Abstract
Background: Fibrodysplasia ossificans progressiva is a rare genetic
disorder of ectopic skeletogenesis associated with dysregulation of bone
morphogenetic protein (BMP) signaling. Hematopoietic cells have been
implicated in the ectopic skeletogenesis of fibrodysplasia ossificans
progressiva, and their replacement has been postulated as a possible cure.
However, the definitive contribution of hematopoietic cells to the
pathogenesis of ectopic skeletogenesis remains obscure.
Methods: We employed both careful clinical observation and in vivo
murine transplantation studies to more precisely determine the contribution of
hematopoietic cells to ectopic skeletogenesis. We identified a patient with
fibrodysplasia ossificans progressiva who had undergone bone marrow
transplantation for the treatment of intercurrent aplastic anemia twenty-five
years earlier and investigated whether the clinical course of the
fibrodysplasia ossificans progressiva had been influenced by bone marrow
replacement or immunosuppression, or both. In complementary studies, we
transplanted hematopoietic stem cells from constitutively expressing LacZ
transgenic mice to identify the contribution of hematopoietic cells to
BMP4-induced heterotopic ossification, a histopathologic model of
fibrodysplasia ossificans progressiva.
Results: We found that replacement of hematopoietic cells was not
sufficient to prevent ectopic skeletogenesis in the patient with
fibrodysplasia ossificans progressiva but pharmacologic suppression of the
apparently normal donor immune system following transplantation in the new
host modulated the activity of the fibrodysplasia ossificans progressiva and
diminished the expression of skeletal ectopia. In complementary murine
transplantation studies, we found that cells of hematopoietic origin
contributed to the early inflammatory and late marrow-repopulating stages of
BMP4-induced heterotopic ossification but were not represented in the
fibroproliferative, chondrogenic, or osteogenic stages of heterotopic
ossification. Interestingly, both recombinant human BMP4 induction in an
animal model and the dysregulated BMP signaling pathway in a patient with
fibrodysplasia ossificans progressiva were sufficient to recruit at least two
populations of cells, one of hematopoietic origin and at least one of
non-hematopoietic origin, that contribute to the formation of an ectopic
skeleton.
Conclusions: Taken together, these findings demonstrate that bone
marrow transplantation did not cure fibrodysplasia ossificans progressiva in
the patient in this study, most likely because the hematopoietic cell
population is not the site, or at least not the dominant site, of the
intrinsic dysregulation of the BMP signaling pathway in fibrodysplasia
ossificans progressiva. However, following transplantation of bone marrow from
a presumably normal donor, immunosuppression of the immune system appeared to
ameliorate activation of ectopic skeletogenesis in a genetically susceptible
host. Thus, cells of hematopoietic origin may contribute to the formation of
an ectopic skeleton, although they are not sufficient to initiate the process
alone.
Clinical Relevance: Therapeutic regulation of hematopoietic and
osteogenic cell populations involved in fibrodysplasia ossificans progressiva
lesions holds promise for treatment of fibrodysplasia ossificans progressiva
and possibly other disorders of heterotopic ossification.
The formation of a skeleton and its regeneration following injury
are complex developmental processes requiring the precise genetic control of
numerous morphogenetic signaling
pathways1. While the
cellular and molecular events of embryonic and postnatal skeletogenesis are
similar, inflammation is thought to play a critical role in the latter, but
not the former,
process2. The bone
marrow microenvironment contains cells responsible not only for the
maintenance of the skeleton but also for its repair and regeneration following
injury. Early in fracture-healing, circulating cells of hematopoietic origin
contribute to the fracture hematoma, and if this inflammatory phase is
disturbed, fracture-healing is
impaired2.
Circulating inflammatory cells of hematopoietic origin have also been
implicated in the induction of heterotopic ossification, but their precise
contribution and physiologic role remain
enigmatic3,4.
Recent studies have shown that primitive adult hematopoietic stem cells can
function as osteoblast
precursors5,6
and that osteogenic cells can be found in the peripheral blood of normal
individuals7 as well
as in patients who have fibrodysplasia ossificans
progressiva8.
Whether cells of hematopoietic origin are either necessary or sufficient for
heterotopic skeletogenesis, however, is presently unknown.
