Sharon A. Perlman, Ann Steele, Stephen P. Coburn, William H. McAlister, Steven Mumm and Michael P. Whyte

Pediatric Research Institute (R.A.C.), Cardinal Glennon Children’s Hospital, St. Louis, Missouri 63110; Center for Metabolic Bone Disease and Molecular Research (D.W., S.M., M.P.W.), Shriners Hospitals for Children, St. Louis, Missouri 63131; All Children’s Hospital (S.A.P., A.S.), University of South Florida, St. Petersburg, Florida 33713; Department of Chemistry (S.P.C.), Indiana University-Purdue University, Fort Wayne, Indiana 46805; Mallinckrodt Institute of Radiology (W.H.M.), Washington University School of Medicine at St. Louis Children’s Hospital, and Division of Bone and Mineral Diseases (S.M., M.P.W.), Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. Michael P. Whyte, Shriners Hospitals for Children, 2001 South Lindbergh Boulevard, St. Louis, Missouri 63131. E-mail: mwhyte{at}shrinenet.org.


   Abstract
Top
 Abstract
Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 
Background: Hypophosphatasia (HPP) is a rare, heritable, metabolic bone disease due to deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase. The infantile form features severe rickets often causing death in the first year of life from respiratory complications. There is no established medical treatment. In 1997, an 8-month-old girl with worsening and life-threatening infantile HPP improved considerably after marrow cell transplantation.

Objective: Our aim was to better understand and to advance these encouraging transplantation results.

Design: In 1999, based on emerging mouse transplantation models involving implanted donor bone fragments as well as osteoblast-like cells cultured from bone, we treated a 9-month-old girl suffering a similar course of infantile HPP.

Results: Four months later, radiographs demonstrated improved skeletal mineralization. Twenty months later, PCR analysis of adherent cells cultured from recipient bone suggested the presence of small amounts of paternal (donor) DNA despite the absence of hematopoietic engraftment. This patient, now 8 yr old (7 yr after transplantation), is active and growing, and has the clinical phenotype of the more mild, childhood form of HPP.

Conclusions: Cumulative experience suggests that, after immune tolerance, donor bone fragments and marrow may provide precursor cells for distribution and engraftment in the skeletal microenvironment in HPP patients to form tissue-nonspecific isoenzyme of alkaline phosphatase-replete osteoblasts that can improve mineralization.


   Introduction
Top
 Abstract
 Introduction
Case Report
 Materials and Methods
 Results
 Discussion
 References
 
HYPOPHOSPHATASIA (HPP) IS the inborn error of metabolism that features deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) presenting with rickets or osteomalacia (reviewed in Refs. 1 and 2). All affected individuals carry one or two loss-of-function mutations within the TNSALP gene alleles (3). This experiment of nature, inherited as either an autosomal dominant or autosomal recessive trait, reveals a crucial role for TNSALP in skeletal mineralization (1, 2).

In the healthy human skeleton, TNSALP functions as an ecto-enzyme anchored to the outer surface of specialized buds of osteoblast and chondrocyte plasma membranes called matrix vesicles, the structures within which hydroxyapatite crystals first form (4, 5). Here, inorganic pyrophosphate (PPi), pumped extracellularly by the membrane channel protein ANK (6) or produced extracellularly by nucleoside triphosphate pyrophosphatase (NTP-PPi-ase or PC-1), is hydrolyzed by TNSALP. The reaction removes this potent inhibitor of hydroxyapatite crystal nucleation and growth (7, 8, 9, 10, 11) while providing inorganic monophosphate ions (Pi) that perhaps further condition mineralization (12). In HPP, and in tnsalp knockout mice, hydroxyapatite crystals nucleate within matrix vesicles (primary mineralization), but excess extracellular PPi hinders hydroxyapatite crystal growth and proliferation after these structures rupture (secondary mineralization) (4, 10).

Five clinical forms of HPP are distinguished primarily by the affected subject’s age at diagnosis: the earlier the presentation, generally the more severe the skeletal disease and biochemical manifestations (1, 2). Nearly all babies with perinatal HPP die in utero or shortly after birth. Those with infantile HPP present before 6 months of age with rickets, failure to thrive, or vitamin B6-dependent seizures (2), and approximately 50% succumb to respiratory failure because of poor lung development or progressive hypomineralization of the rib cage (1). These two, the most severe forms of HPP, are transmitted as autosomal recessive traits with considerable TNSALP mutation heterogeneity (13).

There is no established medical treatment for HPP (1). Augmenting circulating alkaline phosphatase (ALP) activity into, or even above, the normal range for several months using intravenously (iv) administered ALP from various tissue sources has had no convincing beneficial effect (14, 15, 16). Perhaps TNSALP must be attached to the extracellular surfaces of matrix vesicles and osteoblasts to hydrolyze TNSALP substrates in vivo, including PPi (17, 18). Alternatively, great excesses of TNSALP expressed outside the skeleton, or infused into the circulation, might be needed to reduce PPi accumulation in the skeletal microenvironment (11).

In 1997, an 8-month-old girl with infantile HPP underwent, after full myeloablation, bone marrow transplantation (BMT) using T-cell-depleted marrow from her 5/6 HLA-matched sibling (19). This therapeutic trial followed preliminary, favorable findings concerning BMT for tnsalp knockout mice (20). During the first 6 months post BMT (pBMT), the patient showed clinical and radiographic improvement without correction of the biochemical features of HPP. However, clinical deterioration with skeletal demineralization occurred 13 months pBMT (21 months of age). Therefore, she then received, by iv infusion, bone marrow cells that had been expanded ex vivo (19). Six months later, considerable, lasting clinical and radiographic improvement ensued, still without correction of her biochemical abnormalities. Her osseous tissue has not been studied for donor stromal cell engraftment (19).

