Human Adipose-Derived Endothelial Progenitor Cells Accelerate Epithelialization of Radiation Ulcers in Nude Mice.

BACKGROUND: Cotransplantation of adipose-derived stem cells (ASCs) and endothelial progenitor cells has shown superior angiogenic effects compared with ASCs alone in recent animal studies. However, endothelial progenitor cells could only be collected from blood vessels or bone marrow. Thus, the authors have established a method for purifying adipose-derived endothelial progenitor cells (AEPCs). The authors hypothesized that AEPCs would enhance the therapeutic effect of ASCs on radiation ulcers. METHODS: Seven-week-old male nude mice (BALB/cAJcl-nu/nu) were irradiated on the dorsal skin (total 40 Gy); 12 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 plus AEPCs 2 × 10 5 ( n = 4) or 5 × 10 5 ( n = 5)], or only vehicle ( n = 7). The nonirradiated 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 the ASC-treated group (14 ± 0 days versus 17 ± 2 days; P < 0.01). Engraftment of the injected cells could not be confirmed. Only the nonirradiated mice had significantly higher vascular density (0.988 ± 0.183 × 10 -5 /µm -2 versus 0.474 ± 0.092 × 10 -5 /µm 2 ; P = 0.02). CONCLUSION: 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 their combination with ASCs accelerated epithelialization of radiation ulcers in nude mice. The authors suggest that administration of humoral factors secreted from AEPCs (eg, treatment with culture-conditioned media) could be used for the same purpose.

Irradiation affects adipose-derived stem cells and wound healing depending on radiation dose and frequency.

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.

Purification and characterization of human adipose-resident microvascular endothelial progenitor cells.

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.

Selective Proliferation of Highly Functional Adipose-Derived Stem Cells in Microgravity Culture with Stirred Microspheres.

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.

Sodium Alginate as a Potential Therapeutic Filler: An In Vivo Study in Rats.

Filler injection demand is increasing worldwide, but no ideal filler with safety and longevity currently exists. Sodium alginate (SA) is the sodium salt of alginic acid, which is a polymeric polysaccharide obtained by linear polymerization of two types of uronic acid, d-mannuronic acid (M) and l-guluronic acid (G). This study aimed to evaluate the therapeutic value of SA. Nine SA types with different M/G ratios and viscosities were tested and compared with a commercially available sodium hyaluronate (SH) filler. Three injection modes (onto the periosteum, intradermally, or subcutaneously) were used in six rats for each substance, and the animals were sacrificed at 4 or 24 weeks. Changes in the diameter and volume were measured macroscopically and by computed tomography, and histopathological evaluations were performed. SA with a low M/G ratio generally maintained skin uplift. The bulge gradually decreased over time but slightly increased at 4 weeks in some samples. No capsule formation was observed around SA. However, granulomatous reactions, including macrophage recruitment, were observed 4 weeks after SA implantation, although fewer macrophages and granulomatous reactions were observed at 24 weeks. The long-term volumizing effects and degree of granulomatous reactions differed depending on the M/G ratio and viscosity. By contrast, SH showed capsule formation but with minimal granulomatous reactions. The beneficial and adverse effects of SA as a filler differed according to the viscosity or M/G ratio, suggesting a better long-term volumizing effect than SH with relatively low immunogenicity.

Hair Regeneration Potential of Human Dermal Sheath Cells Cultured Under Physiological Oxygen.

