Nature Medicine
5, 309 - 313 (1999)
doi:10.1038/6529
Transplantability and therapeutic effects of bone marrow-derived mesenchymal
cells in children with osteogenesis imperfectaEdwin M. Horwitz1, Darwin J. Prockop2, Lorraine A. Fitzpatrick3, Winston W. K. Koo4, Patricia L. Gordon1, Michael Neel1, Michael Sussman5, Paul Orchard6, Jeffrey C. Marx1, Reed E. Pyeritz2
& Malcolm K. Brenner11 Cell and Gene Therapy Program, St. Jude Children's
Research Hospital, 332 North Lauderdale, Memphis,
Tennessee 38105, USA 2 Allegheny University of the Health Sciences,
15th and Vine Streets, Philadelphia,
Pennsylvania 19102, USA 3 Mayo Clinic, 200 First Street SW, Rochester,
Minnesota 55905, USA 4 Wayne State University, 4707 St. Antoine
Boulevard, Detroit, Michigan 48201, USA 5 Shriner's Hospital for Children, 3101
SW Sam Jackson Park Road, Portland, Oregon
97201, USA 6 University of Minnesota, 420 Delaware
Street, SE, Minneapolis, Minnesota 55455,
USA
Correspondence should be addressed to Edwin M. Horwitz In principle, transplantation of mesenchymal progenitor cells would attenuate
or possibly correct genetic disorders of bone, cartilage and muscle, but clinical
support for this concept is lacking. Here we describe the initial results
of allogeneic bone marrow transplantation in three children with osteogenesis
imperfecta, a genetic disorder in which osteoblasts produce defective type
I collagen, leading to osteopenia, multiple fractures, severe bony deformities
and considerably shortened stature. Three months after osteoblast engraftment
(1.5−2.0% donor cells), representative specimens of trabecular bone
showed histologic changes indicative of new dense bone formation. All patients
had increases in total body bone mineral content ranging from 21 to 29 grams
(median, 28), compared with predicted values of 0 to 4 grams (median, 0) for
healthy children with similar changes in weight. These improvements were associated
with increases in growth velocity and reduced frequencies of bone fracture.
Thus, allogeneic bone marrow transplantation can lead to engraftment of functional
mesenchymal progenitor cells, indicating the feasibility of this strategy
in the treatment of osteogenesis imperfecta and perhaps other mesenchymal
stem cell disorders as well.Bone marrow contains not only precursors for the hemopoietic system, but
also cells that can give rise to mesenchymal lineages, including bone, cartilage
and muscle1,
2,
3,
4,
5,
6,
7,
8. In preclinical models, transplanted
marrow-derived mesenchymal cells migrated to and became incorporated into
bone and muscle of the recipient animals, indicating that these cells have
a 'homing' capacity8,
9,
10,
11. In principle, therefore, bone
marrow transplantation could be used to correct a far wider range of inherited
and acquired disorders than now occurs. Support for this has come almost exclusively
from murine models, leaving several principal questions unanswered. It is
unknown, for example, whether mesenchymal cells from human marrow are capable
of engrafting in allogeneic hosts and whether such cells can differentiate
and function normally in vivo. To begin to address these questions,
we undertook bone marrow transplantation in three patients with osteogenesis
imperfecta.
Osteogenesis imperfecta (OI) is a genetic disorder of mesenchymal cells
in which generalized osteopenia leads to bony deformities, excessive fragility
with fracturing, and short stature. The underlying defect is a mutation in
one of the two genes encoding type I collagen, the primary structural protein
of bone12. There is no cure for OI, nor is there any proven
therapy for alleviating its symptoms12,
13,
14; although pamidronate,
one of the bisphosphonate compounds, may have some therapeutic potential15. We chose this disorder to validate the principle of mesenchymal
progenitor cell transplantation because engraftment of mesenchymal cells in
a murine model of OI produced a small but appreciable improvement in the disease
phenotype16. Here we demonstrate that marrow-derived mesenchymal
cells can indeed engraft in humans and generate donor-derived osteoblasts
that function sufficiently well for 6 months, to attenuate the biochemical,
structural and clinical abnormalities associated with OI.
