Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of
diseases. In addition, autologous marrow transplants potentially provide opportune means of
delivering genes in transfected, engrafting stem cells. However, relatively little is known about
the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and
with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone
marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers
of donor cells being detected by fluorescence in situ hybridization in whole femoral sections.
Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.
Key words:
 |
Introduction |
Marrow transplants are routinely used to correct a
wide variety of diseases. In addition, autologous
marrow transplants potentially provide opportune means of
delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic
origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage.
The bone marrow is encompassed by bone, a rigid structuring matrix that is subject to perpetual remodelling
through the combined actions of osteoclasts, which erode
matrix, and osteoblasts, which secrete it. Although osteoclasts are of hemopoietic origin (1, 2), osteoblasts are believed to be the progeny of mesenchymal stem cells (3, 4).
Differentiation involves the formation of fibroblast-like
progenitor cells, which can also give rise to cartilage, muscle, fat cells, tendon fibroblasts, and possibly other fibroblast
types (5). Cells committed to the osteoblast lineage can
be found in the periosteum as well as in the endosteum and
the bone marrow (7).
Osteoblasts are organized in layers at the bone surface,
working in a coordinated fashion to secrete new bone matrix. This matrix subsequently mineralizes extracellularly.
When osteoblasts become trapped in newly formed mineralized matrix and remain viable, they further differentiate
into osteocytes. Osteocytes, although they have no opportunity to divide or secrete further appreciable quantities of
bone matrix, play a significant role in the regulation of
bone matrix turnover. These cells lie in small cavities or lacunae within the bone matrix, and are connected to other
osteocytes through gap junctions at the end of cell processes, which radiate through tiny channels, or canaliculi.
Other osteoblasts become flattened on the bone surface,
and are known as bone lining cells.
In this study, we have used a male into nonablated female transplant model to show that at the individual cell
level in situ a whole bone marrow transplant contains cells
that engraft, differentiate, are capable of bone formation,
and reside within the bone tissue. Significant numbers of
donor cells were detected by fluorescence in situ hybridization (FISH) in whole femoral sections, as both osteocytes
encapsulated by mineralized matrix and residing within the
bone lacunae and as flattened bone-lining cells in the periosteum.
| |
Materials and Methods |
Mice.
6-8-wk-old BALB/c H-2D mice were purchased from
Charles River Labs. and were housed in a conventionally clean facility for at least 1 wk. All mice received mouse chow and acidified
water ad libitum. The BALB/c H-2D strain was chosen because
of its negligible immunoreactivity to the H-Y (Y chromosome-
associated histocompatibility) antigen (12, 13).
Cell Suspensions and Transplants.
Marrow was collected by
flushing femurs and tibiae with cold PBS supplemented with 5%
heat-inactivated FCS (Hyclone Labs.). The cells were filtered
through a 40-µm filter (), washed, and resuspended for injection in 0.4 ml of PBS per recipient.
Transplants involved a single or three daily intravenous infusions into nonablated female recipients of the same age. After 6 wk
or 6 mo, the presence of donor cells in the marrow was quantitated by Southern blot analysis and the spatial distribution of engrafted cells in the bone tissue was analyzed on whole femoral
marrow sections using FISH.
Southern Blot Analysis.
Marrow DNA extracts were prepared,
digested, and separated by gel electrophoresis, and analyzed by
Southern blot as previously described (14). The presence of Y
chromosome-specific DNA sequences was assessed using a pY2-cDNA probe (donated by I. Lemischka, Princeton University,
Princeton, NJ) (15). Sample loading variability was assessed and
adjusted by reprobing membranes with IL-3 cDNA (donated by
J. Ihle, DNAX, Palo Alto, CA). Probes were labeled with 32P.
The percentage of male DNA was quantified by PhosphorImager (Molecular Dynamics) analysis.
FISH.
