**7. Studies on Nervous System**

Study of the regeneration of nervous system after irradiation was first started by transplantation of oligodendrocyte progenitor cells [58,59]. The results showed significant remyelation of radiation-induced demyelinated rat spinal cord.

Later, regenerative properties of transplantation of two types of neural stem cell were studied in a rat model of radiation myelopathy [60]. Twelve millimetre of rats' spinal cord was irradiated with 22 Gy gamma rays. This was ED<sup>100</sup> in six months in this model of radiation myelopathy. Neuroepithelial stem cells were obtained from the hipocampal proliferative analogue on embryonic day 14 from an H-2kb-tsA58 transgenic mouse. It was believed that both cell types were multipoint stem cells because they were 90% nestin positive in culture and they had been shown to differentiate into neurons, oligodendrocytes, and astrocytes. Stem cells were transplanted, intradurally, three months after spinal cord irradiation. While control animals developed front leg paralysis within 183 days after

irradiation, 30% of animals in stem cell transplanted group stayed paralysis free until day 200.

Wei et al. [61] using rat cervical spinal cord irradiation model irradiated 20 mm of cervical spinal cord of rats and injected one million UC-MSCs through the tail vein at 90 days after injection followed by three weekly injections. These authors demonstrated that multiple injections of stem cells significantly improved neuron survival and locomotor recovery at 180 days post irradiation.

In a rat model of cranial irradiation [62], human embryonic stem cells were transplanted to the hippocampus of athymic nude rats two days after 10 Gy cranial irradiation. This resulted in a significant cognitive improvement four months after irradiation compared to the controls that did not receive stem cell transplantation. The same authors observed similar results in the same model after transplantation of human neural stem cells [63]. These authors reported equivalent cognitive restoration with both types of stem cell transplantations [64]. Efficacy of stem cell therapy in amelioration of radiation-induced brain damage is reviewed by Chu et al. [65].

#### **8. Studies on the Gut**

Semont et al. [66] studied the regenerative effects of transplantation of human BM-MSCs in NOD/SCID mice. Transplantation was by infusion and the results were assessed by functional and histological assessment of the jejunum. The results demonstrated both structural and functional improvements by MSC transplantation.

The effect of autologous bone marrow derived stem cell treatment was studied in a pig model of irradiation proctitis, developed by 4MV photons [67]. It was demonstrated that repeated administration of mesenchymal stem cells resulted in reduction of collagen deposition and radiation-induced fibrosis. Reduction in expression of inflammatory cytokines both systemically and in rectal mucosa were also observed.

In a rat model of colorectal cancer, transplantation of allogenic MSCs significantly improved normal tissue damage induced by radiotherapy [68]. This study also demonstrated that MSC transplantation increased the tumour-free survival of the animals. The number of tumour free animals was higher than expected while the incidence and size of the tumours were reduced.

In our own laboratory (unpublished work), the effect of transplantation by ip injection of human ADSCs on gut was studied in rats. In this study, four cm of rats' distal colon were irradiated with 11 Gy 250 kV X-rays while the rest of the animal was shielded. Twenty four hours after irradiation, the animals were grouped into six groups and treated. Group 1: unirradiated controls received only one ml PBS injection, Group 2–6 received radiation followed by one ml saline injection (radiation only- Group 2), two million ADSCs suspended in one ml PBS (Group 3), two million ADSCs lysate in one ml saline (Group 4). One ml conditioned media collected from 2 million ADSC cultures (Group 5) injected ip and finally conditioned media administered three times 24 h, 72 h, and 120 hrs after irradiation (Group 6). The results were assessed by counting the number of crypts per circumference by light microscopy nine days after irradiation. As expected, radiation only reduced the number of crypts significantly compared with unirradiated control group. Injection of 2 million intact ADSCs, lysate, or a single dose of conditioned media increased the number of crypts almost equally. However, the best result was obtained by three consecutive injections of conditioned media. Comparable results obtained from injection of intact MSCs or the lysate of the equivalent number of cell indicates the possibility of a paracrine effect. This was also confirmed that the outcome of conditioned media injection that usually contains mi-RNA, a number of proteins, and biologically active lipids was more effective than the intact stem cells injections.

