Rescuing Self: Transient Isolation and Autologous Transplantation of Bone Marrow Mitigates Radiation-Induced Hematopoietic Syndrome and Mortality in Mice

The inflamed bone marrow niche shortly after total body irradiation (TBI) is known to contribute to loss of hematopoietic stem cells in terms of their number and function. In this study, autologous bone marrow transfer (AL-BMT) was evaluated as a strategy for mitigating hematopoietic form of the acute radiation syndrome by timing the collection phase (2 h after irradiation) and reinfusion (24 h after irradiation) using mice as a model system. Collection of bone marrow (BM) cells (0.5 × 106 total marrow cells) 2 h after lethal TBI rescued different subclasses of hematopoietic stem and progenitor cells (HSPCs) from the detrimental inflammatory and damaging milieu in vivo. Cryopreservation of collected graft and its reinfusion 24 h after TBI significantly rescued mice from lethal effects of irradiation (65% survival against 0% in TBI group on day 30th) and hematopoietic depression. Transient hypometabolic state (HMS) induced 2 h after TBI effectively preserved the functional status of HSPCs and improved hematopoietic recovery even when BM was collected 8 h after TBI. Homing studies suggested that AL-BMT yielded similar percentages for different subsets of HSPCs when compared to syngeneic bone marrow transfer. The results suggest that the timing of collection, and reinfusion of graft is crucial for the success of AL-BMT.

The inflamed bone marrow niche shortly after total body irradiation (TBI) is known to contribute to loss of hematopoietic stem cells in terms of their number and function. In this study, autologous bone marrow transfer (AL-BMT) was evaluated as a strategy for mitigating hematopoietic form of the acute radiation syndrome by timing the collection phase (2 h after irradiation) and reinfusion (24 h after irradiation) using mice as a model system. Collection of bone marrow (BM) cells (0.5 × 10 6 total marrow cells) 2 h after lethal TBI rescued different subclasses of hematopoietic stem and progenitor cells (HSPCs) from the detrimental inflammatory and damaging milieu in vivo. Cryopreservation of collected graft and its reinfusion 24 h after TBI significantly rescued mice from lethal effects of irradiation (65% survival against 0% in TBI group on day 30th) and hematopoietic depression. Transient hypometabolic state (HMS) induced 2 h after TBI effectively preserved the functional status of HSPCs and improved hematopoietic recovery even when BM was collected 8 h after TBI. Homing studies suggested that AL-BMT yielded similar percentages for different subsets of HSPCs when compared to syngeneic bone marrow transfer. The results suggest that the timing of collection, and reinfusion of graft is crucial for the success of AL-BMT.
Keywords: autologous bone marrow transfer, hematopoietic stem cells, homing, ionizing radiation, mitigation, hematopoietic syndrome inTrODUcTiOn Matched allogenic bone marrow transfer (AG-BMT) has long been established and used for treating radiation-induced hematopoietic depression (1). However, the Chernobyl and other experiences showed its utility only for a fraction of victims exposed to radiation (2). Victims exposed to lower doses (<6 Gy) may not benefit from bone marrow transplantation (BMT) because at these doses, the residual hematopoietic stem cells (HSCs) would eventually cause donor marrow rejection (3,4). Considering the limited use of AG-BMT, strategies must be developed for treating radiationinduced bone marrow (BM) aplasia and hematopoietic form of the acute radiation syndrome (hARS). Previous studies on dogs, non-human primates (NHPs), and humans have reported that if a sufficient number of HSCs could be protected (by shielding the femur), it is possible to ameliorate the myeloablative effects of chemotherapy or radiation (5)(6)(7). These studies prompted researchers to explore the efficiency of AL-BMT in the management of radiation-induced BM aplasia. This efficiency is based on the fact that quiescent HSCs survive high doses of radiation and is contributed by the heterogeneity of the absorbed dose because of the geometry of the exposure and dose attenuation related to the body's thickness (8). The residual functional HSCs isolated soon after irradiation can be expanded and reinfused at later time points to achieve hematopoietic recovery. Contrary to AG-BMT, AL-BMT may accelerate hematopoietic recovery without causing graft-versus-host disease complications. In addition to its application in high-dose exposure patients, AL-BMT may be effective in enhancing hematopoietic recovery against moderate to sublethal radiation doses, where AG-BMT is not desired.
