Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors

Coculture with adipose-derived stem cells (ADSCs) can stimulate proliferation and migration of melanocytes. To enhance outcomes of skin disorders caused by melanocyte loss or death, mixed transplantation with ADSCs has been suggested. However, role of cocultured ADSCs in proliferation and migration of melanocytes remains unclear. This study determined the effect of ADSCs on production of growth factors and expression levels of intergrins in primary culture of adult human melanocytes with or without ADSCs and in nude mice grafted with such melanocytes. Higher amounts of growth factors for melanocytes, such as bFGF and SCF were produced and released from ADSCs by coculturing with melanocytes. Relative levels of integrins β1, α5, and α6 as well as adhesion to fibronectin and laminin were increased in melanocytes cocultured with ADSCs. Such increases were inhibited by neutralization of bFGF or SCF. Relative levels of bFGF, SCF and integrins were increased in nude mice skin after grafting with melanocyte+ADSC cocultures. Collectively, these results indicate that ADSCs can stimulate proliferation and migration of melanocytes by increasing expression of integrins in melanocytes through upregulation of production/release of melanocyte growth factors such as bFGF and SCF.

Adipose-Derived Stem Cells Improve Efficacy of Melanocyte Transplantation in Animal Skin

Vitiligo is a pigmentary disorder induced by a loss of melanocytes. In addition to replacement of pure melanocytes, cocultures of melanocytes with keratinocytes have been used to improve the repigmentation outcome in vitiligo treatment. We previously identified by in vitro studies, that adipose-derived stem cells (ADSCs) could be a potential substitute for keratinocytes in cocultures with melanocytes. In this study, the efficacy of pigmentation including durability of grafted melanocytes and short-term safety was examined in the nude mouse and Sprague-Dawley rat after grafting of primary cultured human melanocytes, with or without different ratios of primary cultured human ADSCs. Simultaneous grafting of melanocytes and ADSCs, which were separately cultured and mixed on grafting at the ratios of 1:1, 1:2, or 1:3, showed better efficacy than that of pure melanocytes. Grafting of melanocytes cocultured with ADSCs resulted in a similar outcome as the grafting of cell mixtures. Skin pigmentation by melanocytes : ADSCs at the ratios of 1:1 and 1:2 was better than at 1:3. No significant difference was observed between the 1-week and 2-week durations in coculturing. Time-course microscopic examination showed that the grafted melanocytes remained a little longer than 6-week post-grafting. No inflammatory cell infiltration was observed in the grafted skin and no melanocytes were detectable in other organs. Collectively, grafting of melanocytes and ADSCs was equally safe and more effective than grafting of melanocytes alone. Despite the absence of significant differences in efficacy between the group of 1:1 and that of 1:2 ratio, 1:2 ratio for 1-week coculturing may be better for clinical use from the cost-benefit viewpoint.

Myogenic Differentiation of Human Adipose-Derived Stem Cells

OBJECTIVE: Cell therapy has been extensively studied as a gene complementation approach in muscular dystrophy including Duchenne muscular dystrophy (DMD), and adipose tissue has recently been identified as a uniquely abundant and adequately accessible source of pluripotent cells. In the present work, we investigated myogenic potentials of adipose-derived stem cells (ADSCs) depending on culture media and isolation with using surface markers. METHOD: Human ADSCs were obtained by liposuction and cultured in two different media; control and myogenic media. In addition we attempted to isolate ADSCs by utilizing surface markers: CD45 and CD133. The following observations were made to evaluate myogenic differentiation as the expression of myogenic regulatory factors (MyoD, Myf-5 and Myf-6) and desmin by RT-PCR and immunoflurescence study. RESULTS: Conversion of ADSCs to myogenic phenotype was observed by indirect immunoflurescence study of MyoD and Myf-5 in regardless of media type and isolation method. In addition mRNA of MyoD and Myf-5 were positive in both culture media, and there were no differences of MyoD and Myf-5 responses between CD45- and CD45-CD133-ADSCs. However, secondary myogenic regulatory factor (Myf-6) was not expressed constantly, and desmin were negative in all cultural condition. CONCLUSION: Our findings suggest that human ADSCs might have myogenic potentials. However, further studies are needed to express the secondary myogenic regulatory factors and proteins in myoblasts.

The effect of LH Receptor in the Pregnancy of Poor Responders / 대한불임학회지

OBJECTIVES: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. MATERIALS AND METHODS: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by beta-actin. Statistical analysis was performed by using Chi(2) test, Student's t-test and Pearson correlation. RESULTS: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). CONCLUSION: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

The effect of renal calcium excretion on bone mineral metabolism of postmenopausal women in Korea / 대한내과학회지

BACKGROUND: Although all postmenopausal women are estrogen deficient, women who have postmenopausal osteoporosis may have some defects, in addition to estrogen deficiency, that explain for their higher rates of bone resorption and greater bone loss, relative to those who do not. To test the hypothesis that one of the defects is an impairment in renal calcium conservation, we have investigated relationship between urinary calcium excretion and bone mineral metabolism of postmenopausal women in Korea. METHODS: We have measured 24-hour urinary calcium level; serum osteocalcin level, serum alkaline phosphatase level; urine deoxypyridinoline level; and bone mineral density in 224 early postmenopausal, 158 late postmenopausal and 145 premenopausal women. RESULTS: 33.0 percent (74/224) of early postmenopausal women in Korea had urinary calcium excretion exceeding 4 mg/kg per day. The early postmenopausal women had higher (p < 0.05) values for mean urinary calcium to creatinine ratio of 0.241+/-0.008 mg/mg of creatinine vs. 0.209+/-0.010 mg/mg of creatinine and higher (p < 0.001) mean serum calcium level of 9.15+/-0.02 mg/dL vs. 8.92+/-0.03 mg/dL than the premenopausal women. Hypercalciuric group of early postmenopausal women had higher (p < 0.05) values for mean urine deoxypyridinoline level (8.6+/-0.4 nMol/mMol Vs. 7.7+/-0.2 nMol/mMol), higher (p < 0.05) mean serum alkaline phosphatase level (73.4+/-2.3 U/L Vs. 67.7+/-1.4 U/L) and lower (p < 0.05) mean bone mineral density of femur neck (0.785+/-0.012 g/cm2 Vs. 0.815+/-0.008 g/cm2) than the normocalciuric group. CONCLUSION: The early postmenopausal women had larger numbers of hypercalciuric women and higher values for urinary calcium excretion than the premenopausal women. Hypercalciuric group of early postmenopausal women had higher values for biochemical markers of bone turnover and lower bone mineral density of femur neck than the normocalciuric group. We suggest that hypercalciuria could be accounted for the partial cause of postmenopausal osteoporosis, but further studies are needed to elucidate the direct effect about that.