In situ detection of individual cells using FISH on
sections of paraffin-embedded whole murine femurs was done as
previously described (16). In brief, mice were perfused with 4%
paraformaldehyde at physiological pressure into the descending
aorta. 5-µm sections were permeabilized using proteinase K enzymatic digestion. Sections were hybridized with a digoxigenin-labeled Y chromosome-specific painting probe (17) and stained
using antidigoxigenin rhodamine-Fab fragments. Levels of autofluorescence were determined by counter-staining in 0.2 µmol
DAPI (4,6-diamidino-2-phenylindole) (see Fig. 1 d). Sections
were mounted in Vectashield (Vector Labs.).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
FISH of femoral
marrow sections. Panel A, male
control showing positive Y
chromosomes (m; deep red/orange). Panel B, female control
(f ); autofluorescence appears yellow. Panel C, transplant recipient 6 wk after transplant of 1.2 × 108 male marrow cells. Sections
are viewed using a dual red and
green filter, and therefore have
no counter-staining. Marrow chimerism was 23.9 ± 2.7% (n = 5),
as determined by Southern blot
analysis (Fig. 2). Panel D, same
as C viewed using a DAPI filter.
Note complete lack of auto fluorescence in positive cell. bm, bone
marrow; d, positive bone cell
of donor origin; e, endosteum;
p, periosteum. Bars, 20 µm.
|
|
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Percentage of male DNA in marrow of individual recipient
female mice determined by Southern blot. Male was taken as 100% and
female as 0%. Loading variability was corrected using an IL-3 probe.
|
|
| |
Results |
To accurately assess the extent of donor chimerism and
the histological location of engrafted cells within the bone
tissue, cells of donor origin were detected by FISH, using a
Y chromosome-specific painting probe, in whole femoral sections.
Distribution of Engrafted Cells.
6 wk after a marrow transplant, cells of donor origin were detected as osteocytes encapsulated in lacunae (Figs. 1 and 3 A). As osteocytes are
terminally differentiated bone cells within the bone matrix
itself, these cells could not have been transplanted during
the original infusion but must be the progeny of engrafted, functionally active donor cells. In addition, donor cells
were evident both as flattened lining cells on the periosteal
bone surface and as cells within the endosteal growth region
(Fig. 3, D and G). It is also important to note that because
whole marrow was transplanted, a significant proportion of
marrow cells in the recipients were also of donor origin (refer to the legends of Figs. 1 and 3 for exact proportions).
View larger version (63K):
[in this window]
[in a new window]
|
Fig. 3.
Femoral marrow sections from female recipients 6 wk after transplant of 1.8 × 108 male marrow cells. Marrow chimerism was 31.3 ± 2.5%
(n = 13), as determined by Southern blot analysis. (A, D, and G) FISH labeling for the Y chromosome-specific probe; d, positive bone cells of donor origin (enlarged in the inserts). A, osteocyte; D, bone-lining cell; G, bone cell. (B, E, and H) Map of cells present both in A, D, and G and the respective
H and E-stained serial sections. C, osteocyte of donor origin within a bone matrix lacuna. F, bone-lining cell of donor origin (enlarged in the insert)
within the periosteum; I, bone cell of donor origin (enlarged in the insert) within the endosteum growth region. Bars, 20 µm.
|
|
A technical limitation of FISH is the removal of the actual bone matrix during the denaturation process. As a consequence, cells at the endosteal marrow surface were not
scored, avoiding any possible confusion between positive
bone marrow cells and positive bone cells once the bone
matrix was removed. To confirm that the cells analyzed
were both of donor and bone origin, serial, longitudinal
femoral sections were cut and every second section was
stained by FISH and every alternate section was stained
with hematoxylin and eosin (H and E). Individual cells
could then be identified in the bone lacunae, periosteum,
or endosteal growth region on the H and E sections (Fig. 3,
C, F, and I). Due to the thickness of each section (5 µm), a
significant number of cells were present in both the FISH
and serial H and E sections. For ease of identification, these
cells are pictorially represented in each figure (Fig. 3, B, E,
and H).
Engraftment Period.