Yet, most reports show that the stroma after conventional BMT or after iv infusion of mesenchymal cells expanded ex vivo remains host, despite full engraftment of donor hematopoietic stem cells (HSCs) (21, 22, 23, 24). Additionally after 1997, the cellular origin of osteoprogenitors became clearer (25), and in mouse models replacement of marrow stroma could be partially achieved using donor bone fragments placed intraperitoneally (ip) or subcutaneously (sc) in the recipient (26, 27). Donor stromal cells migrated from the bone fragments to the recipient bone and other tissues, including the thymus (28).

Therefore, in 1999, we attempted this type of approach to introduce TNSALP-replete osteoblasts to treat a second severely affected girl with infantile HPP. Our strategy was to administer a heterogeneous population of cells, including the use of bone fragments, by three different routes (ip, sc, and iv) to enhance their migration and homing to the stroma. We hoped that engraftment of these cells within the skeletal microenvironment would allow precursor cells to replicate and to differentiate into functional osteoprogenitor cells.


   Case Report
Top
 Abstract
 Introduction
 Case Report
Materials and Methods
 Results
 Discussion
 References
 
A 5-month-old girl was admitted with vomiting, failure to thrive, hypotonia, hypercalcemia, and renal insufficiency. She had widely patent cranial sutures, a full anterior fontanel, diminished muscle tone, bowed femurs, and cried when touched.

Ultrasound at 3.5 months gestation had revealed bowing of her long bones, raising a concern about osteogenesis imperfecta (OI). Delivery was at term by cesarean section. Radiographs showed curved lower extremities but no fractures.

At age 6 wk, limb radiographs revealed worse skeletal disease. By age 4 months, she had gained only 1 kg. Abnormal growth plates (physes) and poor skeletal mineralization were consistent with rickets.

Upon hospitalization at age 5 months, serum biochemical studies (Table 1Go) showed distinctly low total ALP activity of 24 IU/liter (normal range for infants, 130–350), high Ca concentration of 14 mg/dl (8.5–10.5), ionized Ca 1.88 mmol/liter (1.15–1.35), Pi 3.3 mg/dl (4.0–7.0), magnesium 2.1 mg/dl (1.4–1.9), and renal compromise with creatinine (crt) 1.1 mg/dl (0.3–0.5). The urine Ca/crt ratio showed hypercalciuria that fluctuated between 0.6 and 1.0 mg/mg (normal, <0.5). Both serum parathyroid hormone at 7 pg/ml (normal, 10–67) and 1,25-dihydroxyvitamin D at 28 pg/ml (normal, 27–71) were physiologically suppressed by the hypercalcemia. She was anemic. Radiographs documented significant undermineralization of the calvarium, giving the illusion of widely separated cranial sutures. Long bones were poorly mineralized, with bowing of the femurs and humeri. Long bone metaphyses were frayed with a cupped appearance (Fig. 1AGo). Computerized tomography showed nephrocalcinosis bilaterally, kyphosis, and generalized skeletal hypomineralization.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Biochemical studies

 

Figure 1
View larger version (52K):
[in this window]
[in a new window]

  		FIG. 1. Radiographs of the right knee illustrate the severity of the skeletal disease before (A–C) compared with after (D–G) BT. The radiographs at –4 months (A), –2 months (B), and –11 d (C) show significant deterioration of the skeleton with disappearing epiphyses, and the changes are severe through d +17 pBT (1 month pBMT) (D). However, the radiographs at +3.5 months (E), +13.5 months (F), and +6 yr (G) show significant progressive improvement in the physeal widening and reappearance of the epiphyses as early as +3.5 months pBT (E). Metaphyseal mineralization continues to improve afterward, but lucent defects, characteristic of childhood HPP, persist after +6 yr (G).

 
Infantile HPP was confirmed when three ALP substrate levels were found elevated: plasma pyridoxal 5'-phosphate (PLP) (Table 1Go) and urine PPi and phosphoethanolamine (data not shown).

The hypercalcemia was attributed primarily to diminished mineral uptake by the skeleton and managed initially with iv fluids and furosemide. Then, renal function improved (serum crt 0.5 mg/dl). Her diet was changed to a low Ca preparation, Similac PM60/40 (Ross Products, Abbott Laboratories, Abbott Park, IL); i.e. from 46 to 38 mg Ca per 100 ml. Prednisone was not given. However, when supplemental fluids and furosemide were discontinued, serum Ca rebounded to 13 mg/dl. Salmon calcitonin (4 IU/kg twice daily sc) and iv fluids improved the hypercalcemia. When discharged after 11 d, serum Ca was 11.1 mg/dl and Pi 5.3 mg/dl, and urine Ca/crt ratio remained elevated at 2.4. Calcitonin therapy was continued, and oral Pi supplementation consisted of NeutraPhos (sodium/potassium phosphate) at 2 mmol three times daily (30 mg/kg/d) to compensate for the reduced Pi content of the special formula (19 vs. 31 mg/100 ml in conventional infant formula); urinary Ca/crt eventually fell to 0.62. Because of persistent tachypnea and tachycardia attributed to worsening anemia, she received a red blood cell transfusion.

Readmission 2 wk later (age 5.5 months) with projectile vomiting and decreased activity revealed fullness of the anterior fontanel, widely split cranial sutures, obvious rachitic rosary, and pain on manipulation of her extremities. Serum Ca was elevated to 12.2 mg/dl, Pi 5.3 mg/dl, blood urea nitrogen 17 mg/dl, crt 0.5 mg/dl, and albumin 4.2 gm/dl. Serum total ALP was 39 IU/liter and 1 wk later 24 IU/liter. To further manage her hypercalcemia, she received iv pamidronate (0.5 mg/kg/d) for 3 d after discontinuing calcitonin. Etidronate (EHDP), 5 mg/kg by mouth twice daily, was then started.

