齋藤 夏美

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.

Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs). However, no standardized method has been established for the isolation and purification of adipose-derived EPCs (AEPCs). The aim of this study was to establish a method for the isolation and purification of AEPCs. The stromal vascular fraction (SVF) was extracted from human lipoaspirates, and the CD45-CD31+ fraction of the SVF was collected by magnetic-activated cell sorting (MACS). The CD45-CD31+ fraction was cultured for 4.5 days, followed by a second MACS separation to collect the CD31+ fraction. Purified AEPCs were expanded without being overwhelmed by proliferating ASCs, indicating that a high level (> 95%) of AEPC purification is a key factor for their successful isolation and expansion. AEPCs exhibited typical endothelial markers, including CD31, von Willebrand factor, and the isolectin-B4 binding capacity. AEPCs formed colonies, comparable to cultured human umbilical vein endothelial cells (HUVECs). Both AEPCs and HUVECs formed capillary-like networks in the tube formation assay, with no significant difference in network lengths. We are the first to establish a purification and expansion method to isolate these cells. Because adipose tissue is a clinically accessible and abundant tissue, AEPCs may have potential advantages as a therapeutic tool for regenerative medicine.

Therapeutic effects of adult stem-cell transplantations are limited by poor cell-retention in target organs, and a reduced potential for optimal cell differentiation compared to embryonic stem cells. However, contemporary studies have indicated heterogeneity within adult stem-cell pools, and a novel culturing technique may address these limitations by selecting those for cell proliferation which are highly functional. Here, we report the preservation of stemness in human adipose-derived stem cells (hASCs) by using microgravity conditions combined with microspheres in a stirred suspension. The cells were bound to microspheres (100-300 μm) and cultured using a wave-stirring shaker. One-week cultures using polystyrene and collagen microspheres increased the proportions of SSEA-3(+) hASCs 4.4- and 4.3-fold (2.7- and 2.9-fold increases in their numbers), respectively, compared to normal culture conditions. These cultured hASCs expressed higher levels of pluripotent markers (OCT4, SOX2, NANOG, MYC, and KLF), and had improved abilities for proliferation, colony formation, network formation, and multiple-mesenchymal differentiation. We believe that this novel culturing method may further enhance regenerative therapies using hASCs.