Objective To suggest an alternative strategy for deriving histocompatible stems cells without undertaking genetic manipulation. Design Prospective approach using an animal model. Setting Stem cell and bioevaluation laboratory, Seoul National University. Animal(s) F1 (C57BL6 × DBA2) and outbred (ICR) mice. Intervention(s) Ovarian stroma cells of less than 40 μm in diameter were subcultured with fibroblast monolayer, and colony-forming cells were characterized. Main Outcome Measure(s) Stemness, genotype, and imprinted gene methylation. Result(s) Two-lines of colony-forming cells were established, which expressed markers specific for embryonic stem cells (ESC) and formed embryoid bodies and teratomas. Complete matching of microsatellite markers with the cell donor strain confirmed their establishment from ovarian tissue, and identification of both homozygotic and heterozygotic chromosomes raised the possibility of their derivation from parthenogenetic oocytes. However, the use of cells smaller than mature oocytes for primary culture, the difference in imprinted gene methylation compared with parthenogenetic ESCs, and failure to establish the ESC-like cells by primary follicle culture collectively suggested the irrelevancy to gametes. Conclusion(s) Coculture of adult ovarian cells with somatic fibroblasts can yield colony-forming cells having ESC-like activity, which may provide an alternative for establishing autologous stem cells from adults that can be obtained without genetic manipulation.

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Subcutaneous and visceral adipose tissues show a different risk effect on metabolic disorders because they have distinct cellular properties. We isolated stem cells from the separate human adipose tissues to investigate that subcutaneous and visceral fat depots have metabolic differences. Adipose-derived stem cells (ASCs) were characterized by immunophenotype and differentiation potentials into adipogenic, osteogenic, and chondrogenic lineages. Although subcutaneous and visceral ASCs (S-ASC and V-ASC) express same surface markers (CD31 , CD34 , CD45 , CD73 , CD90 , and CD105 ) and have differentiation potentials, S-ASCs had higher capacity to proliferate and to differentiate into adipogenic lineage than V-ASCs. Next, we identified that S-ASC and V-ASC were genetically distinct based on microarray analysis. Among a total of 810 genes detected in ASCs of both depots, the differentially expressed genes were involved in energy and lipid metabolism. These data show the existence of the intrinsic difference between S-ASC and V-ASC and suggest the differences of anatomically separated adipose tissue. On the basis of the differentially expressed gene profiles between S-ASC and V-ASC, we suggested significant evidence that adipose tissues originating from different anatomic regions are distinguished at the level of the undifferentiated stem cells such as mature adipocytes. V-ASCs had the upregulated clusters of genes related to lipid biosynthesis and metabolism. By contrast, S-ASCs highly expressed genes involved in DNA-dependent transcription, contributing to proliferation. We provide further insights for ASCs with the different origins to understand fat accumulation and distribution and a possibility of ASCs as a therapeutic target against metabolic disorders or cancer.

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This study was conducted to improve establishing autologous embryonic stem cells (ESCs) by culture of preantral follicles and parthenogenetic activation of oocytes. First, paternal inheritance of the follicle donor was changed without altering maternal heredity by employing B6CBAF1 instead of B6D2F1 mice. A significant increase in the establishment of parthenogenetic ESCs was detected after the change, and a different gene expression pro-file was detected in the ESCs established. Among 62 stemness-related genes showing different expression level between two strains, 35 (56.5%) were lower in the rarely established ESCs (B6D2F1) than in the easily established ESCs (B6CBAF1). Several paternally expressed genes were aberrantly expressed in the rarely established ESCs. Second, the establishment of parthenogenetic ESCs in B6D2F1 was significantly improved when preantral follicles were cultured in glutathione (GSH)-containing medium. In the ESCs derived from GSH-treated follicles, 77% of the 62 genes showing the difference increased their expression. Translation of several proteins related to stemness (Wnt-1, beta-catenin, p-p44/42, and smad) was similar between the parthenogenetic ESCs established after GSH treatment and the control E14 ESCs. We concluded that change in genetic inheritance and exposure of in vitro-growing ovarian follicles to GSH contributes to improving establishment of parthenogenetic ESCs, which may help increase the feasibility of the established lines for patient-specific, stem cell therapy.

The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.