Evaluation of mesenchymal stem cells in treatment of infertility in male rats
© Hassan and Alam; licensee BioMed Central. 2014
Received: 4 March 2014
Accepted: 30 October 2014
Published: 23 November 2014
The present study aimed to elucidate the therapeutic effects of mesenchymal stem cells (MSCs) derived from the bone marrow of rats (BM) against toxic effects of lead (Pb) on the male gonads of experimental rats.
The experimental animals were exposed to lead in the form of lead nitrate (LN) one quarter of the LD50. The efficacy of MSCs to reduce gonado-totoxicity induced by lead nitrate at 21, 30 and 60 days, was evaluated experimentally in male rats.
The results showed that testosterone levels and semen quality ameliorated following treatment with MSCs. Also, superoxide dismutase, glutathione peroxidase and catalase levels were increased 21, 30 and 60 days post treatment of MSCs. Moreover, a decrease in genomic DNA alteration and percentage of fragmented DNA was recorded after MSCs treatment. Lead nitrate caused degeneration, necrosis, interstitial edema, and reduction in spermatogenic activity in some seminiferous tubules. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment with MSCs. Histological examination of testis showed deformities in morphology of testis in test animals with gross damage within the seminiferous tubules in Lead nitrate group. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment of MSCs.
It was concluded that lead is a gonadotoxic with a tendency of suppressing semen characteristics and testosterone levels of animals, the presence of MSCs was found to alleviate the toxic effects of lead. We conclude that MSCs derived from the bone marrow of rats can be an effective therapy of LN induced gonado toxicity, thus can contribute to the treatment of infertility.
Metals are unique environmental toxicants as they tend to possess bioaccumulative, immutable and non-biodegradable properties and pose a serious threat to eco-biological systems . Lead (Pb) is one of the well-known ubiquitous non-essential metals with wide applications for many centuries, which is released into the environment by several routes, but principally by industrial, mining and hunting activities . Exposure to lead is implicated in serious health hazards in animals and humans due to its toxicity and its ability to accumulate in living organisms . The deterioration of male reproductive health is one of the major manifestations of occupational and/or environmental exposure to Pb toxicity . Earlier studies have demonstrated that lead can pass through the blood-testis barrier, accumulate in the testis and/or epididymis and affect the germinal cells at different levels of differentiation (spermatogonia, primary spermatocytes, spermatids or spermatozoa) . Lead-exposed battery factory workers have shown a decrease in sperm count, density, motility and semen volume [5, 6]. In addition, studies of Biswas and Ghosh  demonstrated that lead exposure reduces the activity levels of testicular steroidogenic enzymes in rats.
Some studies suggested that oxidative stress is a potential contributor to lead toxicity and that lead directly or indirectly changes the pro-oxidant and antioxidant balance in the biological system by the generation of more reactive oxygen species (ROS), which elicits oxidative damage of proteins, lipids and DNA [8–10]. Antioxidant defenses, such as catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), are involved in counteracting the toxicity of ROS . Under normal conditions, these antioxidants protect the cells and tissues from oxidative damage. Enhanced generation of ROS can overwhelm cells intrinsic antioxidant defenses and result in a condition known as ‘oxidative stress’. Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Consequently, it has been suggested that metal-induced oxidative stress in cells can be partially responsible for the toxic effects of heavy metals .
Bone marrow stem cells, including hematopoietic stem cells and bone marrow-derived mesenchymal stem cells (MSCs/BM), are pluripotent and can self-renew. MSCs/BM are characterized by their accessibility, ease of culture and proliferation in vitro, potential to modulate tissue repair and biological stability in long-term culture . In addition, MSCs have been adapted in andrology research on erectile dysfunction and infertility as potential therapeutic agents. The studies related to this area showed that MSCs derived from human fetal lung and umbilical cord can differentiate into sperm-like cells [14, 15]. The differentiation of MSCs into germ cells, Sertoli cells and Leydig cells was also demonstrated in busulfan-treated infertile mice . These insights hold promise to inform strategies for the directed differentiation of stem cells and to offer the potential for novel metabolic or pharmacological therapies to enhance regeneration and the treatment of degenerative disease .
Recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer. Various anti-oxidative and anti-stress mechanisms of stem cells have also been characterized [18, 19].
The effects of MSCs on lead-induced reproductive toxicity have not yet been reported. The present study was undertaken to investigate the ability of MSCs to modify (1) the oxidative stress and (2) the suppressed reproduction induced by lead in male rats.
Materials and methods
Anesthetic procedures and handling of animals were approved by, and complied with, the ethical guidelines of the Medical Ethical Committee of the National Research Centre in Egypt (Approval number: 10031).
Adult male albino rats weighing 160 ± 10 g were used in the present study. The animals were kept in wire bottomed cages in a room under standard conditions of illumination with a 12-hour light–dark cycle at 23 ± 1°C. They were provided with tap water and a balanced diet ad libitum.
Preparation of bone marrow-derived MSC (MSCs/BM)
Seventy male rats were randomly divided into two main groups as follows: (1) the normal control group (GI) (N = 10): (normal saline (NS) received 0.4 ml of physiological saline via the right tail vein; and (2) the lead treated group (LN) (GII) (N = 60): animals were injected intravenously (through the tail vein) with a single dose of lead nitrate (LN, 23.3 mg/kg body weight (bw)) about ¼ LD50 according to the Agency for Toxic Substance and Disease Registry (ATSDR) . GII was divided into two subgroups, GII(A) treated with 1 × 106 MSCs per rat  one week after a single dose of LN (23.3 mg/kg bw) about ¼ LD50 (N = 30) while GII(B) animals did not receive MSCs (N = 30). Ten animals only were tested at 21, 30 and 60 days, respectively, post-injection.
Preparation of tissue homogenate
The excised testicular tissue was washed with distilled water for the removal of blood, after which the fatty parts were removed. Tissues were homogenized in ice-cold 50 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM EDTA. The supernatant was separated by centrifugation at 1,000 g for 20 minutes at 4°C. The supernatants were used for the analysis of all antioxidant enzymes.
Estimation of lipid peroxidation and assay of antioxidant enzymes in testes
Malondialdehyde (MDA) in the testis homogenate was assayed colorimetrically according to the method of Ohkawa et al. , in which MDA is determined by using 1 ml of trichloroacetic acid (10%) and 1 ml of thiobarbituric acid (0.67%).
SOD was determined according to the method described by Misra and Fridovich . The activity of CAT, expressed as units/mg protein, was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme . The determination of testicular glutathione peroxidase (GPx) activity was carried out according to the method of Chiu et al. .
Analysis of sperm parameters
Epididymal sperm were obtained by chopping cauda epididymis in 5.0 ml of Ham’s F12 medium. The sperm were counted using a Neubauer Chamber as describe by Belsey et al. . Progressive sperm motility was evaluated by a previously described method  within five minutes following their isolation from cauda epididymis at 37°C and the data were expressed as percent motility. The morphological abnormalities in sperm were enumerated by the methodology reported by Hemavathi and Rahiman  using light microscopy.
Isolation of genomic DNA from caudal epididymal spermatozoa was carried out using the protocol of Gebert et al. . Briefly, sperm cells were lysed in 500 μl of a buffer consisting of 50 mM Tris–HCl at pH 8.0, 100 mM NaCl2, 100 mM EDTA, 1% SDS and treated with 2.5 μl of Triton X-100 (Merck, Darmstadt, Germany), 21 μl of dithiothreitol (1 M) (Sigma-Aldrich Chemie) and 40 μl of proteinase K (10 mg/ml). DNA precipitation was performed in a saturated sodium chloride solution with subsequent addition of 100% ethanol (Roth, Hamburg, Germany).
The concentration of DNA and its relative purity were determined using a spectrophotometer based on absorbance at 260 and 280 nm, respectively. The integrity of extracted genomic DNA was verified by electrophoresis in 0.8% agarose gel using a DNA molecular weight marker (Eurblio, Paris, France).
