Chinese Medical Journal 2014;127 (3) 475 Original article Allogeneic compact bone-derived mesenchymal stem cell transplantation increases survival of mice exposed to lethal total body irradiation: a potential immunological mechanism

Qiao Shukai, Ren Hanyun, Shi Yongjin and Liu Wei

Keywords: mesenchymal stem cells; transplantation; total body irradiation; colony-forming unit /; immunological mechanism

Background Radiation-induced injury after accidental or therapeutic total body exposure to ionizing radiation has serious pathophysiological consequences, and currently no effective therapy exists. This study was designed to investigate whether transplantation of allogeneic murine compact bone derived-mesenchymal stem cells (CB-MSCs) could improve the survival of mice exposed to lethal dosage total body irradiation (TBI), and to explore the potential immunoprotective role of MSCs. Methods BALB/c mice were treated with 8 Gy TBI, and then some were administered CB-MSCs isolated from C57BL/6 mice. Survival rates and body weight were analyzed for 14 days post-irradiation. At three days post-irradiation, we evaluated IFN-γ and IL-4 concentrations; CD4+CD25+Foxp3+ regulatory T cell (Treg) percentage; CXCR3, CCR5, and CCR7 expressions on CD3+ T cells; and splenocyte T-bet and GATA-3 mRNA levels. CB-MSC effects on bone marrow hemopoiesis were assessed via colony-forming unit granulocyte/macrophage (CFU-GM) assay. Results After lethal TBI, compared to non-transplanted mice, CB-MSC-transplanted mice exhibited significantly increased survival, body weight, and CFU-GM counts of bone marrow cells (P<0.05), as well as higher Treg percentages, reduced IFN-γ, CXCR3 and CCR5 down-regulation, and CCR7 up-regulation. CB-MSC transplantation suppressed Th1 immunity. Irradiated splenocytes directly suppressed CFU-GM formation from bone marrow cells, and CB-MSC co-culture reversed this inhibition. Conclusion Allogeneic CB-MSC transplantation attenuated radiation-induced hematopoietic toxicity, and provided immunoprotection by alleviating lymphocyte-mediated CFU-GM inhibition, expanding Tregs, regulating T cell chemokine receptor expressions, and skewing the Th1/Th2 balance toward anti-inflammatory Th2 polarization. Chin Med J 2014;127 (3): 475-482

