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DSUP modified mesenchymal stem cells exert significant radiation protective effect by enhancing the hematopoietic niche

Stem Cell Research & Therapy volume 16, Article number: 216 (2025) Cite this article

Abstract

Background

Radiation induced hematopoietic failure was the primary cause of death after exposure to a moderate or high dose of whole body irradiation, causing increased challenge for nuclear or radiological treatment, so it is an urgent need to develop radioprotectors for attenuating hematopoietic damage caused by acute radiation syndrome (ARS). Given the excellent therapeutic effects and special benefits of mesenchymal stem cells (MSCs) in radiation damaged hematopoietic stem/progenitor cells (HSPCs) recovery and hematopoietic niche reconstruction, enhancing the hematopoietic niche with the radiotolerance MSCs can be an alternative solution to prevent and attenuate hematopoietic radiation damage, which needs to be studied.

Methods

Here, we constructed MSCs modified with Damage Suppressor Protein (DSUP), a radiotolerance gene identified from tardigrade Ramazzotius varieornatus, and verify its radiation protection effect in HSPCs-MSCs co-culture model in vitro and radiation damaged mice model in vivo.

Results

Our results showed that DSUP protein had no significant toxic side effects on the basic stemness properties and differentiation potential of MSCs, and significantly enhanced the radiation tolerance and DNA protection ability of MSCs. Compared with the control (CON) group MSCs, the DSUP modified MSCs after radiation damage suffered less DNA damage, preserved most of proliferation activity and migration ability. In the HSPCs-MSCs co-culture model, DSUP modified MSCs have significant protective effect on HSPCs by providing a functional hematopoietic niche after radiation damage. The DSUP group irradiated HSPCs exhibited more rapid recovery, the higher HSPCs ratio and better hematopoietic differentiation potential. In animal studies, pre infusion of DSUP modified MSCs reduce irradiated mice mortality rate, reduce hematopoietic failure incidence, and provide a protective effect against radiation injury by protecting hematopoietic microenvironment and promoting HSCs recovery. DSUP modified MSCs can be used as a radioprotector and existed significant radiation protection effect for ARS at doses below 7 Gy total-body irradiation (TBI) of X-ray in both immunodeficient and immunocompetent mice models.

Conclusions

DSUP modified MSCs may serve as a new radioprotector for ARS. DSUP modified MSCs could attenuate radiation damage of HSPCs and promote HSPCs rapid recovery as well as hematopoietic reconstruction by providing a more functional niche after radiation, thereby reducing the occurrence of hematopoietic failure and improving survival rate.

Introduction

Ionizing radiation (IR) is widely applied in different fields such as medical care [1, 2], extraterrestrial exploration [3], industrial production [4] and military [5,6,7]. IR is known to exhibit potential hazard to human life and health, such as damage to body or mortality due to radiotherapy, exposure to solar and galactic radiation, nuclear power plants leakage and even atomic bomb explosions [3]. Based on the radiation dose and critically damaged organs due to radiation, acute radiation syndrome (ARS) is categorized into bone marrow (BM) type, intestinal type and cerebral type [8]. Previous studies and reported clinical cases indicated that vast majority of patients have slightly delayed mortality with zero possibility for long term survival after the treatment for the extremely severe BM type, intestinal type and cerebral types of patients. Often patients exposed to 2–10 Gray (Gy) whole-body radiations during ARS clinical treatment experience immediate problems like BM failure [9]. The hematopoiesis system are the most sensitive tissues/organs to total body irradiation. Ionizing radiation not only directly damages hematopoietic stem cells (HSCs), causing apoptosis, aging or genomic instability of HSCs, but also affects the BM microenvironment, destroys the supporting cells and matrix structures in the niche, and weakens its physiological regulation of HSCs [10]. Specifically, radiation can cause damage to niche cells such as BM stromal cells, endothelial cells [11], and adipocytes [12], a favorable hematopoietic microenvironment is the key to BM hematopoietic reconstitution [13]. Damage to the hematopoietic microenvironment promotes abnormal activation and depletion of HSCs, and leads to premature aging of HSCs. In addition, radiation-induced BM microenvironment remodeling may promote the repair of hematopoietic tissue in the early stage, but may aggravate hematopoietic damage in the long term. Radiation-induced disruption in the BM microenvironment can intensify the injury to BM HSCs and obstruct the regeneration of hematopoietic tissue through multiple mechanisms. Therefore, disrupting and reshaping BM microenvironment play a significant role in both the damage and recovery of HSCs after radiation exposure [14, 15]. Furthermore, the destruction of HSCs and BM microenvironment can trigger a sharp decline in white blood cells (WBC) and platelets (PLTs) counts, as the residual HSCs affected by radiation cannot proliferate and differentiate into downstream hematopoietic cells in a timely manner. This ultimately causes acute hematopoietic failure in the patient, leading to death [16].

At present, in addition to traditional BM transplantation and hematopoietic cytokine therapy, the therapeutic effects of mesenchymal stem cells (MSCs) on ARS are also increasingly attracting attention. Compared to other stem cells, MSCs have more tissue sources such as BM, umbilical cord blood, placenta or fat tissue, and they are easier to be isolated and expanded in vitro. Several studies reported that MSCs had shown good performance in radiation damage repair treatment of various tissues and organs, such as hematopoietic injury [17], liver injury [18], gastrointestinal injury [19], lung injury [20], arterial injury [21], and brain injury [22]. In addition, MSCs exhibit special properties in the therapy of hematopoietic damage, including secreting hematopoietic growth factors, low immunogenicity for allogeneic transplantation, and reconstructing hematopoietic microenvironment [23].

In above studies, MSCs were used as radiomitigator to repair tissue damage after radiation exposure, and the Food and Drug Administration (FDA, USA) approved drugs for preventing or rescuing cells from radiation damage are also mainly radiomitigators (e.g., GM-CSF, G-CSF, etc.) and radionuclide scavengers(e.g., Prussian blue, Calcium and zinc DTPA, etc.) [24]. Amifostine as a radioprotector is not suitable for ARS prevention due to its limited clinical indications and significant toxicity / side effects [25]. Therefore, radioprotectors for ARS and other IR-related injuries are still designated as orphan drugs. So, we wondered whether there is a possibility to enhance the radiation tolerance of MSCs by genetic modification, so that they can serve as a radioprotector for ARS. These radiation tolerant MSCs may provide a protective effect on the hematopoietic microenvironment by pre-infusion before radiation, and after radiation injury the hematopoietic niche still can exist with functional MSCs to promote the residual HSCs recovery and hematopoietic reconstruction. This is of great significance to military personnel and first responders conducting nuclear accident handling and search-and-rescue missions in hazardous areas [26].

In this study, we utilized a unique DNA-associated protein from the tardigrade R. varieornatus called damage suppressor protein (DSUP) [27] to improve radiation resistance of MSCs. It is reported that DSUP could bind to DNA in a non-specific manner and physically shield DNA from direct radiation and in direct effect of reactive oxygen species (ROS) damage [28]. Here, we constructed DSUP modified MSCs and verified their radiation resistance and DNA protection effects. More importantly, the DSUP modified MSCs did not show any significant toxic side effects on the self-renewal and differentiation capacity, while they exhibited a good protective effect on irradiated hematopoietic stem/progenitor cells (HSPCs) in the co-culture model. Compared with the control group, HSPCs co-cultured with DSUP modified MSCs showed a significant increase in the number and proportion of HSPCs recovery after radiation, and the hematopoietic differentiation capacity of the DSUP group recovered HSPCs was closest to that of non-irradiated HSPCs. Our in vivo studies also showed that pre-infusion DSUP modified MSCs could significantly reduce the incidence of hematopoietic failure and promote the recovery of residual HSPCs after radiation injury. It suggested that DSUP modified MSCs have significant protective effect on HSPCs by maintaining a functional hematopoietic microenvironment after radiation. The DSUP modified MSCs may be used as a radioprotector to reduce hematopoietic damage of ARS, and it may provide new methods for the development of new radioprotectors.

