Bone Marrow Stromal Cell Transplantation Mitigates Radiation-Induced Gastrointestinal Syndrome in Mice
et al. (2011) Bone Marrow Stromal Cell Transplantation Mitigates Radiation-Induced Gastrointestinal
Syndrome in Mice. PLoS ONE 6(9): e24072. doi:10.1371/journal.pone.0024072
Bone Marrow Stromal Cell Transplantation Mitigates Radiation-Induced Gastrointestinal Syndrome in Mice
Subhrajit Saha 0
Payel Bhanja 0
Rafi Kabarriti 0
Laibin Liu 0
Alan A. Alfieri 0
Chandan Guha 0
Jan-Hendrik Niess, Ulm University, Germany
0 1 Department of Radiation Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, United States of America, 2 Department of Pathology, Albert Einstein College of Medicine, Montefiore Medical Center , Bronx, New York , United States of America
Background: Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS), resulting from direct cytocidal effects on intestinal stem cells (ISC) and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT) could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome. Methodology/Principal Findings: Autologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy) or abdominal irradiation (16-20 Gy) in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p,0.0007 and p,0.0009 respectively) beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated. Conclusion/Significance: Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated host ISC niche, thus providing a platform to discover potential radiation mitigators and protectors for acute radiation syndromes and chemo-radiation therapy of abdominal malignancies.
Funding: The work has been supported by 1 RC2 AI087612-01 and 1U19AI091175-01 on Centers for Medical Countermeasures against Radiation from the
National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
Accidental or intended radiation exposure in a mass casualty
setting presents a serious and on-going threat. At radiation doses of
3 to 8 Gy, morbidity and lethality is primarily caused from
hematopoietic injury and victims can be rescued by bone marrow
transplantation (BMT). However, with exposure to larger doses,
victims suffer irreversible hematopoietic and gastrointestinal injury
and usually perish despite supportive care and BMT. While BMT
may have some benefit in mitigating hematopoietic syndrome,
currently there are no approved medical countermeasures to
alleviate radiation-induced gastrointestinal syndrome (RIGS).
RIGS results from a dose-dependent, direct cytocidal and
growth inhibitory effects of irradiation on the villous enterocytes,
crypt intestinal stem cells (ISC) [1,2,3], the stromal endothelial
cells  and the intestinal subepithelial myofibroblasts (ISEMF)
. Subsequent loss of the mucosal barrier results in microbial
infection, septic shock and systemic inflammatory response
syndrome. The cells in the ISC niche, consisting of micovascular
endothelial cells, mesenchyme-derived ISEMF  and pericryptal
macrophages  provide critical growth factor/signals for ISC
regeneration and intestinal homeostasis . Of these, ISEMF
continuously migrate upward from the crypt base to the villous tip
along with ISC and transit amplifying enterocytes, establishing
signaling crosstalk and regulating ISC self-renewal and
differentiation [5,8]. ISEMF interacts with pericryptal macrophages with
subsequent release of PGE2 that could reduce radiation-induced
apoptosis of enterocytes [9,10]. Pericryptal macrophages form
synapses with crypt stem cells and secretes growth factors to
stimulate ISC proliferation  upon activation of Toll-like
receptors sensing the entry of bacteria and other intestinal
Since RIGS results from a combination of radiation-induced
loss of crypt progenitors and stromal cells along with aberrant
signaling in the ISC niche, we rationalized that the acute loss of
stromal cells in the ISC niche would require rapid compensation
of their functions. This could be best achieved with cell
replacement therapies that restore the ISC niche after irradiation
so that the stromal cells can secrete growth factors and provide
necessary signals for survival, repair and regeneration of the
irradiated intestine. Earlier reports demonstrated that donor bone
marrow-derived cells could contribute to multiple lineages in the
gastrointestinal tract and facilitate intestinal regeneration in
patients with graft-versus-host disease and ulcer  and in
animal models of colitis . Because of ease in cell culture and its
ability to differentiate into multiple tissue lineages, transplantation
of bone marrow-derived mesenchymal stem cells (MSC) has been
a very attractive option for a wide range of clinical applications
, such as, severe treatment-resistant graft-versus-host diseases
of the gut . Besides trans-differentiating into ISEMF and
stimulating ISC proliferation, MSC transplantation has also been
shown to reprogram host macrophages to induce an
antiinflammatory response and thereby minimizing sepsis in a murine
model of colitis . Intravenous injection of MSC resulted in
enhanced engraftment in irradiated organs, including, small
intestine with subsequent increase in the regeneration of the
intestinal epithelium and accelerated recovery of the villi
postradiation in mice models . Genetic modification of donor
MSCs with superoxide dismutase  or CXCR4  transgene
augments the engraftment and mitigation of intestinal radiation
injury. However, till date, transplantation of whole bone marrow
or MSC has not been successful in ameliorating RIGS and
improve survival of mice that received .10 Gy of irradiation in a
single fraction [16,17,18]. We reasoned that the failure of
cellbased therapies in ameliorating RIGS after lethal doses of
irradiation is because of absence of important cellular components
of the ISC niche, including endothelial cells and macrophages, in
the donor MSC population. Since bone marrow could provide a
source of all the major cell types in the ISC niche, namely, ISEMF,
endothelial cells and macrophages, we amplified the stromal cell
population by culturing freshly isolated bone marrow cells in
mesenchymal basal medium and collected the adherent stromal
cells for transplantation into mice exposed to lethal doses of whole
body or abdominal irradiation. In this report, we demonstrate that
bone marrow-derived adherent stromal cell transplantation
(BMASCT), 24 hours following exposure to lethal AIR of 16
20 Gy, stimulated ISC regeneration, restored the functional
integrity of the villi, dampened inflammatory response and
mitigated RIGS in C57Bl/6 mice.