otal body irradiation (TBI) is an effective therapeutic and chemokine profiles, and genetic levels.11,12 therefore, Ttool to kill cancer cells in certain blood cancers and to it is necessary to understand the specific biological prevent immune rejection in bone marrow transplantation characteristics of CB-MSCs. patients. However, TBI is associated with several limitations, such as bone marrow failure and acute radiation MSCs possess self-renewal ability and the potential syndrome, which cause harmful adverse effects and to differentiate into a variety of mesenchymal increase the risk of death.1,2 TBI-containing conditioning lineage tissues.13,14 Importantly, MSCs express major regimens also significantly increase the incidence of histocompatibility complex (MHC) class I antigens at a graft-versus-host disease following allogeneic stem cell low level, but do not express MHC class II antigens at all; transplantation (allo-HSCT).3 There is currently no effective they also lack the B7 family co-stimulatory molecules that treatment against these TBI-induced injuries. are required to initiate an immune response.15 Based on this hypo-immunogenicity, administered MSCs may cross MHC Mesenchymal stem cells (MSCs) are pluripotent non- barriers without meeting with immunological rejection. hematopoietic progenitor cells that were first identified Many reports indicate that MSCs could exert profound in the bone marrow by Friedenstein et al.4 Since then, anti-inflammatory and immuno-suppressive effects on they have also been isolated from other tissues, including several immune cells via a variety of mechanisms, notably fat, skeleton, umbilical cord blood, and amniotic fluid.5,6 cytokine and chemokine secretion, apoptosis induction, and Several recent studies have independently reported MSC 7-10 DOI: 10.3760/cma.j.issn.0366-6999.20132001 purification from human or mouse trabecular bone. Their Department of Hematology, Peking University First Hospital, Beijing findings and our previous work confirm that the MSCs 100034, China (Qiao SK, Ren HY, Shi YJ and Liu W) obtained from collagenase-treated bone fragments are of Correspondence to: Dr. Ren Hanyun, Department of Hematology, similar or greater amount and purity compared to their Peking University First Hospital, Beijing 100034, China (Tel: 86-10- marrow-derived counterparts.8,10 MSCs from different 83575082. Fax: 86-10-66551811. Email: [email protected]) This study was supported by the National Natural Science Foundation sources also differ in surface immunophenotype, cytokine of China (No. 30940030, No. 81070448, and No. 81370667). 476 Chin Med J 2014;127 (3) expansion of Tregs.16,17 The treated bone fragments were washed three times with PBS and placed in non-coated plastic flasks with MSCs can reportedly protect irradiated mice by inducing C57BL/6 MSC Growth Medium (Cyagen, Guangzhou, hematopoiesis and reducing bone marrow cell apoptosis.18 China) supplemented with 10% fetal calf serum, 100 Shim et al19 found that treatment with umbilical cord µg/ml penicillin, 10 µg/ml streptomycin, and 1 mmol/L blood-derived MSCs had regenerative effects on white L-glutamine. The mixture of fragments and released cells blood cells, lymphocytes, and of irradiated was incubated at 37°C in a humidified atmosphere with 5% mice, and suggested that MSC treatment is superior CO2. The medium was changed every 3−4 days. Adherent to G-CSF treatment for hematopoietic reconstitution. cells were harvested by trypsin digestion and passaged until However, very little is known about the alterations of about 70% confluence. The cells of passage 5 (P5) were T-cell function in mice after lethal TBI, and whether these harvested for use in the following experiments. changes further aggravate hematopoietic toxicity. Several studies reported increased levels of IFN-γ and TNF-α in The harvested P5 C57BL/6 MSCs were stained with irradiated mice. As these cytokines are produced by Th1- fluorescein-conjugated monoclonal antibodies against type cells, we hypothesized that the T cells of irradiated mouse CD45, CD34, CD29, CD44, CD117, and Sca-1 mice were abnormally activated after lethal TBI, leading to (Biolegend, San