Pluripotency of spermatogonial stem cells injected into mice which were nearly killed by gamma radiation

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 119-127 This paper is available online at PLURIPOTENCY OF SPERMATOGONIAL STEM CELLS INJECTED INTO MICE WHICH WERE NEARLY KILLED BY GAMMA RADIATION Nguyen Thi Trung Thu1, Nguyen Lai Thanh2, Vu Dinh Chat2 and Dao Thi Sen1 1Faculty of Biology, Hanoi National University of Education 2Faculty of Biology, University of Science, Vietnam National University Abstract. Spermatogonial stem cells (SSCs) are pluripotent and have properties similar to embryonic stem cells. In this experiment, we collected SSCs from young mouse testis and injected it into mature female mice which were irradiated with gamma rays to bring them to a near death state. The mice were irradiated with a dose of 900R to delete marrow, hematopoietic stem cells (HSCs). SSCs were injected into irradiated mice to determine potential differentiation of SSCs. The result showed that SSCs could prolong the life of the mice and increase the weight of mice that survived the radiation. Upon examination, we found cell division and an increase in the number of bone marrow cells. To prove the existence of transplanted SSCs in the irradiated mice, we isolated the DNA of some tissues and checked for the presence of Y chromosomes in females. Those organs which most often had SSCs were the heart, kidney and bone marrow; the liver and brain had the least amount of SSCs. This demonstrates that introduced SSCs divided and differentiated into some types of cells which replaced depleted cells. However, further study should be done to determine the potential differentiation of SSCs in irradiated animals. Keywords: Spermatogonial stem cells, radiated mice, gamma ray. 1. Introduction Stem cell technology has improved to open the possibility for new, promising medical procedures and interesting results have been obtained in the treatment of Received March 20, 2012. Accepted July18, 2012. Biology Subject Classification: 362 196. Contact Nguyen Thi Trung Thu, e-mail address: trungthuhnue@gmail.com 119 Nguyen Thi Trung Thu, Nguyen Lai Thanh, Vu Dinh Chat and Dao Thi Sen dysfunctional diseases and drug testing. Until now, people with damaged organs have been receiving organs from another body. However, the number of organs available is limited and when transplanted into patientss, these organs have to be accepted by the immune system of the recipient. Embryonic stem cells are obtained from human embryos, and this process is an ethical issue. Finding stem cells in a recipient’s body would eliminate the ethical consideration. Many experiments show that cells from bone marrow can differentiate into organs such as heart muscle [4], liver [6] or ectoderm of neural tissue [10]. SSCs are stem cells which exist in male’s testis. SSCs can grow, divide and differentiate into sperm. This can be considered a potential stem cell source. However, little study has been done in this area and that has mostly focused on the differentiation of SSCs in germline cells [9]. Shinohara’s study in 2004 showed that SSCs have pluripotency and can convert into embryonic stem cells in suitable conditions [6, 7]. Studies on pluripotency of SSCs in Vietnam are very rare, with most focusing on monopotency and the creation of germline cells to treat infertility. We chose to investigate Pluripotency of spermatogonial stem cells injected into mice which were nearly killed by Gamma radiation in order to evaluate the use of SSCs to benefit irradiated female mice in terms of: survival time, body weight, histological integrity and the presence of Y chromosomes. 2. Content 2.1. Time and place of study This study was conducted in an Animal cell technology laboratory at the Faculty of Biology, University of Natural Science, Vietnam National University, Hanoi, from 3/2009 to 11/2010. 2.2. Materials and methods *Materials Animals: White mice (Mus musculus), Swiss strain were obtained from the National Institute of Hygiene Epidemiology. They were mature females aged 2 - 3 months (28 - 32g) and immature males (16 - 18g). * Methods Method of feeding mice: Mice were kept in clean, dry cages, 30 × 50 × 20 cm in size, 4 mice per cage at 25 - 28◦C and 45 - 55% relative humidity. Food for the mice was provided by the National institute of Hygiene Epidemiology with the following composition: protein (22 - 24%), lipid (5 - 6%), starch (45 - 55%) and fiber (4 - 5%). Method of irradiating mice: The entire bodies of female mice were irradiated with 120 Pluripotency of spermatogonial stem cells injected into mice which were nearly killed... gamma