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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2131
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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.


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  		TABLE 1. Biochemical studies

 

Figure 1
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  		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
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  		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
 

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