Materials and methods

Cell culture

hMSCs isolation and culture were performed according to the established standard operating procedure (SOP) in our laboratory [29, 30].The approved procedures involving human subjects were approved by the Ethics Committee at the Third Affiliated Hospital of Sun Yat-sen University (Approval number: 2017–19). Written informed consent were obtained from all the patients who participated in the study. The 293 T cells used in this study were maintained in our laboratory culture collection. The mHSPCs were extracted from C57BL/6 mice and cultured according to the standard procedure [31]. All cells were cultured at 37 degrees centigrade with 5% CO2. The reagents used in this study was listed in Table S1, Supporting Information.

MSCs transfection and purification

SFFV-DSUP-CopGFP-Puro was constucted by inserting the DSUP gene sequence into a SFFV- CopGFP-Puro skeleton vector (gifted by Dr. Hao Yan: Beijing Institute of Radiation Medicine, Beijing). pMD2.G and psPAX2 second-generation lentiviral packaging system were used to co-transfect 293 T cells and extracted the concentrated venom for transfection into human or mouse MSCs. The MSCs transfection was performed using the EnvirusTM-LV reagent (Engreen Biosystem, Beijing, China), and transfection was carried out overnight in the incubator at 37 degrees centigrade with 5% CO2. Transfection status of MSCs was observed under a fluorescent microscope post 48–72 h. Post transfection, the MSCs medium containing 1ug/mL puromycin was used for culture and screened. After 48 h of screening, the medium was replaced with puromycin-free medium. After screening, MSCs were cultured for 2–3 days before been sorted for GFP-positive MSCs by FACS Aria instrument (BD Bioscience, San Jose, CA). High-purity DSUP-MSCs were obtained. And the CON group cell lines were prepared in similar manner.

Animal studies

Male Immunodeficiency Rag2/IL2rg-KO (BALB/c) mice of ages 5–8 weeks which for radiation protection experiment and Male C57BL/6 (C57) mice ages 6–8 weeks which for mHSPCs extraction were both with reliable health and immune records.Mice in this study were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China) and the Vital River Laboratories (Beijing, China), housed under SPF conditions with temperature control (22 ± 1 degrees centigrade), humidity control (60 ± 10%), a 12 h light and 12 h dark cycle, adlibitum water and conventional mouse chow. Mice with no apparent abnormalities in appearance or behaviour were selected and allocated to groups randomly using a random number generator. The order of treatments and measurements for each mouse was randomly assigned. For the radiation protection assay, CON / DSUP modified human or mouse MSCs were pro-infuse into two groups of mice via tail vein (1 × 106 cells / per mouse). After 24 h, mice were anesthetized with 0.5% pentobarbital sodium (anesthesia dose 50 mg/kg of mice body weight, intraperitoneal injection) to minimize physical discomfort, stress and fear during the radiation process. All mice were irradiated with 4.5–7 Gy X-ray using Small Animal Irradiator (Rs2000; RadSource). After radiation, mice were promptly transported back to the animal room and monitored until they regained consciousness. Then, detected the hemogram, body weight, temperature, and survival rate of each group of mice at different time points. During the experiment timeline, dead mice were immediately fixed with BM tissue. After 20 days of radiation, survived mice were sacrificed to collect data. (The study adopted carbon dioxide asphyxiation method, where the experimental animals are placed in a closed box and carbon dioxide is released to the box according to the SOP plan).

Specifically, one femur was fixed for pathology analysis, while the other femur was grinded and digested to obtain BM microenvironment cells for flow cytometry analysis, and two tibias were collected BM mononuclear cells for flow cytometry analysis. The BM pathology of each mouse was analyzed by H&E staining, and percentage of BM karyocyte cells was calculated as the area percentage occupied by karyocyte in the BM cavity. Using ImagePro Plus 6.0 software (Media Cybernetics, Inc., Maryland) three random fields of view were analyzed and calculated separately the total area of karyocyte (S1) and total area of red blood cells (S2) in the BM cavity, and BM karyocyte cells percent = 100% * S1 / (S1 + S2). The components of BM karyocyte cells and hMSCs detection in survival mice were analyzed by flow cytometry. The work has been reported in line with the ARRIVE guidelines 2.0.

Mouse BM MSCs isolation and culture

The mouse BM-derived MSCs are isolated according to the protocol of Nature Protocols [32]. Briefly, Aseptic isolation of mouse limb bones, removal of hematopoietic cells from BM, cutting of bone fragments and digestion with digestive enzymes, and inoculation of bone fragments into mouse MSCs culture medium for isolation and culture. Cultivate for 7–10 days to collect BM-derived MSCs crawled out of bone slices and perform passage expansion. Mouse BM MSCs were expanded in α-MEM medium supplemented with 2 mM L-glutamine, 100 µg/ml penicillin and 100 µg/ml streptomycin, 10 ng/ml mouse basic fibroblast growth factor (bFGF, Novoprotein), 1% HEPES buffer and volume fraction 10% Gibco FBS (suitable for MSCs).

Flow cytometry

Specifc surface markers of MSCs were detected by Guava easyCyte Flow Cytometer (Luminex, China) as previously described [29]. Data was analyzed by Flowjo software (Trees tar Inc., USA). The antibodies used in this study were listed in Table S1, Supporting Information.

Triple lineage differentiation

Gelatin-coated two 24-well plates were washed and cleaned for 1 h, and the cells were inoculated therein, and were subjected to lipid and osteogenic induction according to the human umbilical cord MSCs osteogenic induction differentiation kit and human umbilical cord mesenchymal stem cell adipogenic induction differentiation kit (Cyagen,USA), respectively, with regular exchange of fluid to be stained and observed under the microscope after maturation. In addition, 5 × 105 cells were transferred to 15 mL centrifuge tubes to follow the chondrogenic induction medium according to the human umbilical cord MSCs chondrogenic induction differentiation kit (Cyagen,USA), and the cells were periodically changed to form clusters of cells to be stained and observed under the microscope for chondrogenic induction.

HSPCs culture and radiation treatment

Mouse HSPCs were expanded in PVA medium [33], the umbilical cord blood (CB) was acquired from the Umbilical Cord Blood Bank (Beijing, China). Human CD34+ CB cells were isolated using Human CD34 MicroBead Kit (Miltenyi Biotec). Human HSPCs were expanded in StemSpan SFEM (StemCell Technologies) supplemented with human 50 ng/ml stem cell factor (SCF, Proteintech), 50 ng/ml FMS-like trysine kinase 3 ligand (FLT3L, Proteintech), 50 ng/ml thrombopoietin (TPO, Proteintech), and 1 µM Stemregenin 1 (SR1, MedChemExpress), PVA / SFEM medium was still used when co-culturing with hMSCs [34]. For IR treatment, mouse or human HSPCs were plated into dishes covered with about 50–60% of MSCs and cultured with the PVA/ SFEM culture medium, then immediately received 4 Gy IR (RS-2000, Rad Source Technologies, GA, USA) for radiation damage and further analysis of radiation protection effects.