At age 9.5 months, after discussion with her parents and with Institutional Review Board approval (All Children’s Hospital, University of South Florida, St. Petersburg, FL), she was admitted for marrow cell transplantation. EHDP (taken with seemingly good compliance for 3.5 months) was discontinued 20 d before the procedure. Physical examination was essentially unchanged, except she was less irritable with better control of the hypercalcemia. Renal ultrasound revealed advanced nephrocalcinosis. A fourth radiographic survey showed progression of her severe skeletal disease (Fig. 1Go, A–C) including worsening rickets. The calcaneus and talus of the right foot appeared completely unmineralized. There was now narrowing of her chest, with 12 rib fractures and profound hypomineralization of the thoracic and lumbar vertebrae (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). It was feared this situation would soon be fatal.


   Materials and Methods
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
Results
 Discussion
 References
 
Transplantation

The nonmyeloablative preparative regimen of reduced intensity for transplantation, including anti-thymocyte-globulin, followed previously reported methods (29) (see the supplemental Appendix, published as supplemental data). On the day of BMT, d 0, the patient received her father’s marrow (4/6 match, B and DR mismatch).

On d 16 pBMT (see supplemental Appendix citing Ref. 32), six bone fragments (2 mm x 8–10 mm) were obtained from the donor father. Two fragments were inserted into the patient ip, and two were placed sc near the iliac crest. The remaining two bone fragments were used to culture osteoblasts (30, 31). We henceforth call this procedure bone transplantation (BT).

On d 12 pBT, the patient received the osteoblast-like cells. Additional bone marrow cells were also given, in an attempt to establish bone marrow engraftment, because of failure of hematopoietic engraftment.

SRY determination and PCR analysis

Twenty months pBT, biopsy of the patient’s iliac crest was used to determine whether donor cells were present in her skeleton (see supplemental Appendix citing Ref. 33). Sex-determining region Y chromosome gene (SRY) detection sensitivity is 1/1000 cells (34).

PCR analysis, using short tandem repeats (STR) as previously reported (35), was performed in 2006 to quantitate engraftment using frozen DNA from the patient’s bone marrow and bone obtained 20 months pBT (2001). STR detection sensitivity is 1/100 cells, and quantitation error ±3%.

Biochemical studies

For biochemical study details, see the supplemental Appendix, which cites Refs. 36 and 37 .

TNSALP gene studies

The patient and parents were screened for TNSALP mutations as previously described (38) (see supplemental Appendix).


   Results
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
Discussion
 References
 
Clinical course post marrow infusion and placement of bone fragments

The first pBT radiographs at d +17 showed worsening of the periarticular bone and thoracolumbar kyphosis but some slight improvement in skeletal mineralization (Fig. 1DGo). By d+23, the patient’s appetite increased with a weight gain of 1.2 kg, and she was no longer irritable, although her total serum Ca level was 11.8 mg/dl, ionized Ca was slightly elevated at 1.38 mmol/liter, and Pi was 7.6 mg/dl. Her serum ALP had risen on average by 8 U (range, 34–61 IU/liter; normal, 130–350 IU/liter).

Cytogenetics on d +39 pBT showed no circulating donor cells. Nevertheless, the patient’s serum Ca had decreased, and by 2.5 months pBT, it was normal at 10.5 mg/dl, and Pi was 5.9 mg/dl.

A skeletal survey at 3.5 months pBT showed significant improvement in skeletal mineralization, with less kyphotic curvature and a decrease in the widened (rachitic) growth plates in the shoulders and knees (Fig. 1EGo). Epiphyses that had disappeared were now reappearing. However, extensive metaphyseal cupping and flaring as well as femoral bowing persisted.

Dual-energy x-ray absorptiometry (DXA), 14 months pBT, revealed that the bone mineral density (BMD) and bone mineral content of the hips had increased 16 and 46%, respectively, over the previous 9 months. In the lumber spine, these increases were 60 and 156%, respectively. Her weight was 9 kg (–2.2 SD) and height 80 cm (–1.9 SD). Serum Ca and Pi levels were normal, but her ALP activity remained low at 44 IU/liter. Her legs were slightly bowed.

Radiographic studies and urine chemistries performed at 20 months pBT showed improvement. Nevertheless, there was no change in her serum ALP or plasma PLP level (Table 1Go).

At 4 yr pBT, radiographs revealed continued improvement in the areas of radiolucency, especially in the proximal femurs. Radiolucencies in the proximal tibias and ankles appeared to be slowly improving (Fig. 1Go, E and F), but smaller lucencies persisted 6 yr pBT (Fig. 1GGo).

The patient was studied once in St. Louis, at age 7 yr 5 months (6.5 yr pBT). Stable nephrocalcinosis persisted. Nonprogressive weakness, a feature of HPP, perhaps accounting for delayed gross motor development (rolled over at 18 months, sat up at 2 yr, and walked at 3 yr) remained. Coxa valga and scoliosis (now 47°), diagnosed at age 2 yr, had progressed. Remarkably, there was no atraumatic premature loss of deciduous teeth, a clinical hallmark of pediatric HPP (1, 2). She had significant, progressive genu valgum (17 cm) and could not run. Skull x-rays revealed acquired pansuture closure with prominent gyral impressions of the inner table. Height (Z-score –2.4) and arm span were equal, despite significant scoliosis. DXA revealed L1–L4 BMD Z-score of –2.8 (only slightly low for height). Thus, her phenotype was now that of childhood HPP.

SRY and STR analysis

PCR amplification of genomic DNA for SRY determination (sensitivity 1/1000 cells) at 20 months pBT, using patient bone cleansed free of marrow, revealed a faint band at the SRY 600-bp marker (Fig. 2Go). No band indicative of donor engraftment was found in the patient’s peripheral blood or bone marrow amplified and analyzed simultaneously.