We investigated the effect of oxygen tension on the proliferation and hair-inductive capacity of human dermal papilla cells (DPCs) and dermal sheath cells (DSCs). DPCs and DSCs were separately obtained from human hair follicles and each cultured under atmospheric/hyperoxic (20% O2), physiological/normoxic (6% O2), or hypoxic (1% O2) conditions. Proliferation of DPCs and DSCs was highest under normoxia. Compared with hyperoxia, hypoxia inhibited proliferation of DPCs, but enhanced that of DSCs. In DPCs, hypoxia downregulated the expression of hair-inductive capacity-related genes, including BMP4, LEF1, SOX2, and VCAN. In DSCs, both normoxia and hypoxia upregulated SOX2 expression, whereas hypoxia downregulated BMP4 expression. Microarray analysis revealed that normoxia increased the expression of pluripotency-related genes, including SPRY, NR0B1, MSX2, IFITM1, and DAZL, compared with hyperoxia. In an in vivo hair follicle reconstitution assay, cultured DPCs and DSCs were transplanted with newborn mouse epidermal keratinocytes into nude mice using a chamber method. In this experiment, normoxia resulted in the most efficient induction of DPC hair follicles, whereas hypoxia caused the most efficient induction and maturation of DSC hair follicles. These results suggest that application of physiological/hypoxic oxygen tension to cultured human DSCs enhances proliferation and maintenance of hair inductivity for skin engineering and clinical applications. Impact statement Dermal sheath cells (DSCs) and dermal papilla cells (DPCs) are useful cell sources for cell-based regenerative therapy. This is the first report to describe that low-oxygen conditions are better for DSCs. Normoxic and hypoxic culture of DSCs is beneficial for expanding these hair follicular cells and advancing development of cell-based therapy for both wound healing and hair regeneration. The current study supports that optimized oxygen tension can be applied to use expanded human DPCs and DSCs for skin engineering and clinical applications.

Therapeutic Effects of Human Adipose-Derived Products on Impaired Wound Healing in Irradiated Tissue.

BACKGROUND: Clinical sequelae of irradiation result in tissue devitalization (e.g., ischemia, fibrosis, and atrophy) where wound healing capacity is impaired. Fat-derived products may work to treat such pathology. METHODS: Nonlethal irradiation at various doses (5, 10, and 15 Gy) and frequencies (one to three times on sequential days) was delivered to dorsal skin of nude mice, and subsequent gross and microscopic changes were evaluated for up to 4 weeks. Cutaneous punch wounds were then created to compare wound healing in irradiated and nonirradiated states. Wounds were also locally injected with vehicle, cultured adipose-derived stem cells, centrifuged fat tissue, or micronized cellular adipose matrix, and the therapeutic impact was monitored for up to 15 days. RESULTS: Nude mice given total doses greater than 15 Gy spontaneously developed skin ulcers, and radiation damage was dose-dependent; however, a fractionated irradiation protocol was able to reduce the damage. Histologic assessment revealed dose-dependent dermal fibrosis/thickening and subcutaneous atrophy. Dose-dependent (5 to 15 Gy) impairment of wound healing was also evident. At the highest dosage (15 Gy three times), open wounds persisted on day 15. However, wounds injected with cultured adipose-derived stem cells were nearly healed on day 12, and those treated with injection of centrifuged fat or micronized tissue healed faster than untreated controls (p < 0.05). There was no significant differences between treated groups. CONCLUSIONS: Tissue devitalization by irradiation was dose-dependent, although fractionated protocols helped to reduce it. Adipose-derived stem cells and other fat-derived products harboring adipose-derived stem cells successfully revitalized irradiated tissues and accelerated wound healing.

Biological Properties and Therapeutic Value of Cryopreserved Fat Tissue.

BACKGROUND: Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study was to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting. METHODS: Lipoaspirates were harvested from six women and preserved using two cryopreservation methods: (1) simple cooling to -80°C (cryo-1); or (2) programmed cooling to -196°C (cryo-2). Fresh fat, cryo-1 fat, and cryo-2 fat were analyzed both in vitro and in vivo. RESULTS: Immunohistochemistry of both types of cryopreserved adipose tissue revealed that most adipocytes were necrotic. The cell number and viability of stromal vascular fraction cells were significantly decreased in cryo-1 fat (1.7 × 10 cells, 42.6 percent viable) and cryo-2 fat (2.0 × 10 cells, 55.4 percent viable), compared with fresh fat (3.9 × 10 cells, 90.6 percent viable). Although adipose-derived stem cells were cultured successfully from all fats, functional adipose-derived stem cells from cryopreserved fats were much fewer, with comparable multilineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, 3 months after transplantation, all of the cryopreserved fats maintained their volume to some extent; however, the cryopreserved fats were mostly filled with dead tissue and produced significantly lower engraftment scores than fresh fat. CONCLUSIONS: Most adipocytes were killed in the process of cryopreservation and thawing. Adipose-derived stem cells were isolated from cryopreserved fat, but the number of functional adipose-derived stem cells was very limited in both cryopreservation methods. After grafting, cryopreserved fat was retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.