rays from Co60 using a Chisobalt machine administering a 109.33R/minute dose (Hanoi Institute of Cancer). Method of isolating SSCs from young mouse testis: SSCs were isolated from young mouse testis using the method of Nagy and et al.. Method of injecting SSCs into irradiated mice: After 0.5 day, irradiated mice were injected with a 50 µl suspension of young mouse testis that contained approximately 5 × 105 cells into the tail vein. Method of defining the life expectancy and the average weight of mice: Keeping track of the number of mice that died each day until every mouse of the group had died. Irradiated mice were weighed each day until they died. Method of obtaining a bone marrow specimen:When a mouse appeared to be close to death, we cut out the mouse thighs and removed the bone tube. A small needle was used to draw fluid out and it was scanned on a template. Methanol was dripped into the fluid for a minute until it formed a shape, it was then dyed in mother Gemsa for 3 minutes, and next stained with Gemsa from 15 to 20 minutes using the usual method. Method of extracting DNA genome from bone marrow, kidney, brain, heart and liver of irradiated mice: Samples were ground and placed in liquid nitrogen in 2 mL Eppendorf tubes. The process of separating DNA genome was done under the guidance with iNtRON Biotechnology firm’s G-spinTM Genomic DNA Extraction (17041) kit. Experimental design: Table 1. Experiment arrangement Group Characteristic Number Biological control group (BC) Mice weren’t irradiated 900R dose and injected 40 Irradiated control group (IC) Mice were irradiated 900R dose and injected PBS (phosphate Buffered Saline) 40 Irradiated treated group (IT) Mice were irradiated 900R dose and injected 50µl suspension of young mouse testis 40 2.3. Result and discussion 2.3.1. Isolating SSCs from young mouse testis To obtain SSCs for injecting into irradiated mice, we collected a suspension from the testis of immature mice. This was done because in immature mice, most of the cells obtained were SSCs and first spermatocytes at the start of meiosis. In addition, the potency of SSCs in young mice was higher than that in mature mice. The cell count given in cell/mL suspension is shown in Table 2. 121 Nguyen Thi Trung Thu, Nguyen Lai Thanh, Vu Dinh Chat and Dao Thi Sen Table 2. The number of cells from the testis of immature male mice Immature mouse 1 2 3 Average The number of cells in testis suspension (x106 cells/mL suspension) 18 15 17 16.67 ± 1.53 Table 2 shows that there was little difference in the number of cells in the testis suspension of the three immature mice. The average number of cells in the testis suspension was 16.67.106 cells/mL. After they were isolated, the SSCs were immediately injected into irradiated mice at a dose of 5.105 cells/50 µl testis suspension. 2.3.2. The ability of SSCs to extend the lifetime of irradiated mice Resilience of irradiation damage to SSCs was shown in survival rate of mouse groups (Table 3). Table 3. The average lifetime and survival of mouse groups after being irradiated for 20 days Group Dose (R) Average lifetime (days) Survival rate after 10 days (%) Survival rate after 20 days (%) BC 0 30 ± 0 100 100 IC 900 8.4 ± 0.83 30 0 IT 900 16.1 ± 1.08 80 30 With an 900R dose, the IC group lived for an average of 8.4 days, while mice in the IT group lived for 16.1 days. The percent of mice which survived after 10 days and after 20 days in the IC group were 30%, and 0% respectively; for those in the IC group it was 80% and 30%. It has proven that SSCs could extend the lifetime and restore some lesions in irradiated mice. The result is quite consistent with the study of Nguyen Mong Hung and et al. [3]. 2.3.3. The ability of SSCs to recover weight in irradiated mice Body weight is also an important indicator to assess the vulnerability of mice which undergo irradiation and the resilience of mice which has been administered SSCs because it reflects the functioning of organs and the whole body. The change in average weight of mice after irradiation and treatment with SSCs is shown in Figure 1. Figure 1 shows that in the IC group, body weight decreased continuously until death (19.53 g). This might be due to radial damage which could not be restored resulting in death [2]. In the IT group, body weight decreased 1.33 g after irradiation for 5 days, but increased 4.17 g in the next 20 days (although it was still lower than in BC group). This 122 Pluripotency of spermatogonial stem cells injected into mice which were nearly killed... demonstrates that SSCs injected into mice helped mice restored their bodies; yet in the efirst days SSCs caused