However, isolating a sufficient number of hematopoietic stem and progenitor cells (HSPCs) from irradiated victims remains an important concern for achieving successful engraftment and hematopoietic recovery after AL-BMT. The ex vivo expansion of the collected graft in the presence of antiapoptotic cytokines has been suggested to be critical because the reinfusion of the isolated BM cells without expansion results in poor engraftment and limited hematopoietic recovery (9)(10)(11). However, compromised long-term (LT) hematopoietic potential because of residual DNA damage in the isolated HSPCs remains an important concern related to these approaches (12). Moreover, the infusion of the BM several days after the radiation exposure delays effective hematopoietic recovery because BMT performed within 24 h has so far been the most effective in treating radiation-induced hematopoietic depression (13,14). Moreover, studies on NHPs have shown that the BM collected 2 h after total body irradiation (TBI) is ideal for effective ex vivo expansion and engraftment (15). The BM collected at later time points resulted in the poor recovery of functional HSPCs because of a disturbed milieu and initiation of a large apoptotic burst (15). The requirement of BM collection within 2 h of radiation exposure restricts its use in cases of accidental or nuclear eventualities. The development of strategies or agents effective in preventing the loss of HSPCs both in terms of their number and functional status well beyond 2 h will increase its practical applications. Numerous investigators have reported that shortly after irradiation (from few hours to first few days), free radical-mediated oxidative stress, inflammation, and bystander effects prolong the effects on hematopoietic depression by causing the loss of cells that are not directly affected by radiation (16)(17)(18). Keeping these results in mind, in this study, we applied AL-BMT in a murine model and determined whether transient hypometabolic state (HMS) induced soon after irradiation preserves the functionality of HSPCs until 8 h after TBI.
Non-human primate models are more relevant to humans in terms of stem cell biology (19); however, they extensively restrict the detailed analysis of different aspects of AL-BMT. Mice have been used to understand the different aspects of BMT and homing (20). However, unlike large mammals, in rodents, the AL-BMT process involving a surgical procedure and postoperative care is extremely challenging and results in high mortality rates (21,22). Moreover, irradiated mice surgically operated for BM aspiration has a risk of combined radiation injuries, which is known to accelerate radiationinduced death (23). However, in the past decade, significant advances have been made in the life saving and aseptic surgery in rodents, including mice (24). In our laboratory, we have standardized the surgical procedure and postoperative care for irradiated mice. We used mice as a model for investigating the radiomitigative efficacy of AL-BMT.
In this study, we revealed that BM collection within 2 h after TBI followed by cryopreservation for 22 h and reinfusion 24 h after TBI into the same mice rescues from the lethal effects of TBI by enhancing the hematopoietic recovery. These results suggest the efficiency of this approach, at least in the murine model, without requiring ex vivo graft expansion. Efforts to prolong the BM collection phase by inducing transient HMS (for a total of 8 h after TBI) have not been successful in terms of survival advantage. However, the evaluation of the BM and peripheral blood counts of the surviving animals suggest that HMS effectively preserved the functional status and number of HSPCs collected even 8 h after TBI.

Mouse experiments
Animal handling and experiments were conducted in accordance with the guidelines of the Committee on the Ethics of Animals Experiments, Institute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organization, Delhi, India (Institutional Ethical Committee approval number: INM/IAEC/2013/03/04; Protocol no.: TD-10018; GO/a/99/ CPCSEA). Inbred female mice (C57BL/6J) aged 10-12 weeks (weight: 25 ± 2 g) were used in this study. The animals were kept at the Experimental Animal Facility, INMAS, at an ambient temperature (Ta) and relative humidity of 23-25°C and 55%, respectively. Unlimited mouse chow (Golden Feeds, Delhi, India) and tap water were provided ad libitum, and the animals were maintained on a 12-h light:dark cycle. All efforts were made to minimize animal number and suffering.