BMASCT mitigates RIGS and improves survival of mice
after lethal doses of irradiation
Mortality from acute radiation syndromes results from
dosedependent radiation injury to various organs. While BMT is
effective in improving survival of mice exposed to doses up to 8
9 Gy, it is relatively ineffective as the sole treatment with higher
doses of exposure. We have previously demonstrated that a whole
body exposure of $10.4 Gy induces RIGS and 100% mortality
within 1015 days in C57Bl/6 mice . In order to confirm that
RIGS is induced after exposure to a single fraction of Whole Body
Irradiation (WBI) of 10.4 Gy, we examined whether BMT can
improve the survival of C57Bl/6 mice. While 100% of the
untreated animals died within 10 days, animals receiving BMT
had only 20% survival (Fig. 1A), indicating that whole marrow
that contained primarily CD45+ve hematopoietic cells (Figure S1)
failed to rescue these animals from RIGS. We, then, examined
whether transplantation of bone marrow-derived stromal cells that
have been enriched for MSC, Endothelial Progenitor Cell (EPC)
and macrophages upon culture in mesenchymal basal medium
could mitigate radiation injury in these animals. Fig. 1A
demonstrates that BMASCT rescued 100% of the irradiated
animals (p,0.0009), indicating that stromal cell therapy may
provide factors to repair and regenerate the intestinal epithelium
damaged by irradiation.
To limit the exposure of the bone marrow to irradiation while
escalating the dose to intestine, we delivered Abdominal
Irradiation (AIR) (1220 Gy) after shielding the thorax, head
and neck and extremities, as described previously [19,20]. AIR did
not significantly impact the peripheral blood count at day 5
(Figure S2) post-exposure, indicating that the bone marrow was
not severely damaged by AIR. Control animals that received
either, PBS, or culture medium died within 10 days after exposure
to AIR$16 Gy with characteristic signs and symptoms of RIGS,
including, diarrhea, black stools and weight loss. In contrast,
animals that received AIR+BMASCT had well-formed stools,
maintained body weight (24.160.7 g in AIR+BMASCT versus
16.2161.8 g in AIR cohort, p,0.001) and had 100% survival
beyond 25 days (18 Gy AIR, p,0.0007, Fig. 1B). At 20 Gy,
BMASCT rescued 40% of the animals with survival greater than
25 days, while irradiated animals without BMASCT died within 5
days (median survival time of AIR cohort, 360.5 d versus
AIR+BMASCT cohort, 1261.8 d; p,0.01, Fig. 1C).
Transplantation of CD45+ hematopoietic cell-enriched bone marrow
derived non-adherent cell (BMNAC) and whole bone marrow
cells failed to rescue AIR-treated mice (Fig. 1BC,E & Figure S1),
indicating that stromal cells were responsible for the salvage of
Both myeloid and non-myeloid cell populations are
needed for RIGS mitigation
Flow cytometric analysis of donor cells demonstrated that Bone
marrow-derived adherent stromal cell (BMASC) population
included, primarily MSCs (CD105+CD452 41.2%61.8;
CD29+CD452 39.8%61.2), macrophages (CD11b+F480+
19.2%61.2) and EPCs (CD133+CD34+CD4522.6%60.89) and
CD45+ hematopoietic cells (Fig. 1D). CD44 and Sca1 staining
further confirmed the presence of MSC population (Figure S3). To
evaluate the individual roles of CD11b+ macrophage-enriched
cells versus CD11b2 MSC-enriched stromal cell fraction (Figure
S4) in RIGS mitigation, BMASC population was fractionated by
cell sorting using anti-CD11b-magnetic beads, followed by
transplantation 24 hrs post-AIR. Transplantation of either the
macrophage-enriched (78.1%62.8 F480+ cells), MSC-deficient
(,1.5% CD105+ve cells) CD11b+ve BMASC or
macrophagedeficient (0.68%60.03 F480+ cells), MSC-enriched (6871%
CD105+ve) CD11b2ve BMASC cell population mitigated only
3040% of the animals irradiated with 18 Gy AIR (Fig. 2AB,
Figure S4). Survival was salvaged to 100% when the CD11b+ and
CD11b2 populations were admixed and transplanted, indicating
that the combination of macrophages and bone marrow stromal
cells, including MSC and EPC fractions was necessary for RIGS
BMASCT induces structural and functional regeneration
Histomorphological evaluation after hematoxylin-eosin staining
demonstrated that the AIR+BMASCT-treated animals exhibited
an increase in the overall size of the crypts and maintained villous
length (Fig. 3A, Figure S5 & S10). The percentage of the BrdU+ve
Figure 1. AC. BMASCT improves survival of C57Bl/6 mice following AIR. Kaplan-Meier survival analysis of mice (n = 25) receiving BMASCT,
24 and 72 hrs after irradiation, showed 100% survival after (A) 10.4 Gy WBI (p,0.0006) and (B) 18 Gy AIR (p,0.0007); and 40% survival after (C) 20 Gy
AIR (p,0.01). Whole bone marrow, BMNAC and culture media failed to improve survival. DE. Flow cytometric characterization. (D) BMASC and
(E) BMNAC population using MSC-specific (CD105+CD452/CD29+CD452), macrophage-specific (CD11b+F480+) and endothelial-specific
crypt epithelial cells synthesizing DNA was significantly enhanced
in this cohort of mice at 3.5 days post-irradiation (AIR+BMASCT,
42.8262.01 versus AIR, 23.4361.66; P,0.04; Fig. 3B and E).