Diego, CA) and incubated for 30 minutes increased Th1 activity relative to Th2, and thus increased in the dark, followed by flow cytometry analysis. To secretion of Th1-type cytokines. These cytokines are also evaluate their osteogenic, adipogenic, and chondrogenic negative hematopoietic regulatory factors, which could differentiation, P5 MSCs were seeded at 1 000 cells/cm2 directly cause damage to hematopoietic stem cells (HSC); and incubated for over 24 hours before changing to the in this case, CB-MSC transplantation could not only induction media (Cyagen, Guangzhou, China) following the attenuate radiation-induced HSC injury, but also mediate manufacturer’s protocol. The multilineage differentiation additional protective mechanisms by anti-inflammatory and abilities of MSCs were assessed based on histochemical immunosuppressive effects. staining and differentiation-specific gene expressions measured by reverse transcription PCR (RT-PCR) under In this study, we observed the effects of lethal irradiation different induction conditions, as described previously.20 on hematopoiesis and the immune system, and investigated Table 1 shows the differentiation-specific gene primers. whether CB-MSCs induced immune-protective effects against radiation-induced hematopoietic toxicity by Splenocyte proliferation assays regulating immune function in irradiated mice. The results Confluent P5 MSCs were used as a feeder layer. Varying showed increased Th1 activity after lethal TBI, which could numbers of MSCs (104, 5×103, and 103 per well) were inhibit bone marrow hematopoiesis. Furthermore, CB- seeded in a 96-well plate with complete medium, and MSC transplantation significantly improved hematopoietic maintained for 24 hours to allow MSC adherence. Then, we recovery through the direct protection of HSC, and exerted added 105 splenocytes (SPs) isolated from BALB/c mice additional immune-protective influence by reversing and a final concentration of 10 µg/ml phytohemagglutinin the Th1 immunity-mediated negative regulation of hematopoiesis. Table 1. Primers for RT-PCR analysis of differentiation-specific gene expression and real-time RT-PCR analysis of transcription METHODS factor genes Genes Primer sequences Animals Bone-specific genes OCN Sense: 5′-GACCATCTTTCTGCTCACTCTG-3′ Healthy female C57BL/6 (H-2b) and male BALB/c (H-2d) Antisense: 5′-GTGATACCATAGATGCGTTTGTAG-3′ mice were purchased from the Animal Experiment Center OPN Sense: 5′-CAGTGATTTGCTTTTGCCTGTTTG-3′ of Peking Medical University. The C57BL/6 mice used for Antisense: 5′-GGTCTCATCAGACTCATCCGAATG-3′ Adipose-specific genes MSC cultivation were 6−8 weeks old and 18−23 g each. LP Sense: 5′-GAGGACACTTGTCATCTCATTC-3′ The BALB/c recipient mice were 8−10 weeks old and Antisense: 5′-CCTTCTTATTGGTCAGACTTCC-3′ 23−25 g each. All experimental protocols were approved Adipsin Sense: 5’-ATGGTATGATGTGCAGAGTGTAG-3′ Antisense: 5′-CACACATCATGTTAATGGTGAC-3′ by the Animal Care and Use Committee of Peking Medical Cartilage-specific genes College. Collagen II Sense: 5′-GGCTTAGGGCAGAGAGAGAAGG-3′ Antisense: 5′-TGGACAGTAGACGGAGGAAAGTC-3′ Aggrecan Sense: 5′-CTAAGTTCCAGGGTCACTGTTAC-3′ Isolation and identification of murine CB-MSCs Antisense: 5′-TCCTCTCCGGTGGCAAAGAAG-3′ The femurs and tibias were aseptically separated from Transcription factor genes C57BL/6 mice with ophthalmic scissors, and the T-bet Sense: 5′-TCA ACC AGC ACC AGA CAG AG-3′ Antisense: 5′- AACATCCTG TAA TGG CTT GTG -3′ metaphysis were removed. Bone marrow was flushed out GATA-3 Sense: 5′-CTTATCAAGCCCAAGCGAAG - 3′ and bone cavities were washed thoroughly with phosphate- Antisense: 5′-CCCATTAGCGTTCCTCCTC-3′ buffered saline (PBS) using a syringe. The compact bones Housekeeping gene 2 β-tubulin Sense: 5′-GGT AACAACTGGGCGAAGGG-3′ were cut into small fragments (1−2 mm ) and treated with Antisense: 5′-CATGACGCTAAAGGAGTTCA-3 0.1% collagenase II (Sigma, St. Louis, MO) for 2 hours OCN: osteocalcin; OPN: osteopontin; LP: lipoprotein lipase; Collagen II: collage at 37°C with gentle agitating in plastic culture dishes. type II; T-bet: T-box expressed in T-cells; GATA-3: GATA-binding protein-3. Chinese Medical Journal 2014;127 (3) 477