The possibility of the paracrine effect was indicated in a similar study [69] where the effectiveness of secretions of human UC-MSC to prevent radiation-induced intestinal injury was investigated in BALB/C mice after 10 Gy cobalt irradiation. In this study, UC-MSCs were expanded under hypoxic conditions. Multiple injections of the hypoxic conditioned media was delivered to the animals after irradiation for seven days. This treatment improved the structure of the intestine, decreased diarrhoea, and increased the survival rate.

Paracrine effect of stem cell transplantation was also shown in a study by Chen et al. [70] where conditioned media obtained from rat bone marrow MSCs were injected into rats just before irradiation. The results indicated that the conditioned media injection increased the expression of anti-inflammatory cytokines and reduced the expression of inflammatory cytokines.

In a recent study [71], total body irradiated mouse, at a dose of 7 Gy (60Co γ-rays), received intravenous injections of one million human placenta-derived stem cells for 10 days after irradiation and compared with another group of animals that received radiation only. Ten days after irradiation, radiation-induced small intestinal damage was compared with that of a control group. It was shown that stem cell transplantation significantly improved (*p* < 0.01) the outcome of radiation enteropathy or lethal radiation syndrome. It was also shown that stem cell transplantation exerted inhibitory actions on inflammatory cytokines and assisted the re-establishment of epithelial homeostasis.

In a rat model of colonic anastomosis performed by irradiation [72], it was shown that transplantation of rat ADSCs promoted anastomotic healing of the irradiated colon through enhanced vessel formation and reduced inflammation. In this study, the ADSC injections were delivered several times before and after the surgical procedure.

Sémont et al. [66,73] described the effects of MSCs as a consequence of their ability to improve the renewal capability of the small intestine epithelium. They also suggested that MSC treatment favours the re-establishment of cellular homeostasis by both increasing endogenous proliferation processes and inhibiting radiation-induce apoptosis of the small intestine epithelial cells.

MSC treatment decreased the interactions between mast cells and nerve fibers and reversed mechanical visceral hypersensitivity [74]. These authors suggest that the mechanism of effect is that the MSCs release cytokines and growth factors, such as interleukin (IL)-11, human hepatocyte growth factor, fibroblast growth factor-2, and insulin-like growth factors. Each of these factors have been described earlier as facilitating intestinal mucosa repair, either through enhancement of cell proliferation or inhibition of epithelial cell apoptosis [66,69,73,74].

#### **9. Studies on the Liver**

Prevention of radiation-induced liver damage was the subject of study well before the establishment of mesenchymal cells as stem cells. In an earlier work [75], lethally irradiated mice were treated with syngeneic fetal liver cells that resulted in longer survival.

Later, the effects of BM-MSC transplantation on irradiated liver was studied in NOD/SCID mice [76]. In this study, animals received 10.5 Gy of <sup>60</sup>Co gamma rays, followed by intravenous delivery of 5 million human BM-MSCs five hours after irradiation. This study demonstrated that MSC transplantation reduced radiation-induced apoptosis and significantly reduced the transaminase values (AST and ALT) compared with irradiated but not transplanted animals.

In a study of the effects of hepatic irradiation on transplanted BM-MSCs in cirrhotic rats and the underlying mechanism by which mesenchymal stem cells (MSCs) relieve liver fibrosis [77], the BM-MSCs from male rats were injected via portal vein into two groups of thioacetamide-induced cirrhotic rats. The right hemiliver of one cirrhotic rat group was irradiated (15 Gy) four days before transplantation. It was shown that the transplantation of MSCs alleviated liver fibrosis and reduced expression of transforming growth factor-β1, Smad2, and collagen type I. In addition, hepatic irradiation promoted homing and repopulation of MSCs and enhanced the effect of BM-MSCs in improving thioacetamide-induced liver fibrosis in rats. The authors concluded that BM-MSCs may function by inhibiting transforming growth factor-β-Smad signaling pathway in the liver.