Figure 2
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 2. SRY determination using PCR analysis of DNA obtained from bone, bone marrow, and peripheral blood at +1.6 yr pBT. Small arrows to lanes 2 and 4 demonstrate a faint band from the patient’s bone sample. Lane 1, Recipient bone at suboptimal PCR conditions (60 C); lane 2, repeat of recipient bone DNA under optimal PCR conditions, with band at 600 bp at 55 C; lane 3, same as lane 1; lane 4, same as lane 2; lane 5, donor/father’s peripheral blood lymphocytes, showing a very bright band at 600 bp (long arrow); lane 6, recipient peripheral blood lymphocytes; lane 7, recipient bone marrow; lane 8, negative control, unrelated healthy female; lane 9, DNA ladder, 1000 bp on the bottom to 50 or 30 bp at the top.

 
In 2006, there was no evidence by STR analysis of donor engraftment, on a recipient DNA marker background, in this bone biopsy specimen from 2001. However, the sensitivity is established to be 1%, so lower levels of engraftment would not be detected.

TNSALP mutation analysis

Two mutations were found in the patient [c.571G>A, p.E191K (paternal) and c.1289A>G, p.N430S (maternal)] (supplemental Fig. 2). See the supplemental Appendix, which cites Refs. 39 and 40 , for details.


   Discussion
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
References
 
There is no proven medical treatment for HPP (1). Attempts to bypass the osteoblast defect by iv infusion of various types of soluble ALP have generally failed to improve the disorder’s clinical or radiographic features or to reduce endogenous phosphoethanolamine, PPi, or PLP (14, 15, 16). In 1995, however, we reported that pregnant women who are carriers for severe HPP circulate substantial levels of the placental ALP isoenzyme during the third trimester and correct their hypophosphatasemia and plasma PLP and urinary PPi levels, with an abrupt return to the baseline after the placenta is delivered (41). Here, plasma membrane-anchored placental ALP (or substantial levels of circulating placental ALP) may be bioactive. The impact of pregnancy on the HPP skeleton is not known. Hence, it was uncertain whether treatment of HPP would require TNSALP be attached to cellular surfaces (19), or whether especially high circulating ALP levels would enable skeletal mineralization. A decade later, in 2005, it was shown that tnsalp knockout mice transfected with tnsalp (controlled by the ApoE promoter and a liver-specific enhancer) had very high (>10-fold) circulating levels of TNSALP and corrected excess osteoid without ectopic calcification (11). Elevated skeletal levels of osteopontin (another mineralization inhibitor) were also reported in tnsalp knockout mice (7). Murshed et al. (11) concluded from this, and other experiments, that tnsalp coexpressed on osteoblast/chondrocyte plasma membranes together with a fibrillar collagen-rich scaffold accounts for physiological mineralization. However, their study did not exclude improved mineralization from tnsalp anchored within the liver itself correcting excessive levels of PPi systemically.

In general, the clinical severity of HPP correlates inversely with the patient’s age at presentation and with the age-adjusted serum TNSALP activity, and directly with the plasma PLP concentration (1). Serum total ALP levels seemed slightly higher, but did not correct, in our patient pBT (ranging from 32–64 IU/liter) and were consistent with values encountered in the childhood form of HPP (1). They were in keeping with engraftment of small numbers of donor osteoblasts. Perhaps, TNSALP increases within the skeleton alone accounted for the clinical and radiographic improvement. In fact, as had been observed for the first patient to undergo marrow cell transplantation (MCT) for HPP, our patient had considerable clinical and radiographic improvement without consistent decreases in her elevated plasma PLP level (19). This suggests that positive effects occurred locally at mineralizing surfaces, rather than from systemically lowering TNSALP substrates. In fact, healthy osteoblasts in culture can cause mineralization without detectable changes in extracellular PPi (18). Hence, lowering PPi at mineralization sites seems more important than the endogenous burden of PPi. Additionally, PPi levels in the urine may not fully reflect endogenous PPi levels because some results from renal biosynthesis (20).

Perhaps replacing marrow stroma and/or osteoblasts using TNSALP-replete donor cells will be necessary to allow TNSALP to act at mineralization sites. Conventional BMT, involving iv infusion of marrow, has not replaced recipient stroma even when the host stroma is intrinsically abnormal (21, 22, 23, 24). This failure occurs although bone marrow contains mesenchymal stem cells, also known as marrow stromal cells (MSCs) (42). In 2002, Lee et al. (24) studied the osteoporosis that follows conventional BMT for malignancy and found no evidence, 1 yr later, of donor-derived osteoblasts.

In 1999, Horwitz et al. (43) performed full myeloablative BMT for six children with severe OI and later infused them iv with gene-marked donor cells that were mostly nonadherent in culture. Their engraftment never exceeded 1%, despite good HSC engraftment, and was only temporary (42). In fact, for severe OI from structural defects in type I collagen, it may be necessary to transplant in utero (44), i.e. before the skeleton is fully formed, when tolerance still exists, and provide a relatively greater number of normal osteoblasts (45). Similar considerations could apply for perinatal HPP.

Friedenstein et al. (46), and later Krebsbach et al. (47), demonstrated two new important requirements for the proliferation and differentiation of these multipotential cells. They cloned marrow stromal fibroblasts (MSFs) from the adherent cell layer and showed that bone formation was achieved only when these MSFs were in a three-dimensional vehicle (a diffusion chamber) under the kidney capsule or sc. MSFs administered iv failed to form osteoblasts or bone.

In 2002, Koc et al. (21) attempted a new approach to replace host stroma in lysosomal storage diseases. They had found stroma remained host 1–14 yr after HSC engraftment after conventional BMT (22), and despite HSC enzyme replacement, halting glycosaminoglycan deposition in soft tissues, little improvement occurred in the skeletal dysplasia. Accordingly, Koc et al. (21) infused iv donor MSCs from bone marrow expanded 5,000- to 10,000-fold ex vivo, together with donor bone marrow. The skeletal disease did not improve, and only two of 12 patients reportedly showed 1–2% "donor" MSCs (23).

