Chronic systemic administration of serotonergic ligands flibanserin and 8-OH-DPAT enhance HPA axis responses to restraint in female marmosets.

BACKGROUND: Flibanserin, a novel serotonin (5-HT)(1A) agonist and 5-HT(2A) antagonist, has been shown to increase sexual desire and reduce distress in women with Hypoactive Sexual Desire Disorder (HSDD). In marmoset monkeys, flibanserin has demonstrated pro-social effects on male-female pairmates, while the classic 5-HT(1A) agonist 8-OH-DPAT suppresses female sexual behavior and increases aggressive interactions between pairmates. Activation of 5-HT(1A) and 5-HT(2A) receptors is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. This study aims to characterize the effects of repeated flibanserin and 8-OH-DPAT administration on the marmoset HPA axis and to elucidate endocrine correlates of altered marmoset pair behavior. METHODS: Adrenocorticotropic hormone (ACTH) and cortisol were examined at baseline and during 5-HT(1A) agonist and restraint challenges in 8 female marmoset monkeys receiving daily flibanserin (15mg/kg) and an additional 8 female marmosets receiving 8-OH-DPAT (0.1mg/kg) for 15-16weeks. Corresponding vehicle treatments were administered in a counterbalanced, within-subject design. All females were housed in stable male-female pairs. Treatment-induced changes in ACTH and cortisol levels were correlated with previously assessed marmoset pair behavior. RESULTS: While morning basal cortisol levels and HPA responses to a 5-HT(1A) agonist challenge were not altered by chronic flibanserin or 8-OH-DPAT, both treatments increased the responsiveness of the marmoset HPA axis to restraint. Enhanced ACTH responses to restraint correlated with reduced sexual receptivity and increased aggression in 8-OH-DPAT-, but not in flibanserin-treated female marmosets. CONCLUSIONS: Unaltered HPA responses to a 5-HT(1A) agonist challenge after chronic flibanserin and 8-OH-DPAT treatments indicate little or no de-sensitization of the HPA axis to repeated 5-HT(1A) manipulation. Chronic 8-OH-DPAT, but not flibanserin, leads to aggravated ACTH responses to stress that may contribute to anti-sexual and anti-social behavior between 8-OH-DPAT-treated females and their male pairmates. Despite similar flibanserin and 8-OH-DPAT induced ACTH responses to restraint stress, flibanserin-treated females show unchanged cortisol profiles. This is possibly due to flibanserin's regional selectivity in 5-HT(1A) activation and concurrent 5-HT(2A) inhibition. The contrasting restraint-related cortisol responses emulate contrasting behavioral phenotypes of diminished pair-bond of 8-OH-DPAT-treated females compared to the more affiliative pair-bond of flibanserin-treated females.

Chemically defined conditions for human iPSC derivation and culture.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.