Fibrodysplasia ossificans progressiva is a rare and disabling genetic
disorder of progressive endochondral heterotopic ossification associated with
profound dysregulation of bone morphogenetic protein (BMP) signal
transduction4,9-21.
Recently, the genetic cause of fibrodysplasia ossificans progressiva was
identified as a recurrent mutation in the glycineserine (GS) activation domain
of ACVR1, a BMP type-I receptor; this mutation was identified in all
individuals with classic fibrodysplasia ossificans
progressiva22.
Patients who have fibrodysplasia ossificans progressiva essentially form two
skeletons—a normotopic one during embryogenesis and a heterotopic one
postnatally12,14,23.
BMPs, potent secreted skeletogenic morphogens, are necessary and sufficient
to induce the entire cascade of endochondral osteogenesis at ectopic
sites24,25
and induce a hematopoietic microenvironment that supports the growth of stem
cells26. BMPs are
chemotactic for hematopoietically derived mononuclear
cells27 that are
found at sites of BMP-induced heterotopic
ossification28 and
have profound inductive effects on mesenchymal stem-cell
populations25.
Hematopoietically derived mononuclear cells are also found in the earliest
preosseous lesions of patients with fibrodysplasia ossificans
progressiva28-31.
Furthermore, BMP4-induced heterotopic ossification is histologically identical
to fibrodysplasia ossificans progressiva
lesions28,32.
During the past decade, numerous studies have suggested involvement of the
hematopoietic system in the pathogenesis of fibrodysplasia ossificans
progressiva4,29-31.
Lymphocytes derived from patients with fibrodysplasia ossificans progressiva
overexpress BMP44,
are present in early fibrodysplasia ossificans progressiva
lesions29,30,
are associated with the death of skeletal
muscle19,29,
and dysregulate multiple BMP
antagonists13. The
occurrence of flare-ups following soft-tissue
trauma12,33,
viral infections34,
and
immunizations35,36
and the beneficial response of early flare-ups to
corticosteroids31
further support involvement of the immune system and hematopoietic system in
the pathogenesis of fibrodysplasia ossificans progressiva. As a result, some
have suggested that bone marrow transplantation might cure fibrodysplasia
ossificans
progressiva37-40.
To more precisely determine the contribution of specific stem-cell subsets
to the pathogenesis of fibrodysplasia ossificans progressiva, we employed both
careful clinical observation and in vivo murine transplantation studies. On
identifying a patient with fibrodysplasia ossificans progressiva who had
undergone bone marrow transplantation for the treatment of intercurrent
aplastic anemia twenty-five years earlier, we investigated whether the
clinical course of the disease had been influenced by bone marrow
transplantation or immunosuppression, or both. In complementary studies, we
transplanted stem cells from constitutively expressing LacZ transgenic mice to
identify specific bone-marrow-derived cells in lesions of BMP4-induced
heterotopic ossification.
A three-year-old boy was diagnosed with fibrodysplasia ossificans
progressiva on the basis of pathognomonic findings of congenital malformations
of the great toes and progressive heterotopic ossification in characteristic
anatomic
patterns15,41-43.
He had severe progressive heterotopic ossification of the axial skeletal
muscles and associated connective tissues consistent with the known patterns
of fibrodysplasia ossificans progressiva
progression42.
During the first decade of life, fibrodysplasia ossificans progressiva
lesions formed in the cervical, scapular, and paraspinous soft tissues,
limiting movement of the neck, shoulders, and back. Despite this, he was able
to walk, ride a bicycle, and engage in usual childhood activities. Easy
bruisability, nosebleeds, and pallor developed at the age of ten years. The
peripheral blood cell counts fell, and examination of bone marrow revealed
trilineage hypoplasia consistent with the diagnosis of severe aplastic anemia.
There was no known exposure to marrow toxins. A high-resolution karyotype was
normal. During the clinical presentation of the aplastic anemia, the
fibrodysplasia ossificans progressiva flare-ups diminished greatly in
intensity and frequency (Fig.
1).
The patient underwent transplantation of allogenic bone marrow from his
HLA-identical older sister following his immunoablation with cyclophosphamide,
but stable engraftment was not established. He received a second transplant
from the same donor following additional ablative therapy with nitrogen
mustard and treatment with antithymocyte globulin. The blood cell counts
recovered smoothly. Several months following the second bone-marrow
transplantation, a karyotype evaluation confirmed a female karyotype in 100%
of the peripheral blood cells
evaluated41.
