This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

Buerger's disease is a rare and severe disease affecting the blood vessels of the limbs. Adipose tissue-derived mesenchymal stem cells (ADSCs) have the potential to cure Buerger's disease when developed as a stem cell drug. In the present study, we conducted a prospective, nonrandomized, no placebo-controlled, phase I/II clinical trial with a 2-year follow-up questionnaire survey. A total of 17 patients were intramuscularly administered autologous ADSCs at a dose of 5 million cells/kg. The incidence of adverse events (AEs), adverse drug reaction (ADR), and serious adverse events (SAEs) was monitored. No ADRs and SAEs related to stem cell treatment occurred during the 6-month follow-up. In terms of efficacy, the primary endpoint was increase in total walking distance (TWD). The secondary endpoint was improvement in rest pain, increase in pain-free walking distance (PFWD), toe–brachial pressure index (TBPI), transcutaneous oxygen pressure (TcPO₂), and arterial brachial pressure index (ABPI). ADSCs demonstrated significant functional improvement results including increased TWD, PFWD, and rest pain reduction. No amputations were reported during the 6-month clinical trial period and in the follow-up questionnaire survey more than 2 years after the ADSC injection. In conclusion, intramuscular injection of ADSCs is very safe and is shown to prompt functional improvement in patients with severe Buerger's disease at a dosage of 300 million cells per 60 kg of body weight. However, the confirmatory therapeutic efficacy and angiogenesis need further study.

Advanced maternal age (AMA) has become prevalent globally. With aging, weakened antioxidant defense causes loss of normal function in the ovary and uterus due to oxidative stress. Here, we aimed to improve embryo development in AMA mice by intravenous injection (IV) of human adipose stem cell conditioned medium (ASC-CM) at various frequencies and intervals as an antioxidant intervention. Four- and six-month-old female ICR (Institute of Cancer Research) mice were randomly divided into groups IV treated with human ASC-CM under different conditions, and in vitro and in vivo embryo development were evaluated. Consequently, compared to the control group, blastocyst formation rate of parthenotes was significantly promoted in 4-month-old mice and the mean number of implanted fetuses after natural mating was significantly increased by approximately two-fold in 6-month-old mice. Through gene analysis, the anti-apoptotic and anti-oxidative effects of human ASC-CMs were confirmed in the ovaries and uterus of pregnant mice at both ages. In particular, ovarian expression of gpx1 and catalase drastically increased in 6-month-old mice. Furthermore, the levels of gpx1 and catalase were further increased, with a high frequency of injection regardless of age. Thus, we demonstrated for the first time the anti-oxidative effect of human ASC-CM administration against ovarian aging and the optimal injection condition.

The quality of embryos produced by assisted reproductive techniques should be advanced by the improvement of in vitro culture conditions for successful implantation and pregnancy maintenance. We investigated the anti-oxidative effect of human adipose stem cell (ASC) conditioned medium with its optimal basal medium, Dulbecco′s modified Eagle′s medium (DMEM-CM), or keratinocyte serum-free medium (KSFM-CM) as supplements during in vitro culture (IVC) of in vitro fertilized mouse embryo. At first, preimplantation embryo development was evaluated in KSFM-CM and DMEM-CM supplemented cultures at various concentrations. The blastocyst (BL) and hatched BL formation rates were significantly increased in 5% DMEM-CM, while no difference was observed from KSFM-CM. Next, comparing the efficacy of KSFM-CM and DMEM-CM at the same concentration, DMEM-CM enhanced the developmental rate of 16 cells, morula, BL, and hatched BL. The expression level of reactive oxygen species decreased and that of glutathione increased in BL cultured with DMEM-CM, which confirms its anti-oxidative effect. Furthermore, apoptosis in BL cultured with DMEM-CM was reduced compared with that in KSFM-CM. This study demonstrated that the comparative effect of human ASC-CM made of two different basal media during mouse embryo IVC and anti-oxidative effect of 5% DMEM-CM was optimal to improve preimplantation embryo development.

Oxidative stress is a major cause of damage to the quantity and quality of embryos produced in vitro. Antioxidants are usually supplemented to protect embryos from the suboptimal in vitro culture (IVC) environment. Amniotic membrane-derived mesenchymal stem cells (AMSC) have emerged as a promising regenerative therapy, and their paracrine factors with anti-oxidative effects are present in AMSC conditioned medium (CM). We examined the anti-oxidative potential of human AMSC-CM treatment during IVC on mouse preimplantation embryo development and antioxidant gene expression in the forkhead box O (FoxO) pathway. AMSC-CM (10%) was optimal for overall preimplantation embryo developmental processes and upregulated the expression of FoxOs and their downstream antioxidants in blastocysts (BL). Subsequently, compared to adipose-derived mesenchymal stem cell (ASC)-CM, AMSC-CM enhanced antioxidant gene expression and intracellular GSH levels in the BL. Total antioxidant capacity and SOD activity were greater in AMSC-CM than in ASC-CM. Furthermore, SOD and catalase were more active in culture medium supplemented with AMSC-CM than in ASC-CM. Lastly, the anti-apoptotic effect of AMSC-CM was observed with the regulation of apoptosis-related genes and mitochondrial membrane potential in BL. In conclusion, the present study established AMSC-CM treatment at an optimal concentration as a novel antioxidant intervention for assisted reproduction.