little change and so body weight decreased. Figure 1. The change in mouse weight 25 days after irradiation 2.3.4. Results seen in bone marrow specimens Blood cells have a relatively short life span of a few days to several months. Bone marrow is the only organ that can produce blood cells. Mice were irradiated for the purpose of deleting bone marrow or making HSCs unable to proliferate. To determine the ability of SSCs to change the mice’s hematopoietic system, we took bone marrow specimens of most of the nearly dead mice. The result is shown in Figure 2. Figure 2. Morphology of mice’s bone marrow in the IC group (100x) Observation of IC group’s bone marrow after 5 days of irradiation (Figures 2a, 2b) showed that the number of cells in the IC group decreased rapidly and by the time of death very few cells remained. We also noticed defections in the center of the cells 123 Nguyen Thi Trung Thu, Nguyen Lai Thanh, Vu Dinh Chat and Dao Thi Sen that were caused by the irradiation. This large number of defective cells demonstrated that the radiation had a powerful effect on mice and caused irreversible damage to their hematopoietic system [5]. By the eighth day of irradiation, all of the mice were dead. Mouse bone marrow specimen in the IT group after 5 days (Figure 3a) and 10 days (Figure 3b) of irradiation. Figure 3. Morphology of mice’s bone marrow in the IT group (100x) The number of blood cells after 5 days of irradiation in the IT group was higher than that in the IC group, until 10 days after irradiation the number of blood cells increased significantly. The nucleus of the cells was large and round, and there were fewer defective cells. In addition, we saw a proliferation in number of cells in the bone marrow of the IT group (Figure 4). Figure 4. Morphology of mice bone marrow in the IT group after 10 days of irradiation This demonstrated that SSCs had an effect on the recovery of the mice’s hematopoietic system. 124 Pluripotency of spermatogonial stem cells injected into mice which were nearly killed... 2.3.5. Determining the presence of SSCs in irradiated mouse organs * Result of isolating genomic DNA from mouse organs The organ samples obtained from dead mice of the IT group (the heart, liver, kidney, brain and bone marrow) were processed to isolate genomic DNA. PCR was conducted on a primer pair of the 18S ribosome (in nuclear DNA) to check isolated DNA quality with the 321bp band and PCR was also done with the primer pair of Sry gen to check Sry operation with the 418 bp band. The electrophoretic result is shown in Figure 5. Figure 5. Electrophoretic image of PCR products which tested for quality of DNA isolated from mouse organs 1. Brain; 2. Bone marrow; 3. Kidney; 4. Heart; 5. Mouse testis (to check operation of SRY gene); 6. Spleen; 7. Liver; 8. Control - (H2O) The result showed that most of the genomic DNA that was isolated was equally good in expression at the 18S ribosome (321 bp) and the Sry gen (418 bp). * The presence of Y chromosome in mouse organs DNA samples which were of similar quality were used to check for the presence of Y chromosomes in female mice injected SSCs, looking at the Sry gene primer (418 bp band). Only organs which had a high concentration of SSCs or a significant proliferation of transplanted SSCs showed positive results. The results of the tests are shown in Fig. 6. Electrophoretic results showed that the presence of the Sry gene varied in organs and was not entirely consistent in the IT group. In the heart, kidney and bone marrow, the frequency of the Sry gene were higher than in the liver, spleen and brain. Because the former have open capillaries, SSCs could penetrate easily. As in the liver, spleen and brain, there were very few products of the Sry gene. This can be explained considering that the brain has a closed capillary system while the liver and spleen have an open capillary system making it possible for large macrophages to attack and delete strange elements (SSCs). 125 Nguyen Thi Trung Thu, Nguyen Lai Thanh, Vu Dinh Chat and Dao Thi Sen Figure 6. Electrophoretic results of multiplex PCR products to for the presence of SSCs in organs 1 and 7. Control -; 2 and 10. Control + (with DNA from testis); 3 and 13. Marker 200bp; 4. Heart; 5. Bone marrow 1 6. Kidney; 8. Liver; 9. Brain; 11. Bone marrow; 12. Spleen Thus, mobility and integration of SSCs in mouse bodies is complex and its difficult to predict the destination of SSCs because these cells are capable of differentiating into several cell types in the body and residing in tissues and organs, carried there by the bloodstream. 3. 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