Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid.

BACKGROUND: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. METHODS: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. RESULTS: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. CONCLUSIONS: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.

The growth inhibitory activity of the Cimicifuga racemosa extract Ze 450 is mediated through estrogen and progesterone receptors-independent pathways.

Despite the wide use of Cimicifuga racemosa (CR) extract to treat symptoms associated with menopause and other gynecological disorders, very little is known about its mechanism of action. Therefore, we studied in this report the antiestrogenic and antiproliferative effect of a new CR ethanolic extract, Ze 450, in a MCF-7 cell clone that does not proliferate in response to 17beta-estradiol (E(2)). Using this cell line, we have found that the extract inhibited cell proliferation and showed antiestrogenic activity using an ERE-luciferase reporter assay. The growth inhibitory activity was different from the antiestrogenic activity since the CR extract also inhibited the growth of the ER-negative human breast cancer cell line T-47D. Also, we evaluated the effects of this CR extract on the transcriptional regulation of genes involved in cell cycle progression in the ER-negative cell lines 293T and T-47D and we found that this extract markedly inhibited the luciferase activity driven by the cyclin D1 promoter and increased the transcriptional activity of the p21 gene promoter. Finally, we observed that our CR extract bound to the progesterone receptor B1 but did not show progestin-like activity in the T-47D cell line. These findings provide new mechanistic insights into the antiproliferative activities of CR in ER-positive and ER-negative tumour cell lines and highlight their potential in the management of climacteric disorders in women with a history of breast cancer.