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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1106-1108
CORRESPONDENCE
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Letter |
To the editor:
Role of muscle-derived cells in hematopoietic reconstitution of
irradiated mice
In the process of embryonic development, all cells arise from
one common cell, the fertilized egg. Similarly, all
mesodermal tissues are derived from a common undifferentiated ancestor.
It is not known if this common ancestor differentiates into
stem cells for each type of tissue and then disappears, or whether multipotential stem cells can persist in an undifferentiated state, and
depending upon specific environmental conditions, function as a stem
cell for many different tissues. The genesis of hematopoietic stem
cells during early vertebrate development has intrigued investigators for several centuries. The steps that lead to embryonic hematopoiesis and the existence of hematopoietic stem cells in fully developed adults
remain to be determined. One of the major impediments to studying the
development of the hematopoietic system has been the absence of
suitable experimental systems. We have found that muscle-derived cells
can reconstitute hematopoiesis in lethally irradiated mice. To
further explore this phenomenon, we have conducted a series of
experiments described below.
In the first set of experiments, female Kunming mice were irradiated
with 8.5 Gy, and then divided into 5 groups. Group 1 received no
additional treatment. Group 2 was injected on day 2 after irradiation
with 0.2 ml of muscle cell culture medium. Group 3 was injected on the
day of irradiation with 0.2 ml of muscle cells (10,000 cells/ml) obtained from male mice. Group 4 was injected on day 2 after
irradiation with 0.2 ml of muscle cells (10,000 cells/ml). Group 5 was
injected on day 2 after irradiation with 0.2 ml of muscle cells (10,000 cells/ml) that had been cocultured with bone morphogenetic protein 2 (BMP2) for 24 hours prior to injection. All mice from Groups 1, 2, and
5 died, while 20% of the mice from Group 3 and 53% of the mice from
Group 4 survived. In a second set of experiments, female mice were
similarly irradiated and then divided into 4 groups. On day 2 after
irradiation, Group 1 received 0.2 ml of peripheral blood (PB) obtained
from male mice, Group 2 received 0.2 ml bone marrow (BM) cells (10,000 cells/ml) obtained from male mice, Group 3 received 0.2 ml BM (100,000 cells/ml) and Group 4 received 0.2 ml BM (100,000 cells/ml) that had
been incubated with BMP2 for 24 hours. All mice from Groups 1 and 2 died, while 80% of mice from Group 3 and 87% from Group 4 survived. In additional experiments that looked at the time course of
hematopoietic reconstitution, surviving mice injected with muscle cells
following irradiation were found to have male-derived cells present in
their BM by day 9 after irradiation. Female-derived blood cells could be detected beginning at day 21. In addition, bone marrow cells obtained from all surviving mice showed the presence of the
Y-chromosome-specific sry gene. No male-derived cells were
found in any of the mice from the control groups. These results
indicate that some muscle-derived cells have the capacity to
reconstitute hematopoiesis in lethally irradiated mice, and that these
cells express receptors for BMP2, which can block this reconstitution
process. In contrast, BMP2 did not affect hematopoietic reconstitution
by BM-derived cells.
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Materials and methods |
Experimental animals: Female and male Kunming mice were
obtained from the Department of Experimental Animals, Institute of Hematology, Chinese Academy of Medical Sciences. Weight of female mice:
35g, weight of male mice: 20g.
Culture of muscle cells: Male Kunming mice were sacrificed and
muscle tissue was removed from the femurs under sterile conditions. The
muscles were cut into small cubes and transferred to 10 ml test tubes
for incubation in 4% trypsin-Hanks balanced salt solution at 37°C
for 10 minutes. A 5 ml aliquot was then transferred to a fresh tube,
and 5 ml RMPI 640 culture solution was added to stop the
trypsin reaction. An additional 5 ml of 4% trypsin was added to the
first tube to continue the reaction. After repeating this process 5-6 times, the collected cells were centrifuged and resuspended in RPMI
1640 with 10% serum.
Muscle-derived cell rescue of irradiated animals: To determine
the ability of muscle-derived cells to rescue lethally irradiated mice,
75 Kunming female mice were irradiated with 8.5 Gy by Gamma cell 40 (cerium) and then divided into 5 groups of 15 mice each. Group 1:
irradiation only. Group 2: injected through tail vein on day 2 after
irradiation with 0.2 ml muscle cell culture medium. Group 3: injected
on day of irradiation with 0.2 ml muscle cells (10,000 cells/ml). Group
4: injected on day 2 after irradiation with 0.2 ml muscle cells (10,000 cells/ml). Group 5: injected on day 2 after irradiation with 0.2 ml
muscle cells (10,000 cells/ml) that had been cocultured with BMP2 for
24 hours. The survival of these animals was then compared with animals
rescued with BM as described below. The results of these studies were
used to determine the minimum number of muscle-derived cells needed for rescue in the engraftment studies described below.