(PHA, Sigma, St. Louis, MO) to each well, such that SPs 1×105 cells/ml in a 12-well plate with Methylcellulose were in direct contact with confluent MSC feeder cells. The Culture Medium-M3534 (StemCell Technologies, BC). co-culture ratios of MSC/SP were 1:10, 1:20, and 1:100, The cultures were incubated at 37°C with 5% CO2 under respectively. As controls, 105 SPs were cultured alone saturated humidity for 7 days, and then CFU-GM colonies with or without PHA. Cultures were set up in triplicate were quantified by counting colonies of ≥50 cells using an and maintained at 37°C in a humidified atmosphere with inverted microscope. Assays were performed on at least

5% CO2 for 66 hours. Then we added 10μl MTS-PES three independent occasions and plated in triplicate for each (Promega, Madison, WI) to each well for and incubated experiment. for 6 more hours. A microplate reader was used to measure the absorbance value at 490 nm for each well, which To determine whether CB-MSCs accelerated CFU-GM represented the SP proliferation. formation by directly acting on T cells, we designed a relevant in vitro co-culture system. The experiment included Experimental groups four groups: irradiated BMC+SPs, BMC+irradiated SPs, We used a clinically relevant animal model to investigate BMC+irradiated SPs+MSCs (with 5×104CB-MSCs added), the effects of CB-MSCs on mice exposed to lethal TBI. and BMC+SPs. Briefly, BMC and SP were isolated from BALB/c recipient mice received 8 Gy of total body normal age-matched BALB/c mice (with or without X-ray irradiation (0.5 Gy/min). At 6 hours after TBI, irradiation of 8 Gy) and were mixed at a 1:3 ratio. The some irradiated mice were injected with 1.0×107 CB- cell mixtures from each group were then plated at a final MSCs via tail veins. The animals were divided into three concentration of 1×105 BMC/ml in a 12-well plate with groups: simple irradiation (n=10); irradiation and MSC Methylcellulose Culture Medium-M3534. Cultures were transplantation (n=10); and healthy age-matched control maintained in a humidified atmosphere at 37°C and 5%

BALB/c mice (n=10). Mice were subsequently housed in CO2 for 7 days, and then CFU-GM colonies were counted. sterilized microisolator cages, and received normal chow Experiments were performed in triplicates. and autoclaved hyper-chlorinated water. Survival rates and body weight changes were assessed for 14 days after TBI. Statistical analysis All statistical data are presented as mean±standard At 3 days post-irradiation, peripheral blood, splenocytes, deviation (SD). Treatment effects among different groups and bone marrow were extracted from each surviving were compared using a one-way analysis of variance. animal. Plasma samples were prepared for IFN-γ and IL-4 Survival rates were compared using a log rank (Mantel- level measurements. Blood cell samples were used to detect Cox) test. All data were analyzed using GraphPad Prism Tregs and the expressions of CXCR3, CCR5, and CCR7. Version 5.0 (San Diego, CA, USA) and P<0.05 was Splenocytes were isolated to assess the mRNA levels of considered statistically significant. T-bet and GATA-3. Bone marrow cells (BMC) were used for CFU-GM assay. RESULTS

Cytokine levels, Tregs, and chemokine receptor CB-MSC morphological features, immunophenotype, expression on T cells and multilineage differentiation The protein levels of IFN-γ and IL-4 were measured by After about 24 hours of incubation, spindle-shaped cells ELISA following the manufacturer’s protocol (R&D with plastic adherence features were generated from the Systems, Minneapolis, MN). Tregs and chemokine receptor inoculated compact bone fragments. After three passages, protocols were evaluated by flow cytometry. Prior to the obtained spindle cells were homogeneous and possessed flow cytometric analysis, the blood samples were treated strong adherence and trypsinization capacities (Figure 1A). according to the manufacturer’s protocol and stained with Flow cytometry results indicated that P5 MSCs expressed fluorescein-conjugated monoclonal antibodies against high levels of CD44, CD29, CD34, and Sca-1, but did mouse CD4-CD25-FOXP3, CD3ε, CXCR3, CCR5, and not express CD45 and CD117 (Figure 1B). P5 MSCs CCR7 (Biolegend, San Diego, CA, USA). successfully differentiated into osteogenic, adipogenic, and chondrogenic cells under different in vitro induction Measurement of T-bet and GATA-3 mRNA conditions, showing positive staining results with alizarin Total RNA was isolated from splenocytes using Trizol red, oil red O, and alcian blue, respectively (Figure 2A). reagent (Invitrogen, Carlsbad, CA, USA), and then cDNA The differentiated cells also showed differentiation-specific was synthesized from 2 µg total RNA using a first-strand gene expressions; OCN and OPN represented bone- cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). T-bet specific genes, LP and adipsin represented adipose-specific and GATA-3 were quantified relative to β-tubulin, using an genes, and collagen II and aggrecan represented cartilage- ABI 7500 and SYBR Green chemistry. PCR primers were specific genes (Figure 2B). Together, these data clearly designed based on the corresponding mouse gene structure, demonstrated that the cells obtained from the compact bone for which the sequences are listed in Table 1. Real-time RT- fragments fulfilled the standard definition of MSCs. PCR assays were performed in triplicates. CB-MSCs inhibited splenocyte proliferation triggered CFU-GM assay by PHA in vitro Bone marrow cells were plated at a concentration of Absorbance values were significantly lower for the MSC/ 478 Chin Med J 2014;127 (3)