Acute and chronic graft-versus-host disease developed after the
transplantation, and the patient was treated with prednisone, cyclosporine,
and methotrexate for fourteen years. When the patient was twenty-four years of
age, all medications were tapered and finally discontinued, without recurrence
of the graft-versus-host disease.
Flare-ups of fibrodysplasia ossificans progressiva were notably absent
following the second bone-marrow transplantation and for the ensuing fourteen
years while the patient received immunosuppressive medications. Over the
fifteen months following cessation of immunosuppression, mild flare-ups of
fibrodysplasia ossificans progressiva slowly returned in areas previously
involved by the disease. During this time and for the subsequent decade, the
patient did not return to the transplantation clinic for any medical
follow-up. Two years following cessation of all immunosuppressive therapy,
there were severe flare-ups of fibrodysplasia ossificans progressiva in
previously uninvolved areas, including the jaw, elbows, hips, and knees,
without a history of soft-tissue trauma or other precipitating events. Despite
two operative procedures to remove heterotopic bone, supported by preoperative
prophylactic radiation therapy and nonsteroidal anti-inflammatory medications,
heterotopic bone rapidly recurred. The fibrodysplasia ossificans progressiva
continued to progress, so that by the age of thirty-five, twenty-five years
following the bone marrow transplantation and eleven years following the
cessation of the immunosuppression, most of the axial and appendicular joints
were ankylosed by ribbons, sheets, and plates of heterotopic bone
(Fig. 1). The patient first
came to the attention of the fibrodysplasia ossificans progressiva medical
community at the age of thirty-five. Subsequently, the bone marrow donor was
seen and examined. She was well with healthy offspring and no evidence of any
musculoskeletal or immunological disorders.
Patient Protocols
The severe aplastic anemia was treated with a standard
protocol41. The
patient was followed in an outpatient clinic, where measurements of the
hematologic recovery, graft-versus-host disease, opportunistic infection, and
heterotopic ossification were performed and
recorded41.
Immunosuppressive therapy for the graft-versus-host disease consisted of
cyclosporine, methotrexate, and prednisone. Medications were adjusted to
provide clinical relief at the lowest effective
doses41. Blood
samples were obtained after informed consent was given in accordance with
institutional guidelines and institutional review board approval.
Cell Separations Prior to Analysis of Donor/Host Chimerism
Blood samples were combined with immunomagnetic beads (Dynal, Oslo,
Norway), which are superparamagnetic particles with monoclonal antibodies
coupled to their surfaces, and then were sorted and collected with use of a
magnetic field to recover cells attached to the specific monoclonal antibody.
Monoclonal antibodies used for cell subset separation from peripheral blood
were CD3 (pan T), CD14 (monocytes), CD15 (granulocytes), CD4 (CD4+ T-cells),
CD8 (CD8+ T-cells), and CD19 (B-cells).
Analysis of Donor/Host Chimerism
DNA was isolated from whole blood, from sorted cells, or from epithelial
cells from buccal swabs with use of Qiagen mini-columns (Hilden, Germany).
Chimerism analysis was performed by comparing informative short tandem repeat
(STR) loci between the patient's DNA before bone marrow transplantation and
his DNA after it.
The AmpFISTR Identifiler PCR Amplification Kit (ABI [Applied Biosystems],
Foster City, California), an STR multiplex assay, was used to amplify fifteen
tetranucleotide repeat loci and the amelogenin gene marker in a single
amplification tube. All sixteen primer sets were first tested for informative
polymorphisms. In our patient, six loci were informative. The GeneScan
software program (ABI) was used to compare the informative alleles between the
recipient and donor specimens. Alleles were differentiated by the number of
copies of the repeat sequence contained within the amplified region of each
STR. Alleles were distinguished from one another with use of an ABI Prism 3100
Genetic Analyzer, and chimerism analysis was reported as the percentage of
donor or recipient cells in the respective cell subsets tested.