animal irradiation
Total body irradiation was performed in partially restrained mice by using a 60 Co γ-ray irradiator (Bhabatron-II Teletherapy Unit, Panacea Medical Technologies, Bangalore, India). For radiomitigation studies, the mice were exposed to 8.5 Gy (LD100/30) at a dose rate of 1 Gy/min on a rotating chamber, and the irradiation field size was 35 cm × 35 cm. The animals were partially restrained to avoid cuddling and shielding while radiation exposure; they were exposed in a group of 4. The exposure rates in the teletherapy unit were mapped by an Atomic Energy Regulatory Board-authorized health physicist by using a calibrated RadCal 0.6-cm 3 therapy grade ion chamber/electrometer system. All irradiations were consistently provided between 12:00 and 01:00 p.m. to minimize chronosensitivity (25). The irradiated animals were monitored daily and those showing a combined score higher than 8 (posture, eye appearance, and activity scores) were humanely euthanized to minimize the pain and distress (26).

induction of hMs in Mice
Numerous small molecules, including adenosine receptor agonist-like adenosine monophosphate and 6 N-cyclohexyl adenosine, are known to induce reversible HMS (27)(28)(29). By using a drug repurposing approach in conjunction with cheminformatics, the in silico screening of many Food and Drug Administration-approved small molecules and drug-like molecules [LOPAC 1280, JHCCL(v2), Enzo FDA library] yielded a small molecule having adenosine receptor agonist action (hereafter referred to as SJNP-1). SJNP-1, clinically used for treating depression, was used in this study to induce HMS. HMS was induced 2 h after TBI by intraperitoneally (i.p.) administering 100 mg/kg body weight (b.w.) of SJNP-1 (Sigma-Aldrich, St. Louis, MO, USA) prepared in phosphate-buffered saline (PBS). Thereafter, the animals were kept in an incubator at 15°C (for maintaining HMS) for 6 h. They were then maintained at room temperature for recovery. The core body temperature (Tb) was manually measured on an hourly basis for the entire duration of the study (8 h) at different time points by using a rectal probe (RET 3, Braintree Scientific Inc., MA, USA).

BM aspiration from Femur, cryopreservation, and al-BMT
For AL-BMT, the BM from the right femur of live mice was aspirated, 2 h after TBI or 8 h after induction of HMS essentially by following a previously reported protocol, with a few modifications (24). Before surgical manipulation, 10-to 12-week-old mice were anesthetized with 80 and 10 mg/kg b.w. of ketamine and xylazine, respectively, and fur in the area where the incision was to be made was gently clipped. The entire femur and tibia were disinfected with povidone-iodine (Betadine) scrub. Thereafter, an incision was made covering the femur and tibia joint, and muscles were gently spread to clearly observe the condyle. The area was disinfected with 5% H2O2. Subsequently, by using a wet 0.5-mL syringe fit with a 27-G needle, a hole was gently drilled by turning clockwise and counter clockwise while applying pressure. The needle was inserted into the distal femur above the patella through the patellar tendon, and the BM was aspirated (~8 μL). Consistently, the aspiration was performed only once, and the tendon and overlying muscles were gently positioned and sutured to close the incision. The mice were placed on a heating pad (28°C) to recover and were returned to animal housing on complete recovery. The collected BM was washed in PBS, and the pellet was resuspended in 500 µL of cryopreserving solution [90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] and frozen by sequentially reducing the temperature to a final storage temperature of −80°C. The cell count and viability were assessed through flow cytometry, as previously reported (30). For AL-BMT, which was conducted at 24-h after irradiation, the cryopreserved cells were washed two times with PBS to remove traces of DMSO and serum. The final pellet was suspended in 150 µL (mean cell count: 0.5 × 10 6 cells) of PBS and injected via the tail vein into the same mice. For assessing the effects of cryopreservation on the quality and quantity of different subsets of HSPCs, the BM was harvested from a separate group of untreated animals and either cryopreserved for 24 h at −80°C or freshly processed, without cryopreservation. The harvested BM was processed and stained with different surface markers for enumerating the different subsets of HSPCs. The mitochondrial membrane potential (MMP) and apoptosis were analyzed in different subsets of HSPCs.

radiomitigation-30-Day survival assay
After performing AL-BMT, the animals were housed and maintained at the experimental animal facility. All animals received ciprofloxacin (45 mg/kg b.w./day; i.p.) for three consecutive days after bone marrow transfer. The kinetics of the overall survival, defined as the time from the date of irradiation to the date of death of the examined mice, was analyzed using the (two-tailed) log-rank test. The 30-day survival rate was compared between the autologous transfer and radiation-only control groups.