Figure 2. The specific histochemical staining (A) and lineage- specific gene expressions (B) of MSCs after committed induction. A: a, Alizarin red staining after osteoblastic induction; b, Oil red O staining after adipocytic induction; c, Alcian blue staining after chondrocytic induction. B: a, Bone-specific genes; b, Fat-specific genes; c, Cartilage-specific genes. Lane 1: uninduced MSCs, Lane 2: induced MSCs. OCN: osteocalcin; OPN: osteopontin; LP: lipoprotein lipase; Collagen II: collage type II.

MSC transplantation had similar manifestations during the first five days post-radiation, including rapid weight loss, lassitude, ruffled fur, and a hunched posture; none had diarrhea. After the first 5 days, body weights began to Figure 1. The morphological features (A) and phenotypic gradually rise among the mice with MSC transplantation. characterization (B) of MSCs cultured from C57BL/6 mice The median survival times in irradiated mice with using a collagenase-treated bone fragment method. and without MSC transplantation were 8 and 13 days, respectively. At 13 days post-irradiation, the mice without SP 1:10 and 1:20 co-culture groups than for SPs alone MSC transplantation had all died, while 5/10 mice with with or without PHA (P<0.01). The absorbance was also MSC transplantation remained surviving at 14 days post- significantly lower for the MSC/SP 1:100 group than for radiation. Our data showed that TBI alone significantly SPs with PHA (P<0.01), but did not significantly differ reduced the survival rate compared to that of the healthy compared to SPs without PHA (P>0.05). These findings control group (respectively, 0/10 vs. 10/10 surviving mice indicate that CB-MSCs could inhibit PHA-stimulated at 14 days). More importantly, CB-MSC transplantation splenocyte proliferation in a MSC dose-dependent manner, significantly increased the survival rate of irradiated mice and that the PHA-stimulated effect could be completely (5/10 mice surviving) compared with irradiation alone neutralized by small amounts of MSCs (MSC/SP ratio of (P<0.05, Figure 4A). Figure 4B shows body weight 1:10, Figure 3). changes at different time-points after TBI. CB-MSC transplantation significantly reduced the weight loss of Changes in survival rate and body weight mice at 7 and 10 days post-TBI (P <0.05 and P<0.01). Mice subjected to simple irradiation with and without CB-MSCs facilitated Treg generation and altered chemokine receptor expression on T cells The Treg percentage was significantly lower in the simple irradiation group than in controls (P<0.05), and was significantly higher with CB- MSC transplantation compared Figure 3. Effect of CB-MSCs on splenocyte proliferation potential. Splenocytes were co- to irradiation alone (P<0.01); cultured with MSCs at different ratios for 72 hours, and then analyzed by MTS/PES assay with however, Treg percentage did measurement of absorbance at 490 nm. Data represent mean±standard deviation (SD) of three * † not significantly differ between independent experiments. P<0.01 with respect to PHA-stimulated splenocytes; P<0.01 with the MSC-infused group and respect to splenocyte cultures alone. Figure 4. CB-MSC transplantation after TBI increased survival rate (A) and reduced weight loss controls (P>0.05, Figure 5). We (B). Survival rate was determined using a log rank (Mantel-Cox) test. Post-TBI weight is shown as a also analyzed the expression percentage of pre-irradiation weight. *P<0.05 and †P<0.01 with respect to the simple irradiation group. profiles of CXCR3, CCR5, Chinese Medical Journal 2014;127 (3) 479

reversed the irradiation-induced T cell phenotypes changes by up-regulating CCR7 and down- regulating CCR5 and CXCR3 expressions.