Random amplified polymorphic DNA analysis
Sequence of selected random primers, number of total bands and percentage of polymorphisms calculated from treated and control sperm cells
Sequence (5′- 3′)
Total number of band studied
Number of polymorphic bands
Size range (bp)
CAG GCC CTTC
GTG ACG TAGG
TCG GCG ATAG
CTT GCG ATC C
Agarose gel electrophoresis for DNA fragmentation
DNA was isolated from rat testis using proteinase K and RNase A with the methods of Gilbert et al. . To estimate DNA damage, 5 μg of rat DNA was separately loaded on 1.5% agarose gel containing 1.0 μg/ml ethidium bromide including DNA standards (0.5 μg per well). Electrophoresis was performed for 45 minutes at 100 volts. After electrophoresis, the gel was studied under a gel doc system and was photographed with a digital camera.
At 21, 30 and 60 days post treatment, blood samples were collected from the retroorbital plexus in plastic centrifuge tubes, left to clot at 4°C for 30 minutes and serum obtained by centrifugation at 3,000 rpm for 20 minutes. Quantitative measurement of serum testosterone was carried out by solid phase radioimmunoassay (RIA) using components of a commercial kit (Coat-A-Count, Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) with modifications described by Richards et al. .
Serum protein assay
The total protein level was determined according to the method of Bradford .
Pieces of testes were fixed in 10% neutral-buffered formalin that was embedded in paraffin and the deparaffinized sections were stained routinely with hematoxylin and eosin as described by Bancrofet and Stevens .
All values are expressed as mean ± SE. Statistical analysis was performed with two way analysis of variance (ANOVA) followed by Duncan’s test. P values <0.05 were considered to be statistically significant.
Testicular oxidative stress
LN exposure significantly decreased (P < 0.05) total protein level at 21, 30 and 60 days. Meanwhile, after 30 and 60 days of MSCs treatment the level of total protein was increased significantly as compared to the untreated rats (Figure 3).
Testosterone level and total protein
Lead nitrate-induced changes in sperm characteristics and their response to administration of MSCs/BM in rats after 21, 30 and 60 days of exposure
Mean control value
LN + MSCs/BM
Sperm count (10 6 /ml)
Sperm motility (%)
Abnormal sperm (%)
Sperm morphology (%)
64.77c ± 0.11
Random amplified polymorphic DNA-PCR
To analyze instability in the genome of treated rats compared to non-treated controls using RAPD-PCR fingerprinting, four random 10-mer primers were used to amplify genomic DNA samples. RAPD primers generated strong banding patterns in all samples tested, and the presence of changes in the RAPD profiles obtained from the exposed animals depended on the primer used. Profiles generated by these primers revealed differences between control and exposed rats, with visible changes in the number and size of amplified DNA fragments.
The RAPD profiles obtained with the RAPD primers exhibited bands between 63 to 1,301 bp in length. In a total of 44 bands scored, 35 bands were polymorphic giving 79.5% polymorphism. Primers OPA08 and OPA12 amplified the minimum and maximum number of bands which were 8 and 13 bands, respectively.
Effect of stem cells and lead on DNA fragmentation
The effect of environmental lead on the male reproductive system in which the testicular spermatogenesis and spermatozoa within the epididymis are the major targets for lead action to produce toxicity on reproduction has been a major area of concern for several years . Although the exact mechanism of LN-induced toxicity is not completely understood, cumulative data has shown that oxidative stress plays an essential role in its toxicity. Lead administration disrupts the testes spermatogenesis process via mechanisms that involve the induction of lipid peroxidation, depletion of ROS scavengers and disruption of testicular antioxidant enzyme activity .
In the present study, lead exposure correlated with increased levels of oxidative stress biomarkers in the testis of rats, represented by decreased levels of antioxidant enzymes (SOD, CAT, and GPx) and an increase of testicular MDA. MDA is one of the major products of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation. In general, SOD is the first line of defense against oxidative stress  and plays a pivotal role in dismutation of superoxide anions to hydrogen peroxide, and CAT neutralizes hydrogen peroxides to molecular oxygen and water . The decrease in these enzymes in Pb treated rats clearly postulates improper dismutation of superoxides and improper decomposition of H2O2. The production of ROS is a normal physiological event in various organs including the testis. On the other hand, overproduction of ROS can be harmful to sperm and, subsequently, to male fertility . It has been demonstrated that lead toxicity leads to free radical damage via two separate pathways : 1) the generation of ROS, including hydroperoxides, singlet oxygen and hydrogen peroxide; and 2) the direct depletion of antioxidant reserves . Marchlewicz et al.  and Sainath et al.  demonstrated that Pb toxicity caused testicular oxidative stress by increasing the levels of lipid peroxidation and decreasing the activities of SOD and CAT in testes, which is inconsistent with the findings of the present study.