Analysis of Donor/Host DNA for Fibrodysplasia Ossificans Progressiva
Gene Mutation
DNA was isolated from whole blood or from epithelial cells from buccal
swabs. Donor and host DNA was screened for the canonical ACVR1 (c.617G>A;
R206H) mutation with use of exon-specific primers as previously
described22. DNA
sequence analysis of genomic DNA was conducted on an ABI3730 sequencer through
the University of Pennsylvania School of Medicine DNA Sequencing Facility.
Sequence data were analyzed with use of 4Peaks software, version 1.6
().
Bone Marrow Transplantation in a Murine Model of BMP-4-Induced
Heterotopic Ossification
All animal studies were approved by the Institutional Animal Care and Use
Committee. Following irradiation with 1000 cGy, twenty-six C57BL/6 mice each
received a transplant of 5 × 106 bone marrow cells harvested
from seven Rosa26R-LacZ transgenic
mice44. Sixteen
weeks following transplantation, stable engraftment was confirmed through
density isolation of the mononuclear cells obtained from the peripheral blood
of two mice. These cells were fixed in 2% paraformaldehyde/0.25%
glutaraldehyde and then stained with X-gal solution and post-fixed in 2%
paraformaldehyde/0.25% glutaraldehyde. The cells were then spun onto glass
slides for counting. All cells from five high-power fields (400×) were
counted and evaluated for ß-galactosidase activity. The protocol was
repeated for the remaining twenty-four mice at the time of the matrigel plug
recovery. The average peripheral engraftment was 97%. Heterotopic ossification
was induced in the ventral abdominal musculature by percutaneous delivery of
BMP4 (50 µg/mL) carried in matrigel in a standard BMP-induced model of
heterotopic
ossification28.
Eight matrigel implants were recovered at four, seven, and fourteen days
following implantation, and the presence of bone-marrow-derived cells was
detected by histochemical staining for ß-galactosidase
activity44.
Donor/Host Chimerism in the Patient with Fibrodysplasia Ossificans
Progressiva
We investigated whether the presence and functional status of
normal, donor-derived hematopoietic stem cells correlated with the presence of
active disease following bone marrow transplantation in our patient with
fibrodysplasia ossificans progressiva and aplastic anemia. The assessment of
engraftment twenty-five years following the second bone-marrow transplantation
was done with molecular analysis rather than karyotype analysis; it compared
genomic DNA from a buccal swab (epithelial cells; pre-transplantation) with
that in peripheral blood (hematopoietic stem-cell-derived;
post-transplantation) from the patient.
To distinguish donor from host cell origin, an analysis of highly
polymorphic STR alleles that are specific to an individual patient or to the
donor is usually performed. Alternatively, the identity of cell origin can be
based on the patterns of pre-transplantation and post-transplantation samples
from the patient. In such situations, it is routine to use epithelial cells as
the pre-transplantation sample. However, no pre-transplantation sample from
our patient had been retained. Therefore, a buccal swab, which would contain
only cells of host origin, was obtained from the thirty-five-year-old patient
and was used as the "pre-transplantation" sample. The patient's
peripheral blood cells, which could contain cells of donor as well as host
origin, were used as the post-transplantation sample. STR alleles that are
found in the post-transplantation sample but not in the pre-transplantation
sample would represent alleles derived from the bone marrow donor. The
molecular analysis of genomic DNA reconfirmed persistence of complete
engraftment with 100% donor cells in all hematopoietic cell lineages
twenty-five years following bone marrow transplantation
(Table I).
Since the patient had severe progression of fibrodysplasia ossificans
progressiva despite complete and sustained engraftment with normal donor
hematopoietic cells, we inferred that the molecular abnormality responsible
for fibrodysplasia ossificans progressiva must be expressed, and its
consequences must be initiated, in a non-hematopoietic mesenchymal
cell—i.e., one that was not replaced by hematopoietic repopulation in
this patient. However, since, despite engraftment, the fibrodysplasia
ossificans progressiva was clinically quiescent for as long as the patient was
immunosuppressed, we inferred that normal donor hematopoietic cells must
interact with the genetically abnormal non-hematopoietic cells of the patient
to cause the pathophysiology of fibrodysplasia ossificans progressiva.
