齋藤 夏美

Background: Radiotherapy is a well-established cancer treatment, but its detrimental effects on normal tissue have been significantly underestimated. Late radiation-induced tissue damage leads to dysfunctions like loss of elasticity and delayed wound healing, which negatively affect the prognosis and quality of life of cancer survivors. Our previous research demonstrated that local prophylactic administration of adipose-derived stem cells (ASCs) immediately after irradiation could prevent late radiation-related disorders. This study aims to explore the effects of systemic administration of ASCs for future clinical applications. Methods: Nude mice were irradiated on their dorsal skin (40 Gy total: 2 Gy × 20, simulating clinical radiotherapy). Human ASCs were administered systemically (via intravenous or intraperitoneal injection, once or twice). Control groups included a non-irradiated group and a vehicle-treated radiotherapy group. After 6 months, full-thickness skin wounds were created on the mice’s dorsal skin and observed for 28 days. The wound healing process was assessed, and tissue samples were analyzed using immunohistochemistry. Results: Radiotherapy caused subcutaneous fat atrophy and fibrosis at 6 months, which was effectively prevented in the ASC-treated groups. Wound healing was faster in the ASC-treated groups than in the vehicle-treated groups, though not as fast as in the No-radiotherapy group. ASC-treated groups exhibited less fat layer atrophy and fibrosis than vehicle-treated groups. Engraftment of ASCs was confirmed in the fatty layer of the irradiated skin. Conclusions: Prophylactic systemic administration of ASCs immediately after radiotherapy can prevent tissue damage and dysfunction associated with radiation therapy.

Background: The pathogenesis of deterministic radiation damage is not clearly understood, but it has been reported that fibroinflammatory pathways are up-regulated. We hypothesized that the number of adipose-derived stem/stromal cells (ASCs) decline after radiotherapies, preventing normalization of fibrosis and angiogenesis, resulting in chronic radiation damages that progress over time. Methods: Dorsal skin of 8-week-old male BALB/cfC3H mice was irradiated with 10 Gy weekly for 4 weeks. At 1, 3, 6, 9, and 12 months after radiotherapy (n = 5, 5, 5, 5, and 4), tissue hemoglobin oxygen saturation, and time until epithelialization were evaluated. Skin biopsies were measured for thickness and CD34⁺/isolectin⁻ stem/stromal cell count. Nonirradiated (NRT) controls were evaluated at each time point as well (n = 5 each). Results: Compared with NRT controls, time until epithelialization was significantly longer at 1 month (28 ± 3, P < 0.01); not statistically different at 3 months (16 ± 2, P = 0.32); and lengthened over time at 6 months (20 ± 2, P = 0.21), 9 months (28 ± 2, P < 0.01), and 12 months (26 ± 3, P < 0.01), as did tissue oxygen saturation. The number of CD34⁺/isolectin^– ASCs decreased over time, at 1 month (5.3 ± 1.3, P = 0.01), 3 months (6.0 ± 1.4, P = 0.03), 6 months (4.0 ± 0.8, P < 0.01), 9 months (1.7 ± 0.5, P < 0.01), and 12 months (0.3 ± 0.5, P < 0.01). The subcutaneous fatty layer was significantly thinner at 3 months (116 ± 33, P < 0.01), 6 months (147 ± 22, P = 0.02), 9 months (52 ± 12, P = 0.04), and 12 months (89 ± 19, P = 0.04), but not at 1 month (141 ± 18, P = 0.43). Conclusions: After 6 months postirradiation, the number of ASCs continued to decline over time, accompanied by irreversible progression of fibrosis, atrophy, and ischemia, which resulted in impaired wound healing.

INTRODUCTION: A specialized device equipped with a sharp blade filter has been developed to enable more efficient purification of a micronized cellular adipose matrix (MCAM) containing stem cells. The aim of this study is to compare the characteristics and functions of the population of stromal cells (mSVF) and cultured cells (mASCs) purified using this device with those of cSVF and cASCs obtained through conventional enzymatic purification. METHODS: Cell viability, proliferation capacity and yield were assessed. Characterization of stem cell potency was performed by analyzing cell surface markers including CD34, a marker of activated adipose-derived stem cells. The trilineage differentiation potential was evaluated using RT-PCR and histology. RESULTS: The yield rate of mSVF obtained from MCAM was significantly higher than that with the conventional method, although use of the device resulted in a slight decrease in cell viability. After culture, mASCs exhibited a remarkable clonogenic potential and significantly higher cell proliferation potential than cASCs. The mASCs also displayed a distinct pattern of ASC cell surface markers, increased expression of genes related to CD34, high pluripotency, and a high trilineage differentiation ability. CONCLUSION: The specialized device enhanced the yield of SVF and produced cells with high proliferation rates and characteristics that include expression of stem cell markers.