Hematopoietic colony formation assay

In vitro colony-forming unit (CFU) assay was performed to detect the clonogenic potential of mHSPCs using MethoCult™ M3434 Methylcellulose-Based Medium (Stem Cell Technologies, Vancouver, Canada), and hHSPCs using MethoCult™ H4434 Methylcellulose-Based Medium (Stem Cell Technologies, Vancouver, Canada). After 7–10 days of culture, the hematopoietic colonies were collected and washed then the number of different hematopoietic colonies were counted. After, we detected different lineages surface markers of hematopoietic colony cells by flow cytometry in each group.

Statistical analysis

All data were shown as Mean ± SEM. Image IOD, fluorescence intensity and positive cell ratio was analyzed using Image Pro Plus software (version 6.0, Media Cybernetics, USA). Statistical analyses and plots were performed with GraphPad Prism 8.0 software (GraphPad Software, USA). Euclidean distance was used to evaluate the similarity analysis between two groups, the smaller the Euclidean distance, the higher the similarity between two groups. Survival curves of two groups was analyzed by Log-rank test. The differences between two groups of change curves over time were analyzed by two-way ANOVA test. For comparisons of the mean between two groups, statistical analysis was performed unpaired two-tailed Student’s t-test as indicated in the bar graph. Statistical significance was set at p < 0.05.

Results

Construction of DSUP-modified MSCs

To obtain DSUP modified MSCs, the available DSUP gene sequence of Ramazzottius varieornatus from National Center for Biotechnology Information (NCBI:LC050827) was used. The constructed SFFV-DSUP-CopGFP-Puro vector contained not only DSUP gene sequence, but also had flag tag, puromycin resistance gene and CopGFP gene to facilitate the selection and purification of transfected MSCs (Fig. 1A). The control group vectors were not inserted with the DSUP gene. We utilized these vectors to prepare Lentivirus concentrates from 293 T packaging system, and was transfected into MSCs. The CopGFP percentage of transfected MSCs at the first round was about 30–40%. Flow sorting of CopGFP-positive cells and screening with puromycin enriched the CopGFP-positive MSCs (around 90%) (Fig. 1B). CopGFP protein was found to be mainly expressed in the nucleus in the DSUP group, suggesting that the DSUP protein was mainly concentrated in the nucleus, which is consistent with previous studies [35] (Fig. 1C). In order to clarify the integration and expression of the target gene in modified MSCs, DSUP gene fragments were initially detected by PCR gel electrophoresis and DSUP group showed a clear positive DSUP gene band between 1200 and 1500 bp (Fig. 1D), which was consistent with the length of the DSUP gene fragment (1450 bp). Further sequencing of the integrated DSUP gene revealed the similarity to the reported gene used from the NCBI (Figs. S1, S2), indicating the successful integration of DSUP gene fragments at the genomic level. The mRNA expression of DSUP gene in the DSUP group MSCs expressed highly DSUP mRNA (Fig. 1E). Finally, the expression of DSUP protein was detected using immunofluorescence staining. Flag tag was labeled at the tail of DSUP to identity DSUP protein. About 80% of the DSUP group MSCs successfully expressed DSUP and it was used further for subsequent experiments (Fig. 1F, G). The above results evidently proved that DSUP-integrated MSCs could stably express DSUP protein.

Fig. 1
figure 1

Construction of DSUP modified hMSCs. A Schematic diagram of DSUP lentiviral vector backbone; B GFP ratio of lentiviral transfected hMSCs before and after sorting by flow cytometer, bar = 25 μm; C GFP immunofluorescence of hMSCs after sorting and purification; D Detection of DSUP target gene fragments in each group of hMSCs (1200–1500 bp); E qRT-PCR to detect the expression of DSUP mRNA in each group of hMSCs. The mRNA expression level of wildtype MSCs was normalized to 1, n = 3, Mean ± SEM, **p < 0.01, by Student’s t-test. F–G Immunofluorescence detection of DSUP-Flag fusion protein expression in each group of MSCs, DSUP-Flag was fluorescently labeled in red, and GFP protein was fluorescently labeled in green, with bar = 100 μm. Statistical analysis of the proportion of DSUP-Flag expression in each group of MSCs was plotted, n = 3, Mean ± SEM, ***p < 0.001, by Student’s t-test

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DSUP protein did not significantly affect the basic properties of MSCs.

As DSUP is a heterozygous gene from tardigrade Ramazzotius varieornatus, though it was expressed in 293 T cells without significant cytotoxicity, but its expression in cortical neurons exhibited cytotoxicity [36]. Therefore, we constructed DSUP-expressed MSCs to investigate whether DSUP protein could significantly impact the basic properties of MSCs. According to the minimal criteria for defining multipotent mesenchymal stromal cells [37], they need to exhibit the ability to adhere and express CD105, CD73, and CD90, but should not express hematopoietic cell surface molecules, such as CD45, CD34, CD19, and HLA-DR, and lastly, they need to have the ability to undergo triple lineage differentiation, that is to say, the ability to differentiate into osteoblastic, adipogenic, and chondrogenic cells, under standard culture conditions. We examined three basic properties of MSCs in each group. Firstly, when cultured in serum-free MSCs medium, DSUP group MSCs could normally adhere and no significant difference in the cell morphology with that of the WT and CON group MSCs (Fig. S3A). Also, the expression of CD19, CD31, CD34, CD45 and HLA-DR markers in all the groups were almost negative (the expression ratio was less than 5%), while the expression of CD73, CD90, CD105, CD29 and CD44 markers were significantly positive (the expression ratio was more than 95%), suggesting that the DSUP protein did not alter the expression of surface markers in MSCs (Fig. S3B). Meanwhile, these three groups of MSCs all performed very normally in adipogenic, osteogenic, and chondrogenic induction differentiation, and the triple lineage differentiation results in all groups without significant differences (Fig. S3C). In summary, DSUP protein expressed in MSCs had no significant negative effect on the three basic properties of MSCs.

DSUP modified MSCs have stronger radiation tolerance

In order to clarify the role of DSUP on the radiation tolerance of MSCs, each group of MSCs were treated with 12 Gy X-ray radiation, and the cell morphology changes as well as the cell proliferation were detected after irradiation (Fig. 2A, B). The results showed that the proliferation was significantly inhibited in each group MSCs after irradiation, but the cell counts in DSUP group gradually increased after 4 days, which was significantly higher than that of the WT and CON groups. EdU (5-ethynyl-2-deoxyuridine) staining was used to detect the S phase proliferative cells. EdU-positive cells proportion and mean fluorescence intensity in the WT and CON groups decreased significantly after 24 h post radiation. Though the EdU mean fluorescence intensity was decreased in the DSUP group MSCs, but the decrease in EdU-positive cells proportion was not found significant (Fig. 2C, D). In addition, given that migration ability is an important characteristic for the function of MSCs, we found that the migration ability was significantly reduced in the WT and CON groups MSCs at 24 h after radiation, while it was not significantly affected in the DSUP group MSCs (Fig. 2E). In summary, the DSUP group MSCs exhibited significantly higher level of proliferation and migration abilities than those of the WT and CON groups MSCs post radiation damage, suggesting that DSUP protein holds the capacity to enhance significantly the radiation resistance in MSCs.