cytological and histological evaluation of BM
To determine the effects of AL-BMT on recovery from hematopoietic depression, BM cellularity was evaluated in BM smears of the surviving animals. For the irradiation alone group, BM smears were prepared from the animals having a combined distress score of more than 8 (the dying animals; on the 13th day). These smears were prepared using a paint brush and stained with May-Grünwald-Giemsa stain. For histology, femurs from different groups of animals were dehydrated and processed following routine procedures and stained with hematoxylin and eosin (H&E). Hematological profiling was performed within 1 h of collection of blood by using an automatic hematology analyzer (Celltac α, Nihon Kohden, Japan). resUlTs rescuing self: BM aspirated from lethally irradiated animals within 2 h and readministered after 24 h can rescue animals from irradiation-induced lethality Total body irradiation (8.5 Gy; dose rate: 1.0 Gy/min) resulted in 100% lethality in 30 days (mean survival time: 12 days; Figures 1A,C). The surgical procedure performed on irradiation control mice, to assess the effects of irradiation on survival, did not result in any changes in radiation-induced mortality (data not shown). The mice that received the cryopreserved unprocessed total BM (0.5 × 10 6 nucleated cells/mice), 24 h after TBI, showed 65% survival advantage after 30 days compared to 0 survival in the 8.5 Gy irradiation and surgically manipulated group (BM aspiration procedure; hereafter referred as TBI + Surg), which received a vehicle. To assess the efficiency of transient HMS on extending the duration before the BM collection phase, the effect of SJNP-1 was investigated (Figures 1B,C). The administration of a single dose of SJNP-1 (100 mg/kg b.w.) 2 h after TBI followed by placing them at a Ta of 15°C resulted in a gradual reduction in Tb gradually and nadir (~32°C) reached 2 h after administration ( Figure S1 in Supplementary Material). The reduced Tb remained consistent throughout the experiment (6 h). The Tb of the animals quickly returned to normal levels within 2 h of shifting from 15°C to room temperature. Furthermore, the induction of HMS 2 h after TBI followed by BM aspiration after 8 h under general anesthesia did not induce any immediate death and showed a mortality trend similar to that observed in the TBI alone or TBI + Surg group. However, the AL-BMT of aspirated BM (collected 8 h after HMS) reinfused 24 h after TBI resulted in a survival advantage (16%) in comparison to the 0 survival observed in the TBI + HMS + Surg group (Figure 2). Thereafter, to determine the optimal time point, AL-BMT experiments were performed using the BM collected at different time points after TBI (Figure 2). The results indicated that the AL-BMT of BM grafts collected at different "delayed" time points after TBI (6, 12, or 24 h after TBI) did not exhibit any mitigative effects, and all animals succumbed to TBI-induced lethality ( Figure 2B).

al-BMT ameliorates irradiation-induced leukocytopenia, erythropenia, and Thrombocytopenia
After demonstrating the radiomitigative potential, we determined the effects of AL-BMT on recovery from radiation-induced leukocytopenia, erythropenia, and thrombocytopenia (Figure 3). TBI caused a significant loss (93 compared with 0% loss in the untreated control group) of white blood cells (WBCs) on day 13. However, AL-BMT significantly mitigated radiation-induced loss   of WBC (25%; Figure 3A). The TBI + HMS + Surg group showed a similar trend of leukocytopenia and erythropenia and a decline in thrombocytes, as also shown by the TBI + Surg and TBI alone groups (data not included). We consistently observed mice death during the night, which complicated blood collection from a large number of samples. Similarly, the surviving animals (16%), which received the BM collected 8 h after HMS induction, showed effective recovery compared with those in the TBI + Surg group, which received a vehicle, at 24 h, in place of BM ( Figure 3A). We evaluated two major subsets of WBCs, which play a significant role in establishing a competent immune status and facilitating recovery from hARS: lymphocytes and granulocytes (Figures 3B,C). In the untreated control group, lymphocytes and granulocytes accounted for 78 and 22% of total WBCs, respectively. The TBI + Surg group revealed a significant loss of both lymphocytes and granulocytes compared with the vehicletreated un-irradiation control group (Figures 3B,C). Moreover, a slight myeloid bias (M-bias) with WBCs constituting 60 and 40% of lymphocytes and granulocytes, respectively, was evident. The animals that underwent AL-BMT also showed a slight M-bias with the lymphoid (57%) to myeloid (42%) ratio. However, the surviving animals (16%) that received the BM collected at 8 h after HMS induction showed a strong myeloid shift with WBCs comprising 16 and 84% of lymphocytes and granulocytes, respectively. As was observed with WBC, the RBCs and the total hemoglobin content have also exhibited a similar trend (Figures 3D,E). Similarly, the TBI + Surg group showed a significantly reduced (90 ± 1.5% loss) platelet count compared with the vehicle-treated un-irradiation control group (Figure 3F). AL-BMT effectively recovered the platelet count (10% loss compared with 90% loss in the IR group) on day 30 as compared with the count in the vehicle-treated un-irradiation control group. Similar to WBCs, the animals that received AL-BMT by using the BM collected hours after HMS induction, showed complete recovery; the number of platelets was found similar in this group and the untreated control group (Figure 3F).