Mutational Analysis of the Fibrodysplasia Ossificans Progressiva Gene
(ACVR1) in the Patient and in the Bone Marrow Donor
The discovery of the fibrodysplasia ossificans progressiva gene (ACVR1) and
the recurrent nature of the mutation (c.617G>A; R206H) in all classically
affected individuals was reported during the time that this manuscript was
being reviewed22.
During the period of revision of the manuscript, we were able to obtain DNA
samples from the normal donor's peripheral blood as well as from the
peripheral blood and from the buccal swab (endogenous fibrodysplasia
ossificans progressiva DNA) of the patient with fibrodysplasia ossificans
progressiva. DNA sequence analysis confirmed the canonical heterozygous
c.617G>A; R206H mutation in ACVR1 from DNA isolated from the patient's
buccal swab (endogenous fibrodysplasia ossificans progressiva DNA). The
617G>A nucleotide mutation was not identified in the DNA isolated from the
peripheral blood of the normal donor or the host (donor blood following stable
engraftment). These results established the molecular basis of the patient's
fibrodysplasia ossificans progressiva, demonstrated that it was identical to
that in all other reported cases of classic fibrodysplasia ossificans
progressiva22,
confirmed that the patient's clinically normal sister did not harbor the
mutation, and established that there was complete stable engraftment following
transplantation from a normal related donor who did not have fibrodysplasia
ossificans progressiva.
Hematopoietic Contributions to BMP4-Induced Heterotopic
Ossification
We used a previously described in vivo model system in which heterotopic
ossification is induced by recombinant human BMP4 (rhBMP4)
implants28. To
better define the contribution of hematopoietic stem-cell populations to the
histopathology of heterotopic ossification, we induced osteogenesis with
rhBMP4 in mice that had previously undergone transplantation of bone marrow
from a genetically engineered LacZ-labeled transgenic donor.
Bone-marrow-derived cells in the resulting chimeric mice could therefore be
identified by ß-galactosidase expression. By four days following
implantation of BMP4, a fibroproliferative response was observed at the site
of implantation, and ß-galactosidase expression was detected in
infiltrating inflammatory cells but not in the fibroproliferative component of
the early lesion. By the seventh day, a chondrogenic response was present, and
chondrogenic cells remained negative for ß-galactosidase. By fourteen
days, an ossicle had formed, containing ß-galactosidase-labeled
bone-marrow elements surrounded by a shell of immature unlabeled bone. These
findings in the murine model corresponded to the previously described
histopathology of lesion formation in humans with fibrodysplasia ossificans
progressiva (Fig.
2)28,29.
These results indicate that, in this murine model of BMP4-induced
heterotopic ossification, hematopoietic cells contributed to the early
inflammatory stage and to the late marrow-repopulating stage of the developing
lesions and suggest that non-hematopoietic osteogenic connective-tissue
progenitors give rise to the fibroproliferative, chondrogenic, and osteogenic
cells of the heterotopic skeletal
anlagen45-47.
Heterotopic ossification, either induced by rhBMP4 in murine models
or occurring in clinical flare-ups of fibrodysplasia ossificans progressiva,
is a complex process of endochondral skeletogenesis that involves the creation
of fibroproliferative and chondrogenic anlagen and their eventual replacement
with
bone28,32,48.
Osteogenesis is therefore a later stage in the process of endochondral
ossification, whether it occurs embryonically in the formation of the
skeleton, postnatally during fracture repair, or postnatally in the formation
of an ectopic skeletal element. Skeletogenesis can also occur in the absence
of osteogenesis. This is best exemplified in the elasmobranchs, such as the
shark, which have cartilaginous skeletons but lack bone, and in the RUNX2
knockout mice, which have cartilaginous skeletons but lack all evidence of
bone
formation49.
Our data from bone-marrow-transplantation studies of mice and from a
twenty-five-year follow-up of a unique patient with fibrodysplasia ossificans
progressiva who had undergone bone marrow transplantation for treatment of
intercurrent aplastic anemia suggest that the early inflammatory and late
bone-marrow-repopulating stages of heterotopic skeletogenesis have
hematopoietic contributions. In contrast, the skeletal anlagen—the
fibroproliferative and cartilaginous primordia of ectopic
skeletogenesis—are derived primarily from as yet undefined osteogenic
connective-tissue progenitor cells in the target tissues of muscle and fibrous
connective tissue and are not primarily of hematopoietic origin
(Fig.