Fig. 2
figure 2

Effect of DSUP on the proliferative and migratory abilities of hMSCs after radiation treatment. A Cell morphology of MSCs in each group 0–6 days after 12 Gy X-ray radiation treatment; B Changes in the cell density of MSCs in each group after radiation in Fig. A. For DSUP compared with WT group, *p < 0.05, **p < 0.01, ***p < 0.001; For DSUP compared with CON group, ###p < 0.001, n = 3, Mean ± SEM, by Student’s t-test; C Statistical plots of the EdU fluorescence staining proportion and mean fluorescence intensity of MSCs in each group before and 24 h after 12 Gy radiation, n = 3, Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t-test; D Immunofluorescence staining images of MSCs in each group before and after 12 Gy radiation with EdU, nuclei labeled with DAPI and EdU labeled with red fluorescence on a scale of 100 μm; E Images and statistical analysis results of transwell migration assay of MSCs in each group without radiation and after 12 Gy radiation for 24 h. n = 3, Mean ± SEM, **p < 0.01, by Student’s t-test. Scale bar is 200 μm

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DSUP attenuates apoptosis and DNA damage of MSCs under radiation and ROS injuries

Radiation-induced cellular damage is mainly caused by the direct action of high-energy radiations and the indirect action of water irradiation reaction, especially the water irradiation reaction induced ROS damage accounts for about two-thirds of cellular DNA damage [38]. Firstly, after 12 Gy of X-ray radiation, there was an obvious change in the cell morphology in each group MSCs, with increased cell volume and length (Fig. S4A). Secondly, apoptosis was detected by Annexin V/7-AAD staining before and after radiation in each group. However, only a slight increase in the apoptosis percentage (about 5%) was observed with no significant difference between each group of MSCs after 48 h of radiation (Fig. S4B). Probably, the 12 Gy of radiation was not enough to induce higher level of apoptosis in MSCs. Further experiments were conducted to simulate the indirect effects of radiation using oxidative stress damage by H2O2 [39]. ROS damage studies showed that after 24 h H2O2 treatment, the apoptosis percentage of DSUP group MSCs (about 12–20%) was significantly lower than that of the WT and CON groups MSCs (about 70–80%) (Fig. 3A, B), indicating that DSUP protein could significantly reduce the ROS damage-induced cell apoptosis in MSCs.

Fig. 3
figure 3

Validation of the anti-apoptotic and DNA protection ability of DSUP-modified MSCs against ROS and radiation damage. A Cell morphology images of MSCs in each group 24 h after 15 mmol H2O2 treatment. Scale bar is 200 μm; B Flow cytometry detection of apoptosis and statistical histograms of MSCs in each group in Figure A. Early apoptosis was labeled with Annexin V, and late apoptosis was labeled with 7-AAD, n = 3, Mean ± SEM, **p < 0.01, ***p < 0.001, by Student’s t-test; (C) Comet assay images and statistical plots of MSCs in each group after treatment with 12 Gy X-ray radiation, n = 50, Mean ± SEM, ***p < 0.001, by Student’s t-test. Scale bar is 100 μm; D Immunofluorescence images of γH2AX in MSCs of each group after 12–24 h treatment with 12 Gy X-ray radiation, scale bar is 50 μm. Statistical analysis of γ-H2AX foci expression rate plotted by mean fluorescence intensity of cell nuclei reflecting γ-H2AX foci expression rate, n = 3, Mean ± SEM, **p < 0.01, ***p < 0.001, by Student’s t-test

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Several studies reported that the radiation protection function of DSUP is mainly realized by physically shielding DNA from direct radiation and indirect ROS damage [27, 28, 35]. The neutral comet assay and γH2AX immunofluorescence staining were utilized to detect DNA double-strand breaks (DSBs) in each group MSCs. The comet assay results showed that the fragmented DNA proportion in the DSUP group MSCs was significantly lower by about 50% than that in the CON group after 12 Gy of X-rays radiation (Fig. 3C). In addition, similar results were observed after ROS oxidative stress (H2O2) treatment between the two group MSCs. The fragmented DNA proportion was reduced by about 25% in the DSUP group MSCs than that in the CON group (Fig. S4C). In addition, the expression of γH2AX in irradiated MSCs was detected by immunofluorescence staining. Considering that many γH2AX foci fused into a whole under 12 Gy radiation, it was difficult to be counted one by one, so we analyzed the γH2AX foci mean fluorescence intensity in the nucleus to calculate the proportion of γH2AX-positive cells. The γH2AX-positive cells proportion was significantly lower in the DSUP group than that of the CON group at both 12 and 24 h after radiation (Fig. 3D). Thus, it suggests that DSUP can protect the DNA of MSCs in the presence of direct radiation and indirect ROS damage, and thereby improving the survival rate of irradiated MSCs.

DSUP modified MSCs significantly improve the recovery of irradiated HSPCs by enhancing hematopoietic microenvironment

The hematopoietic system is highly sensitive to radiation injury, and the primary cause of death for ARS patients is radiation-induced hematopoietic failure [40]. Radiation disrupts the hematopoietic microenvironment in the BM, causing apoptosis to HSPCs and senescence of HSCs niche [16]. MSCs play a significant role in the hematopoietic microenvironment for HSCs self-renewal and hematopoietic differentiation, and radiation-induced impairment of MSCs function is one of the important causes for HSCs senescence and dysfunction [41]. Many studies have shown that MSCs contributes to the recovery and hematopoietic reconstruction of HSCs after radiation injury [16, 42]. In this study, a co-culture system of MSCs and HSPCs was used to simulate the hematopoietic microenvironment of BM. HSPCs could be expanded and cultured in vitro with the PVA system for more than 30 days [33], and a co-culture model of HSPCs and MSCs was constructed by inoculating HSPCs into dishes covered with about 50–60% of MSCs and cultured with the PVA system. The HSPCs and MSCs in the co-culture model were then irradiated with 4 Gy of X-rays, which could kill most of the HSPCs at this dose. Next, the cell counts, cell viability, morphological changes, Lin−Sca1+c-Kit+ (LSK) ratios, and hematopoietic colony-forming capacity of HSPCs were detected at different time points after radiation damage (Fig. 4A). The results showed that the number of irradiated HSPCs co-cultured with DSUP group MSCs increased from day 3 and gradually became significantly different from that of the WT and CON group (Fig. 4B). On day 9 the HSPCs count of DSUP group was significantly higher (about 8 times) than that of the WT and CON groups (Fig. 4C). Moreover, the cell viability of the DSUP group HSPCs was always significantly higher than WT and CON groups during day 1–9 after radiation (Fig. 4D). We further analyzed the morphology and composition of HSPCs in each group on day 9 after radiation. DSUP group irradiated HSPCs morphology was closest to the non-irradiated control (NC) group HSPCs, while the WT and CON groups HSPCs showed more significant differentiation and aberration damage (Fig. 5A). Flow cytometry was performed to detect the LSK proportion of each group irradiated hematopoietic cells. The results showed that the LSK proportions of all three irradiated groups cells were lower than that of NC group cells, among which the DSUP group LSK proportion (about 45–52%) recovered the highest and was significantly different from the other two groups LSK proportions (about 10–30%) (Fig. 5B, C). We also used human CD34+ HSPCs to verify the radiation protective effect of DSUP modified MSCs, hHSPCs and MSCs were co-cultured in SFEM medium and irradiated with 4 Gy of X-rays. Consistent with the results of mHSPCs, DSUP group human HSPCs count was significantly higher than CON group during day 2–8 after radiation (Fig. S5). So, DSUP modified MSCs could significantly improve the recovery of irradiated HSPCs in the co-culture model by providing a radiation tolerance hematopoietic microenvironment.