However, administration of MSC/BM extract significantly prevented the influence of lead on the antioxidative system. It decreased MDA and concomitantly increased the activities of SOD, CAT and GPx levels in testes tissue. These results clearly demonstrate the anti peroxidative role of the MSC/BM. The in vivo protection by MSC/BM against lead-induced oxidative damage may be because of its free radical scavenging potential. The specific responses of MSCs to oxidative stress may play a crucial role in the regulation of tissue homeostasis as well as regeneration of organs after oxidative injury . It could also be because of direct scavenging/neutralization of the free radical or induction of endogenous antioxidant enzymes, such as CAT and SOD. El Far et al.  reported that transplantation of MSCs can correct and reverse the imbalance between ROS and antioxidant defense in favor of antioxidant defense by restoring and augmenting its capacity as well as modulating lipid peroxidation.
The sperm endpoints, such as epididymal sperm count and sperm motility, were used as important indicators to detect adverse effects of various factors on spermatogenesis . In the present study, epididymal sperm count and sperm motility decreased in rats at different time intervals of exposure to lead as compared to control rats. These results are in agreement with earlier reports . Moreover, Leiva et al.  reported a reduction in epididymal sperm number and daily sperm production in male rats treated with lead acetate and explained this reduction in sperm number by positing that lead acetate administration inhibited spermatogenesis by reducing the length of the stages related to spermiation and onset of mitosis. The deterioration in the selected sperm characteristics might be due to increased oxidative stress during Pb intoxication. Oliveira et al.  showed that lead had adverse effects on the sperm in male rabbits. It increased the percentage of sperm shape abnormalities. These findings coordinate with results reported by those who found a decrease in the percentage of sperm motility and intact acrosomes in mice treated with lead acetate. Also, Mendiola et al.  reported a significant positive association between the percentage of immotile sperm and seminal plasma levels of lead and cadmium in men. Moreover, Leiva et al.  reported a reduction in epididymal sperm number and daily sperm production in male rats treated with lead and explained this reduction in sperm number by the fact that lead acetate administration inhibited spermatogenesis by reducing the length of the stages related to exposure to lead and induction of genetic damage. Spermiation and onset of mitosis [49, 52, 53] indicated that lead acts as a spermicidal agent in the case of high exposure for a long time. Our experiments showed that the toxic effects of lead on the reproductive system in male rats were dose-dependent.
Poor sperm quality caused by oxidative stress due to generation of ROS has been reported to result in infertility . Several studies suggest a correlation between increased ROS production and decreased sperm motility [55, 56]. It is hypothesized that H2O2, one of the lipid peroxidation products, might diffuse across the membrane and affect the vital enzymes in the sperm , thereby resulting in decreased sperm motility. Spermatogenesis occurs in the testis and its duration varies among species, for example, about 52 days in rats  and 64 days in humans . Spermatozoa are generated in the testes and are transported to the epididymis for concentration and maturation. Spermatogenesis is a prolonged process spanning 40 to 50 days in rodents . Agarwal et al.  and Manivannan et al.  showed that from 20 to 60 days the seminiferous tubules are in an active developing stage with cellular units in which the sections exhibited round-shaped seminiferous tubules and many newly formed spermatogenic cells arranged properly inside the tubules At 60 days, all stages of spermatogenesis were clearly visible and spread in the lumen of the tubules as was seen in the corresponding control. The total duration of spermatogenesis, which takes approximately 4.5 cycles, lasts from 30 to 75 days in mammals [58, 60]. Although strain or breed differences can be found in the literature among members of the same species , the duration of the spermatogenic cycle has been generally considered constant for a given species. According to a study utilizing xenogenic spermatogonial transplantation has demonstrated that the spermatogenic cycle duration is under the control of the germ cell genotype .