BACKGROUND: Co-transplantation of adipose-derived stem cells (ASCs) and endothelial progenitor cells (EPCs) has shown superior angiogenic effects than ASCs alone in recent animal studies. However, EPCs could only be collected from blood vessels or bone marrow. Thus, we have established a method for purifying adipose-derived endothelial progenitor cells (AEPCs). We hypothesized that AEPCs would enhance the therapeutic effect of ASCs on radiation ulcer. METHODS: Seven-week-old male nude mice (BALB/cAJcl-nu/nu) were irradiated on the dorsal skin (total 40 Gy) and twelve weeks later 6 mm diameter wounds were created. The mice were then treated with subcutaneous injection of human ASCs (1×10 5, n = 4), human AEPCs (2×10 5 or 5×10 5, n = 5), combinations of those (ASCs 1×10 5 + AEPCs 2×10 5 (n = 4) or 5×10 5 (n = 5)), or only vehicle (n = 7). Non-irradiated group was also prepared as a control (n = 6). The days required for macroscopic epithelialization was compared and immunostaining for human-derived cells and vascular endothelial cells was performed at Day 28. RESULTS: AEPC-ASC combination-treated groups healed faster than ASC-treated group (14 ± 0 vs 17 ± 2 days, p < 0.01). Engraftment of the injected cells could not be confirmed. Only the non-irradiated mice had significantly higher vascular density (0.988 ± 0.183 vs 0.474 ± 0.092 ×10 -5µm -2, p = 0.02). CONCLUSIONS: The results suggested therapeutic potentials of AEPCs and an enhanced effect of combination with ASCs. This study is a xenogenic transplantation model and further validation in an autologous transplantation model is needed. CLINICAL RELEVANCE STATEMENT: Human AEPCs and its combination with ASCs accelerated epithelialization of radiation ulcer in nude mice. It was also suggested that administration of humoral factors secreted from AEPCs, e.g. treatment with culture conditioned media, could be used for the same purpose.

BACKGROUND: Radiation therapies are often associated with permanent devitalization in the surrounding tissue. We hypothesized that stem cells are damaged depending on each irradiation dose and frequency of fractionated radiotherapies, which results in impaired tissue function including wound healing capacity. METHODS: To test the hypothesis, susceptibility of human adipose-derived stem cells (ASCs) to a single irradiation (0-10 Gy) was assessed in vitro. In vivo chronic radiation effects were also assessed on the mouse dorsal skin (N=4-5) for 6 months after a total of 40 Gy irradiation (0 Gy as control) using one of three fractionated protocols (2 Gy daily for 20 days, 10 Gy weekly for 4 weeks, or 10 Gy monthly for 4 months). Oxygen partial pressure, oxygen saturation of hemoglobin, and dorsal skin viscoelasticity were periodically measured, and wound healing and tissue immunohistology were compared at 6 months. RESULTS: A single irradiation of cultured human ASCs resulted in a dose-dependent increase in cell death up to 2 Gy but with no further increases between 2 and 10 Gy. Most of the apoptotic ASCs were in the proliferation phase. Among the three in vivo irradiation protocols, the 2 Gy×20 group had the most severe chronic tissue damage (i.e., skin dysfunction, subcutaneous atrophy, and depletion of CD34+ stem cells) 6 months after the irradiation. Wound healing was also impaired most significantly in the 2 Gy×20 group. CONCLUSIONS: These results have important clinical implications for surgeons and radiotherapists such as the timing of surgical interventions and the optimization of fractionation protocols.Clinical Relevance Statement: Irradiation damages stem cells depending on the radiation dose and frequency. Using the ultimately optimized protocol, we can minimize the long-term functional deficits of radiated tissue without losing anti-cancer efficacy of radiation therapy.