The numbers of regenerative crypt microcolonies per unit
intestinal cross sectional area at 3.5 days post-irradiation serves
as a surrogate indicator of crypt regenerative response
postirradiation [1,21,22]. The crypt microcolony count was increased
significantly in AIR+BMASCT cohort, compared with those that
received AIR alone (AIR+BMASCT, 12.561.2/mm versus AIR,
6.860.8, p,0.004, Fig. 3D), indicating intestinal regenerative
response following BMASCT. Consistent with the regenerative
response, immunohistological analysis demonstrated the presence
of nuclear b-catenin in the AIR+BMASCT-treated animals, while
cytosolic staining was predominant in the animals receiving AIR
(Fig. 3C), suggesting that BMASCT activates the Wnt b-catenin
pathway in crypt cells to stimulate proliferation post-irradiation.
We performed xylose absorption test and determined the
functional recovery of the intestinal villi in RIGS. Since xylose is
not metabolized in the body, serum xylose level is a good indicator
of the intestinal absorptive capacity in animals fed with a test dose
of xylose . Compared to animals that received AIR alone,
xylose absorption was significantly improved in animals that
received BMASCT at 7 d post AIR (AIR+BMASCT, 7265.5 g/ml
vs. AIR, 3562.7 g/ml; p,0.004; Fig. 3F), indicating quick
functional restitution of the intestinal villi.
BMASCT promotes survival of irradiated Lgr5-positive
crypt base columnar cells
We examined the effect of AIR on the number of
Lgr5EGFP+ve crypt base columnar cells, the putative ISC population
[3,23], in the jejunum of Lgr5-EGFP-IRES-creERT2 transgenic
mice by detecting EGFP expression using confocal microscopy.
While these cells are present at 1 d post-AIR, they are absent at
3.5 d post-AIR (Fig. 4A). Flow cytometric analysis confirmed the
gradual loss of Lgr5+ve crypt ISCs following irradiation exposure
(5.17%61.8 at 1 d vs. 0.89%60.15 at 3.5 d; p,0.001; Fig. 4B). In
contrast, BMASCT increased the number of Lgr5-EGFP+ve
CBCs at 3.5 d post-AIR (Fig. 4A). Flow cytometric analysis
confirmed that BMASCT increased the number of irradiated
Lgr5-GFP+ve crypt cells at 3.5 d post-AIR (9.27%61.75, vs.
0.89%60.15; (p,0.0003; Fig. 4B), possibly by providing signals
for survival and growth. This provides us with a potential window
of radiation mitigation, whereby BMASCT rescued lethally
irradiated mice within 24 hrs of irradiation, but not after 72 hrs
BMASCT restores the ISEMF and pericryptal macrophages
in the irradiated ISC niche
ISEMF and pericryptal macrophages provide the epithelial
mesenchymal cross-talk signals for growth, differentiation and cell
fate determination to ISCs [6,8,9]. Immunohistochemistry and
confocal microscopy demonstrated that 18 Gy AIR reduces the
number of a-SMA+, desmin2ve ISEMF (Fig. 5A) and F480+ve
pericryptal macrophages (Fig. 5B). BMASCT restored the
aSMA+, desmin2 ISEMF (Fig. 5A) and increased the number of
pericryptal and subepithelial macrophages in the lamina propria
(AIR+BMASCT, 7266.4/hpf versus AIR, 1563.2/hpf; p,0.003;
Fig. 5B,C) of irradiated mice. Transplantation of the CD11b2ve
fraction of BMASC restored the ISEMF population (Fig. 5A),
whereas transplantation of the CD11b+ve fraction exhibited an
increase in the number of intestinal macrophages (p,0.006,
Fig. 5B,C), which further suggests that transplantation of both
CD11b+ and CD11b2 fractions restores the ISC niche for RIGS
BMASCT induces secretion of intestinal growth factors
and anti-inflammatory cytokines
We examined the engraftment and repopulation of the donor
cells in various organs by transplanting dipeptidyl peptidase IV
(DPPIV)-proficient BMASC in DPPIV-deficient C57Bl/6 host.
