Changes in the content of estrogen α and progesterone receptors during differentiation of mouse embryonic stem cells to dopamine neurons

Abstract

Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinson's disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor alpha (ER-alpha) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-alpha expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-alpha. These results show a differential expression pattern of ER-alpha and PR isoforms during neuronal differentiation of ESC.

Activated Notch1 is a stronger astrocytic stimulus than leukemia inhibitory factor for rat neural stem cells

“…We performed immunoblots as described (Diaz et al, 2007;MolinaHernández and Velasco, 2008) using the Phospho-STAT3 kit from Cell Signaling following the recommendations provided by the manufacturer. Briefly, neural progenitors/NSC were grown to subconfluency and stimulated with CNTF or LIF for 30 min.…”

“…Reported procedures were followed (Velasco et al, 2003;Diaz et al, 2007). In brief, fixed cells were blocked in 10% normal goat serum (Microlab, México) and permeabilized with 0.3% Triton X-100 in PBS.…”

Stem Cells with Neurogenic Potential and Steroid Hormones

“…Different intracellular pathways can be activated in mouse or human cells just before differentiation, which might result in up-or down-regulation of pr expression. Increases in PR have been reported also for a 5-stage dopaminergic differentiation protocol in mESC: PR was detected by immunofluorescence in just a few scattered pluripotent cells, and both PR-A and PR-B measured by immunoblot increased 2-and 3-fold, respectively, when cells were at the final stage where terminally differentiated neurons are present [77]. The functional significance of stimulation with progesterone (1 nM -10 μM) on dopamine neuron differentiation was tested in this system by incubating mESCderived NSC.…”

“…Undifferentiated mouse and human ESC express transcripts for ER and , as well as their corresponding polypeptides [76,77,90]. Investigators found only ER mRNA in mESC, and transfected these cells with the coding sequence for ER ; this ectopic expression of ER in pluripotent mouse cells did not induce differentiation.…”

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

“…11,12 Large doses of progesterone for up to days after injury have been shown to limit CNS damage, reduced loss of neural tissue, and improve functional recovery. In addition, the progesterone derived neurosteroid allopregnenolone has been shown to be even more efficacious.…”