Engraftment of hemopoietic and mesenchymal cells
Three children with severe deforming OI (Table )
were intravenously infused with unmanipulated bone marrow from HLA-identical
or single-antigen-mismatched siblings after they had received ablative conditioning
therapy. All three showed engraftment with hemopoietic donor cells. Patient
1 had a mixed hemopoietic chimerism (21% donor cells) that was stable for
more than 6 months. In patients 2 and 3, more than 99% of the hemopoietic
cells analyzed were of donor origin. Osteoblasts were cultured from fresh
bone biopsy specimens. The individual adherent cells in the cultures had typical
osteoblast morphology, expressed alkaline phosphatase and produced stainable
matrix. Flow cytometric analysis of these cells indicated a lack of contaminating
lymphohemopoietic cells (Fig. 1). Fluorescence in
situ hybridization to detect the Y chromosome in osteoblasts collected
from patient 1 on day 101 after transplantation showed that 1.5% of the cells
were of donor origin. DNA polymorphism analysis of osteoblasts from patient
2, collected on day 80 after transplantation, demonstrated 2.0% donor cells
(Fig. 2). Osteoblasts could not be grown from patient
3, precluding evaluation of engraftment with donor-derived mesenchymal cells.
 | | |
Changes in bone histology and mineral content
Engraftment was associated with improvements in bone histology (
Fig. 3). A specimen of trabecular bone taken before transplant from
the iliac wing of patient 1 contained numerous, disorganized osteocytes, enlarged
lacunae and relatively few osteoblasts (Fig. 3a).
The bone had the characteristic appearance of high bone turnover, including
woven bone, which is characteristic of OI (refs. 17, 18, 19)(Fig.
3e). Fluorescence microscopy of the same specimen showed a distorted
pattern of tetracycline labeling (Fig. 3c), consistent
with the disorganized formation of new bone and poor mineralization. In contrast,
similar specimens taken on day 216 after transplantation, near the site used
previously, showed a reduced number of osteocytes, linearly organized osteoblasts
and evidence of lamellar bone formation (Fig. 3b
and f). Because of fragmentation of the specimens,
full histomorphometric analysis could not be done; however, using a magnification
of  100, we counted the number of osteoblasts per high-power field in
biopsy specimens taken both before and after transplantation (five fields
each). There were 4.6  1.8 (s.e.m.) osteoblasts per high-power field
in the sample taken before transplantation, compared with 16.0 3.0
in the sample taken after transplantation (P = 0.005, t-test).
Fluorescence microscopy showed linear, single and double tetracycline labeling,
indicative of improved bone formation and mineralization. Similar histologic
changes were apparent in biopsy specimens from patients 2 and 3, taken from
the iliac wing opposite the initial specimen (not shown).
| | Figure 3. a, Biopsy specimen of trabecular bone before transplantation,
stained with Goldners-Masson trichrome. | | |  | The calcified tissue appears blue-green, and the uncalcified tissue is
red-brown. Numerous, randomly arranged osteocytes (OC) are present in large
lacunae. There are also peritrabecular marrow fibrosis, a paucity of osteoblasts
relative to the specimens after transplantation and an incompletely calcified
area of bone matrix. b, A specimen after transplantation stained with
Goldners-Masson trichrome, taken near the site shown in Fig.
2a. There are fewer osteocytes, and there is a small section
of lamellar bone (L), indicating normalization of the remodeling process.
Original magnification, 88. c, Fluorescence photomicrograph
of the tetracycline-labeled trabecular bone specimen (same section as in Fig. 2a). The labeling is poorly defined, indicating
disorganized formation of new bone and abnormal mineralization. d,
A contrasting specimen after transplantation with definitive, crisp, single
and double tetracycline labeling, indicative of considerably improved new
bone formation and mineralization. Original magnification, 56.
e, Trabecular bone specimen before transplantation, stained with toluidine
blue and photographed under polarized light to emphasize the woven (w) texture
of the bone, a characteristic feature of patients with osteogenesis imperfecta17,
18,
19. f, Bone specimen after transplantation stained
with toluidine blue and photographed under polarized light, demonstrating
lamellar bone (L) formation, and linearly arranged osteoblasts (OB) in areas
of active bone formation along the calcified trabecular surface. Original
magnification, 88.
 Full Figure and legend (94K) |
| |
There was also an increase in the total body bone mineral content, as determined
by measurements with dual energy X-ray absorptiometry. The three patients
accumulated 21−29 g of bone mineral (median, 28 g) during the first
100 days after transplantation in the absence of substantial weight gains
and with only small changes in body length (Fig. 4a).
In contrast, normal children would not be predicted to show changes in bone
mineral content in only 100 days without substantial changes in body mass20.
| | |
The most salient result was a 77% increase in total body bone mineral content
(21.6 g, from 28.0 to 49.6 g) in patient 1, who grew 2.5 cm and did not gain
weight during the first 100 days after transplantation. Patient 2 had a less
salient but still considerable 45% gain (29.8 g, from 67.0 to 96.8 g). This
13-month-old boy grew 2.0 cm and did not gain weight after transplantation.
Although patient 3 lacked interpretable absolute measurements
of total body bone mineral content, because of the presence of three intramedullary
rods, he did gain 28.6 g of bone mineral with minimal weight gain and grew
0.5 cm, in agreement with findings in patients 1 and 2.