Bone marrow rescue of irradiated animals: BM and PB cells were
obtained from male Kunming mice, and mononuclear cells were isolated by
Ficoll centrifugation. Sixty female Kunming mice were irradiated with
8.5 Gy by Gamma cell 40 (cerium) and divided into 4 groups of 15 mice
each. Group 1: injected on day 2 after irradiation with 0.2 ml PB cells
(10,000 cells/ml). Group 2: injected on day 2 after irradiation with
0.2 ml BM cells (10,000 cells/ml). Group 3: injected on day 2 after
irradiation with 0.2 ml BM cells (100,000 cells/ml). Group 4: injected
on day 2 after irradiation with 0.2 ml BM cells (100,000 cells/ml)
incubated with BMP2 for 24 hours.
Pathology studies: The femurs and spleens of all dead animals
were fixed in 10% formaldehyde, sectioned, and stained with hematoxylin and eosin for histological studies.
Source of engrafted cells in animals rescued with muscle-derived
cells: To determine the source of engrafted cells in animals rescued by injection of muscle-derived cells, 60 female Kunming mice
were divided into two groups of 30 mice each. The animals were
irradiated as described above. The first group did not receive additional cells for rescue. The second group was injected with 0.2 ml
of muscle-derived (10,000 cells/ml) as described above. Threemice from
each group were evaluated every 3 days by C band chromosome analysis
and Y chromosome polymerase chain reaction (PCR) (see below).
C band chromosome analysis: BM cells were harvested from
sacrificed mice, cultured at 37°C for 2-3 weeks, and then incubated with colchine. C-band analysis was then performed.
Y-chromosome polymerase chain reaction: PCR was performed using
BMcells obtained from all expired female mice to determine if any
male-derived cells were present. Processed DNA samples were amplified
in 50ul containing 20 pmols of mouse Y chromosome-specific primers and
mouse PDGF B receptor-specific primers,1 as
well as 1.5mM MgCl, 50 mM KCl, 10 mM Tris-Hcl, pH 8.3, 0.2 mM dNTPs, and 1.5 units Taq polymerase (SABC, China). Y-chromosome
specific primers: (sense primer) CTG CTG TGA ACA GAC ACT AC;
(anti-sense primer) GAC TCC TCT GAC TTC ACT TG. Mouse PDGF B receptor
primers: (sense primer) CAT TGG CTC CAT CCT GCA TA; (anti-sense primer) GGA TAA GCC TCG AAC ACC AC. PCR was initiated at 94°C for 4 minutes, followed by 30 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 2 minutes, followed by a final cycle of
72°C for 5 minutes. Amplification with Y-chromosome specific
primers results in a 722 bp fragment corresponding to the sry
locus sequence 256-978. Amplification with PDGF B receptor primers
generates a fragment of approximately 750 bp corresponding to the
mouse PDGF B receptor cDNA sequence 948-1166, which contains an intron.
All PCR amplifications included a male (positive) and female (negative) control.
| |
Results and discussion |
Rescue of irradiated mice: In the first set of experiments
in which muscle-derived cells were injected into irradiated mice, all
animals from Groups 1, 2, and 5 died between days 9-13 after irradiation. In contrast, 3/15 mice from Group 3 and 8/15 mice from
Group 4 have survived for over 2 months (Table
1). In the second set of experiments in
which peripheral blood or BM cells were injected into irradiated mice,
all animals from Groups 1 and 2 died between days 9 -13 after
irradiation. In contrast, 12/15 mice from Group 3 and 13/15 mice from
Group 4 have survived (Table 2). Bone
marrow and spleen sections were obtained from all dead mice from both
sets of experiments. All animals studied exhibited empty BMs and
splenic atrophy. No differences were seen among the different study
groups. In addition, Y-chromosome specific PCR was performed on BM
tissue obtained from the dead mice. No male-derived cells were detected
in the BMs of any dead mice.
Source of engrafted cells in animals rescued with muscle-derived
cells: On day 3 after irradiation, only female-derived cells were
present in BM by C-band chromosome analysis. On day 9, 1.7% of cells
were of male origin. From days 12-18, in general, only male-derived
cells were present, with only a few female-derived cells found
. On day 21, female-derived cells were again present. In
the irradiation-only control group, male-derived cells were never
found. Similarly, PCR amplification of the Y-chromosome specific
sry gene revealed the presence of male-derived cells in BM from
day 9 after irradiation and beyond.