Recovery from radiation-induced hARS depends on the number of surviving and functional HSCs in the BM, which undergoes division and forms multilineage progenitors that further differentiate into varied lineages. After demonstrating the improved recovery of the different subsets of hematopoietic cells in peripheral blood, the cellular status of the BM was assessed ( Figures S3A-L    The evaluation of histopathological changes in longitudinally sectioned and H&E-stained femurs in the vehicle-treated mice showed that TBI caused aplasia in the surviving animals with a few islands of nucleated hematopoietic cells and massive deposition of adipose cells on day 14 ( Figure S3J in Supplementary Material). The animals that received the BM collected 2 h after TBI showed significant recovery and increased cellularity ( Figure  S3K in Supplementary Material). While the animals that received BM grafts collected 8 h after HMS induction revealed the highest recovery from irradiation-induced aplasia and adipogenesis ( Figure S3L in Supplementary Material).

isolation and cryopreservation effectively Preserves Different subsets of hsPcs
The number of functional HSPCs in the aspirated BM samples is critical for achieving successful engraftment and hematopoietic recovery; hence, this number in aspirated grafts was determined. The procedure involved the collection of BM cells followed by cryopreservation for 22 h before reinfusion. Therefore, assessing the effects of the procedure on the viability and number of the different subsets of HSPCs was important. The freezing method used in this study resulted in a significant loss of viability (91% of cells were viable, as determined using the 7-AAD uptake assay) in BM-MNCs compared with freshly processed BM samples (97% viable) ( Figure S2 in Supplementary Material). However, TBI did not aggravate the effects of cryopreservation and did not result in any further increase in the number of dead cells when harvested 2 h after TBI (90% of cells were viable compared with 91% for the BM aspirated from the un-irradiation control animals and processed similarly). Subsequently, the effects of cryopreservation on the loss of different subsets of HSPC were evaluated ( Figure S4 in Supplementary Material). The cryopreservation of the BM aspirated from the vehicle-treated un-irradiated animals for 22 h resulted in a significant loss of HPCs and LT-HSCs compared with that of freshly processed BM samples obtained from vehicle-treated un-irradiated animals ( Figures S4B,E  Previously, studies have investigated the effects of cryopreservation, a known inducer of apoptosis, in various cell types (36)(37)(38). In concordance with the previous reports, cryopreservation induced significant increases in apoptosis levels in different subsets of HSPCs ( Figures S6B-D  To ascertain that the timely removal of BM cells circumvents the excessive loss of HSPCs because of a disturbed BM milieu, the number of surviving and functional HSPCs in aspirated and cryopreserved BM samples vis-à-vis that of different HSPCs present 24 h after irradiation in vivo was determined. The aspiration of BM samples 2 h after TBI followed by cryopreservation resulted in a statistically significant preservation of the different subsets of HSPCs compared with the number of HSPCs present 24 h later in the BM of TBI-exposed animals (Figures 4A,B). However, compared with freshly processed BM harvested from the vehicle-treated un-irradiation control animals, the number of different HSPCs were significantly low ( Figure 4B).