Fig. 4
figure 4

Comparison of HSPCs recovery after radiation injury in each group MSCs and HSPCs co-culture model. A Schematic diagram of MSC-HSPCs co-culture model and radiation injury recovery experimental; B Cell morphology images of MSC and HSPCs co-culture models in each group 1–9 days after radiation injury, fibrous adherent cells are MSCs, round suspended cells are hematopoietic stem/progenitor cells, scale bar is 100 μm; C–D Change curves of total hematopoietic cell count and cell viability after radiation injury in each group of co-culture model, DSUP vs. WT group, ***p < 0.001; DSUP vs. CON group, ###p < 0.001, n = 3,Mean ± SEM, by Student’s t-test

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Fig. 5
figure 5

Comparative analysis of HSPCs-related properties of radiation co-culture models in each group. A Morphological maps and Giemsa staining images of each group HSPCs in co-culture model after 9 days of radiation, NC group is the same batch of unirradiated mHSPC cells, bright field image scale = 100 μm, Giemsa image scale = 20 μm; B Flow cytometry t-SNE downscaling plots of LSK ratios of each group HSPCs in Figure A. Lin− cells are shown in blue and LSK cells are shown in red; C Statistical analysis plot of the LSK ratios in each group of HSPCs, n = 3, Mean ± SEM, *p < 0.05, **p < 0.01, by Student’s t-test; D Statistical plot of the hematopoietic colonies counts of each group HSPCs in MSCs-HSPCs radiation co-culture model, n = 4, Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t-test; E Typical images of hematopoietic colony formation of HSPCs in the radiation co-culture model for each group, scale bar = 500 μm; F Statistical plots of the different lineages hematopoietic colonies counts of HSPCs in each group of Figure E, n = 4, Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t-test; G Statistical plot of the total cell counts from hematopoietic colonies of HSPCs in each group of Figure E, n = 4, Mean ± SEM, **p < 0.01, ***p < 0.001, by Student’s t-test

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DSUP group HSPCs in radiation co-culture model have stronger hematopoietic differentiation capacity

Studies have shown that after radiation injury, even if the surface markers are not significantly different from normal HSPCs, their multidirectional differentiation potential tends to be significantly reduced and the hematopoietic differentiation profile is significantly biased [43]. In order to compare the hematopoietic differentiation potential of recovered HSPCs in each radiation co-culture model, the hematopoietic differentiation ability of each group HSPCs was further examined by hematopoietic colony formation assay. First, the total number of hematopoietic clones formed by each group of HSPCs were compared. Non-irradiated NC group hematopoietic colonies count was the highest among the four groups. DSUP group hematopoietic colonies count was slightly lower than that of the NC group, but was significantly higher than that of the WT and CON groups (Figs. 5D, E, S6). Further analysis of the different lineages hematopoietic clones in each group revealed that there was no significant difference in GEMM colonies counts among the four groups. For the CFU-E counts: NC > DSUP ≈ WT > CON; For the BFU-E counts: NC ≈ DSUP > WT ≈ CON; and for the CFU-GM counts: NC > DSUP ≈ WT ≈ CON (Fig. 5F). Due to the various sizes of different lineages of hematopoietic clones, the comparison only from the number of clones has remained biased. The size of the hematopoietic colony represents the cells number it contains, also reflects the proliferation and differentiation capacity of HSPCs. Therefore, we collected and counted the hematopoietic colonies cells in each group. The cell count of the DSUP group colonies was significantly higher than that of the WT and CON groups, but has no significant difference compared to the NC group (Fig. 5G).

In the radiation group, the cell morphology showed different degrees of aberrations and vacuoles, resulting in not meeting the typical characteristics of various lineage hematopoietic cells (Fig. S7A). In order to more accurately analyse hematopoietic lineage profile in each group, flow cytometry was used to detect the different lineage typical surface markers, such as CFU-E labeled with Ter119, BFU-E labeled with CD71, CFU-GM labeled with Gr1 and CD11b, and CFU-GEMM could be considered as consisting of c-Kit+ cells, CFU-E, BFU-E and CFU-GM (Table S3). Hematopoietic lineage profile of DSUP group was closer to that of NC group, followed by the WT and CON groups (Fig. S7B). The count of different lineage markers positive in hematopoietic cells from each group was calculated. The similarity analysis results showed that compared with the NC group, the positive cell counts expressing different markers in the DSUP group was more similar to that of the NC group (Fig. S8). Next, we used the different markers positive cells counts and different lineages hematopoietic colonies counts in each group to estimate the average cell number of each hematopoietic colony (Table S3). The similarity analysis results showed that compared with the NC group, the average cell number of each hematopoietic colony in the DSUP group was highly similar to that in the NC group (Fig. S9). In summary, the hematopoietic differentiation potential and the hematopoietic colonies lineages profile of DSUP group HSPCs were similar to those of the NC group HSPCs. whereas the hematopoietic colony formation ability in the WT group and the CON group was significantly decreased, and the lineage differentiation profiles were biased at different degrees. The hematopoietic differentiation capacity of the restored HSPCs in the DSUP group was the most similar to that of the non-irradiated HSPCs.

DSUP modified MSCs significantly reduced the hematopoietic failure incidence of radiation damaged mice

To investigate the radiation protective effect of DSUP modified MSCs on HSPCs in radiation damaged animals, we pre infused CON / DSUP modified human MSCs into Rag2/IL2rg-KO immunodeficient mice (106 cells / per mouse) and performed 4.5 Gy X-ray radiation damage on the following day. At this radiation dose, the mortality of Rag2/IL2rg-KO immunodeficient mice is about 40–60%, and the main cause of death was radiation induced hematopoietic failure [44]. Then, we detected the mice weight, body temperature, hemogram, survival rate, BM pathology and hematopoietic recovery of two groups of irradiated mice (Fig. 6A). In both the groups after radiation, the peripheral blood red blood cell (RBC) count of the surviving mice continued to decrease, and WBC and PLTs count continued to decrease for about 2 weeks before they began to recover. No significant change in the hemogram between the two groups of irradiated mice (Fig. 6B). In addition, no significant difference in the weight and body temperature change curves between the two groups (Fig. S10A, B). However, the survival rate of the DSUP group irradiated mice (91.6%) was significantly higher than that of the CON group irradiated mice (50%) (Fig. 6C). Further pathological analysis showed that the BM karyocyte cells percentage in the 6 CON group dead mice were significantly reduced and reached its lowest value on about 14–15 days after radiation, but in the other 6 surviving mice they were recovered to 69.02 ± 6.60% as observed on 20 days after radiation. In contrast, only one DSUP group mouse died after radiation, and the BM karyocyte cells percentage in the other 11 surviving mice had recovered to 81.98 ± 2.04%, which was significantly higher than that of the CON group surviving mice (Fig. 6D, E). Compared to the CON group, the DSUP group mice had a higher survival rate with better hematopoietic recovery post radiation, with significant reduction in radiation induced hematopoietic failure.