Bone cells of donor origin were
easily detected both 6 wk and 6 mo after transplant, and no
difference in the incidence of these cells was observed at either of these time points. In all sections analyzed after a
transplant of 120 -180 × 106 marrow cells (resulting in
19.2 ± 2.9 to 31.3 ± 2.5% marrow chimerism), at least one
positive cell was detected. In greater than two-thirds of the
sections analyzed (n = between 2 and 9 sections for 3 and 2 individual mice at 6 wk and 6 mo, respectively), three or
more positive cells were detected. The maximum number
of positive cells observed in the bone matrix and the periosteal region of any individual, centrally cut longitudinal
section was 8, of which a significant number were in the
matrix itself. With a section being 5 µm thick, and the central region of a femur from a 12-wk-old mouse ~2-mm-thick, there is a conservative estimate of up to 3,200 of
these positive cells per femur. A section contained 2,870 ± 114 (n = 8) osteocytes, and therefore there was a maximum of 3 donor cells per 1,000 osteocytes.
Even up to the age of 6 mo, a mouse grows significantly.
Between the ages of 6 and 12 wk, the period of the 6-wk
transplant, this growth resulted in a significant 16.5% increase in body weight (18.3 ± 0.2 g and 21.9 ± 0.4 g,
SEM, respectively; P < 0.001 using a Wilcoxon rank-sum
test, n = 15 and 13 respectively). During this period of
time, bone growth, or specifically bone remodelling by resorption and formation, resulted in a 30.3% increase in
femoral mass (30.4 and 43.6 mg, respectively), as well as a
significant increase in size (Fig. 4). Consequentially, by 12 wk
of age there is essentially complete turnover of the bone
present at 6 wk of age, allowing osteocytes of donor origin
to be found anywhere within the bone matrix. In addition,
mice transplanted at 6 wk of age and analyzed at 6 mo of
age would have many complete turnovers of bone matrix
during the transplant period. This suggests that a cell of donor origin detected as an osteocyte in the bone matrix 6 wk
after transplant is extremely unlikely to be present 6 mo after transplant. Cells present in the bone matrix 6 mo after transplant are most likely to have been derived from cells
that were not actively producing bone 6 wk after transplant; instead, the donor osteocytes detected 6 mo after
transplant probably arose from an originally transplanted
osteoprogenitor.
View larger version (89K):
[in this window]
[in a new window]
|
Fig. 4.
Femurs isolated from a 6-wk-old (top) and a 12-wk-old
(bottom) mouse, demonstrating a period of substantial growth. Bar, 2 mm.
|
|
| |
Discussion |
Our data demonstrate that whole marrow contains cells
of the bone lineage that can engraft post-intravenous infusion, form bone, and give rise to osteocytes and bone lining
cells. These are easily detectable in the femur 6 wk and 6 mo
after transplant into nonablated mice. This occurs long-term in vivo, without any in vitro culturing manipulation.
The long-term stability in the incidence of bone cells of
donor origin is consistent with the stable bone marrow chimerism that we have previously observed over this period
of time (18).
The data support the suggestion by Pereira et al. (19) and
Prockop (20) that transplanted cells with bone forming capabilities engraft in the marrow of recipient mice and participate in normal biological turnover, in which the marrow provides a continual source of osteoblastic progenitor
cells. The data also supports the hypothesis proposed by
Horwitz et al. (21) that whole marrow contains enough of
these cells to be able to replace sufficient osteoblasts to be
clinically useful in diseases such as osteogenesis imperfecta,
where marrow ablation is standard preparative treatment. Other strategies for the therapeutic use of putative "mesenchymal stem cells" have involved the direct injection of
isolated and culture-expanded bone marrow cells into bone
lesions or joints. These techniques were shown to promote
lesion repair and joint resurfacing at the site of injection but
did not have systemic effects (7, 22).
Marrow stromal cells produce bone matrix when placed
in diffusion chambers and implanted into the peritoneum
(9, 10). Other studies have shown that fibroblast colonies,
or clones originating from single stromal cells (CFU-f ),
have high osteogenic potencies (21) and can form bone
(23). However, stromal cells are not repopulated in radiochimeras reconstituted by marrow transplantation (24).