surgical stress reduces the homing of Different subsets of hsPcs both in al-BMT and sg-BMT setups
Successful engraftment depends on the number of HSPCs homed in the BM niche, and the performance of small grafts can be improved by enhancing their homing potential (39,40). A study suggested that AG-BMT and SG-BMT have a similar homing potential (41). After demonstrating the survival advantage of AL-BMT in mice, the homing potential of different subsets of HSPCs was evaluated in a similar setup. Numerous factors, including stress, influence the homing of stem cells and successful outcomes of bone marrow transfer (42,43). Therefore, the effects of the surgical procedure under anesthesia on the homing of HSPCs were investigated ( Figure 5A). Surgical stress affected the homing of CFSE-labeled BM-MNCs, the Lin − fraction, and the different subsets of HSPCs in the tibia and femur at 24 h after BM transfer compared with syngeneic transplantation performed without surgical manipulations (Figures 5B-G). Moreover, the transfer of autologous CFSElabeled BM cells revealed reduced homing in the tibia and femur compared with SG transfer performed without surgery (Figures 5B-G). SG-BMT involving a surgical procedure and AL-BMT revealed similar homing potential (Figures 5B-G). Consistent with these homing results, evaluation of radiomitigative action of using survival as an end point revealed that both SG-BMT (with surgery) and AL-BMT groups have shown similar radiomitigative action ( Figure S8 in Supplementary Material). While, SG-BMT done in a setup without the involvement of surgical procedure resulted in an increased survival clearly suggesting the negative impact of surgical procedure ( Figure S8 in Supplementary Material).
To determine whether the transplanted autologous BM contributed to the overall postirradiation hematopoietic recovery, the dilution of CFSE fluorescence, indicating the divisional history of cells, was assessed 24 h after transfer (Figure 6). The CFSE dilution assay clearly indicated that among the different HSPCs analyzed in this study, all HPCs underwent division, as evident from the single peak without any shoulder (Figure 6A). Similarly, KSL, ST-HSC, and LT-HSC populations revealed significantly diluted CFSE fluorescence (a shift toward the left), but a significant shoulder was evident, indicating heterogeneity in   DiscUssiOn AL-BMT, despite its limitations, merits attention as an effective option for managing hARS in patients overexposed to radiation. Considering the relevance of AL-BMT, several researchers have proposed preserving the BM of nuclear power plant workers and first responders who are at the highest risk of radiation overexposure (44). However, any such efforts would rarely be cost-effective and may not be a feasible option for underdeveloped and developing countries. Advancements made in the past decade in the field of BM collection (45) and cryopreservation have warranted investigating AL-BMT as an effective strategy for managing hARS. The radiomitigative action of AL-BMT, demonstrated in this study, can be attributed to the transient isolation of residual HSC (which survived from radiation effects) from inflamed BM environment and protracted oxidative stress until the BM niche becomes conducive for engrafting and reconstitution. We supported this assumption by demonstrating that the isolation and cryopreservation of BM grafts more efficiently preserves different HSPCs than those of the BM of an irradiated animal, at least during the first 24 h after lethal TBI. This result is in concordance with a recent report by Ishida et al (32) who showed that elevated tumor necrosis factor alpha (TNF-α) levels in inflamed BM contribute to the compromised reconstitution of donor BM. However, such a direct comparison between in vivo and a vial containing BM frozen to −80°C is complex considering the number of influencing variables involved. The results could be skewed because of the mobilization of the residual HSPCs to extramedullary hematopoietic sites, which led to a reduced number of critical HSPCs in the BM collected 24 h after irradiation (46). The time point selected for reinfusing the cryopreserved BM was based on a time course study of radiation-induced proinflammatory cytokines, including TNF-α, which peaked after 2-4 h and became normal by 24 h (47)(48)(49). Notably, the reconstituted hematopoiesis post AL-BMT showed a shift toward the myeloid lineage, and the M-bias was strongly evident in the mice that received the BM collected 8 h after the induction of hypometabolism. A recent study suggested that the transfer of LT-HSCs pretreated with an antioxidant, N-acetyl cysteine, led to myeloid reconstitution (32). The M-bias observed in this study could partially be explained by considering that SJNP-1, which was used for inducing HMS, is an effective free radical scavenger and antioxidant. It must be validated whether the treatment of HSCs with antioxidants leads to a selective protection or proliferation of the M-bias HSPCs. In this study, ciprofloxacin was administered as a part of supportive care after surgical manipulation. Antibiotics are typically known to offer radioprotection, raising question about their contribution to overall radiomitigative action (50). However, the contribution of three suboptimal doses of ciprofloxacin can be ruled out because it was reported to be an ineffective mitigator (50).