Fig. 6
figure 6

DSUP modified MSCs significantly reduced the incidence of hematopoietic failure in irradiated mice. A Schematic diagram of 4.5 Gy X-ray radiation damage assay in mice model after infusion of DSUP modified MSCs. B Hemogram change curve of CON / DSUP group mice after 4.5 Gy radiation (including RBC, WBC, and PLTs). Mean ± SEM, n = 12, ns: no significant difference, by two-way ANOVA. C Survival rate curve of CON / DSUP group mice after radiation, n = 12, *p = 0.026, analyzed by Log-rank test. D BM pathological section of femoral shaft in CON / DSUP group mice after radiation, the BM collection time of each mice after radiation was marked above each image. The red collection time represents the death time of mice after radiation, while the green collection time (D20) represents the sacrifice time of surviving mice after 20 days of radiation. The mean of BM karyocyte cells percent was marked in the bottom left corner of the image, bar = 200 μm. E BM karyocyte cells percent of CON / DSUP group irradiated mice at collection time point of Figure D. The difference between the two groups dead mice cannot be statistically analyzed due to the too small sample size of the DSUP group dead mouse, while the difference between the two groups surviving mice was analyzed, Mean ± SEM, *p < 0.05, by Student’s t-test

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DSUP modified MSCs significantly promoted HSPCs recovery of radiation damaged mice by enhancing hematopoietic niche

In addition, we also analyzed the composition of BM hematopoietic cells and stromal cells of surviving mice after 20 days of radiation. The flow cytometry results showed that a small amount of human MSCs could be detected in the non-hematopoietic cells subpopulations (mCD45 negative cells) of BM karyocyte cells in mice in both the groups (Fig. 7A). Further fluorescence imaging of these karyocyte cells also revealed a small number of hCD90/hCD105/hCD73 triple positive human MSCs (Fig. 7B). Finally, we compared the cell count and composition of tibias BM mononuclear cells between the NC group mice and the two radiation group mice. The results showed that the BM mononuclear cells count in two radiation groups mice were both significantly lower than that in the NC group mice, but there was no significant difference between the CON and DSUP group irradiated mice (Fig. 7C). We further evaluate the hematopoietic recovery of two group irradiated mice by comparing the proportion of HSPCs, lineage negative progenitor cells (Lin− progenitors), c-Kit positive progenitor cells (c-Kit+ progenitors) and mature hematopoietic cells in each group mice [45] (Figs. 7D, S10C). HSPCs proportion in both the radiation groups mice were both significantly lower than that in the NC group mice, and HSPCs proportion in the DSUP group mice was also significantly higher than that in the CON group mice. Meanwhile, the proportion of Lin− progenitors and c-Kit+ progenitors in the DSUP group mice were both lower than those in the CON group mice, but the proportion of mature hematopoietic cells in the DSUP group mice was significantly higher than that in the CON group mice. However, no significant difference in the proportion of Lin− progenitors, c-Kit+ progenitors and mature hematopoietic cells between the DSUP and NC group mice (Figs. 7E, S11). Incidence of hematopoietic failure in DSUP group irradiated mice was lower, as the BM recovery percent and HSPCs proportion were higher, as well as the distribution of different stages of hematopoietic cells in it was closer to the level of NC group mice. DSUP modified MSCs was able to promote the regeneration and recovery of residual HSPCs in vivo by providing a functional hematopoietic microenvironment after radiation, and reduced the mortality as well as hematopoietic failure incidence, thus exhibiting significant radiation protection effects.

Fig. 7
figure 7

DSUP modified MSCs promoted the recovery of residual HSPCs in irradiated mice. A Typical flow cytometry plots of human MSCs in the mCD45− cells subpopulations of BM karyocyte cells in two groups irradiated mice. B Representative fluorescent images of human MSCs detected in the Fig. 7A, scale bar = 10 μm. C Statistical plots of tibias BM mononuclear cells count in three groups mice, Mean ± SEM, ***p < 0.01, by Student’s t-test. D Typical flow cytometry t-SNE downscaling plots of different stages of hematopoietic cells proportion in each group mice, HSPCs: Lin−c-Kit+CD150+ cells, Lin− progenitors: Lin− cells, c-Kit+ progenitors: c-Kit+ cells, mature cells: Lin+c-Kit− cells. (E) Statistical plots of the proportion of HSPCs, Lin− progenitors, c-Kit+ progenitors and mature hematopoietic cells in each group mice detected in Fig. 7 D, Mean ± SEM, *p < 0.05, **p < 0.01, by Student’s t-test

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The radiation protective effect of DSUP modified MSCs in immuno-healthy mice

In order to demonstrate the protective effect of DSUP modified MSCs to immuno-healthy mice exposing various doses of radiation, rather than limited to immunodeficient mice. We used immuno-healthy C57BL/6 mice to verify the effect of DSUP modified hMSCs, C57BL/6 mice were pre infused CON / DSUP modified hMSCs and treated total-body irradiation (TBI) of X-ray at sublethal dose of 6 Gy and lethal dose 7 Gy [46, 47]. The results show that the survival rate of DSUP group mice was slightly higher than that of CON group mice at both radiation doses (survival rate increased 17.5% and 12.5% in 6 and 7 Gy doses, respectively), but there was no statistical difference (Fig. S12A, B). In addition, there was no significant difference in the hemogram curves between the two groups of irradiated mice (Fig. S12C). Obviously, the radiation protective effect of DSUP modified human MSCs in immuno-healthy mice is significantly lower than that in immunodeficient mice. The reason for protective effect declining may be due to immune rejection of xenogeneic human MSCs in immuno-healthy mice. Research shows that allogeneic MSCs can survive for over 120 days in immunodeficient mice, while they only survive about 20 days in immuno-healthy mice. In addition, the greater the genetic background difference, the faster the immune clearance rate [48]. Due to the radiation protection effect of DSUP modified hMSCs mainly depends on their continuous interaction with HSPCs and hematopoietic niche, the rapid immune clearance of hMSCs would significantly weaken their protective effect in immuno-healthy mice. In addition, we found almost no human derived MSCs in the BM cavities of the two groups of irradiated mice, which supports our hypothesis (Fig. S12D). Therefore, it is highly likely that the rapid immune clearance of human DSUP-MSCs in immuno-healthy mice reduces their hematopoietic radiation protection effect.