Why processed MSCs, or marrow MSCs, ultimately fail to engraft after iv infusion may be due to 1) loss of adhesion molecules for migration and homing to the marrow (48), 2) MSCs in culture differentiate and lose their ability to self-renew and undergo apoptosis (49), and 3) rejection (50, 51, 52).

Accordingly, in 1999, for our current HPP patient, we followed the mouse model of Ikehara and Good (see Ref. 26), who demonstrated that cells from bone fragments placed ip will migrate to and replace bone marrow stroma as well as appear in other tissues (27, 28). These migrating cells were macrophage negative (anti-Mac-1–), but donor positive (anti-H-2Kd+). Additionally, El-Badri et al. (31) had reported that cotransplantation of osteoblast-like cells cultured from bone and infused iv with HSCs could substitute for ip bone fragments to enhance engraftment. Thus, we used both donor bone fragments placed ip and sc as well as osteoblasts propagated ex vivo and given iv, hoping to engraft osteoblast progenitor cells as well as HSCs into our patient’s skeleton.

For our patient, despite unsuccessful HSC engraftment after BMT (see below), bone fragments ip and sc and cultured osteoblasts iv (BT) seemed to reverse her skeletal deterioration. However, there is uncertainty concerning any MSC engraftment. The bands (Fig. 2Go) corresponding to donor SRY were faint, suggesting limited donor engraftment in bone only. Donor MSC engraftment was not revealed by PCR for STRs performed 6 yr later using original DNA. This would suggest that very minimal engraftment (<1%) can significantly improve the clinical consequences of TNSALP deficiency. Seven years later, her phenotype is childhood HPP (Fig. 1GGo). This novel transplantation procedure seems to have been beneficial. Similar skeletal improvements occurred in the first HPP transplantation patient (19). Surprisingly, this second patient after BT did not lose deciduous teeth prematurely, a complication seen in even the most mildly affected children with HPP (odontohypophosphatasia). Indeed, for both transplantation patients, the disease phenotype changed from infantile to childhood HPP.

Perhaps, in both patients, improvement occurred spontaneously or as a result of the transplantation regimen itself (19). However, spontaneous improvement after significant skeletal deterioration during the first year of life, noted in both patients, has not been observed in infantile HPP (1). We have previously reviewed, for the first patient, which factors in a transplantation regimen could affect the HPP skeleton (19). It seems improbable that other pyrophosphatases (11) were enhanced by BT. EHDP for hypercalcemia, however, may have been an extenuating factor in our patient. Bisphosphonates (pamidronate and then EHDP) were administered before BMT for her symptomatic hypercalcemia accompanying a demineralizing skeleton (53) up to 20 d before BMT. Positive effects of bisphosphonates (including EHDP), i.e. increasing the number of colony-forming units-fibroblast colonies and differentiation and proliferation of osteoblasts both in vitro and in vivo, have been reported in young and aged mice (54, 55). However, no reports concern bisphosphonate exposure in HPP; and EHDP, which resembles PPi, in high doses reversibly inhibits mineralization (56, 57). Conceivably, EHDP exacerbated our patient’s skeletal deterioration, but improvement in her radiographs was not seen until 170 d, not sooner, after the drug was stopped.

Our secondary, but unsuccessful, aim was to facilitate HSC engraftment by cotransplanting MSCs, as others have subsequently reported in clinical trials (58) and animal models (26, 27, 28, 31, 59, 60). In most genetic diseases treated with transplantation (e.g. Hurler syndrome and thalassemia), HSC engraftment is especially difficult despite a full myeloablative preparative regimen (61, 62). In our patient, failure of HSC engraftment, despite MSC cotransplantation, was possibly explained by the nonmyeloablative regimen, compounded by a mismatched, T-cell-depleted transplant, plus the small dose of infused CD34+ cells. Full engraftment of HSCs occurred in the first transplanted HPP patient despite a mismatch at DR and T-cell depletion, but appeared to be facilitated by her having had a full myeloablative regimen.

In the first HPP patient treated by MCT (19), full HSC engraftment may have subsequently allowed engraftment of the MSCs in the second marrow cell infusion given 13 months later (i.e. tolerance). Full engraftment of donor MSCs from the first marrow cell infusion is inhibited by host cells that initially survive such transplantation and play a role in posttransplantation rejection, namely host macrophages, T and B cells (63), dendritic cells (64), and especially natural killer (NK) cells (50, 51, 52). Recent evidence shows a major histocompatibility complex restriction between both MSCs and HSCs and the host immune system, such that a genotypic mismatch may impair MSC (50, 51, 52, 65) as well as HSC (26, 27, 66) engraftment. Gurevitch et al. (67) demonstrated that MSCs could be transplanted only in mice that had first been engrafted with compatible donor HSCs, whereas third-party stromal cells were rejected or failed to engraft. Tolerance to MSCs may be variable, depending on the recovery of donor NK cells after transplantation (66, 68). Replacement of the stroma can be accomplished in part by placement of bone tissue ip, sc, and directly into bone, as was demonstrated in animal models (26, 27, 28) and reported in two other transplantation patients (35). Alternatively, a second marrow infusion to establish sufficient numbers of MSCs, vis-à-vis the first MCT for HPP (19), is best performed later in the presence of mixed chimerism without any evidence of graft-vs.-host disease (GvHD) (69).