Spermatozoa are generated in the testes and are transported to the epididymis for concentration and maturation. Spermatogenesis is a prolonged process spanning 40–50 days in rodents. Manivannan et al.  showed that from 20 to 60 days the seminiferous tubules showed active developing stage with cellular units the sections exhibited rounded shape seminiferous tubules and many newly formed spermatogenic cells arranged properly inside the tubules At 60 days period all stages of spermatogenesis were clearly visible and spreaded in the lumen of the tubules as were seen in the corresponding control. The total duration of spermatogenesis, which takes approximately 4.5 cycles, lasts from 30 to 75 days in mammals [58, 62]. Although strain or breed differences can be found in the literature among members of the same species, the duration of the spermatogenic cycle has been generally considered constant for a given species. A recent study utilizing xenogenic spermatogonial transplantation has demonstrated that the spermatogenic cycle duration is under the control of the germ cell genotype .
In the present investigation, reduction in sperm number and motility was associated with an increase of sperm abnormalities in rats exposed to LN, which suggests the lead may impair the spermatogenesis or damage the genetic material of spermatogonia and spermatocytes by crossing the blood-testis barrier and gaining access to germinal cells. Furthermore, Hsu et al.  and Acharya et al.  declared that ROS generation causes chromosomal aberrations in germ cells by mutating certain gene segments involved in the maintenance of normal sperm structure, resulting in a deformed sperm population and/or drastically minimizing sperm count. The degraded sperm characteristics caused by lead administration may be due to a low testosterone concentration as observed in this study since a high level of testosterone is critically required for normal spermatogenesis, development, maintenance of sperm morphology and normal morphology and physiology of somniferous tubules . In infertility and sterility, stem cell therapy promises to be a potential source of male and female germ cells. Not only embryonic stem cells (ESCs) but also fetal porcine skin stem cells, human fetal lung-MSCs, bone marrow and umbilical cord MSCs were the candidates for germ cell differentiation in vitro[53, 65]. Recently, stem cells experimentally derived from bone marrow have been used in experimental busulfan-treated infertility rodent models . In the present study, the MSC group was associated with a significantly higher sperm count and motility, and a lower percentage of abnormal sperm population along with a concomitant increase in testosterone level, compared to the lead treated rat group. Hence, it could be concluded that MSCs might have a potential role in treating male infertility and testosterone deficiency. It could also be proposed that the beneficial effects of MSCs may be due to differentiation into male germ cells as reported by Nayernia et al. . Yazawa et al.  proved that MSCs have the capacity to differentiate into steroidogenic cells, such as Leydig cells, both in vivo and in vitro. Lue et al.  showed that MSCs/BM, transplanted into testis of a busulfan-treated infertility mouse model, appeared to differentiate into germ cells, Sertoli cells and Leydig cells.
A growing amount of study has provided abundant evidence which has established the fact that metals are capable of interacting with nuclear proteins and DNA causing oxidative deterioration of biological macromolecules . A decrease of total protein content was recorded in this study at different time intervals of lead treatment. Previously, Kansal et al.  reported a decrease in total protein level following LN exposure in mouse liver and kidney. They concluded that protein loss in lead toxicity might decrease the level of specific proteins such as albumin, hormones, hormone and metal binding proteins, enzymes and so on and thereby disturb the homeostasis and rate of metabolic activities.
Sperm DNA damage is a novel indicator of male infertility, which may be caused by an abnormal packaging and segregation of chromatin material, oxidative stress or abnormal cell apoptosis . In the present study, genomic DNA alterations were estimated in epididymal sperm using RAPD profiles that reflect DNA effects in treated rats. Genetic changes in the rat genome included the obvious disappearance of the normal bands and appearance of new PCR products, indicated by the absence and presence of amplified RAPD fragments in DNA from lead exposed rats in comparison to those in the control DNA. The disappearance (deletion) or appearance (insertion) of an amplified RAPD fragment might be associated with DNA damage and mutations (for example, point mutations and large rearrangements) at the primer-template interaction sites, and/or unequal mitotic recombination or other effects (structural effects) which have facilitated primer hybridization . In fact, participation of lead compounds in direct damage to DNA is not yet reported. However, evidence indicates that lead ions can apparently take part in a Fenton reaction to generate damaging oxygen radicals and can cause DNA strand breaks . Also, some indirect mechanisms cause inhibition of DNA polymerase B, by lead induced ROS, possibly indicating the failure of DNA repair mechanisms . In a study performed by Ahmed et al. , it was reported that lead treatment increased the percentage of chromosomal abnormalities in rabbit bone marrow cells.