Although some DPPIV+ve donor cells were noted per intestinal
villi upon DPPIV immunohistochemistry (Figure S7 AB), the
majority of the donor cells were lodged in the lungs (Figure S7 C
D). We, therefore, hypothesized that the regeneration and repair
of the irradiated intestine is possibly mediated by paracrine growth
factors that were secreted by the donor BMASCs. Immunoblot
analysis of the serum of animals that received AIR+BMASCT
showed an increase in serum levels of R-spondin1, FGF2,
PDGFB and KGF by 28 folds at 24 h post-BMASCT, compared to
animals that received AIR alone (Fig. 6A). Interestingly, animals
that received whole BMT did not show an increase in serum
Rspondin1 levels (Figure. S8). While KGF and R-spondin1 can
increase the proliferation of intestinal crypt cells [1,24], FGF2 and
PDGF-B could support the growth of endothelial cells  and
ISEMF , respectively in the ISC niche of
RIGS is associated with a systemic inflammatory response
syndrome (SIRS) resulting from bacterial entry from the denuded
gut lumen and resultant endotoxemia . We performed
multicytokine ELISA in the serum of animals that received AIR alone
and compared them with those that received AIR+BMASCT.
Compared to untreated controls, there was a significant increase in
serum pro-inflammatory cytokines, such as, IL12A (p,0.001),
IL17 (p,0.006) in animals that received AIR (Fig. 6C) or
AIR+BMT (Figure S9B). BMASCT reduced the secretion of these
inflammatory cytokines, while inducing the release of
antiinflammatory cytokines, IL6 (p,0.004) and IL10 (p,0.002)
(Fig. 6B) that may dampen the SIRS in RIGS. AIR+BMASCT
also increased the levels of serum GCSF (p,0.006) and GMCSF
(p,0.007) (Fig. 6D) compared to AIR alone, which could induce
macrophage infiltration and activation in the irradiated intestine
Since BMASCT was postulated to modulate the ISC niche, we
also examined the expression of mRNA level of intestinal growth
factors and inflammatory cytokines from cells isolated from the
crypt region. Quantitative RT-PCR analysis of crypt cell mRNA
from AIR+BMASCT-treated animals showed several fold increase
in expression level of intestinal growth factors, such as, FGF10,
KGF, EGF, FGF2, and anti-inflammatory cytokine, IL-10 with
BMASCT at 24 hr post-AIR, compared to AIR alone (see Tables
S1, S2, S3). While R-spondin1 levels were elevated in the serum,
its expression was absent in the crypt region. In contrast to
BMASCT, whole BMT had lower expression of intestinal survival
and growth factors and chemokines, such as, EGF, FGF10, FGF,
Figure 3. BMASCT mitigates RIGS by promoting structural and functional regeneration of the irradiated intestine. A. H&E staining, B.
Brdu immunohistochemistry, C. b-catenin immunohistochemistry. b-catenin stained green and nucleus was stained with DAPI (pseudo colored with
red). Confocal microscopic images (636) were magnified 2.36 (inset). Note the greater crypt depth (A), increase in crypt cell proliferation (B) and an
increase in nuclear translocation of b-catenin (stained yellow) in AIR+BMASCT cohort compared to other cohorts. D. Number of regenerative crypts,
E. Crypt proliferation rate and F. Xylose absorption assay. A time course study showed significant recovery (p,0.0003) of xylose absorption at 7 days
post-irradiation in AIR+BMASCT-treated animals compared to the AIR cohort.
IGF1, VEGFa, CSF1, CXCL1 and CXCL12 (Table S1). These
results suggested that bone marrow-derived stromal cells could
modulate the regenerative signals in intestinal microenvironment.
Depletion of host macrophages reduces survival of
Pericryptal macrophages play an important role in forming
synapses with ISC and modulating ISC regeneration . To
evaluate the involvement of host macrophages in RIGS mitigation,
we depleted them by administering clodronate-filled liposomes
(clodrosome) intraperitoneally from day 4 pre-AIR to a week
postAIR. The depletion of macrophages (CD11b+F480+) was verified
using FACS analysis of splenocytes and immunohistochemical
staining of intestinal sections (Fig. 7BC). Macrophage depletion
reduced the RIGS-mitigating effect of BMASCT with only 25% of
the animals surviving after 18 Gy AIR, compared to 100%
survival in mice that received AIR+BMASCT (Fig. 7A). This
indicated an essential role of host macrophages in the regenerative
process of irradiated intestines.
Prostaglandin E2 (PGE2) is an essential mediator for
BMASCT-induced RIGS mitigation
Intestinal macrophages have been implicated in inducing the
expression of COX2 for PGE2 synthesis by ISEMF. PGE2 has
been known to be involved in selfrenewal and differentiation
process of hematopoetic stem cell (HSC). Furthermore, PGE2
increased the homing efficiency of HSCs with the selective
induction of short-term-HSC engraftment in murine models .
Moreover, it was shown that PGE2 also inhibits the
radiationinduced apoptosis of intestinal crypt cells by binding to the EP
receptor on ISC [9,10]. To further elaborate on the cross-talk of
pericryptal macrophages and ISEMF in the ISC niche that are
replenished after BMASCT and also involvement of PGE2 in
repair process, we inhibited PGE2 synthesis with COX2 inhibitor
NS398. COX2 inhibition reduced the BMASCT-mediated
survival of irradiated animals to 35% (p,0.008), which was
restored to 80% with dmPGE2 supplementation (Fig. 7D). Tunnel
staining demonstrated that COX2 inhibition significantly
increased the percent of apoptotic cell in crypt of animals that
received AIR+BMASCT (p,0.002) (Fig. 7 EF), which was
reduced with dmPGE2 supplementation (Fig. 7 EF).