2)45-47.
Furthermore, follow-up investigations of the patient with fibrodysplasia
ossificans progressiva described in this study demonstrated that
fibrodysplasia ossificans progressiva-derived hematopoietic cells are not
necessary to induce heterotopic ossification. Rather, heterotopic ossification
in such a patient can occur with normal donor-derived hematopoietic cells.
Examination of our patient also suggested that bone marrow failure associated
with aplastic anemia, myeloablative immunosuppression, and/or chronic
immunosuppressive therapy was correlated with, and likely played a role in,
suppression of the induction of new episodes of heterotopic ossification for
many years. Taken together, these data suggest that suppression of the early
hematopoietic contribution to heterotopic ossification modulates the activity
of the connective tissue progenitors responsible for the formation of the
ectopic skeletal anlagen (Fig.
3). Interestingly, previous work by our group showed that, once
formed, the fibroproliferative cells in the fibrodysplasia ossificans
progressiva lesion produce robust amounts of BMP4 and that overactivity of the
BMP4 pathway is sufficient to drive the process of endochondral ossification
to completion in the absence of an inflammatory
stimulus10,28.
The recent discovery of a recurrent mutation in the activation domain of
ACVR1, a BMP type-I receptor, in all familial and sporadic cases of classic
fibrodysplasia ossificans progressiva suggests a cell autonomous basis for
fibrodysplasia ossificans
progressiva22.
Protein modeling of the mutant receptor predicts destabilization of the GS
activation domain, consistent with constitutive activation of ACVR1 as the
underlying cause of the ectopic chondrogenesis, osteogenesis, and joint
fusions seen in fibrodysplasia ossificans
progressiva22.
Furthermore, these findings allow us to hypothesize that trauma and
inflammation may recruit osteogenic connective-tissue precursors in which the
activating mutation is presumably expressed. These findings are entirely
consistent with in vivo observations in animal models and clinical studies
that showed that, once the endochondral anlagen are induced, abrogation of the
inflammatory response will not inhibit the formation of heterotopic
bone10,14.
Interactions between hematopoietic cells and mesenchymal cells in the
initiation, progression, and sustenance of bone formation in fibrodysplasia
ossificans progressiva probably occur at several steps. In addition to
contributing cells to the early inflammatory and late marrow-repopulating
stages of fibrodysplasia ossificans progressiva and BMP4-induced heterotopic
ossification, inflammatory cells including B-cells, T-cells, monocytes, and
mast cells play a vital role in recruiting and/or activating the connective
tissue precursor population (Fig.
3)3,29,30,50.
Recent studies have strongly suggested that migration of stem cells to sites
of inflammation is a key step in normal and disordered regenerative
responses51.
The status of our patient with fibrodysplasia ossificans progressiva, who
had aplastic anemia prior to transplantation, closely mimics an ablation of
the hematopoietic stem-cell compartment. As the aplastic anemia emerged, the
fibrodysplasia ossificans progressiva became quiescent and remained so for the
next fourteen years, during intense immunosuppression. Data from clinical and
basic-science studies strongly support the role of inflammation in the
induction of heterotopic
ossification3,14,29-31,50,52
and further support the autonomy of the process once inductive inflammatory
events have
occurred10,14.
Another key aspect of our patient's condition is that the clinical course
of fibrodysplasia ossificans progressiva was not worsened by the
graft-versus-host disease that developed following the transplantation. In
fact, there appeared to be a "fibrodysplasia ossificans progressiva
holiday" during emergence of the aplastic anemia and during
immunosuppression following the bone marrow transplantation. The findings
suggest that intensive immunosuppression, with such agents as cyclosporine
and/or methotrexate as well as corticosteroids, could have a strong salutary
effect on the clinical course of fibrodysplasia ossificans progressiva.
However, it is unclear whether immunosuppression in the absence of bone marrow
transplantation would be sufficient to reduce progressive heterotopic
ossification in a patient with fibrodysplasia ossificans progressiva who has
normal hematopoiesis. While it is assumed that the immunosuppressive therapy
had a direct action on the hematopoietic system and an indirect effect on the
connective tissue progenitors in fibrodysplasia ossificans progressiva, a more
direct effect of these drugs on connective tissue progenitors cannot be ruled
out on the basis of this single observation. These questions might best be
addressed in an animal model that precisely mimics the exact fibrodysplasia
ossificans progressiva mutation in ACVR1. The development of such an animal
model is presently under way.