Hematopoietic stem cells are thought to have the property of
self-renewal, and to be able to differentiate into a variety of
hematopoietic lineages. Previous studies have demonstrated that they
have the capacity to differentiate but have been unable to demonstrate
self-renewal. One difficulty has been how to identify a hematopoietic
stem cell, becauseby the time a cell is defined as such, it has already
differentiated into a committed progenitor.
We found that hematopoiesis can be reconstituted in lethally irradiated
mice by infusion of muscle-derived cells. This demonstrates that some
muscle-derived cells have the ability to differentiate into
hematopoietic cells. The addition of BMP2 blocks the ability of these
cells to reconstitute hematopoiesis. This suggests that these cells
express receptors for BMP2, and that BMP2 induces these muscle-derived
cells to differentiate into bone rather than hematopoietic
tissue2,3. Previous studies have demonstrated that BMP2 can
induce muscle cells to differentiate into bone both in vivo and in
vitro. It is possible that these muscle-derived cells can function as
stem cells for both hematopoietic and bone tissue. One outstanding
question is whether muscle tissue is heterogeneous in nature and
contains several types of stem cells for different tissues.
Alternatively, muscle-derived cells may be homogeneous in nature, and
retain the capacity to differentiate into other tissue types under the
appropriate conditions.
We found that administration of BM or PB cells could not save lethally
irradiated mice when administered at the same concentration (10,000 cells/ml) as muscle-derived cells. Rather, a tenfold increase in BM
cells was needed for hematopoietic rescue. This result shows that
muscle-derived cells are more efficient in reconstituting hematopoiesis
than BM cells. In addition, BMP2 did not block BM cells from rescuing
the irradiated mice. Therefore, muscle-derived cells differ from BM
cells. They are not blood cells, but can differentiate into
hematopoietic tissue.
On day 9 after irradiation, male-positive blood cells were present in
the BM of rescued mice. This demonstrates that muscle-derived cells
were able to reconstitute hematopoiesis. However, some mice died
despite being treated in the same way. This shows that while only a few
muscle-derived cells are needed to reconstitute hematopoiesis, the cell number we used was lower than that needed to rescue all the
experimental animals. Additional experiments are needed to identify the
optimal cell number for rescue. The above studies illustrate that
muscle-derived cells provide a highly efficient source of cells that
are able to differentiate into hematopoetic tissue. In addition,
selection of these cells in vitro for transplant may be a better way to
obtain hematopoietic stem cells than BM because muscle tissue is the
biggest tissue in the body, and a donor is not needed.
On day 21 after irradiation, female-positive blood cells were present
in the BM of rescued mice. This demonstrates that the irradiated mice
could renew hematopoiesis by themselves. The time needed for
reconstitution of hematopoiesis in this manner was longer than that
required for reconstitution by the transplanted cells. It is not known
which cells are able to reconstitute hematopoiesis, and why more time
would be needed for this process to occur. If there are hematopoietic
stem cells circulating in the blood of the treated mice, then they
should be able to reconstitute hematopoiesis in a shorter period of
time than the transplanted cells. We hypothesize that there are no true
hematopoietic stem cells circulating in the blood, but rather only
progenitors. The true stem cells appear to be the common ancestors of
other tissues such as bone and blood, which need time to enter BM and
to differentiate into blood cells.
The above studies demonstrate that some muscle-derived cells can
differentiate into blood cells, and that these cells express receptors
for BMP2 on their surface. Further purification of these cells would
provide a new way to perform bone marrow transplant, and to resolve the
difficulty of in vivo proliferation of hematopoietic stem cells.
Wenxin Pang
Institute of Hematology, Peking Union Medical College & Chinese Academy of Medical Sciences
| |
References |
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Yan XQ, Chen Y, Hartley C, McElroy P, Fletcher F, McNiece IK.
Marrow repopulating cells in mobilized PBPC can be serially transplanted for up to five generations or be remobilized in PBPC reconstituted mice.
Bone Marrow Transplant.
1998;21:975-981[Medline]
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Wang EA, Rosen V, D'Alessandro JS, et al.
Recombinant human bone morphogenetic protein induces bone formation.
Proc Natl Acad Sci USA.
1990;87:2220-2224[Abstract/Free Full Text].
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Katagiri T, et al.
The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiated into osteoblastic cells by recombinant human bone morphogenetic protein-2.
Biochem Biophys Res Commun.
1990;172:285-299.
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