This is the first demonstration of RIGS mitigation by
BMASCT, 24 hours after exposure to high doses of either, single
fraction of whole body irradiation (10.4 Gy) or AIR (1620 Gy).
BMASCT restores the ISC niche, including, the pericryptal
macrophages, endothelial cells and ISEMF. In contrast to BMT
that mitigates radiation-induced hematopoeitic syndrome by
donor cell repopulation, BMASCT mitigates RIGS via accelerated
regeneration of irradiated host ISC rather than its replacement
with donor derived cells. This would require the presence of Lgr5+
Figure 4. BMASCT promotes survival of Lgr5-positive crypt base columnar cells following AIR. A. Confocal microscopic imaging of EGFP
expression in the jejunum of Lgr5-EGFP-ires-CreERT2 transgenic mice. Lgr5-EGFP+ve crypt cells are present at 1 d post-AIR but are absent at 3.5 d
post-AIR, indicating the time course of radiation-induced ISC death. BMASCT inhibits the radiation-induced cell loss of Lgr5+ISCs. Confocal
microscopic images (636) were magnified 2.36 (inset). Nucleus was stained with DAPI and pseudo colored with red. B. Flow cytometric analysis of
EGFP expression in crypt cells of Lgr5-EGFP-ires-CreERT2 transgenic mice post-AIR.
Figure 5. BMASCT restores the ISEMF and pericryptal macrophages of the ISC niche, 3.5 days post-AIR. A. ISEMF detection by
immunohistochemistry and confocal microscopy using anti-a-SMA (stained red, indicated with arrow) and anti-desmin (stained green) antibodies.
aSMA+ve and desmin2ve ISEMF were reduced in AIR-treated animals, which was restored by BMASCT. Nucleus was stained with DAPI (blue). B. F480
Immunhistochemistry and confocal microscopic analysis and C. Quantification of Number of pericryptal macrophages. The number of F480+ve
macrophages (green, indicated with arrow) increased at 3.5 d post-AIR in the AIR+BMASCT (p,0.003) and CD11b+ve BMASCT (p,0.006) group,
compared to the AIR cohort, respectively. Nucleus was stained with DAPI (pseudo colored with red). Confocal microscopic images (636) were
magnified 2.36 (inset).
ISCs, which were noted in crypt for 24 hrs post-AIR, thus
affording a time window for effective radio-mitigation. Hence,
BMASCT was successful in rescuing animals up to 24 hrs
postradiation but not at later time points.
Since the majority of the donor cells were lodged in the lungs,
radiation injury was perhaps mitigated by secreted growth factors.
Potential candidates include R-spondin1, KGF, FGF2, PDGF-B,
IL-6, IL-10, G-CSF and GM-CSF. Serum R-spondin1 levels
increased by 810-fold. Human R-spondin1, a 29 kd, 263 amino
acid protein that acts as a specific growth factor of intestinal crypt
cells , has been shown to be a mucosal protective agent in
radiation and chemotherapy-induced mucositis . We have
demonstrated that R-spondin1 can be radioprotective for RIGS
. R-spondin1 binds with high affinity to the Wnt co-receptor,
LRP6, and induce phosphorylation, stabilization and nuclear
translocation of cytosolic b-catenin, thereby activating
TCF/bcatenin-dependent transcriptional responses in intestinal crypt cells
. The presence of nuclear b-catenin in the crypt cells of
AIR+BMASCT-treated animals could represent
R-spondin1mediated Wnt activation in ISC of these animals. BMASCT also
modulated the mRNA expression of several intestinal growth
factors in the crypt cells of irradiated intestine. However,
Rspondin1 was not expressed in the cells of the crypt region.
BMT can rescue animals that develop primarily a
hematopoietic syndrome with exposure to radiation doses #89 Gy in single
fraction. With higher doses of irradiation, intestinal injury sets in
and animals cannot be rescued by BMT alone. Although, bone
marrow-derived, MSCs contribute to intestinal regeneration and
transplantation of these cells ameliorated intestinal injury in
murine models of radiation and chemotherapy-induced injury,
colitis, and autoimmune enteropathy [16,18,31,32], MSC
transplantation alone failed to improve survival of animals exposed to
higher irradiation doses (.9.6 Gy) in a single fraction [16,17,18].
Our study shows that whole bone marrow transplantation cannot
mitigate intestinal injury induced by irradiation ($10.4 Gy).
However, upon amplification of stromal cells in mesenchymal
basal medium culture, and transplantation of a combination of
CD11b+ macrophages and CD11b2 MSC and EPCs could
Figure 6. Serum analysis of intestinal growth factors and cytokines. A. Immunoblot analysis. An increase in the serum levels of R-spondin1,
FGF2, KGF and PDGF-B was noted in AIR+BMASCT cohort compared to AIR. BD. Multi cytokine ELISA. B. Anti-inflammatory cytokines, IL6 (p,0.004)
and IL10 (p,0.002) levels were significantly increased in the AIR+BMASCT, cohort compared to AIR alone. C. Pro-inflammatory cytokines, IL12A
(p,0.001) and IL17 (p,0.006) levels were induced in AIR cohort, compared to AIR+BMASCT treated animals (IL12A, p,0.001; IL17, p,0.008). D.