CB-MSCs induced the shift of Th1/Th2 balance toward Th2 polarization IFN-γ protein level was significantly higher in the simple irradiation group than in healthy controls (P<0.05), and Figure 5. Effects of CB-MSC transplantation on Tregs. A: Representative flow diagrams this irradiation-induced increase of splenocytes labeled for CD4, CD25, and FOXP3; numbers represent percentage of was significantly reduced by CD4+CD25+Foxp3+ Treg cells. a, Simple irradiation group; b, MSC-infused group; c, Control group. + + + MSC transplantation (P<0.05). B: Percentages of CD4 CD25 Foxp3 Treg cells. Data represent mean±SD from four to six mice in IL-4 levels did not significantly each group. *P<0.05 and †P<0.01 with respect to the simple irradiation group. differ among the three groups and CCR7 in peripheral blood T cells. Compared to (P>0.05, Figure 7A). The mRNA level of T-bet was mice that were only irradiated, those who also received significantly higher in the simple irradiation group than MSC transplantation exhibited significantly decreased in controls (P<0.01), and the increase was significantly CXCR3 and CCR5 expressions (P<0.01) and significantly reduced by MSC transplantation (P<0.01). In contrast, increased CCR7 expression (P<0.01) on CD3+ T cells. In GATA-3 mRNA level did not significantly differ among contrast, expressions of CXCR3, CCR5, and CCR7 did the three groups (P>0.05). Correspondingly, the T-bet/ not significantly differ between irradiated mice with MSC GATA-3 ratio was significantly lower in irradiated mice transplantation and healthy controls (P>0.05, Figure 6). with MSC transplantation than without (P<0.05, Figure These results indicate that TBI caused a quantitative defect 7B). These data suggested that TBI accelerates Th1-type of Tregs and abnormal chemokine receptor expression immune responses by promoting inflammatory cytokine profiles, and that CB-MSCs promoted Treg generation and production, while CB-MSCs induces a marked decrease in Th1 immunity, shifting the Th1/Th2 balance toward anti- inflammatory Th2 polarization.

CB-MSCs alleviated splenocy- temediated inhibition of CFU-GM and accelerated hematopoietic recovery Representative CFU-GM from bone marrow cells were shown in (Figure 8 A−C). CFU-GM Figure 6. Effects of CB-MSC transplantation on CXCR3, CCR5, and CCR7 expressions in CD3+ counts in the simple irradiation + T cells. A: Representative examples of CXCR3, CCR5, and CCR7 expressions in CD3 T cells. B: group were significantly lower Percentages of CXCR3, CCR5, and CCR7 expressions in CD3+ T cells. Group a, BALB/c mice that received 8.0 Gy TBI; Group b, BALB/c mice treated with 8.0 Gy TBI followed by 1.0×107 MSCs; than in controls (P<0.01), Group c, healthy age-matched BALB/c mice (each group, n=10). Data represent mean±SD from and irradiated mice with CB- four to six mice in each group. *P<0.01 with respect to Group a. MSC transplantation exhibited significantly higher CFU- GM counts than those without CB-MSC transplantation (P<0.01, Figure 8D). In vitro, the CFU-GM count in the BMC+irradiated SPs group was significantly lower than in the BMC+SPs group (P<0.05). The BMC+irradiated SPs with MSCs group had higher CFU- GM counts than the group without MSCs (P<0.05, Figure Figure 7. A: Serum concentrations of IFN-γ and IL-4. B: T-bet and GATA-3 mRNA expressions 8E). These data indicate that and T-bet/GATA-3 ratios. *P<0.05 and †P<0.01 with respect to the simple irradiation group. CB-MSC transplantation 480 Chin Med J 2014;127 (3)