In conclusion, for infantile HPP that is worsening and will likely prove fatal, considerations for MSC transplantation can now be based on experience from two patients. The goal is to first establish engraftment with HSCs to avoid the potential problem of MSC rejection. In the future, the preparative regimen to establish mixed chimerism might better be achieved by either 1) a full myeloablative transplant or 2) a known reduced-intensity regimen that includes anti-thymocyte-globulin starting at d –21 through d –19 (70) to eliminate host NK cells. Infants born with the worst prognostic indicators, respiratory compromise or vitamin B6-dependent seizures, might be candidates for especially urgent transplantation. When the donor is haploidentical or mismatched, T cells must be depleted to prevent GvHD because of the detrimental effect of GvHD on NK cell replacement (69). After successful HSC engraftment and the establishment of tolerance, MSCs would then be introduced. Because our patient, as well as those with different genetic bone diseases, may take considerable time to arrest and reverse the pathological skeletal process (71), sequential introductions of bone fragments and osteoblasts/MSCs iv in the setting of mixed chimerism may become necessary, depending on the precise nature and severity of the specific disorder.


   Acknowledgments
 
We are grateful to Vivienne Lim and Dawn Russell (Shriners Hospital for Children, St. Louis, MO) for help with preparation of the manuscript and figures, respectively, to Dr. Xiafang Zhang (Washington University School of Medicine, St. Louis, MO) for TNSALP gene analysis, and to Dr. Tom Mueller (Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, FL) for PCR analysis. Shrine volunteer Cindy Webster provided expert secretarial help.


   Footnotes
 
This work was supported by Shriners Hospitals for Children, The Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund, The Hypophosphatasia Research Fund, and The Barnes-Jewish Hospital Foundation.

This work was presented in part at the 43rd Annual Meeting of The American Society of Hematology, December 7–11, 2001, Orlando, FL [Blood 98:796a, 2001], and the 27th Annual Meeting of The American Society for Bone and Mineral Research, September 23–27, Nashville, TN [J Bone Mineral Res 20(Suppl 1):S42, 2005].

The authors have no conflict of interest.

First Published Online May 22, 2007

Abbreviations: BMT, Bone marrow transplantation; BT, bone transplantation; crt, creatinine; DXA, dual-energy x-ray absorptiometry; EHDP, etidronate; GvHD, graft-vs.-host disease; HPP, hypophosphatasia; HSC, hematopoietic stem cell; MCT, marrow cell transplantation; MSC, marrow stromal cell; MSF, marrow stromal fibroblast; NK, natural killer; OI, osteogenesis imperfecta; pBMT, post BMT; Pi, inorganic monophosphate ion; PLP, pyridoxal 5'-phosphate; PPi, inorganic pyrophosphate; STR, short tandem repeats; TNSALP, tissue-nonspecific isoenzyme of alkaline phosphatase.

Received September 28, 2006.

Accepted May 15, 2007.