Myeloid cytokines, GM-CSF (p,0.007) and G-CSF (p,0.006) were increased in AIR+BMASCT group, compared to AIR.
effectively mitigate RIGS. Important differences were noted in the
animals that received BMASCT from BMT. In contrast to the
AIR+BMT cohort, the AIR+BMASCT cohorts had elevated
levels of serum R-spondin1 and expressed various intestinal
growth factors in the crypt cells, suggesting a role of stromal cells in
secreting growth factors and signals for inducing ISC proliferation
in these animals. These stromal cells secrete factors that support
the regeneration of the ISC and its niche. Increased serum levels of
Figure 7. BMASCT promotes signaling cross-talk between macrophages and ISEMF in the ISC niche post-AIR. A. Kaplan-Meier survival
analysis of animals treated with AIR+BMASCT following depletion of host macrophages by clodrosome. Clodrosome treatment reduced the animal
survival after AIR+BMASCT to 25%, indicating host macrophages are needed for mitigation. BC. Flow cytometric (B) and confocal microscopic
evaluation (C) of macrophages. Note depletion of host macrophages post-AIR by clodrosome. DF. Inhibition of COX2 reduced the BMASCT
mediated mitigation of RIGS. D. Kaplan-Meier survival analysis. Administration of COX2 inhibitor, NS398, reduced survival of animals treated with
AIR+BMASCT (p,0.008). Survival was improved to 80% with dmPGE2 supplement. EF. TUNEL staining of crypts. AIR+BMASCT inhibited apoptosis in
the crypts at day 3.5, which was increased by NS398-mediated COX2 inhibition (p,0.002). Supplementation with dmPGE2 restored the anti-apoptotic
effect of BMASCT (p,0.005).
PDGF-B and FGF2, growth factors for ISEMF and EPC
proliferation , along with GMCSF and GCSF [33,34] for
macrophage activation support the involvement of BMASC in
restoring the ISC niche. Several growth factors that could mediate
intestinal regeneration, such as, FGF10, FGF, EGF, IGF1,
VEGFa, CSF1 and CXCL12 were induced in the crypt cells in
BMASCT-transplanted animals. ISEMF residing throughout the
lamina propria and pericryptal region plays a vital role in intestinal
structural regeneration [7,8,25]. Similarly, submucosal
macrophages are activated by the bacterial ligands for Toll-like receptors
(TLR) upon bacterial entry through disrupted intestinal mucosa.
Thus activated macrophages act as mobile cellular transceivers
that transmit regenerative signals to ISCs . Crosstalk between
host macrophages and ISEMF was necessary for RIGS mitigation
by PGE2-mediated inhibition of radiation-induced apoptosis of
crypt cells, also noted in other studies [9,10]. Regenerative role of
PGE2 is very well established in hematopoetic system where it was
reportedly involved in engraftment as well as survival of
transplanted HSCs or cord blood cells [27,35]. Moreover in
embryonic and adult zebrafish model it was shown that PGE2 is
required for Wnt-mediated effects on HSC development and can
enhance Wnt activity in-vivo [27,36]. It was quiet evident in our
observation that PGE2 has a significant role in
BMASCTmediated amelioration of RIGS. Based upon previous studies
[27,35], it is possible that PGE2 could increase the engraftment of
stromal cells. Furthermore, PGE2 from ISC niche may induce
Wnt signaling in ISCs, thereby participating in intestinal
In summary, these experiments point towards a new paradigm
for RIGS mitigation, whereby growth factors secreted after
BMASCT induce regeneration of the irradiated host crypt
progenitors and ISC niche, thereby, accelerating functional
recovery of the intestine in RIGS. By reducing the levels of
proinflammatory cytokines, while inducing anti-inflammatory
cytokines, BMASCT also dampens the SIRS in RIGS. Thus,
BMASCT provides a platform to discover potential biological
agents for mitigation of acute radiation syndromes and for mucosal
radioprotection during chemoradiation therapy of abdominal
Materials and Methods
Five- to 6-weeks-old male C57Bl/6 (NCI-Fort Dietrich, MD),
dipeptidyl peptidase-deficient (DPPIV2ve) (gift from Dr. David
Shafritz, Einstein College, Bronx, NY) Lgr5-EGFP-IRES-creERT2
(Jackson Laboratories, Bar Harbor, Maine) mice were maintained
ad libitum and all studies were performed under the guidelines and
protocols of the Institutional Animal Care and Use Committee of
the Albert Einstein College of Medicine. The animal use protocol
for this study was reviewed and approved by the Institutional
Animal Care and Use Committee (IACUC) of Albert Einstein
College of Medicine (IACUC approval# 20080703).