Clinical correlates of improved osteogenesis
During the 13 months immediately preceding bone marrow transplantation,
patient 1 had 37 documented fractures, and patient 2 had 20. During the first
6 months after transplantation, only three fractures were identified in patient
1 by clinical assessment and radiographic skeletal survey, and only two were
found in patient 2. Patient 3, who had rods in both femurs and in the right
humerus, had three fractures during the 6 months just before transplantation,
but none during the next 6 months.
From 6 to 13 months of age, patients 1 and 2 achieved only 50% and 40%,
respectively, of the normal median growth velocity for age- and sex-matched
children21. After transplantation, patient 1 grew 8.0 cm in
7 months, and patient 2 grew 6.5 cm in 6 months, approximately 100% of the
normal median growth velocity. About two-thirds of this growth occurred after
100 days after transplantation. These results contrast with the usual slowing
of growth in children of this age group who have type III OI (22). Patient 3, who was 19 months older than the other two
patients, failed to grow during the 6 months preceding transplantation, but
during the next 6 months, he grew 1.5 cm, or 38% of the normal median velocity
(Fig. 4b).
Toxicity
Neither patient 1 nor patient 3 had clinically significant toxicity over
the transplantation course. The course for patient 2 was more complex, including
sepsis, pulmonary insufficiency and the development of a bifrontal hygroma.
Although the first two complications are recognized risks of bone marrow transplantation,
an association with bifrontal hygroma has not been described. Whether transplantation
produces unique neurologic complications in children with OI remains to be
determined. All of the complications in patient 2 have resolved, and the child
is doing well.
Discussion
Bone marrow transplantation has not been considered as a means to correct
disorders of mesenchymal cells. Its successful use to treat children with
osteopetrosis23 can be explained by the hemopoietic (rather
than mesenchymal) stem cell origin of the osteoclast, the defective cell in
this genetic bone disorder24. Our study demonstrates that mesenchymal
progenitors in transplanted marrow can migrate to bone in children with osteogenesis
imperfecta, and then give rise to osteoblasts whose presence correlates with
an improvement in bone structure and function. Engraftment of mesenchymal
cells in OI patients might be possible because the normal osteoblasts derived
from the allograft compete successfully with the recipient's cells that express
the genetic defect. Although the percent increases in total body bone mineral
content and actual linear growth velocities differed among the three patients,
the incremental gains in mineral content were similar, in agreement with the
similar doses of nucleated marrow cells received by these patients (Table).
This indicates that increased mineralization may be related to mesenchymal
cell dose. Finally, the accelerated growth velocity shown by each child during
the first 6 months after transplantation contrasts with the typical findings
in OI patients22 and with the usual clinical course in patients
undergoing marrow transplantation for disorders other than OI, in whom there
is maintenance or slowing of growth25,
26,
27,
28.
Selection of OI as a prototypic bone disorder was prompted mainly by observations
that mesenchymal progenitors can migrate to and become incorporated into bone
in murine models11,
16. Moreover, studies of the parents of
probands with lethal OI indicated that some parents were mosaic for the same
mutation in type I procollagen that produced severe OI in the offspring29. The mosaic parents were asymptomatic even though the ratio of
mutated-to-normal alleles in some tissues, including skin fibroblasts, approached
the value of 1:1 seen in the tissues of their affected offspring. This finding
indicates that the severity of the disease is essentially dependent on the
relative balance between the rates of synthesis of mutated and normal pro
polypeptide chains. Indeed, different lines of transgenic mice that expressed
various levels of the same mutated COL1A1 gene showed a range of OI manifestations30. Therefore, even low levels of mesenchymal progenitor cell engraftment
may be sufficient to produce a shift in the balance between the synthesis
of mutated and normal pro chains, thereby converting a severe OI phenotype
to a less-severe one.
This prediction helps to explain how the presence of only 1.5−2.0%
donor mesenchymal cells in our patients could lead to improvements in total
body bone mineral content, body growth, fracture incidence and bone histology.
The principle that low-level correction of a genetic defect can produce clinical
benefit is well-known in such diseases as chronic granulomatous disease31 and osteopetrosis23. Another consideration is that
the presence of a small fraction of normal osteoblasts could alter the osteogenic
microenvironment, as has been suggested in a study of osteopetrosis32.
This explanation might also account for the greater than expected increase
in osteoblasts at 6 months after transplantation.
An alternative is that engraftment may have been short-lived; hence, the
percentage of donor osteoblasts early after engraftment may have been greater
than during the cell sampling interval (at 80−101 days after transplantation).
The increased amounts of normal collagen fibers deposited by these cells might
well have provided a matrix for the deposition of mineral, resulting in stronger
bone that persisted longer than most donor osteoblasts. This could also explain
our observation that each child's total body bone mineral content increased
with minimal changes in height and weight. Although each child did grow between
the time of transplantation and the aborptiometry scan, we attribute the increase
of bone mineral content to enhanced mineralization of existing bone, possibly
because of an improved ratio of normal to mutated collagen.