If marrow fibroblasts are to be used for clinical therapies, transplanting them so that they engraft and are active may
be a major problem (29). Piersma et al. (30) have subsequently demonstrated that CFU-f could be repopulated in
an irradiated host when infused intravenously as part of a
whole marrow transplant, although the exact population
being assayed remains controversial. Keating et al. (31)
showed that the hemopoietic stromal microenvironment, defined as the adherent layer of human long-term marrow
cultures grown from patients who received normal marrow
transplants, progressively became donor with time after
transplant. However, Simmons et al. (32) and Lennon and
Micklem (33) more recently showed that the stromal fibroblastic cells in marrow cultures made from human allogeneic transplant recipients and long-term murine chimeras were mainly or entirely host derived. All these studies were
done in irradiated recipients, involved in vitro manipulation in order to discriminate donor versus host, and did not
go on to show functional feed-out of transplanted donor
cells in vivo. Therefore, it remains to be determined
whether stroma, which has been considered non-transplantable via intravenous infusion, lodges within the marrow when part of a whole bone marrow transplant.
Recently, Pereira et al. (19) showed that 1-5 mo after a
transplant into irradiated hosts of "mesenchymal cells" enriched by adherence to plastic, expanded in culture, and
marked with the human COL1A1 minigene, there was
widespread tissue distribution of connective tissue cells
containing and expressing the gene. Along with many
other tissues, such as brain, spleen, marrow, and lung, the
study showed positive signal for and expression of the gene
by PCR and reverse transcriptase PCR from ground bone. However, these positive results could be due to many different cell types, and they do not definitively prove the
presence of donor bone cells. In the studies of Pereira et al.
(19), the irradiation of recipient mice was necessary to ensure engraftment, and hence limits clinical application of
this strategy. Our data demonstrate that the need for isolation and purification of osteoblastic cells can be circumvented, and preparative marrow ablation treatments are not
required. In addition, the data show conclusively that not
only do these cells successfully engraft when included as part of a normal marrow transplant, but they are then functionally active, as only osteoblasts, which are actively producing bone, can become encapsulated within it and differentiate into osteocytes.
Previous studies have shown that marrow chimerism can
be achieved in mice without the need for marrow ablation
(14, 18). This study provides the first clear demonstration
of the in vivo derivation of osteocytes from a whole marrow transplant into nonablated recipients. With the development of new methodologies and transgenic mouse models, future studies will reveal further critical information at
the individual cell level, as well as possibilities in the treatment of bone disorders by bone marrow transplantation.
Address correspondence to S.K. Nilsson at her present address, Sir Donald and Lady Trescowthick Research
Laboratories, Peter MacCallum Cancer Institute, Locked Bag No. 1, A'Beckett Street, Melbourne, 3000, Australia. Phone: 61-3-9656-1280; Fax: 61-3-9656-1411; E-mail: s.nilsson{at}pmci.unimelb.edu.au
| 1.
|
Chambers, T.J..
1978.
Multinucleate giant cells.
J. Pathol.
126:
125-148
[Medline].
|
| 2.
|
Ash, P.,
J.F. Loutit, and
K.M. Townsend.
1980.
Osteoclasts
derived from haematopoietic stem cells.
Nature.
283:
669-670
[Medline].
|
| 3.
|
Caplan, A.I..
1991.
Mesenchymal stem cells.
J. Orthop. Res.
9:
641-650
[Medline].
|
| 4.
|
Bruder, S.P.,
D.J. Fink, and
A.I. Caplan.
1994.
Mesenchymal
stem cells in bone development, bone repair, and skeletal regeneration therapy.
J. Cell. Biochem.
56:
283-294
[Medline].
|
| 5.
|
Grigoriadis, A.E.,
J.N. Heersche, and
J.E. Aubin.
1988.
Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone.