   References
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 

  1. Whyte MP 2001 Hypophosphatasia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, eds. The metabolic and molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 5313–5329
  2. Whyte MP 1994 Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 15:439–461[Abstract/Free Full Text]
  3. Henthorn PS, Raducha M, Fedde KN, Lafferty MA, Whyte MP 1992 Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. Proc Natl Acad Sci USA 89:9924–9928[Abstract/Free Full Text]
  4. Anderson HC, Hsu HH, Morris DC, Fedde KN, Whyte MP 1997 Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals. Am J Pathol 151:1555–1561[Abstract]
  5. Cai G, Michigami T, Yamamoto T, Yasui N, Satomura K, Yamagata M, Shima M, Nakajima S, Mushiake S, Okada S, Ozono K 1998 Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras. J Clin Endocrinol Metab 83:3936–3942[Abstract/Free Full Text]
  6. Ho AM, Johnson MD, Kingsley DM 2000 Role of the mouse ank gene in control of tissue calcification and arthritis. Science 289:265–270[Abstract/Free Full Text]
  7. Harmey D, Johnson KA, Zelken J, Camacho NP, Hoylaerts MF, Noda M, Terkeltaub R, Millan JL 2006 Elevated skeletal osteopontin levels contribute to the hypophosphatasia phenotype in Akp2–/– mice. J Bone Miner Res 21:1377–1386[CrossRef][Medline]
  8. Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR, Whyte MP 1999 Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 14:2015–2026[CrossRef][Medline]
  9. Wennberg C, Hessle L, Lundberg P, Mauro S, Narisawa S, Lerner UH, Millan JL 2000 Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice. J Bone Miner Res 15:1879–1888[CrossRef][Medline]
  10. Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti E, Camacho NP, Millan JL 2004 Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am J Pathol 164:841–847[Abstract/Free Full Text]
  11. Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G 2005 Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 19:1093–1104[Abstract/Free Full Text]
  12. Robison R, Soames KM 1924 The possible significance of hexosephosphoric esters in ossification. Part II. The phosphoric esterase of ossifying cartilage. Biochem J 18:740–754[Medline]
  13. Mornet E 2006 Tissue non-specific alkaline phosphatase gene mutations database. Accessed October 20, 2006, at http://www.sesep.uvss.fr/database_hypo/hypophosphatasia.html
  14. Whyte MP, McAlister WH, Patton LS, Magill HL, Fallon MD, Lorentz Jr WB, Herrod HG 1984 Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J Pediatr 105:926–933[CrossRef][Medline]
  15. Whyte MP, Magill HL, Fallon MD, Herrod HG 1986 Infantile hypophosphatasia: normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J Pediatr 108:82–88[CrossRef][Medline]
  16. Whyte MP, Habib D, Coburn SP, Tecklenburg F, Ryan L, Fedde KN, Stinson RA 1992 Failure of hyperphosphatesemia by intravenous infusion of purified placental alkaline phosphatase (ALP) to correct severe hypophosphatasia: evidence against a role for circulating ALP in skeletal mineralization. J Bone Miner Res 7:S155 (Abstract)
  17. Fedde KN, Cole DE, Whyte MP 1990 Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline phosphatase in cultured skin fibroblasts. Am J Hum Genet 47:776–783[Medline]
  18. Johnson KA, Hessle L, Vaingankar S, Wennberg C, Mauro S, Narisawa S, Goding JW, Sano K, Millan JL, Terkeltaub R 2000 Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1. Am J Physiol Regul Integr Comp Physiol 279:R1365–R1377
  19. Whyte MP, Kurtzberg J, McAlister WH, Mumm S, Podgornik MN, Coburn SP, Ryan LM, Miller CR, Gottesman GS, Smith AK, Douville J, Waters-Pick B, Armstrong RD, Martin PL 2003 Marrow cell transplantation for infantile hypophosphatasia. J Bone Miner Res 18:624–636[CrossRef][Medline]
  20. Fedde KN, Blair L, Terzic F, Anderson HC, Narisawa S, Millan JL, Whyte MP 1996 Amelioration of the skeletal disease in hypophosphatasia by bone marrow transplantation using the alkaline phosphatase-knockout mouse model. Am J Hum Genet 59:A15
  21. Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W 2002 Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 30:215–222[CrossRef][Medline]
  22. Koc ON, Peters C, Aubourg P, Raghavan S, Dyhouse S, DeGasperi R, Kolodny EH, Yoseph YB, Gerson SL, Lazarus HM, Caplan AI, Watkins PA, Krivit W 1999 Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 27:1675–1681[CrossRef][Medline]
  23. Awaya N, Rupert K, Bryant E, Torok-Storb B 2002 Failure of adult marrow-derived stem cells to generate marrow stroma after successful hematopoietic stem cell transplantation. Exp Hematol 30:937–942[CrossRef][Medline]
  24. Lee WY, Cho SW, Oh ES, Oh KW, Lee JM, Yoon KH, Kang MI, Cha BY, Lee KW, Son HY, Kang SK, Kim CC 2002 The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells. J Clin Endocrinol Metab 87:329–335[Abstract/Free Full Text]
  25. Aubin JE 1998 Bone stem cells. J Cell Biochem Suppl 31:73–82
  26. Ishida T, Inaba M, Hisha H, Sugiura K, Adachi Y, Nagata N, Ogawa R, Good RA, Ikehara S 1994 Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. Complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor. J Immunol 152:3119–3127[Abstract]
  27. Hisha H, Nishino T, Kawamura M, Adachi S, Ikehara S 1995 Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol 23:347–352[Medline]
  28. Li Y, Hisha H, Inaba M, Lian Z, Yu C, Kawamura M, Yamamoto Y, Nishio N, Toki J, Fan H, Ikehara S 2000 Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: a role of stromal cells in positive selection. Exp Hematol 28:950–960[CrossRef][Medline]
  29. Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G, Varadi G, Kirschbaum M, Ackerstein A, Samuel S, Amar A, Brautbar C, Ben-Tal O, Eldor A, Or R 1998 Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 91:756–763[Abstract/Free Full Text]
  30. Robey PG, Termine JD 1985 Human bone cells in vitro. Calcif Tissue Int 37:453–460[Medline]
  31. El-Badri NS, Wang BY, Cherry, Good RA 1998 Osteoblasts promote engraftment of allogeneic hematopoietic stem cells. Exp Hematol 26:110–116[Medline]
  32. Chen XD, Qian HY, Neff L, Satomura K, Horowitz MC 1999 Thy-1 antigen expression by cells in the osteoblast lineage. J Bone Miner Res 14:362–375[CrossRef][Medline]
  33. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN, Fellous M 1990 Genetic evidence equating SRY and the testis-determining factor. Nature 348:448–450[CrossRef][Medline]
  34. Fiegler H, Knabel M, Franz M, Kolb HJ, Just U 2002 Determination of donor-type chimerism using a semi-quantitative PCR-based method in a canine model for bone marrow transplantation. Vet Immunol Immunopathol 84:61–70[CrossRef][Medline]
  35. Cahill RA, Jones OY, Klemperer M, Steele A, Mueller TO, el-Badri N, Chang Y, Good RA 2004 Replacement of recipient stromal/mesenchymal cells after bone marrow transplantation using bone fragments and cultured osteoblast-like cells. Biol Blood Marrow Transplant 10:709–717[CrossRef][Medline]
  36. Cheung CP, Suhadolnik RJ 1977 Analysis of inorganic pyrophosphate at the picomole level. Anal Biochem 83:61–63[CrossRef][Medline]