Irradiation was performed on anesthetized mice (intraperitoneal
ketamine and xylazine 7:1 mg/ml for 100 ml/mouse) using a
320 KvP, Phillips MGC-40 Orthovoltage irradiator at a 50 cm
SSD with a 2 mm copper filter at a dose rate of 72 cGy/min. We
administered WBI (10.4 Gy) or escalating doses of AIR (16
20 Gy) after shielding the thorax, head and neck and extremities
and protecting a significant portion of the bone marrow, thus
inducing predominantly RIGS.
Donor bone marrow cells were harvested using sterile techniques
from the long bones from C57Bl/6 mice and cultured in MSC basal
medium (Cambrex-Lonza, Walkersville, MD) supplemented with
10% heat inactivated FBS, 1% Glutamine, and 1% Penicillin/
Streptomycin for 4 days, followed by collection of adherent
cells as BMASC. BMASC were then subjected to flow
cytometric characterization to determine the percentage of MSC
(CD105+CD452/CD29+CD452), EPC (CD34+CD133+CD452)
and macrophages (CD11b+ F480+). CD11b+ve and CD11b2ve
cells were fractionated using anti-CD11b-magnetic beads (MACS,
Miltenyi Biotec, Auburn, CA), following the manufacturers
protocol. Fractionated and whole BMASC (26106 cells/mice) were
injected intravenously via tail vein to C57Bl6 mice at 24 and
72 s hours after irradiation.
Characterization of RIGS
Animals were sacrificed at 1, 3.5 and 7 days after irradiation for
histopathological analysis to examine apoptosis by TUNEL
staining, regenerating crypt colonies and villi denudation
(Hematoxylin and eosin staining) . To visualize villous cell
proliferation, each mouse was injected intraperitoneally with 120 mg/kg
BrdU (Sigma-Aldrich, USA) 24 hrs prior to sacrifice and
midjejunum was harvested for paraffin embedding and BrdU
immunohistochemistry (Text S1). The crypt proliferation rate
was calculated as the percentage of BrdU positive cells over the
total number of cells in each crypt. A total of 30 crypts were
examined per animal for all histological parameters. A
regenerative crypt was confirmed by immunohistochemical detection of
BrdU incorporation into five or more epithelial cells within each
crypt, scored in a minimum of four cross-sections per mouse. The
number of regenerative crypts was counted for each dose of
irradiation and represented using the crypt microcolony assay
Characterization of ISC
Lgr5+ve ISCs were detected in 4% para-formaldehyde-fixed
sections from Lgr5-EGFP-ires-CreERT2 mouse jejunum by
examining EGFP expression using confocal microscopy, according to
published protcols . GFP expression was also measured by flow
cytometry of crypt cells, isolated from Lgr5-EGFP-ires-CreERT2
mouse intestines, according to method described earlier .
Characterization of ISC niche
ISEMF were stained in formalin-fixed, paraffin-embedded
tissue sections for alpha-smooth muscle actin (a-SMA) and desmin
using Cy3-conjugated mouse anti-a-SMA (1:500; Sigma, St. Louis,
MO) and rabbit anti-desmin (1:250; Abcam, Cambridge MA)
antibody, respectively, with overnight incubation at 4uC followed
by staining with goat anti-rabbit Alexafluor 488 (1:1000;
Molecular Probes, Carlsbad, CA). Pericryptal macrophages were
stained by Alexa Fluor488-conjugated, rat anti-mouse, F480
antibody (1:50; Caltag laboratories, Carlsbad, CA). Images were
captured using a Zeiss SP2 confocal microscope at 636 optical
zoom and the macrophages were counted by using the
VelocitySoft Version 5.0 (Improvision, Waltham, MA) in 10 fields
per mice in various cohorts (n = 3).
Functional regeneration of the irradiated intestines was
determined by measuring intestinal absorption by a xylose uptake
assay [1,37]. Briefly, 5% w/v D-xylose solution was administered
orally by feeding tube (100 mL/mice, n = 5/cohort), followed by
collection of blood 2 hours post-feeding. Plasma xylose levels were
measured by a modified micro-method .
Cytokines and growth factors in blood
Intestinal growth factors, R-spondin1, keratinocyte growth
factor (KGF), basic fibroblast growth factor (bFGF) and platelet
derived growth factor-b (PDGFb) were detected in serum by
immunoblotting using goat polyclonal anti-mouse antibodies to
Rspondin1 (1:200; R & D Systems, Minneapolis, MN), KGF (1:250),
bFGF (1:250) and PDGFb (1:250). Inflammatory cytokines were
measured in the serum using a multianalyte ELISArray kit (SA
Biosciences, Fredrick, MD), according to manufacturers protocol.
Cytokine and growth factors in crypt cells
To compare the mRNA levels of different growth factors and
cytokines in intestine crypt cells from AIR and AIR+BMASCT
treated mice, real time PCR were performed using growth factor
(cat # PAMM-041) and cytokine (cat # PAMM-011) real time
array system from SA Biosciences.
To deplete macrophages liposomal clodronate (Encapsula
NanoSciences, Nashville, TN, USA) (30 mg/kg of body weight)
was injected intravenously from day 4 pre-AIR to a week
postAIR. Plain liposome was injected as control. Neither the
clodronate filled nor the empty liposomes are considered toxic to
Inhibition of COX2
NS-398 (Biomol, Plymouth Meeting, PA) was administered at a
dose of 1 mg/kg of body weight (36/week, ip) started at 1 week
prior to AIR. Animals treated with dmPGE2 (Sigma) received a
dose of 0.5 mg/kg of body weight (36/week, ip) started at 1 week
prior to AIR.