J. Cell Biol.
106:
2139-2151
[Abstract/Free Full Text].
|
| 6.
| Caplan, A.I., D.J. Fink, T. Goto, A.E. Linton, R.G. Young,
S. Wakitani, V.M. Goldberg, and S.E. Haynesworth. 1993. Mesenchymal stem cells and tissue repair. In The Anterior
Cruciate Ligament: Current and Future Concepts. D. Jackson, S. Arnoczky, S. Woo, and C. Frank, editors. Raven
Press, New York. 405 pp.
|
| 7.
|
Wakitani, S.,
T. Goto,
S.J. Pineda,
R.G. Young,
J.M. Mansour,
A.I. Caplan, and
V.M. Goldberg.
1994.
Mesenchymal
cell-based repair of large, full-thickness defects of articular
cartilage.
J. Bone Joint Surg. Am. Vol.
76:
579-592
[Abstract/Free Full Text].
|
| 8.
|
Friedenstein, A.J.,
R.K. Chailakhjan, and
K.S. Lalykina.
1970.
The development of fibroblast colonies in monolayer
cultures of guinea-pig bone marrow and spleen cells.
Cell.
Tissue Kinet.
3:
393-403
[Medline].
|
| 9.
|
Owen, M., and
A.J. Friedenstein.
1988.
Stromal stem cells:
marrow-derived osteogenic precursors.
Ciba. Found. Symp.
136:
42-60
[Medline].
|
| 10.
|
Nakahara, H.,
S.P. Bruder,
S.E. Haynesworth,
J.J. Holecek,
M.A. Baber,
V.M. Goldberg, and
A.I. Caplan.
1990.
Bone
and cartilage formation in diffusion chambers by subcultured
cells derived from the periosteum.
Bone.
11:
181-188
[Medline].
|
| 11.
|
Rodan, G.A..
1992.
Introduction to bone biology.
Bone.
13:
S3-S6
.
|
| 12.
|
Fierz, W.,
M. Brenan,
A. Mullbacher, and
E. Simpson.
1982.
Non-H-2 and H-2-linked immune response genes control
the cytotoxic T-cell response to H-Y.
Immunogenetics.
15:
261-270
[Medline].
|
| 13.
|
Silvers, W.K., and
S.S. Wachtel.
1977.
H-Y antigen: behavior and function.
Science.
195:
956-960
[Abstract/Free Full Text].
|
| 14.
|
Nilsson, S.K.,
M.S. Dooner,
C.Y. Tiarks,
H.-U. Weier, and
P.J. Quesenberry.
1997.
Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse
model.
Blood.
89:
4013-4020
[Abstract/Free Full Text].
|
| 15.
|
Lamar, E.E., and
E. Palmer.
1984.
Y-encoded, species-specific DNA in mice: evidence that the Y chromosome exists
in two polymorphic forms in inbred strains.
Cell.
37:
171-177
[Medline].
|
| 16.
|
Nilsson, S.K.,
R. Hulspas,
H.U. Weier, and
P.J. Quesenberry.
1996.
In situ detection of individual transplanted bone
marrow cells using FISH on sections of paraffin-embedded
whole murine femurs.
J. Histochem. Cytochem.
44:
1069-1074
[Abstract].
|
| 17.
|
Weier, H.U.,
D. Polikoff,
J.J. Fawcett,
K.M. Greulich,
K.H. Lee,
S. Cram,
V.M. Chapman, and
J.W. Gray.
1994.
Generation of five high-complexity painting probe libraries from
flow-sorted mouse chromosomes.
Genomics.
21:
641-644
[Medline].
|
| 18.
|
Stewart, F.M.,
R.B. Crittenden,
P.A. Lowry,
S. Pearson-White, and
P.J. Quesenberry.
1993.
Long-term engraftment
of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice.
Blood.
81:
2566-2571
[Abstract/Free Full Text].
|
| 19.
|
Pereira, R.F.,
K.W. Halford,
M.D. O'Hara,
D.B. Leeper,
B.P. Sokolov,
M.D. Pollard,
O. Bagasra, and
D.J. Prockop.