  37. Mahuren JD, Coburn SP 1990 B6 vitamers: cation exchange HPLC. J Nutr Biochem 1:659–663[CrossRef][Medline]
  38. Mumm S, Jones J, Finnegan P, Henthorn P, Podgornik MN, Whyte MP 2002 Denaturing gradient gel electrophoresis analysis of the tissue non-specific alkaline phosphatase isoenzyme gene in hypophosphatasia. Mol Genet Metab 75:143–153[CrossRef][Medline]
  39. den Dunnen JT, Antonarakis SE 2001 Nomenclature for the description of human sequence variations. Hum Genet 109:121–124[CrossRef][Medline]
  40. Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon-Bouy B, Serre JL, Mornet E 1999 Correlations of genotype and phenotype in hypophosphatasia. Hum Mol Genet 8:1039–1046[Abstract/Free Full Text]
  41. Whyte MP, Landt M, Ryan LM, Mulivor RA, Henthorn PS, Fedde KN, Mahuren JD, Coburn SP 1995 Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy. J Clin Invest 95:1440–1445[Medline]
  42. Prockop DJ 1997 Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74[Abstract/Free Full Text]
  43. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, Muul L, Hofmann T 2002 Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA 99:8932–8937[Abstract/Free Full Text]
  44. Le Blanc K, Gotherstrom C, Ringden O, Hassan M, McMahon R, Horwitz E, Anneren G, Axelsson O, Nunn J, Ewald U, Norden-Lindeberg S, Jansson M, Dalton A, Astrom E, Westgren M 2005 Fetal mesenchymal stem cell engraftment in bone following in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation 79:1607–1614[CrossRef][Medline]
  45. Marini JC 1999 Osteogenesis imperfecta calls for caution. Nat Med 5:466–467[Medline]
  46. Friedenstein AJ, Chailakhyan RK, Gerasimov UV 1987 Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20:263–272[Medline]
  47. Krebsbach PH, Kuznetsov SA, Satomura K, Emmons RV, Rowe DW, Robey PG 1997 Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 63:1059–1069[CrossRef][Medline]
  48. Rombouts WJ, Ploemacher RE 2003 Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia 17:160–170[CrossRef][Medline]
  49. Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R 2000 Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 28:707–715[CrossRef][Medline]
  50. Eliopoulos N, Stagg J, Lejeune L, Pommey S, Galipeau J 2005 Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice. Blood 106:4057–4065[Abstract/Free Full Text]
  51. Poggi A, Prevosto C, Massaro AM, Negrini S, Urbani S, Pierri I, Saccardi R, Gobbi M, Zocchi MR 2005 Interaction between human NK cells and bone marrow stromal cells induces NK cell triggering: role of NKp30 and NKG2D receptors. J Immunol 175:6352–6360[Abstract/Free Full Text]
  52. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L 2006 Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107:1484–1490[Abstract/Free Full Text]
  53. Lteif AN, Zimmerman D 1998 Bisphosphonates for treatment of childhood hypercalcemia. Pediatrics 102:990–993[Abstract/Free Full Text]
  54. Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G 1998 Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone 22:455–461[Medline]
  55. McCarthy EA, Raggio CL, Hossack MD, Miller EA, Jain S, Boskey AL, Camacho NP 2002 Alendronate treatment for infants with osteogenesis imperfecta: demonstration of efficacy in a mouse model. Pediatr Res 52:660–670[CrossRef][Medline]
  56. Fleisch H, Reszka A, Rodan G, Rogers M 2002 Bisphosphonates: mechanisms of action. In: Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. 2nd ed. San Diego: Academic Press; 1361–1385
  57. Fleisch H 1998 Bisphosphonates: mechanisms of action. Endocr Rev 19:80–100[Abstract/Free Full Text]
  58. Koc ON, Gerson SL, Cooper BW, Dyhouse SM, Haynesworth SE, Caplan AI, Lazarus HM 2000 Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307–316[Abstract/Free Full Text]
  59. Almeida-Porada G, Flake AW, Glimp HA, Zanjani ED 1999 Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 27:1569–1575[CrossRef][Medline]
  60. Noort WA, Kruisselbrink AB, in’t Anker PS, Kruger M, van Bezooijen RL, de Paus RA, Heemskerk MH, Lowik CW, Falkenburg JH, Willemze R, Fibbe WE 2002 Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34+ cells in NOD/SCID mice. Exp Hematol 30:870–878[CrossRef][Medline]
  61. Grewal SS, Krivit W, Defor TE, Shapiro EG, Orchard PJ, Abel SL, Lockman LA, Ziegler RS, Dusenbery KE, Peters C 2002 Outcome of second hematopoietic cell transplantation in Hurler syndrome. Bone Marrow Transplant 29:491–496[CrossRef][Medline]
  62. Sodani P, Gaziev D, Polchi P, Erer B, Giardini C, Angelucci E, Baronciani D, Andreani M, Manna M, Nesci S, Lucarelli B, Clift RA, Lucarelli G 2004 New approach for bone marrow transplantation in patients with class 3 thalassemia aged younger than 17 years. Blood 104:1201–1203[Abstract/Free Full Text]
  63. Kraus AB, Shaffer J, Toh HC, Preffer F, Dombkowski D, Saidman S, Colby C, George R, McAfee S, Sackstein R, Dey B, Spitzer TR, Sykes M 2003 Early host CD8 T-cell recovery and sensitized anti-donor interleukin-2-producing and cytotoxic T-cell responses associated with marrow graft rejection following nonmyeloablative allogeneic bone marrow transplantation. Exp Hematol 31:609–621[CrossRef][Medline]
  64. Chklovskaia E, Nowbakht P, Nissen C, Gratwohl A, Bargetzi M, Wodnar-Filipowicz A 2004 Reconstitution of dendritic and natural killer-cell subsets after allogeneic stem cell transplantation: effects of endogenous flt3 ligand. Blood 103:3860–3868[Abstract/Free Full Text]
  65. Roth C, Carlyle JR, Takizawa H, Raulet DH 2000 Clonal acquisition of inhibitory Ly49 receptors on developing NK cells is successively restricted and regulated by stromal class I MHC. Immunity 13:143–153[CrossRef][Medline]
  66. Ruggeri L, Capanni M, Casucci M, Volpi I, Tosti A, Perruccio K, Urbani E, Negrin RS, Martelli MF, Velardi A 1999 Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 94:333–339[Abstract/Free Full Text]
  67. Gurevitch O, Prigozhina TB, Pugatsch T, Slavin S 1999 Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment. Transplantation 68:1362–1368[CrossRef][Medline]
  68. Westerhuis G, Maas WGE, Willemze R, Toes REM, Fibbe WE 2005 Long-term mixed chimerism after immunologic conditioning and MHC-mismatched stem-cell transplantation is dependent on NK-cell tolerance. Blood 106:2215–2220[Abstract/Free Full Text]
  69. Cooley S, McCullar V, Wangen R, Bergemann TL, Spellman S, Weisdorf DJ, Miller JS 2005 KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation. Blood 106:4370–4376[Abstract/Free Full Text]
  70. Shenoy S, Grossman WJ, DiPersio J, Yu LC, Wilson D, Barnes YJ, Mohanakumar T, Rao A, Hayashi RJ 2005 A novel reduced-intensity stem cell transplant regimen for nonmalignant disorders. Bone Marrow Transplant 35:345–352[CrossRef][Medline]
  71. Bianco P, Riminucci M, Gronthos S, Robey PG 2001 Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180–192[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cahill, R. A.
Right arrow Articles by Whyte, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cahill, R. A.
Right arrow Articles by Whyte, M. P.
Related Collections
Right arrow Calcium and Bone Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals