Human mesenchymal stem cells promote survival and prevent intestinal damage in a mouse model of radiation injury

Abstract

Radiation-induced intestinal injury is a complex disease for which there is no effective treatment. Apoptosis of intestinal stem cells (ISCs) is the major cause of tissue damage, and replacing those ISCs with an alternative source of stem cells is gaining increasing interest as a potential therapeutic strategy for this condition. In the present study, we examined the protective effects of human umbilical cord mesenchymal stem cells (hMSCs) against ISC death and intestinal damage in a mouse model of radiation injury. hMSCs were introduced via tail vein injection in male C57BL/6J mice two hours after abdominal irradiation (AIR), and survival rates and weight changes were monitored over time. Cellular apoptosis in intestinal tissue was evaluated 6 h after AIR. Histological changes and survival of Lgr5⁺ ISCs were evaluated at days 3.5 and 5 post-AIR. Intestinal crypts were cultured ex vivo to further characterize the protective effects of hMSCs on ISCs. Ki67⁺ cells, vill⁺ enterocytes, lysozymes, CD31 vascular endothelial cells and α-smooth muscle actin were also examined by immunohistochemistry. Compared to mice treated with PBS after AIR, hMSC-treated mice exhibited improved survival, diminished weight loss, reduced radiation-induced apoptosis, attenuated structural damage of the small intestine, enhanced Lgr5⁺ ISC and Paneth cell survival, as well as increased Ki67⁺ transient amplifying cells and vill⁺ enterocytes. The distribution of CD31 vascular endothelial cells and α-smooth muscle actin demonstrated no significant difference between treatment groups. Our results demonstrate that hMSCs promote survival and attenuate intestinal damage by protecting Lgr5⁺ ISCs in a mouse model of radiation-induced injury.

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Introduction

Radiation damage caused by radiotherapy and nuclear accidents can affect the quality of life, and can even be fatal. According to previous reports, approximately 40 000 patients worldwide develop radiation-induced intestinal damage every year.¹ The gastrointestinal (GI) tract is highly sensitive to ionizing radiation (IR) in a dosage-dependent manner. Exposure to high levels of IR can lead to acute GI symptoms, for which there are no effective prevention or treatment strategies.^2–5 Intestinal epithelial cells (IECs) undergo rapid and continuous renewal, which is under the control of intestinal stem cells (ISCs) located at the bottom of intestinal crypts.^6–8 IR has been shown to inhibit the growth of IECs and ISCs and even induce their apoptosis, which may result in epithelial damage and inflammation, loss of intestinal barrier function and even fatal septicaemia.^9,10

In 1974, mesenchymal stem cells (MSCs) were first identified in human bone marrow by Friedenstein et al.¹¹ MSCs have also been found in many other tissues such as blood vessels, placenta, adipose and umbilical cord tissues.^12–16 Subsequent studies have shown that MSCs have the ability to migrate to sites of tissue damage and differentiate into tissue-specific cell types.^17–19 MSCs may also create a protective microenvironment for the damaged tissue by secreting various growth factors and other molecules.^20–29 Furthermore, MSCs have demonstrated resistance to IR and have retained their stem cell characteristics even after high doses of radiation.¹⁹ MSC-based therapeutics had emerged as an advanced tool to treat a variety of conditions. For example, Prochymal, Provacel and Chondrogen are FDA-approved products for the treatment of graft-versus-host disease, myocardial infarction and meniscus regeneration.³⁰ Moreover, animal model studies have revealed that mice exposed to intestinal irradiation exhibited increased survival and decreased weight loss upon subsequent treatment with MSCs as compared to control mice treated with irradiation alone.^31,32 At the cellular level, upon radiation, intestinal cell apoptosis decreased and the proliferation marker Ki67 increased in mice receiving MSC treatment.³⁰ Taken together, these studies suggest that MSCs may play an important role in the treatment of intestinal injury induced by radiation.

ISC damage is considered one of the main mechanisms of radiation-induced intestinal injury. As such, our study aimed to examine the role of hMSCs in protecting ISCs in a mouse model of radiation-induced intestinal damage. Using immunohistochemistry, we also analyzed Ki67⁺ cells, vill⁺ enterocytes, lysozyme expression, and the distribution of CD31 vascular endothelial cells and α-smooth muscle actin. In summary, we found that human umbilical cord mesenchymal stem cells protect Lgr5⁺ ISC from radiation-induced damage, as well as Ki67⁺ cells, vill⁺ enterocytes and Paneth cells.

Materials and methods

Mouse model of radiation-induced intestinal injury

6–8 week-old male C57BL/6J mice weighing 23–24 g, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (China). All mice were housed on a 12 hour light/dark cycle in a temperature controlled specific-pathogen free environment and fed standard chow and water. All experimental procedures and protocols were conducted in accordance with the guidelines of our local animal care and use committee.

Abdominal irradiation (AIR) was performed on mice at a dosage rate of 1 Gy per minute using a Cr¹³⁷ γ-ray irradiator (Atomic Energy of Canada) at room temperature. Lead shielding was used to protect other parts of the body from irradiation. Mice were irradiated with 11, 13 and 15 Gy, and 13 Gy was selected as the optimal irradiation dose. There were at least 10 mice in each treatment group.

Cell culture

Human umbilical cord mesenchymal stem cells (hMSCs) were gifted by the National Engineering Research Center of Cell Products of Tianjin in China. hMSCs were cultured in DMEM-12 medium supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin/streptomycin without any additional growth factors, in a humidified incubator containing 5% CO₂ at 37 °C. After approximately 5 passages, cells were washed with ice-cold PBS, harvested by trypsinization and then resuspended in ice-cold PBS. Within two hours post-IR, 1 million MSCs or PBS were injected into mice via the tail vein.

Isolation and ex vivo culture of small intestinal crypts

Mouse intestinal crypts were isolated and cultured as previously described.^33,34 Crypts were first dissected from the small intestine, and approximately 500 crypts were suspended in 20 μl of gut media (Advanced DMEM/F12 (Gibco), 50 ng ml⁻¹ EGF (Sigma-Aldrich), 100 ng ml⁻¹ Noggin (R&D Systems), 1 μg ml⁻¹ R-spondin (R&D Systems), 10 mM HEPES (Gibco), 100 U ml⁻¹ penicillin (Gibco), 100 μg ml⁻¹ streptomycin (Gibco), 1% N2 supplement (Invitrogen) and 1% B27 supplement (Invitrogen)). These crypts were mixed with 50 μl of Matrigel (BD Bioscience) and plated in one well of a pre-warmed 24-well plate (Corning). The plate was placed in the 37 °C incubator for 30 min, and 500 μl of gut media was added. Gut media was changed every 4 days.

Apoptosis analysis

After 13 Gy AIR, mice were sacrificed at 6 h and the entire small intestine was harvested for histology. The small intestine was fixed in neutral formalin. Three 5 mm segments of small intestinal tissue were taken from each mouse, dehydrated and embedded in paraffin. Sections of 3 μm were dewaxed, rehydrated and treated with proteinase K for 5 min in a 37 °C water-bath. Sections were then incubated with TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) detection liquid for 1 h at 37 °C. After multiple washes, sections were counterstained with DAPI. Slides were then washed and observed under a fluorescence stereomicroscope.

Immunohistochemistry

Mice were sacrificed at 3.5 and 5 days after AIR, and small intestinal tissue was fixed with neutral formalin. Segments of duodenum, jejunum and ileum were collected, dehydrated and embedded with paraffin. Sections (3 μm thick) were dewaxed and treated with citrate buffer. After antigen retrieval, sections were treated with hydrogen peroxide for 15 min then blocked with serum for 1 h. Sections were incubated with primary antibody overnight at 4 °C, including antibodies against Lgr5 (Abcam), Ki67 (Abcam), lysozyme (Abcam), CD31 (Abcam), α-smooth muscle actin (Abcam) and vill (Abcam). Sections were washed thoroughly with PBS and incubated with secondary antibody for 30 min at 37 °C. After washing, signals were detected using a DAB kit. Sections were counterstained with H&E.

Statistical analysis

All data were obtained from three independent experiments and expressed as the mean ± SEM unless otherwise indicated. The experimental data were analyzed using SPSS17.0 software. One-way ANOVA testing was used to assess differences between groups. P < 0.05 was considered statistically significant.

Results

hMSCs improve survival and prevent weight loss in AIR-treated mice

Unlike humans who are often treated with radiation as a treatment strategy for various disorders, mice exposed to radiation mainly exhibit weight loss and death. To evaluate the therapeutic effect of hMSCs in radiation-induced intestinal injury, we first observed the survival of mice irradiated at 11, 13 and 15 Gy (Fig. 1A). After 15 Gy AIR, all treated mice died within 7 days in both the hMSC group and the PBS control group. In the PBS group, there was a 30% survival rate in mice receiving 13 Gy AIR, whereas the same dosage led to a 100% survival rate in mice in the hMSC group. Exposure to 11 Gy AIR resulted in nearly equal survival rates between the two groups, 90% and 100% respectively. Our findings indicated that hMSCs prevented mortality throughout the 30 d experimental period, suggesting that hMSCs conferred a significant survival benefit in mice.

Fig. 1 hMSCs protect mice from radiation-induced mortality and weight loss. Mice were tail vein-injected with PBS or hMSCs after 11, 13 and 15 Gy AIR. (A) Exposure to 11 Gy or 15 Gy resulted in no significant differences in the survival rate of mice treated with PBS or hMSCs. After 13 Gy, a significant increase in survival was observed in hMSC-treated mice compared to PBS-treated controls (P < 0.05). (B) Body weight changes after radiation. During the course of the experiment, the body weight was measured everyday after AIR. There was no significant difference in mice treated with PBS or hMSCs receiving 11, 13 or 15 Gy AIR.

Weight loss is one of the symptoms of acute GI syndrome caused by radiation (Fig. 1B). Irradiated mice receiving 15 Gy AIR consistently lost weight until death in both the hMSC and PBS groups. The trend in weight loss demonstrated no significant difference. Upon exposure to 11 Gy and 13 Gy AIR, mice consistently lost weight for 5 days in both treatment groups, then began to regain the lost weight. At 10 days, mice lost weight again. In mice exposed to 13 Gy, PBS-treated mice exhibited an increase in weight loss as compared to those in the hMSC treatment group. There was no difference between groups receiving 11 Gy AIR. After 15 days, the weight of mice exposed to 13 Gy returned to normal, however, the weight in the hMSC-treatment group was always higher than the PBS group. After 15 days, mice radiated with 11 Gy still lost weight until reaching a plateau in the PBS group. Taken together, these findings demonstrate that hMSCs may prevent radiation-induced weight loss.

hMSCs reduce radiation-induced apoptosis

Intestinal epithelial cells are highly sensitive to radiation due to their capacity for rapid proliferation. The TUNEL assay was used to evaluate radiation-induced apoptosis. Six hours post-radiation with 13 Gy revealed a four-fold increase in TUNEL-positive cells relative to non-irradiated control mice, mice treated with hMSCs showed a significant reduction in TUNEL-positive cells (Fig. 2), indicating that hMSCs have a protective role in preventing radiation-induced intestinal damage through suppression of apoptosis.

Fig. 2 Radiation-induced apoptosis is reduced in mice receiving hMSCs. 6 hours after AIR, apoptosis was assayed by TUNEL staining. The number of TUNEL positive cells was quantified per field. Results represent fifteen tissue specimens in each group (three mice per group) and are shown as the mean ± SEM. **P < 0.01 bars, 100 μm.

hMSCs attenuate structural damage of the small intestine

Radiation is known to cause severe tissue damage. Therefore, we assessed the effect of radiation on the overall structure of the small intestine at 3.5 and 5 days post-radiation (Fig. 3). H&E staining revealed a change in epithelial structure among the three groups. At day 3.5, the PBS-treated group exhibited sparser, shorter villi throughout the small intestine, whereas radiation-induced epithelial disorganization was reduced in the hMSC group. At day 5, the epithelial layer was slightly repaired, however, sloughing of villi was still observed in mice treated with PBS. In the hMSC-treated group, mice had an increased number of villi which were significantly longer and thicker than those in the PBS-treated group. These results strongly suggest that hMSCs promote the repair of radiation-induced intestinal damage.

Fig. 3 Histological analysis of small intestinal injury in control mice, PBS-treated mice and hMSC-treated mice after AIR. On day 3.5 (A) or day 5 (B), duodenum, jejunum and ileum tissues from each group were stained with H&E. The number of crypts per field was measured and statistically analyzed. Results represent twenty-five tissue specimens in each group (five mice per group) and are shown as the mean ± SEM. **P < 0.01 bars, 100 μm.

We also observed a dramatic reduction of crypts in mice exposed to 13 Gy radiation. These remaining intestinal structures exhibited hypertrophy, which was in accordance with the previous study.³⁵ Compared to the PBS group, there was a significantly higher number of remaining crypts in mice treated with hMSCs.

hMSCs enhance Lgr5⁺ ISC survival

Previous studies have established that Lgr5-positive crypt base columnar cells generate all epithelial lineages over a 60 day period, suggesting that Lgr5⁺ cells represent the stem cell of the small intestine.⁸ 3.5 days after radiation, Lgr5-expressing cells were very sparse in PBS-treated mice as compared to the control group. In contrast, the number of Lgr5-positive cells was significantly increased in mice treated with hMSCs. At 5 days post-radiation, the number of Lgr5⁺ cells was significantly lower than both the control and the hMSC group (Fig. 4A).

Fig. 4 hMSCs protect Lgr5⁺ ISCs from radiation. (A) 3.5 days or 5 days post-exposure to radiation, small intestine samples were collected and immunostained using an antibody against Lgr5. Lgr5-positive cells (highlighted with arrows) were quantified in five crypts per section. Results represent twenty-five tissue specimens in each group (five mice per group) and are shown as mean ± SEM. **P < 0.01 crypts were obtained from each group on day 3.5 (B) or 5 (C) after AIR and cultured ex vivo for 7 days. Isolated crypts containing Lgr5⁺ ISCs generate organoids in vitro (highlighted with arrows). Results represent six samples in each group (three mice per group) and are shown as the mean ± SEM. **P < 0.01 bars, 50 μm (A), 1000 μm ((B) left) and 400 μm ((B) right and (C)).

In order to further demonstrate that hMSCs increase Lgr5⁺ ISC survival, a three-dimensional ex vivo intestinal crypt culture system was used (Fig. 4B and C). Isolated crypts expressing Lgr5 can generate organoids in vitro.³⁴ Compared to the control group, the number of isolated crypts was significantly lower in mice treated with PBS at 3.5 days of exposure to irradiation. Furthermore, isolated crypts cultured ex vivo for 7 days lacked the ability to form organoids. Although the intestinal crypts isolated from mice treated with hMSCs were also fewer than the control mice, the number of resulting organoids was significantly higher than those crypts isolated from PBS-treated mice. Moreover, isolated crypts from PBS-treated mice formed organoids 5 days after radiation. Meanwhile we observed that the number of organoids was less than the other two groups. These results corresponded to the previous immunohistochemistry results.

These results demonstrate that hMSCs have a protective effect on intestinal stem cells against radiation injury.

hMSCs promote Paneth cell survival and increase the proliferation of Ki67⁺ transient amplifying cells and vill⁺ enterocytes

In addition to Lgr5⁺ ISCs, the stem cell niche located at the bottom of intestinal crypts contains Paneth cells, which are known to produce lysozymes.³⁶ Previous studies have shown that the number of Paneth cells decrease in parallel with loss of Lgr5⁺ stem cells.³⁷ Thus, the change in Paneth cells was investigated among control mice, PBS-treated mice and hMSC-treated mice at days 3.5 and 5 after 13 Gy radiation (Fig. 5A). While the number of Paneth cells was significantly reduced in radiation groups compared to the control group, the hMSC-treated group resulted in mice with a higher number of intestinal Paneth cells relative to PBS-treated groups.

Fig. 5 hMSCs promote Paneth cell survival and increase the proliferation of Ki67⁺ transient amplifying cells and vill⁺ enterocytes. At days 3.5 or 5 after AIR, small intestinal tissues from the control group, PBS-treated group and hMSC-treated group were collected and immunostained to identify (A) lysozymes, (B) Ki67⁺ transient amplifying cells and (C) vill⁺ enterocytes. Results represent twenty-five tissue specimens in each group (five mice per group) and are shown as the mean ± SEM. **P < 0.01 bars, 50 μm (A) and 100 μm (B and C).

We also evaluated the proliferation of intestinal epithelial cells using a Ki67 antibody. Ki67⁺ immunostaining identifies proliferating cells, which is indicative of a regenerative response of the epithelial layer. 3.5 days after radiation there was a significant decrease in Ki67 positive cells in the PBS treated mice as compared to control mice, however, treatment with hMSCs leads to an increase in Ki67 staining relative to PBS-treated mice. At day 5, the expression of Ki67 positive cells was increased in mice treated with PBS, but was still lower than the other two groups (Fig. 5B). These results indicate that hMSC treatment promotes cell proliferation in irradiated mice.

The expression of vill⁺ enterocytes was also affected by radiation. Radiation caused villi damage, and as a result, vill⁺ enterocytes were reduced in the two radiation groups. However, mice treated with hMSCs exhibited a significant increase in vill⁺ positive cells as compared to PBS-treated controls (Fig. 5C). Therefore, hMSCs promote the restoration of vill⁺ enterocytes in a mouse model of radiation-induced injury.

CD31 vascular endothelial cells and α-smooth muscle cells are not obviously affected

To demonstrate that the intestinal lesion caused by radiation was mainly associated with intestinal stem cells, we analyzed the expression of CD31 vascular endothelial cells and α-smooth muscle actin. In mice treated with PBS or hMSCs post-radiation, we found no obvious aberrant localization of vascular endothelial cells or smooth muscle cells in the small intestine at days 3.5 and 5 (Fig. 6A and B).

Fig. 6 Localization of vascular endothelial cells and smooth muscle cells in the small intestine. The small intestine was harvested from control, PBS-treated and hMSC-treated mice. Tissue sections were immunostained using antibodies against markers for smooth muscle cells (A) and endothelial cells (highlighted with arrows) (B). Results represent twenty-five tissue specimens in each group (five mice per group) and are shown as the mean ± SEM. **P < 0.01 bars, 20 μm (B) and 50 μm (A).

Discussion

Humans are often exposed to radiation whether through therapeutic use in treating cancer or through environmental exposure in the event of a nuclear accident. Intestinal epithelial cells are radiosensitive, and exposure to radiation leads to acute radiation syndrome (ARS) of the GI tract through the loss of Lgr5⁺ ISCs. The crypt base columnar (CBC) cells reside at the base of the crypt, and Lgr5⁺ CBC cells are the small intestinal stem cells.⁸ Lgr5⁺ ISCs are active ISCs and rapidly cycling, the result of which forms transient-amplifying cells (TACs).^8,38 TACs can differentiate into intestinal epithelial subtypes, i.e. enterocytes, goblet cells and neuroendocrine cells.⁸ Therefore, Lgr5⁺ ISCs may play the key role of maintaining intestinal epithelial homeostasis and regeneration following injury. There has been growing interest to develop therapeutic agents for the management of GI ARS; more specifically to counter the radiation-induced loss of Lgr5⁺ ISC. MSCs have demonstrated the ability to home to injury sites and release beneficial factors.¹⁹ MSCs exhibit a notable capacity to adapt to the requirements of the injured tissue. In addition, MSCs do not elicit a host immune response due to low expression of Major Histocompatibility Complex class II (MHC-II) molecules.^39,40 MSC-based cell therapy is an emerging paradigm gaining increasing attention in drug development. In this study, we demonstrate that human umbilical cord MSCs provide an effective method for the treatment of GI ARS in a mouse model of radiation injury.

