Seung Pyo Gong

Embryonic stem cell-like cells established by culture of adult ovarian cells in mice

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 X DBA2) and outbred (ICR) mice. INTERVENTION(S) Ovarian stroma cells of less than 40 mum 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.

Long-term maintenance of mouse embryonic stem cell pluripotency by manipulating integrin signaling within 3D scaffolds without active Stat3

We engineered an acellular biomimetic microenvironment to regulate stem cell fate and applied it to maintain mouse embryonic stem (ES) cell self-renewal. In the 3D environment formed using hydrogel scaffolds in which specific integrin ligation was provided, Stat3 activation by exogenous leukemia inhibitory factor (LIF) no longer acted as a limiting factor for stem cell self-renewal. Instead, simultaneous stimulation of integrins α(5)β(1), α(v)β(5), α(6)β(1) and α(9)β(1) within the 3D scaffold greatly increased Akt1 and Smad 1/5/8 activation, which resulted in prolonged self-renewal of the ES cells. The ES cells exposed to the combined stimulation of the integrins for 4 wk in LIF-free 3D scaffolds maintained the spherical morphology of cell colonies without losing any activity of pluripotency. In conclusion, cell niche-specific integrin signaling within the 3D environment supported mouse ES cell self-renewal, and the resulting integrin signaling replaced Stat3 with Akt1 and Smad 1/5/8 as critical signals for mouse ES cell self-renewal.

Derivation of developmentally competent oocytes by the culture of preantral follicles retrieved from adult ovaries: maturation, blastocyst formation, and embryonic stem cell transformation

OBJECTIVE To determine whether the preantral follicles in adult ovaries can generate developmentally competent oocytes after in vitro culture. DESIGN Prospective, animal-model study. SETTING Gamete and Stem Cell Biotechnology Laboratory, Seoul National University, Seoul, Korea. ANIMAL(S) B6CBAF1 mice. INTERVENTION(S) Preantral follicles collected from 8-week-old mice were cultured in vitro. MAIN OUTCOME MEASURE(S) Follicle development, embryogenesis, and embryonic stem cell characterization. RESULT(S) A mean of 50.3 preantral follicles were retrieved from one adult animal, which is significantly less than the number (88.7 follicles) retrieved from a prepubertal female. Extension of the culture period greatly improved oocyte maturation; increased follicular growth to the pseudo-antral (89%-91% vs. 32%) or mature oocyte stage (65%-77% vs. 13%) was observed after 12 or 13 days of culture compared with 9 days of culture. Blastocyst formation after parthenogenesis was detected in only one case; in comparison, the use of IVF yielded a large number of embryos that developed into blastocysts. A mean of 14.7 intrafollicular oocytes per animal were produced after 13 days of culture, and 41% of those developed into blastocysts after IVF. Embryonic stem cell-like colonies were established by subculturing the inner cell mass cells from the blastocysts. CONCLUSION(S) Developmentally competent oocytes can be generated by culturing adult preantral follicles. These results may help increase the feasibility of follicle culture systems.

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

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.

Change in gene expression of mouse embryonic stem cells derived from parthenogenetic activation.

BACKGROUND We previously established parthenogenetic mouse embryonic stem cells (ESCs) and this study was subsequently conducted for elucidating the influence of oocyte parthenogenesis on gene expression profile of ESCs. METHODS Gene expression of parthenogenetic ESC (pESC)-1 or pESC-2 was separately compared with that of two normally fertilized ESC (nfESC) lines (B6D2F1 and R1 strains), and quantification of mRNA expression was conducted for validating microarray data. RESULTS In two sets of comparison, reaction of 11 347 and 15 454 gene probes were altered by parthenogenesis, while strain difference changed the expression of 15 750 and 14 944 probes. Level of correlation coefficient was higher in the comparisons between normal fertilization and parthenogenesis (0.974-0.985) than in the comparisons between strains of nfESCs (0.97-0.971). Overall, the expression of 3276-3329 genes was changed after parthenogenesis, and 88% (96/109) of major functional genes differentially (P < 0.01) expressed in one comparison set showed the same change in the other. When we monitored imprinted genes, expression of nine paternal and eight maternal genes were altered after parthenogenesis and 88% (14/16) of these was confirmed by mRNA quantification. CONCLUSIONS The change in gene expression after parthenogenesis was similar to, or less than, the change induced by a strain difference under a certain genetic background. These results may suggest the clinical feasibility of parthenogenesis-derived, pluripotent cells.

Effects of combined antioxidant supplementation on human sperm motility and morphology during sperm manipulation in vitro

OBJECTIVE To evaluate the effect of antioxidant combinations on human sperm motility and morphologic normality during preparation and in vitro incubation to improve sperm quality for assisted reproductive technology (ART). DESIGN Prospective study. SETTING Laboratory. PATIENT(S) Six fertile males, 21 to 25 years of age, after 3 days of sexual abstinence. INTERVENTION(S) Sperm retrieved from patients with normal fertility prepared and incubated in vitro in medium supplemented with taurine, cysteine, and/or glutathione. MAIN OUTCOME MEASURE(S) Motility indices, including motility (MOT), average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and morphologic normality. RESULT(S) Taurine supplementation during sperm preparation had no significant effects on motility or morphologic normality. However, when sperm was incubated for 24 hours under taurine-supplemented conditions, the rates of reduction in MOT, VAP, VCL, and VSL were statistically significantly decreased compared with taurine-free conditions. Morphologic normality was maintained, regardless of taurine treatment. The optimal taurine concentration for improving sperm motility and morphologic normality during preparation and in vitro incubation was 1 mM. Subsequently, combined treatment with 1 mM taurine, 1 mM cysteine, and 1 mM glutathione induced statistically significant synergistic effects on MOT, VAP, VCL, and VSL without any alteration of morphologic normality. CONCLUSION(S) Combined treatment with taurine, cysteine, and glutathione statistically significantly ameliorated the reduction in sperm motility during in vitro manipulation of human sperm.

Transformation of somatic cells into stem cell-like cells under a stromal niche

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast‐derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony‐forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid‐like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle‐related proteins, as well as both homologous and heterologous recombination of genomic single‐nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony‐forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.—Lee, S. T., Gong, S. P., Yum, K. E., Lee, E. J., Lee, C. H., Choi, J. H., Kim, D. Y., Han, H., Kim, K.‐S., Hysolli, E., Ahn, J. Y., Park, I.‐H., Han, J. Y., Jeong, J.‐W., Lim, J. M. Transformation of somatic cells into stem cell‐like cells under a stromal niche. FASEB J. 27, 2644‐2656 (2013). www.fasebj.org

Stem cell engineering: limitation, alternatives, and insight

The 21st century will see improvements in the quality of human life. The development of new therapeutic technologies will prevent prevalent diseases and enable recovery from currently incurable diseases. The development of cell and tissue replacement therapies using stem cells and their progenitors will accelerate the development of causative treatments. The effort expended thus far in developing cell therapies has revealed many technical limitations. Thus, we must explore conceptual changes in the feasibility of stem cell therapy. This paper introduces the current limitations to stem cell engineering and ways to overcome these limitations, which will provide new insight into their clinical application.

Establishment and Characterization of Permanent Cell Lines from Oryzias dancena Embryos

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

Gene expression profiles of human subcutaneous and visceral adipose-derived stem cells.

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.