The occurrence of idiopathic aplastic anemia in our patient with
fibrodysplasia ossificans progressiva was probably coincidental as we have not
seen idiopathic aplastic anemia in any other patient with fibrodysplasia
ossificans progressiva worldwide, although recent reports document the
importance of BMP signaling in regulating the hematopoietic stem-cell
niche53-57.
Flare-ups of fibrodysplasia ossificans progressiva are episodic, and
disability is cumulative throughout
life15,42,43.
Fibrodysplasia ossificans progressiva can remain relatively quiescent for long
periods of time even without palliative
treatments15.
However, two of us (F.S.K. and D.L.G.) have followed more than 500 patients
with fibrodysplasia ossificans progressiva worldwide and have not observed
complete quiescence of disease progression for fourteen years between the ages
of ten and twenty-four years in any affected individual. This is a time of
generally active disease flare-ups in most patients with fibrodysplasia
ossificans
progressiva42,43.
Lessons learned from our unique patient with fibrodysplasia ossificans
progressiva and from our associated murine experiments allowed us to construct
a hypothetical schema in which inflammatory events are central to the
production of heterotopic ossification, whether induced by extrinsic delivery
of rhBMP4 or associated with the intrinsically dysregulated BMP4 signaling
pathway in fibrodysplasia ossificans progressiva
(Fig. 3). This schema suggests
that multiple locations in the inflammatory cascade may be targets for
modulating the activation of connective tissue progenitors
(Fig. 3). However, once the
fibroproliferative stage of endochondral osteogenesis has been induced, the
process of heterotopic ossification is self-sustaining and is unimpaired by
inhibition of the inflammatory
cascade10,29,30.
Thus, inhibition of the inflammatory pathway is probably prophylactic but
likely is not therapeutic after the process is under way. Such
pathophysiologic modeling is supported by a wide array of additional in vivo
experimental
data14.
Many observations support the involvement of bone-marrow-derived cells in
the pathophysiology of fibrodysplasia ossificans
progressiva28-31.
The present findings suggest that the molecular defect or defects causing
clinical fibrodysplasia ossificans progressiva do not need to be
phenotypically expressed in hematopoietic cells to induce the pathological
changes in vivo. As directly and powerfully demonstrated by the case of the
patient described in this report, replacement of genotypically abnormal
hematopoietic stem cells with presumably normal ones by transplantation does
not cure fibrodysplasia ossificans progressiva. The results of our studies
suggest that the failure of a successful bone-marrow transplantation (with
reconstitution of the entire hematopoietic compartment) to prevent or correct
fibrodysplasia ossificans progressiva after withdrawal of immunosuppression
probably occurred either because cells of hematopoietic origin are not the
dominant source of the intrinsic abnormality in signaling that is responsible
for the fibrodysplasia ossificans progressiva phenotype or because cells of
hematopoietic origin are incapable of eliciting a fibrodysplasia ossificans
progressiva lesion in the absence of an intrinsically genetically predisposed
population of connective tissue precursors
(Fig. 3).
The fact that bone marrow transplantation did not cure fibrodysplasia
ossificans progressiva in one patient does not preclude the possibility that
cells of hematopoietic origin (even from normal donor marrow) or circulating
osteogenic cells of stromal origin may contribute to heterotopic osteogenesis
in a genetically susceptible
host7,58-69.
The results of our murine bone-marrow-transplantation experiments are
consistent with the findings of An et
al.26 and provide
strong support for the hypothesis that circulating cells of hematopoietic
origin contribute to the late phase of marrow repopulation but do not
contribute appreciably to the fibroproliferative or chondrogenic anlagen
necessary for the formation of new skeletal elements by endochondral
osteogenesis.