As MSCs can be transplanted across allogeneic barriers without eliciting an obvious immune response, they can exist and support the hematopoietic recovery in irradiated C57BL/6 mice for at least 15–20 days (i.e. the minimum time required the hematopoietic recovery). We constructed DSUP modified mouse MSCs to alleviate the impact of immune rejection to MSCs function and validate their radiation protective effect in C57BL/6 mice. The isolated BM MSCs exhibit a typical adherent fibroblast like morphology and a typical phenotype of mouse MSCs surface markers (The proportion of mCD105, mCD44, mSca1, mCD29 are all greater than 95%, and the proportion of mCD34, mCD45, mCD48, mCD31, and mCD11b are all less than 5%) (Fig. 8A). The transfection and screening for mouse CON / DSUP MSCs remains the same method as before, and we repeated the radiation damage assay in C57BL/6 mice pre infused CON / DSUP modified mouse BM MSCs. The results indicate that the DSUP modified mouse MSCs have significant radiation protective effect in C57BL/6 mice at 6 Gy radiation doses (CON group survival rate: 66% and DSUP group survival rate: 100%), while at 7 Gy radiation doses the survival rate of DSUP group mice was slightly higher than that of CON group mice without significant statistical difference (CON group survival rate: 13.3% and DSUP group survival rate: 31.2%). However, DSUP group mice had a longer median survival period (Fig. 8B). Based on the above animal studies results, it can be concluded that the DSUP modified MSCs have significant radiation protection effect on TBI of X-rays at sublethal dose (including 4.5 Gy in Rag2/IL2rg-KO mice and 6 Gy in C57BL/6 mice). However, the protection effect is not significant for lethal dose radiation damage of 7 Gy. What’s more, we observed that the peripheral hemogram curves of irradiated mice at sublethal dose showed a recovery trend after 2 weeks, while those of irradiated mice at lethal dose still showed no recovery trend after 3 weeks (Figs. 6B, 8C, D and S12C). We speculate that TBI of X-ray at 7 Gy lethal dose or above may cause irreversible damage to mouse HSCs, and it is difficult to reverse HSCs apoptosis and hematopoietic failure solely by protecting the hematopoietic microenvironment after radiation. Therefore, our results demonstrated that DSUP modified MSCs play a radiation protective role by protecting the hematopoietic microenvironment and promoting the recovery of residual HSCs, especially against hematopoietic radiation damage below lethal dose. Although DSUP modified MSCs cannot change the ultimate outcome of radiation damage above lethal dose, they may prolong the median survival period by protecting the hematopoietic microenvironment, which is expected to provide a longer treatment window for HSCs transplantation.

Fig. 8
figure 8

The radiation protective effect of DSUP-modified mouse MSCs in immuno-healthy C57BL/6 mice. A Morphological images and flow cytometry results of mouse BM-MSCs, scale bar = 100 μm. The flow cytometry results of mouse MSCs typical surface markers (mCD105, mCD44, mSca1, CD29 were positive markers, and mCD34, mCD45, mCD48, mCD31, and mCD11b were negative markers). B Survival rate curve of C57BL/6 mice pre infused with edited MSCs and then treated with 6 / 7 Gy X-ray radiation (CON: n = 15; DSUP: n = 16), *p < 0.05, median survival period and survival rates were analyzed by Log-rank test. C–D Hemogram change curve of two group C57BL/6 mice after 6 Gy (C) and 7 Gy (D) X-ray radiation. Mean ± SEM, *p < 0.01, ns: no significant difference, by two-way ANOVA

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Discussion

Tardigrades, also named as water bears, could exhibit extraordinary tolerance to a wide range of physical extremes, such as exposure to high dose of irradiation [49], extreme temperatures [50, 51], high pressure [52] and even exposure to outer space environment [53]. Since the DSUP protein of tardigrade has been identified to be associated with radiation resistance [27], several studies have reported the expression of DSUP protein into other organisms to improve radiation resistance [54]. DSUP has been stably expressed in bacteria [55], nematodes [56, 57], drosophila, and human kidney cell lines [58] exhibiting various degrees of radiation tolerance, indicating that DSUP can be expressed in eukaryotic cells and exerts anti-radiation function. However, the expression in other species may also have unpredictable consequences. For example, the DSUP protein expression in Drosophila melanogaster reduced the level of their locomotor activity [54]. And the DSUP expression in cortical neurons promoted neurotoxicity, leading to neurodegeneration [36]. Therefore, in our study, we analyzed whether DSUP protein would affect MSCs self-renewal and multilineage differentiation potential. Interestingly, DSUP protein exhibited no significant negative effect on the basic stemness properties of MSCs, which is a prerequisite for the application of DSUP modified MSCs as a radioprotector.

Next, we examined the DSUP role towards radiation resistance in the DSUP modified MSCs. As ionizing radiation can cause irreversible damage to cells mainly through two ways: one is through direct radiation effect on DNA structure including single-strand breaks (SSBs) and DSBs, and the other is indirectly by ROS damage generated from radiolysis of water [38, 59]. Interestingly, the cell morphology of MSCs significantly enlarged and elongated in all the groups. Perhaps it may be due to the radiation affecting the expression of proteins involved in MSCs cytoskeleton dynamic regulation [60]. But the apoptosis rate after radiation only increased slightly in each group, and no significant intergroup differences were observed. Severe DNA damage, and nearly complete loss in the proliferation activity and migration ability was observed in the control MSCs group. Interestingly, DSUP group MSCs showed less DNA damage, slightly reduced proliferation activity, and almost no change in migration ability. In addition, H2O2 was used to simulate ROS damage caused by water radiolysis. The DSUP group MSCs exhibited lesser DNA damage with 18% apoptosis rate after H2O2 treatment compared to the control group MSCs. Those results indicated that the DSUP group MSCs was able to exhibit original functional activity post radiation damage. Thus, DSUP expressed MSCs may provide the functional hematopoietic microenvironment to promote the irradiated HSPCs recovery.

In order to further improve the radiation therapy capability of MSCs, many researchers have tried to express specific functional efficacy genes in MSCs by gene editing technology. For example, ginsenoside RG1 gene modified MSCs exerted excellent therapeutic ability against radiation-induced intestinal injury in rat model in vivo as well as small intestinal epithelial cells model in vitro [61]. Similarly, HMGB1-modified MSCs could attenuate radiation-induced vascular injury and enhance the potential of MSCs to differentiate towards the vascular endothelium [62]. Likewise, MnSOD2 modified MSCs could significantly alleviate radiation-induced lung injury [63]. So, genetically modified MSCs can rescue the radiation damage in ARS [61,62,63]. In terms of application purposes, our study differs from the earlier reported studies. Our focus was to achieve a certain degree of radiation prevention and protection by DSUP modified MSCs. Besides, we verified whether improving the radiation resistance of the hematopoietic niche can reduce the occurrence of radiation induced hematopoietic failure. Many studies have reported that the quiescent status of HSCs were more radioresistant than their downstream hematopoietic progenitors [10, 64], even if exposed to 10 Gy or more dose radiation, it is possible to achieve hematopoietic recovery by relying on the minimal self-residual HSCs [65]. The main cause of hematopoietic failure is the depletion of the hematopoietic niche and IR induced HSCs senescence, which pushes the body to spend a longer time for hematopoietic reconstruction [16]. However, ARS patients often experience mortality from other complications before hematopoietic recovery. Therefore, radioprotector that can reduce radiation damage and simultaneously protect the hematopoietic microenvironment function are of great significance for HSCs recovery and hematopoietic reconstruction.

To verify the radiation protective effect of DSUP modified MSCs on HSPCs, it is necessary to establish an appropriate in vitro model of HSPCs and hematopoietic niche. The PVA culture system for functional mouse HSPCs expansion ex vivo for more than 30 days was developed and reported by Wilkinson group [33]. The PVA culture system simulated hematopoietic microenvironment including TPO and SCF cytokine components, as well as matrix components such as PVA and fibronectin. Similarly, our co-culture model of MSCs and HSPCs was designed similar like PVA culture system to simulate the hematopoietic niche, and the protection effect of adhered MSCs on suspended HSPCs can be visually observed after radiation damage. The results in our study suggested that the DSUP group HSPCs have the shorter recovery period, the higher LSK cells ratio, and better hematopoietic colony-forming ability among all radiation groups. Besides, the stemness properties of DSUP group recovered HSPCs are closest to those of the non-radiation NC group HSPCs. Moreover, DSUP modified MSCs also have a similar radiation protective effect on human CD34+ HSPCs.