1995.
Cultured adherent cells from marrow can serve as
long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.
Proc. Natl. Acad. Sci. USA.
92:
4857-4861
[Abstract/Free Full Text].
|
| 20.
|
Prockop, D.J..
1997.
Marrow stromal cells as stem cells for
nonhematopoietic tissues.
Science.
276:
71-74
[Abstract/Free Full Text].
|
| 21.
|
Horwitz, E.M.,
D.J. Prockop,
J. Marini,
L.A. Fitzpatrick,
R. Pyeritz,
M. Sussman,
P. Orchid, and
M.K. Brenner.
1996.
Treatment of severe osteogenesis imperfecta by allogeneic
bone marrow transplantation.
Matrix Biol.
15:
188a
. (Abstr.)
.
|
| 22.
|
Goldberg, V.M., and
A.I. Caplan.
1994.
Biological resurfacing: an alternative to total joint arthroplasty.
Orthopedics.
17:
819-821
[Medline].
|
| 23.
|
Friedenstein, A.J.,
R.K. Chailakhyan, and
U.V. Gerasimov.
1987.
Bone marrow osteogenic stem cells: in vitro cultivation
and transplantation in diffusion chambers.
Cell. Tissue Kinet.
20:
263-272
[Medline].
|
| 24.
|
Friedenstein, A.J.,
K.V. Petrakova,
A.I. Kurolesova, and
G.P. Frolova.
1968.
Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues.
Transplantation.
6:
230-247
[Medline].
|
| 25.
|
Friedenstein, A., and
A.I. Kuralesova.
1971.
Osteogenic precursor cells of bone marrow in radiation chimeras.
Transplantation.
12:
99-108
[Medline].
|
| 26.
|
Knospe, W.H.,
J. Blom, and
W.H. Crosby.
1968.
Regeneration of locally irradiated bone marrow. II. Induction of regeneration in permanently aplastic medullary cavities.
Blood.
31:
400-405
[Abstract/Free Full Text].
|
| 27.
|
Friedenstein, A.J.,
A.A. Ivanov-Smolenski,
R.K. Chajlakjan,
U.F. Gorskaya,
A.I. Kuralesova,
N.W. Latzinik, and
U.W. Gerasimov.
1978.
Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants.
Exp. Hematol.
6:
440-444
[Medline].
|
| 28.
|
Golde, D.W.,
W.G. Hocking,
S.G. Quan,
R.S. Sparkes, and
R.P. Gale.
1980.
Origin of human bone marrow fibroblasts.
Brit. J. Haematol.
44:
183-187
[Medline].
|
| 29.
|
Friedenstein, A.J..
1995.
Marrow stromal fibroblasts.
Calcif.
Tissue Int.
56:
S1-S17
.
|
| 30.
|
Piersma, A.H.,
R.E. Ploemacher, and
K.G. Brockbank.
1983.
Transplantation of bone marrow fibroblastoid stromal
cells in mice via the intravenous route.
Brit. J. Haematol.
54:
285-290
[Medline].
|
| 31.
|
Keating, A.,
J.W. Singer,
P.D. Killen,
G.E. Striker,
A.C. Salo,
J. Sanders,
E.D. Thomas,
D. Thorning, and
P.J. Fialkow.
1982.
Donor origin of the in vitro haematopoietic
microenvironment after marrow transplantation in man.
Nature.
298:
280-283
[Medline].
|
| 32.
|
Simmons, P.J.,
D. Przepiorka,
E.D. Thomas, and
B. Torok-Storb.
1987.
Host origin of marrow stromal cells following
allogeneic bone marrow transplantation.
Nature.
328:
429-432
[Medline].
|
| 33.
|
Lennon, J.E., and
H.S. Micklem.
1986.
Stromal cells in long-term murine bone marrow culture: FACS studies and origin
of stromal cells in radiation chimeras.
Exp. Hematol.
14:
287-292
[Medline].
|
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
