Recent research on stem cell conditioned medium (CM) has been revealed that CM could influence on the embryo development when supplemented to in vitro culture medium. However, the optimal basal medium for CM production has not determined although it is the fundamental factor of CM. The purpose of this study is to examine the effect of human derived adipose stem cell CM (hASC-CM) with different basal medium on mice embryo development after parthenogenetic activation (PA). hASC-CM was collected from 2 kinds of serum free basal medium, DMEM and KSFM, respectively on day 5 from the culture of hASC isolated from human fat tissue. Intra-peritoneal injection of PMSG and hCG was conducted into 7-week-old ICR mice for superovulation. The oocytes were recovered from the oviductal ampulla, 18 h after hCG injection, and denuded using 0.1% hyaluronidase. PA of oocytes was conducted with KSOM media including strontium chloride. The parthenotes were in vitro cultured in 3 groups: 100% KSOM (Control), 75% KSOM + 25% DMEM or KSFM without FBS (DMEM or KSFM group) and 75% KSOM + 25% hASC-CM from DMEM or KSFM (DMEM-CM or KSFM-CM group). Cleavage rate was assessed after 2 days post IVC and blastocyst formation rate was evaluated after 6 days post IVC both using stereomicroscope. Total cell number of blastocysts was counted by Hoechst staining. 1way ANOVA from Graphpad prism 5 was used for statistical analysis and the values are presented as means ± standard error of mean. As a result, blastocyst formation rate of DMEM-CM group (16.09±3.32%, P<0.05) was significantly lower than control and DMEM group (34.43±2.89% and 34.49±5.34%, P<0.05) but cleavage rate and total cell number of blastocysts showed no significant difference among groups. In case of KSFM, there was no significant difference in cleavage rate, blastocyst formation rate and total cell number of blastocysts among the control, KSFM group and KSFM-CM group. The sort of basal medium used for the CM collection affected the development of parthenotes during in vitro culture differently. Therefore, further research should be conducted to find out the alternative basal medium of CM able to improve the embryo development. This research was supported by Nature Cell (#550-20170028), Cooperative Research Program of RDA (CCAR, #PJ013954022018), Research Institute for Veterinary Science and the BK21 plus program.

체외 배양액에 성장호르몬 및 사이토카인의 첨가는 초기배 발육 및 생산된 배반포의 질에 영향을 미칠 수 있다. 본 연구는 돼지 유도만능줄기세포(porcine induced pluripotent stem cell, piPSC)의 조정배지(conditioned medium, CM)가 돼지 난자의 체외성숙 및 단위발생 후 초기배 발육에 미치는 영향을 검토하기 위하여 수행하였다. 난자-난구세포 복합체(cumulus-oocyte complex, COC)는 0(control), 25, or 50%의 줄기세포 배양액(stem cell medium, SM) 또는 CM이 첨가된 체외성숙 배양액으로 배양하였으며, 성숙된 난자는 활성화 유도 후 같은 농도의 SM 또는 CM을 첨가한 체외배양액에서 배양하였다. 체외 성숙율은 CM-25% 그룹에서 대조구보다 유의적으로 높았으나(p<0.05), 다른 SM 또는 CM 처리구와는 차이가 없었다. 배반포 형성율은 CM-25% 그룹(29.2%)에서 대조구(20.7%), SM-50%(19.6%) 및 CM-50%(23.66%) 처리구보다 유의적으로 높았다(p<0.05). 배반포에서의 세포수 및 세포사 비율은 SM-25% 그룹이 대조구에 비하여 유의적인 차이가 나타났다(p<0.05). 난자의 질과 연관되어 있는 유전자들(Oct4, Klf4, Tert 및 Zfp42)의 발현은 CM-25% 그룹에서 대조구보다 유의적으로 증가되었다(p<0.05). 따라서 본 실험의 결과 체외성숙(IVM) 및 체외발달(IVC) 배양액에 25% 수준의 CM의 첨가는 돼지 단위발생 난자의 배발달과 난자의 질적 향상에 기여하는 것으로 사료된다.

The embryogenesis stimulating activity(ESA) had been shown in co-culture of embryos with bovine oviduct epithelial cell(BOEC) and culture in BOEG-conditioned medium. The present study was undertaken to purify and quantify the embryotropic proteins and to determine the optimum concentration of the embryotropic protein for the proper development of embryos. In BOEC-conditioned medium, five major bands of proteins were detected(66, 53, 40, 32 and 24 kDa) by SDS-PAGE. From these proteins, 288pg of protein that had a 32kDa molecular weight was purified by gel filtration column and perfusion chromatography ion-exchange column. When purified protein was supplemented to the in vitro culture media at various concentrations in protein-free media, 2.5g /ml supplement group showed significantly higher rates of embryo development into morula /blastocyst stages than other groups(p<0.05). In conclusion, we purified 32kDa protein from BOEC-conditioned medium and this protein showed optimum embryogenesis stimulating effect at 2.5g /ml.

While adipose-derived stem cell-conditioned medium (ADSC-CM) has been demonstrated to promote skin wound healing, the mechanism regulating this effect remains unelucidated. In this study, we aimed to investigate the role of Ell3 in the wound healing activity of ADSC-CM. In vitro analysis revealed that Ell3 suppression in ADSCs impairs the promotive activity of ADSC-CM on the proliferation and migration of mouse embryonic fibroblasts (MEF) and normal human dermal fibroblasts (NHDF). Consistently, the expression of MMP family genes, which regulate cell proliferation and migration, was significantly suppressed in MEF and NHDF treated with siEll3-transfected ADSC-CM. Proinflammatory cytokines, such as interleukin-1 and interleukin-6, were highly expressed in MEF treated with siEll3-transfected ADSC-CM. The wound healing activity of siEll3-transfected ADSC-CM was significantly lower than that of the control in vivo. Our results suggest that Ell3 may contribute to the inhibition of inflammatory response during skin wound healing.