Interestingly, our study revealed that treatment of irradiated mice with hMSCs promoted survival and reduced overall weight loss as compared to mice treated with PBS. Furthermore, 6 h after irradiation, cell apoptosis reached a peak then gradually reduced over time.³⁷ We observed that hMSCs reduced intestinal apoptosis 6 h after radiation at the site of the crypt, where Lgr5-expressing intestinal stem cells reside.

Within the range of exposure dosages between 8 and 15 Gy, Lgr5⁺ ISC survival gradually deceased with the increase in radiation at 2 days or 3.5 days post-injury. After exposure to 12 Gy, Lgr5⁺ ISC loss was the most profound at 2 days with recovery after 3.5 days. Upon exposure to 15 Gy however, there was insufficient survival of Lgr5⁺ ISCs 1 day post-irradiation, and correspondingly no surviving cells were detectable at 3.5 days.³⁷ Based on our previous work, we selected 13 Gy as the optimal radiation dosage, and determined that the best detection time point was at days 3.5 and 5. Intestinal epithelial cells undergo rapid and continuous renewal via the activation of crypt Lgr5⁺ ISCs.³⁸ ISC loss caused by radiation can lead to disorders of the intestinal structure and function and in some cases even death. Our study showed that hMSCs injected into irradiated mice have a protective effect on the small intestinal structure and Lgr5⁺ ISCs at 3.5 and 5 days after 13 Gy radiation in vivo. Single crypts isolated from the small intestine form organoids when cultured ex vivo.³⁴ Furthermore, we also observed that hMSCs protected Lgr5⁺ cells from radiation using this ex vivo culture method. Paneth cells play a supportive role for Lgr5⁺ ISCs to maintain homeostasis.⁴¹ Radiation-induced damage to Lgr5⁺ cells was accompanied by Paneth cell injury.³⁷ Using immunohistochemistry, we observed that the expression of Paneth cell associated lysozymes sharply decreased after radiation. Conversely, the expression of lysozymes in mice treated hMSCs was significantly higher than that of mice receiving PBS. In hMSC-treated mice, we also observed a significant increase in both Ki67⁺ cells and vill⁺ enterocytes, suggesting that hMSCs promote the survival of Paneth cells as well as enterocytes upon radiation injury.

Previous studies have shown that microvascular endothelial apoptosis correlated with the development of radiation-induced GI syndrome. Inhibiting endothelial injury prevented crypt damage.^42,43 However, the conclusion was controversial.⁴⁴ In our study, we found that radiation has no obvious effect on the distribution of CD31 vascular endothelial cells and α-smooth muscle actin cells between the mice receiving PBS or hMSCs. We speculate that radiation-induced GI syndrome may be attributed mostly to the damage and loss of Lgr5⁺ cells. Future studies will utilize transgenic knockout mice to further decipher the cell types affected by radiation-induced intestinal injury.

Conclusions

This study concludes that hMSCs promote survival and reduce intestinal damage by preventing apoptosis of Lgr5⁺ ISCs in a mouse model of intestinal radiation injury. Furthermore, hMSCs also increase the total number of intestinal crypts and Ki67⁺ transient amplifying cells, and enhance the expression of lysozymes and vill⁺ enterocytes.

Acknowledgements

This study was supported by the Innovation Fund of Chinese Academy of Medical Science and Peking Union Medical College (no. Academy 1511), the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (no. 33320140125), the Special Foundation of the Ministry of Health (no.201002009), National Natural Science Foundation of China (no. 31170804, 31200634, 31300695), the Natural Science Foundation of Tianjin (no. 12JCYBJC15300, 12JCYBJC32900, 13JCYBJC23500, 13JCQNJC11600), the Science Research Foundation for Doctor-Subject of High School of the National Education Department (no. 20121106120044, 20121106120043).

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