The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.
Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.
Our objective was to evaluate the effectiveness of the insulin-sensitizing agent rosiglitazone in obese women with polycystic ovary syndrome (PCOS) and severe insulin resistance. Twelve obese women with PCOS were recruited. All were hirsute and anovulatory with acanthosis nigricans indicating severe insulin resistance. All women were treated with 4 mg of rosiglitazone daily for 6 months. A standard 75-g oral glucose tolerance test with insulin levels was performed before and after the women were treated with rosiglitazone. Glucose and insulin areas under the curve (AUC) were calculated. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate, LH, and 17-hydroxyprogesterone were also measured before and after treatment. The body mass index was determined before and after treatment. There was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during oral glucose tolerance test and basal total testosterone levels. After treatment with rosiglitazone, there were significant decreases in fasting insulin levels (46.0 +/- 6.5 vs. 16.9 +/- 2.0 microU/ml; P < 0.001), insulin AUC (749.3 +/- 136.3 vs. 225.0 +/- 15.7 microU/ml; P = 0.003), fasting glucose levels (90.8 +/- 3.0 vs. 81.8 +/- 1.9 mg/dl; P = 0.003), and glucose AUC (437.9 +/- 25.0 vs. 322.5 +/- 14.7 mg/dl; P < 0.001). Both total testosterone (96.3 +/- 17.3 vs. 56.1 +/- 5.8 ng/dl; P = 0.01) and free testosterone (5.8 +/- 0.6 vs. 3.4 +/- 0.5 pg/ml; P < 0.001) decreased significantly after treatment, although there was no significant change in LH levels. Levels of SHBG increased significantly (18.3 +/- 3.4 vs. 25.8 +/- 6.6 nmol/liter; P = 0.009) after treatment, and dehydroepiandrosterone sulfate levels decreased significantly (P = 0.04). There was no significant change in body mass index (40.4 +/- 2.4 vs. 41.1 +/- 2.7 kg/m(2)). Eleven of the women reverted to regular ovulatory cycles during the treatment period. We conclude that 1) rosiglitazone therapy improves insulin resistance and glucose tolerance in obese women with PCOS; 2) rosiglitazone decreases ovarian androgen production, which appears to be independent of any changes in LH levels; 3) hyperinsulinemia appears to play a key role in the overproduction of ovarian androgens in these women because attenuation of insulin levels is associated with decreased testosterone levels; and 4) short-term rosiglitazone therapy helps restore spontaneous ovulation.
These results indicate that in women with PCOS and severe insulin resistance, insulin sensitivity appears to be the major determinant of adiponectin levels rather than adiposity. Low adiponectin levels may predict women with PCOS who are at high risk for developing type 2 diabetes.
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