Recent data suggest that bone marrow contains a primitive cell able to
generate both the hematopoietic and the osteoblastic
lineages6. While it
may be unlikely that conventional bone-marrow transplantation will provide a
benefit if it is applied similarly to other patients with fibrodysplasia
ossificans progressiva, it may be wise not to categorically dismiss the
potential transplantability and opportunity for repopulation of osteogenic
progenitor cells. It is possible, for example, that osteogenic cells that are
therapeutically relevant are transplantable or may already have been
transplanted but failed to manifest during the observed period of time in the
described patient or in the associated mice experiments for one or more
reasons. For example, preparation of the recipient by irradiation does not
effectively deplete existing osteogenic cells in local tissues, limiting
potential sites for engraftment and leaving any engrafted cells far
outnumbered by the residual native cells. Also, because the rate of turnover
of stem cells upstream of the osteoblastic progenitor pool is much slower than
that of the hematopoietic population, particularly from late adolescence on,
transplantation at an earlier age, when the connective tissue progenitor
population is rapidly expanding, might provide a different downstream result.
Finally, conventional bone-marrow transplantation, which has been optimized to
harvest and transplant cells of the hematopoietic system, has not been
optimized for reconstitution of other connective-tissue progenitor-cell
compartments, such as bone and other tissues. Alternative methods for harvest
and processing of marrow or other tissues may improve the yield and the
engraftment of osteogenic cells.
In addition to cells of marrow origin, pluripotent adult stem cells with
osteogenic capacity have been isolated from many
tissues65,70-75.
Marrow stromal cells of non-hematopoietic origin can contribute to
osteogenesis in mice and humans with osteogenesis
imperfecta62,67,68.
Interestingly, there appears to be an increased number of hematopoietically
derived circulating osteoprogenitor cells during flare-ups in patients with
fibrodysplasia ossificans progressiva, suggesting that circulating
osteoprogenitor cells may be derived from both stromal and hematopoietic
precursors in the bone
marrow8. It would be
interesting to know if the heterotopic bone that formed under the influence of
donor marrow in the patient with fibrodysplasia ossificans progressiva
described here contained any hematopoietic cells of donor origin. We would
predict that such cells might be found in the late remodeling stages of the
mature heterotopic bone. However, such specimens were not available.
The developmental potential of stem cells cultured in vitro and the
endogenous function of stem cells in vivo may be vastly different because the
in vivo potential is more highly regulated in the endogenous stem-cell niche.
Thus, cell lines that can be manipulated to exhibit stem-cell properties in
vitro may not exhibit those same properties in
vivo72. The fact
that BMP4-induced heterotopic skeletogenesis, as seen in our animal model, is
so remarkably similar to fibrodysplasia ossificans progressiva illustrates how
the in vivo responses of tissues can be very similar even when the responses
are elicited by different mechanisms. This is illustrated by the fact that the
local connective-tissue-progenitor response to exogenous application of BMP4
can closely mimic the response of the intrinsically dysregulated population of
connective-tissue precursors in fibrodysplasia ossificans
progressiva28,32.
The question of which cell(s) contribute to the fibroproliferative and
chondrogenic mesenchymal anlagen in fibrodysplasia ossificans progressiva
lesions is fascinating, important, and unresolved. Taken together, our
studies, performed with two independent routes of investigation, support the
belief that such cells are not of hematopoietic origin but arise from a
different pool of connective-tissue progenitors residing in skeletal muscle
and associated connective tissues with possible lineage origins, such as
endothelial cells, smooth muscle cells, satellite cells, neural cells, or
other connective-tissue cells, including cell populations that may transit
through the
circulation14,73.
Therefore, multiple sources of pluripotent stem cells or progenitors may
contribute to the formation of an ectopic skeleton in a patient with
fibrodysplasia ossificans progressiva. Detailed lineage-tracing experiments in
transgenic mice with stable cell-lineage markers will be necessary to
definitively determine the origin of these cells. Such studies are presently
under way.
In summary, we showed that at least two populations of connective tissue
cells, one derived from circulating cells of hematopoietic origin and another
derived from an osteogenic connective-tissue-progenitor population (that gives
rise to the preosseous skeletal anlagen), are necessary to form an ectopic
skeleton. Therapeutic regulation of connective-tissue-progenitor populations
and the use of modulators of inflammation, hematopoietic cell function,
osteogenesis, and BMP signal transduction hold promise for controlling ectopic
organogenesis relevant for fibrodysplasia ossificans progressiva and perhaps
for many common disorders of heterotopic ossification in humans. ?
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