In vitro cell model results demonstrated the radiation protective effect of DSUP modified MSCs on HSPCs, and in vivo animal model experiments, mice pre-infused with DSUP modified MSCs also showed significant radiation protective function. Based on the above results, we speculated that a portion of DSUP modified MSCs transplanted into the hematopoietic niche of mice that can still function in situ microenvironment after radiation injury (similar to the recovery promoting effect on HSPCs in vitro). We could still detect human MSCs in BM stromal cells on 20 days after radiation, and the proportion of residual DSUP modified MSCs in mice BM is higher, indicating that DSUP modified MSCs continued to play a role during the hematopoietic recovery, promoting the regeneration of residual HSPCs and increasing the proportion of HSPCs in BM cells. Thus enabling the DSUP group mice to enter the hematopoietic recovery stage earlier, shortening the time of hematopoietic recovery and reducing the hematopoietic failure incidence as well as the mortality. Therefore, the distribution of different stages of hematopoietic cells recovery ratio in the DSUP group mice on 20 days after radiation is closer to the level of non-irradiated mice.

MSCs have long been reported to be hypoimmunogenic for allogeneic transplantation, but recent studies indicating the generation of antibodies and the immune rejection of allogeneic donor MSCs suggest that MSCs may not actually be immune privileged [66]. Our results also indicated that the immune rejection of xenogeneic DSUP modified MSCs will severely weaken their radiation protection effect, while allogeneic DSUP modified MSCs could already achieve radiation protection by targeting hematopoietic niche. The hematopoietic niche, which include perivascular cells, MSCs, megakaryocytes, adipocytes, macrophages, regulatory T cells (Tregs), etc [67]. MSCs not only promotes HSCs repair, but also promotes recovery of other cellular components in niche through cell replacement or fusion, paracrine activity and exosomes. The recovery of various components in the hematopoietic niche further promotes HSCs regeneration [67, 68]. For example, Megakaryocytes play a crucial role in the HSC niche, secreting IGF1 to promote HSC regeneration post-radiation injury, while also coordinating activation and ferroptosis to protect HSCs [69]. Megakaryocytes also enhance hematopoietic regeneration by promoting osteoblast proliferation and HSC niche expansion via PDGF-BB [70]. In addition, adipocytes expressed a high level of SCF which promoted the HSC regeneration post radiation [12]. Tregs promote HSCs survival post-radiation by transferring cAMP, activating the PKA-CREB pathway, and reducing apoptosis sensitivity, facilitating long-term HSC aging [71], and paracrine secretion of adenosine derived from highly expressed CD150 Tregs promoted hematopoietic regeneration post radiation [72]. Consequently, pre infused anti-radiation DSUP modified MSCs profoundly affect the structure and homeostasis of the BM microenvironment, leading to complex impacts on the survival and regeneration of HSCs after radiation.

MSCs have low immunogenicity and are suitable for allogeneic transplantation, and they can directly participate in tissue differentiation and repair after cell transplantation treatment, and gradually be replaced by endogenous tissue cells at a later stage [73]. Which provides a guarantee for the safety of pre-infusion application for radiation protection. In our study, the prerequisite for DSUP modified MSCs effectiveness is the remaining functional HSCs in BM after radiation. When the vast majority of HSCs apoptosis or exhaustion due to lethal dose radiation damage, it is unrealistic to reverse HSCs apoptosis and restore hematopoietic function in short time solely through the protection and enhancement of the hematopoietic microenvironment. But at least a better hematopoietic microenvironment and longer survival time will be more conducive to the success of future BM transplantation. Therefore, DSUP modified MSCs may serve as an effective protection strategy for ARS and have important significance to first responders for radiation exposure, astronauts under space radiation, cancer patients undergoing radiotherapy, and military in search-and-rescue missions under nuclear attack.

Conclusions

In summary, all above in vitro and in vivo results suggested that DSUP modified MSCs have significant radiation tolerance ability and exhibit significant protective effect on HSPCs by providing a functional hematopoietic niche after radiation damage. Using DSUP modified MSCs as radioprotector to attenuate ARS induced hematopoietic damage is feasible, and this study may provide new ideas and methods for the development of new radioprotectors.

Abbreviations

DSUP:

Damage suppressor protein

MSCs:

Mesenchymal stem cells

ARS:

Acute radiation syndrome

HSCs:

Hematopoietic stem cells

BM:

Bone marrow

HSPCs:

Hematopoietic stem/progenitor cells

IR:

Ionizing radiation

ROS:

Reactive oxygen species

SSBs:

Single-strand breaks

DSBs:

Double-strand breaks

RBCs:

Red blood cells

PLTs:

Platelets

WBC:

White blood cells

LSK:

Lin−Sca1+c-Kit+

Gy:

Gray

γ-H2AX:

Phosphorylation of the Ser-139 residue of the histone variant H2AX

DAPI:

4′,6-Diamidino-2-phenylindole

EdU:

5-Ethynyl-2-deoxyuridine

CON:

Control

WT:

Wild-type

NC:

Non-irradiated control

TBI:

Total-body irradiation

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Acknowledgements

Part of the illustration material provided by Servier Medical Art (https://smart.servier.com) under CC BY 3.0 license. The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by grants from the Nature Science Foundation of China (82273569, 82271467, 82101969).

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  1. Fu-Dong Chen, Biao Zhang and Li-Li Wang have contributed equally to this work.

Authors and Affiliations

  1. Medical School of Chinese PLA: Chinese, PLA General Hospital, Beijing, 100039, China

    Fu-Dong Chen, Ming-Fang Xiong, Li Chen & Jia-Fei Xi

  2. Stem Cell and Regenerative Medicine Lab, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing, 100850, China

    Fu-Dong Chen, Biao Zhang, Ya-Li Jia, Quan Zeng, Tao Fan, Hai-Yang Wang, Ying-Xue Lin, Jun-Nian Zhou, Wen Yue & Jia-Fei Xi

  3. Department of General Medicine, The First Center of the Chinese PLA General Hospital, Beijing, 100853, China

    Fu-Dong Chen, Li-Li Wang & Li Chen

  4. School of Medicine, Nankai University, Tianjin, 300071, China

    Ying-Xue Lin

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Contributions

FDC, BZ and LLW performed the experiments and data analysis. FDC and LC prepared the original draft and edited the manuscript. LC, JFX, WY, JNZ, YLJ, QZ, HYW, TF, MFX and YXL contributed to conceptualization, methodology, supervision, project administration, and funding acquisition. All the authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Wen Yue, Li Chen or Jia-Fei Xi.

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For umbilical cord tissue collection was approved by the Stem Cell Clinical Research Ethics Committee, the Second Affiliated Hospital of Guangzhou Medical University (Date: 05. 02. 2024, No. [2022]001-5) and was conducted following approved institutional guidelines. For animal experiments, all mice were housed and conducted according to protocols approved by the Institutional Animal Care and Use Committee in compliance with Beijing Medical Experimental Animal Care Commission.(Date: 03. 03. 2021, No. IACUC-DWZX-2021-596).

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Chen, FD., Zhang, B., Wang, LL. et al. DSUP modified mesenchymal stem cells exert significant radiation protective effect by enhancing the hematopoietic niche. Stem Cell Res Ther 16, 216 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04300-x

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512