A third explanation for the therapeutic effects observed after low-level
mesenchymal cell engraftment is that the changes were independent of the donor
osteoblasts, and were induced by the allogeneic transplantation procedure
itself. However, this seems unlikely, as similar effects have not been observed
in animal models11,
16, and total-body irradiation and the cytotoxic
drugs used for allogeneic transplantation generally have inhibitory (not stimulatory)
effects on growth and development of children24,
25,
26,
27,
28.
The ultimate value of bone marrow transplantation for OI will depend on
the ability to infuse adequate quantities of mesenchymal progenitors and to
devise clinical protocols that are both safe and easy to follow. It will also
be important to extend follow-up studies to learn whether early improvements
in bone structure and function reflect the activity of self-renewing stem
cells or merely that of progenitors with limited proliferative capacity. Finally,
the therapeutic activity of donor mesenchymal progenitor cells in patients
with OI indicates that bone marrow transplantation may also be feasible in
other disorders originating in mesenchymal progenitors, such as hypophosphatasia33 or possibly even muscular diseases8.
Methods
Patients and transplantation procedure.
All three children
with OI were enrolled (with informed parental consent) in a clinical trial
that had been approved by the Institutional Review Board of St. Jude Children's
Research Hospital. Each patient had a mutation of either the COL1A1
or COL1A2 gene that is associated with severe deforming (type III)
OI and had physical features indicative of poor bone growth and development
(Table).
The bone marrow transplantation conditioning regimens received by these
patients are outlined in the Table. Moderate-dose
total-body irradiation was added to the regimen for patient 2 because of a
mismatch at the HLA DR 1 allele with his sibling donor. Mesna was always
administered with cyclophosphamide, and phenytoin with busulfan. Unmanipulated
bone marrow freshly harvested from a sibling donor was intravenously infused
into each patient. Chemoprophylaxis against graft-versus-host disease consisted
of intravenous cyclosporine (2.5 mg/kg every 12 hours), begun 2 days before
transplantation.
Growth evaluation.
Each patient was measured from crown
to heel by the same investigator (P.L.G.) before and at 6 months after transplantation.
Growth velocity was determined as the difference between measurements at these
two intervals, which were chosen to avoid any confounding effects of growth
spurts and plateaus. Angulations of the bony deformities, which can alter
direct measurements of body length, were unchanged over the 6-month observation
period, as indicated by a radiographic skeletal survey.
Bone histologic studies.
Patients received 3-day courses
of tetracycline at approximately 3 weeks and 1 week before biopsy. A 5.0-mm
core of iliac bone was taken before and 6 months after transplantation with
a trephine inserted through a 1.5 cm incision, from patients sedated by general
anesthesia. Histologic changes were determined on sections 5  m in thickness
of polymethyl methacrylate-embedded samples, using a Zeiss microscope.
Mesenchymal cell cultures.
Osteoblasts from bone biopsies
were prepared and maintained in culture as specifically described for this
cell type by Robey and Termine34. Bone fragments were dissected
from soft tissue, progressively 'minced' to a fine granular consistency, digested
with collagenase and placed into culture. Flow cytometric analysis indicated
a lack of lymphohemopoietic cells in the osteoblast preparations (
Fig. 1).
Chimerism studies.
For sex-mismatched donor-recipient
pairs, peripheral blood or cultured mesenchymal cells (passage 1) were analyzed
by fluorescence in situ hydridization to determine the sex chromosome
ratio, according to the manufacturer's suggestions (VYSIS, Downers Grove,
Illinois). For sex-matched pairs, chimerism was determined by analysis of
DNA polymorphisms between donor and recipient (PCR amplification of a variable
number of tandem-repeat sequence) by collaborators at the Molecular Diagnostics
Laboratory, University of Minnesota.
Dual-energy X-ray absorptiometry.
These measurements
of total body bone mineral content were done on a whole-body scanner with
a pediatric platform (Hologic QDR 2000 Densitometer; Hologic, Inc., Waltham,
Massachustetts), as described20,
35,
36.
Received 15 July 1998; Accepted 30 December 1998
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Acknowledgments
We acknowledge J. Marini for discussions throughout this study; and S.
Nooner, W. Cabral, B. Hopkins and M. Kinnarney for assistance. We also thank
J. Gilbert for his editorial review and J. Johnson for assistance in preparation
of this manuscript. This work was supported in part by NHLBI Clinical Investigator
Development Award #K08 HL 03266, by Cancer Center Support CORE Grant P30 CA
21765, by the Hartwell Foundation, and by the American Lebanese Syrian Associated
Charities (ALSAC).
|
) and after (
) transplantation.