During spermatogenesis, apoptosis in testicular germ cells is recognized as an important physiological mechanism to limit the germ cell population to numbers that the Sertoli cells can support . In addition to its role in normal testicular physiology, apoptosis of germ cells has been recently reported as a mechanism responsible for the toxic damage to spermatogenesis . Apoptotic DNA fragmentation observed in our study by gel electrophoresis after LN treatment was also reported in another study . Such evidence may be due to a direct effect of Pb (II) on the DNA structure, oxidative mechanisms  or indirectly due to another mechanism involving the activation of caspases in the process of cell death . In addition, impaired spermatogenesis is clearly depicted in the testicular histology. Exposure of adult male rats to lead can seriously alter the testicular tissues which started the changes with vacuolar degeneration until necrosis and atrophy of seminiferous tubules; the changes were time dependent according to the experimental groups. In addition, epididymal change in our results showed that some of the epididymal tubules were free of sperm which is considered an important contributory factor in infertility caused by lead. These findings support the results from other reports that lead alters the testes and reproductive tract in an animal model treated with lead .
It can be seen from our results that MSCs can ameliorate alteration in genomic DNA and protect the testis tissues from apoptotic damage induced by lead. Also, MSCs relatively improve the histopathological changes induced in the testis of rats. This protection is multifactorial, including modulating the oxidative stress reaction, tissue damage and repair. In this respect, El-Attar et al.  have reported that pretreatment with MSCs attenuates lipopolysaccharide-induced acute lung injury in rats through inhibition of neutrophilic recruitment, inflammation, oxidative stress and apoptosis. One theory of tissue repair holds that organ injury is ‘sensed’ by stem cells that migrate to the site of damage and differentiate into organ-specific cells, promoting structural and functional repair [80, 81]. LN administration induces severe damage to testicular cells, resulting in DNA and protein damage and apoptosis. Because these dead cells are not able to divide, other cells must replace them to repair the tissue and maintain organ homeostasis . Cakici et al.  reported that MSCs were found both outside of the basal compartment and in the seminiferous tubules, supporting the idea that MSCs might have functioned in reestablishment of spermatogenesis in two ways: MSCs’ differentiation into sperm or maintenance of the spermatogonial stem cells. These results show that the MSCs could be both a rich and functional source for infertility treatment.
In conclusion, the fertile status of LN treated male rats was recovered by treatment with MSCs in this study. Animals treated with MSCs showed mild degeneration in a few seminiferous tubules after 30 days. There was no histopathological alteration as recorded post 60 days of MSCs treatment, supporting the idea that MSCs might have functioned in the reestablishment of spermatogenesis by differentiation into sperm. MSCs have tremendous potential for regenerative medicine; MSCs/BM are capable of differentiating into germ cells and Leydig cells in the testis. In the present study, MSCs modulated the decline of serum testosterone levels induced by LN and approached within control values, especially at 60 days. Because Leydig cells are responsible for testosterone production, stem cell transplantation may replace the need of life-long testosterone supplementation in male hypogonadism. In addition, MSCs modulated DNA apoptosis in sperm and testicular tissues. These results show that MSCs could be both a rich and functional source for the treatment of infertility.
bone morphogenetic protein-2
(Dulbecco’s) modified Eagle’s medium
mesenchymal stem cells
random amplified polymorphic DNA
reactive oxygen species
real time polymerase chain reaction
sodium dodecyl sulfate
- Taq DNA:
Thermus aquaticus deoxyribonucleic acid
transforming growth factor β3.
The authors are grateful to Dr. Adel M. Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, and Cairo University for his help in the examination of the histopathological slides and for his valuable comments. The authors also thank Dr. Laila A. Rashed, Professor of Medical Biochemistry Department, Faculty of Medicine, and Cairo University for critical feedback and for expert technical assistance, especially in the stem cells field.
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