Kaplan-Meier Survival analysis
Mice survival/mortality in different treatment group was
analyzed by kaplan-Meier as a function of radiation dose using
Graphpad Prism-4.0 software for Mac.
Statistical analysis of digital images
Sampling regions were chosen at random for digital acquisition
for data quantitation. Digital image data was evaluated in a blinded
fashion as to any treatment. A two-sided students t-test was used to
determine significant differences between experimental cohorts
(P,0.05) with representative standard errors of the mean (SEM).
Figure S1 Flowcytometric characterization of freshly
isolated bonemarrow cells for expression of MSC
specific (A) (CD105+ CD452) (B) (CD29+ CD452), (C)
macrophage specific (CD11b+F480+) and (D) EPC
specific (CD133+ CD34+CD452) markers. It was noted that
bone marrow cell were primarily enriched with CD45+
hematopoetic cells (AB).
Figure S2 Blood count was performed with the help of
ANTECH DIAGNOSTICS (LAKE SUCCESS, NY) to
evaluate the effect of abdominal irradiation (AIR) on
hematopoesis. Absence of any significant changes in (A)
differential count and (B) number of RBC and among the
irradiated and transplanted group in comparison to untreated
control group suggested AIR could not affect the bone marrow.
Figure S3 Expression of different MSC surface markers
CD105, CD29, CD44, SCA1 in BMASC population.
Staining for IgG isotype fluorescence was used as a control.
Isotype control for CD105, CD29, CD44 and SCA-1 are rat
IgG2a, hamster IgG, rat IgG2bK and rat IgG2aK respectively.
Figure S4 Flowcytometric charaterization of CD11b2ve
(AB) and CD11b+ve (CD) BMASC population for
CD105 and CD29 (MSC marker) expression. It was noted
that CD11b2ve BMASC population was primarily enriched with
CD105 and CD29 positive cells.
Figure S6 Kaplan-Meier survival analysis. Mice (n = 15)
receiving first dose of BMASC at 72 h post AIR follwed by second
dose failed to mitigate RIGS in contrast to BMASCT at 24 h
follwed by 72 hr second where 100% survival were noted.
Figure S7 Transplanted BMASC were primarily
detected in intestine and lung. BMASC from DPPIV positive wild
type mice were transplanted to DPPIV negative mice exposed to
AIR. (A&C) DPPIV immunohistochemistry followed by confocal
micrscopic analysis. DPPIV positive BMASC (stained green) were
found primarily in the lung (C) and intestine (A). Nucleus was
stained with DAPI and pseudo colored with red. (B&D)
Quantification of engrafted DPPIV+ve cells. Significantly higher
number of engrafted cells in lung (p,0.002) (B) and in intestine
(p,0.004) (D) was noted at 1day post AIR compared to 3.5 day
post AIR. Confocal microscopic images (636) were magnified
2.36 and presented in inset. The number of DPPIV positive cells
were counted using volocity soft version 5 (Improvision). Based on
the intensities, number of cells were determined by scoring at least
10 fields from each animal (n = 3). Resolution of the images were
same for both experimental and control groups.
Figure S8 Immunoblot analysis of intestinal growth
factors in serum. An increase in the serum levels of
RFigure S9 AB. Multi cytokine ELISA. A.
Anti-inflammatory cytokines, IL6 (p,0.004) and IL10 (p,0.002) levels were
significantly increased in the AIR+BMASCT-treated animals,
compared to AIR alone. Induction of anti-inflammatory cytokine
IL6 (p,0.007) and IL10 (p,0.005) was also observed in the
animals treated with AIR+CD11b+ve BMASCT. Transplantation
of freshly isolated bone-marrow cells could not increase the
antiinflammatory cytokine level. B. AIR+BMASCT reduces the
proinflammatory cytokine levels (IL12A, IL17), compared to AIR
alone. Transplantation of freshly isolated bone-marrow cells could
not reduce the pro-inflammatory cytokine level compare to AIR
alone. C. AIR+BMASCT induces the GMCSF and GCSF levels
compared to AIR alone. Transplantation of freshly isolated
bonemarrow cells did not induce the GMCSF and GCSF level.
Figure S10 BMSCT maintains villus length after
radiation injury. Low magnification images (106) of jejunal
crosssections showed the reduction of villi length and thickness (H&E
staining) with the decrease in Brdu positive crypt cells in irradiated
cohort (18 Gy AIR) compared to 18 Gy+BMASC group.
Table S3 Median survival time of animals exposed to
18 Gy AIR and 10.4 Gy WBI followed by cell
transplantation. Please note the clear difference of median survival time of
the animals exposed to 18 Gy AIR compared to 10.4 Gy WBI.
Conceived and designed the experiments: SS PB RK LL AA CG.
Performed the experiments: SS PB RK LL. Analyzed the data: SS PB RK
CG. Contributed reagents/materials/analysis tools: SS PB RK CG. Wrote
the paper: SS PB AA CG.
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