In this study, we used a clinically established approach, reversible hypothermia or HMS, for preserving the BM in vivo and as a possible strategy for extending the collection phase after TBI. The induction of the HMS state, followed by the collection of grafts under general anesthesia resulted in reduced survival advantage, which could be attributed to the inability of the rodent model to survive the prolonged period of stress. This assumption is in accordance with previous reports that the prolonged maintenance of animals in HMS increases mortality (27). However, considering the efficient hematopoietic recovery observed in the residual animals, validating the usefulness of reversible HMS in larger mammals is important. In this study, we observed a significant survival advantage with 0.43% (5 × 10 5 cells) of the BM grafts (assuming tibia and femurs constitute 25% of total BM in C57 Bl/6J mice) (51) collected and reinfused without further refinement. This result is in agreement with previous reports showing that 2.5 × 10 5 BM nucleated cells per mice can render protection against lethal doses of ionizing radiation (52,53). However, because cryopreservation significantly induced early apoptotic events (positive for annexin V staining) and by assuming that the apoptotic fraction will be eventually eliminated in vivo, on an average the mice have received ~830, 110, 80, and 25 HPC, KSL, ST-HSC, and LT-HSC, respectively, which in an ideal scenario should be able to reconstitute hematopoiesis and radioprotect mice (31). However, they rarely engraft with absolute efficiencies, and typically, a much higher number of KSL cells (2,000 cells) or LT-HSCs (50 KSL CD34 − cells) in isolation are required for achieving radioprotection (54). Moreover, we showed that the surgical procedure significantly reduced the homing of different HSPCs, further complicating the situation. The protective effect observed in this study could be attributed to the use of total BM containing different committed progenitors and the most primitive HSCs. The committed progenitors (ST-HSCs and HPCs) facilitate the initial engraftment and LT-HSCs ensure durable engraftment.
The CFSE dilution assay, an effective method for investigating the division of the transplanted cells, clearly suggested that the transplanted BM cells are functional and dividing, which must have contributed to the hematopoietic recovery. This is consistent with previous observations that 2 × 10 5 of total BM [considering that KSL cells account for 0.1% (nearly 200 KSL cells) of BM nucleated cells] can protect mice from radiation-induced mortality (53). However, the duration of this study enabled the assessment of ST reconstitution only, and the effect of AL-BMT on LT reconstitution must be investigated. Moreover, a relatively lower radiation dose was used in this study, which probably requires a lesser number of HSPCs compared with 2,000 KSL cells or 2 × 10 5 BM cells containing 200 KSL cells for conferring protection against supralethal doses (9.8 Gy). Notably, unlike anti-apoptotic cytokine-treated BM, which raises concerns about the persisting mutations and compromised LT repopulation (12), the untreated BM used in this study can be expected to effectively perform LT repopulation. However, assessing the persisting DNA damage by using markers, such as gamma-H2AX, or chromosomal aberrations in transplanted cells can be informative.
Bone marrow mononuclear cells are established as a source of mesenchymal stem cells (MSCs) and when cotransplanted, they enhance the engraftment of HSPCs (55)(56)(57). In this study, we used total BM without further processing, and enumeration studies revealed 0.3% of MSCs in the samples (data not shown). Although the number of MSCs was much lower than the minimum threshold (58) and MSCs were unlikely to affect the hematopoietic recovery solely by themselves, their contribution to HSPC engraftment cannot be ruled out. Studies conducted using a baboon model have reported that the cografting of MSCs improves the hematopoietic potential of small grafts (9).
In conclusion, this study proposes that AL-BMT is a viable strategy and that the timing of collection and reinfusion can alleviate the requirement of ex vivo expansion. The efficiency of AL-BMT in cases of radiation overexposure must be assessed. However, we suggest that people with early symptoms of large total absorbed doses (exposures of more than 4 Gy) typically present with vomiting within 1 h (59) and may be triaged to undergo BM collection and cryopreservation. Studies must validate reversible HMS as a strategy for prolonging the BM collection phase, which would markedly improve the clinical utility of AL-BMT.