Figure 8. Effect of CB-MSCs on bone marrow CFU-GM. Representative CFU-GM from bone marrow cells in the following groups: simple irradiation mice (A), MSC-transplanted irradiated mice (B), and normal control mice (C). (D) Effect of CB-MSC transplantation on bone marrow CFU-GM. (E) CB-MSCs ameliorated the lymphocyte-mediated inhibition of CFU-GM. BMC and SPs were mixed at a 1:3 ratio. Data represent mean±SD of three independent experiments. *P<0.05 with respect to the irradiated (BMC+SP) group; †P<0.05 with respect to the BMC+irradiated SP group. stimulates CFU-GM formation and accelerates Our data is the first demonstration of the therapeutic use hematopoietic recovery in mice post-TBI. The irradiated of CB-MSCs in a mouse model of radiation-induced splenocytes directly suppressed CFU-GM formation injury. CB-MSCs transplantation increased the survival from normal bone marrow cells, and CB-MSC co-culture rate and reduced weight loss after lethal TBI. All 10 mice reversed this inhibition of CFU-GM mediated by activated in the simple irradiation group died within 13 days post- lymphocytes after irradiation. irradiation. In contrast, 5 of the 10 irradiated mice that received CB-MSC infusion were still alive at 14 days post- DISCUSSION irradiation. Our GM-CFU assay results showed that CB- MSC transplantation enhanced clonogenic survival of bone In the present study, we successfully isolated and marrow progenitor cells and attenuated radiation-induced expanded MSCs from the compact bone of C57BL/6 mice hematopoietic toxicity in vivo. The radio-protective role using a collagenase-treated bone fragment method. The of MSCs was clearly primarily associated with damaged standard method of whole bone marrow adherence yields tissue repair by virtue of their self-renewal and multipotent relatively heterogeneous MSCs due to hematopoietic cells differentiation capacities. However, the data from our in contamination; comparatively, the bone fragment method vitro experiments indicates that the radiation-activated has the distinct advantages of being a simple, stable, high- splenocytes directly inhibited CFU-GM formation from yield procedure with good repeatability.21 In contrast to a normal bone marrow cells, and that CB-MSCs significantly majority of previous studies, our present findings showed alleviated the lymphocyte-mediated inhibition of CFU- GM formation. These results clearly demonstrated that CB- that CB-MSCs from C57BL/6 positively expressed CD34 MSCs have an additional immuno-protective role after TBI antigen. However, our results are in agreement with those 22 through their anti-inflammatory and immune suppressive of Peister et al, which showed positive CD34 expression capacities. in MSCs from C57BL/6 but not BALB/c and DBA1 mice. Thus, we believe that CD34 antigen is heterogeneously Mechanically, our study further found that the serum IFN-γ expressed among MSCs from different strains of mice, and level was significantly higher in the simple irradiation group among different species. than in healthy controls, and that this TBI-induced increase was significantly decreased by MSC transplantation. Xun It is generally believed that MSCs dramatically inhibit et al25 reported that serum and colonic levels of TNF-α T cell proliferation triggered by various stimuli. In 23,24 and IL-1α were significantly increased after TBI when agreement with previous studies, we found that CB- compared with non-TBI regimens. These findings suggest MSCs suppressed PHA-stimulated-splenocyte proliferation that T cells, and maybe other tissues, are abnormally in vitro. Absorbance values were significantly lower in activated shortly after TBI, leading to a relatively increased MSC co-culture groups than in non-MSC treated groups Th1-type immune response. Tregs are thought to play a regardless of PHA stimulation, and a relatively small critical role in immune response suppression and immune amount of MSCs (MSC/SP over 1:100) was necessary to tolerance regulation.26,27 English et al28 reported that inhibit the proliferative response. human Tregs were induced when CD4+ T cells were co- Chinese Medical Journal 2014;127 (3) 481 cultured with MSCs, and that they exerted suppressive hematopoietic toxicity and increased the survival of mice activity in mixed lymphocyte cultures. Several in vivo exposed to lethal TBI. Mechanically, CB-MSCs alleviated studies have demonstrated the protective effects of MSCs lymphocyte-mediated inhibition of CFU-GM and induced by directly inducing Treg generation in transplant models additional immuno- protective effects by expanding the and in models of allergic and autoimmune disease.29-33 Tregs, regulating chemokine receptor expression, and Accordingly, here we found that Tregs were reduced in skewing the Th1/Th2 balance toward anti-inflammatory the simple irradiation group compared to healthy controls, Th2 polarization. CB-MSC transplantation may be an and that MSC transplantation significantly increased the effective therapeutic approach for diseases related to percentage of Tregs compared with the simple irradiation radiation-induced injury. group. These data suggest that Treg expansion might be a mechanism behind the radio-protective role of MSCs. REFERENCES

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