Abstract
Chenodeoxycholic acid (CDCA), a farnesoid X receptor (FXR) ligand, is a member of the nuclear receptor family and is probably involved in regulating the cellular activities of embryonic stem (ES) cells. Recently, although it was reported that the FXR ligand can mediate differentiation, apoptosis, and/or growth arrest in several cell types, it is still not well known how CDCA mediates effects in ES cells. Therefore, we investigated the direct effect of CDCA on mES cells. Feeder-free mES cells were treated in a dose-dependent manner with CDCA (50, 100, and 200 μM) for 72 h, and then a 100 μM CDCA treatment was performed for an additional 72 h. We analyzed the morphology, cell growth, cell characteristics, immunocytochemistry, and RT-PCR. In CDCA-treated cells, we observed the disappearance of pluripotent stem cell markers including alkaline phosphatase, Oct4, and Nanog and a time- and dose-dependent increase in expression of nestin, PAX6, and α-smooth muscle actin, but not α-fetoprotein. The 100 μM CDCA-treated cells in their second passage continued this differentiation pattern similar to those in the controls. In conclusion, these results suggest that CDCA can guide mES cells by an FXR-independent pathway to differentiate into ectoderm and/or mesoderm, but not endoderm.
Highlights
It was reported that the farnesoid X receptor (FXR) ligand can mediate differentiation, apoptosis, and/or growth arrest in several cell types, it is still not well known how Chenodeoxycholic Acid (CDCA) mediates effects in embryonic stem (ES) cells. erefore, we investigated the direct effect of CDCA on mouse ES (mES) cells
For direct differentiation of mES cells by CDCA, feeder cells were removed by plating on nongelatin coated dish for 1 h, which allowed the feeder cells to adhere, while most of the mES cells stayed in the suspension. e suspended mES cells were once transferred onto a new 0.1% gelatin-coated dish for propagation in the presence of 1,000 U/mL of leukemia inhibitory factor (LIF). e feeder-free mES cells were subcultured a er 24 h. en the cells were incubated under different conditions for 72 h. e cells were incubated in (i) basal medium, (ii) basal medium supplemented with 1,000 U/mL of LIF, and (iii) basal medium supplemented with 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich Corp) in the absence of LIF
We investigated the effect of CDCA, an FXR ligand, on mES cell differentiation under feeder-free conditions
Summary
Since the establishment of embryonic stem (ES) cell lines [1, 2], it has been known that ES cells have the capacity for self-renewal and pluripotency, with the ability to differentiate into multiple cell types in vitro and in vivo. ese characteristics of ES cells make them a valuable model system for differentiation study and cell-based regeneration therapies.Numerous reports have documented the differentiation of ES cells into speci c cell types, such as neurons [3], cardiomyocytes [4], adipocytes [5], endothelial cells [6], hepatocytes [7], keratinocytes [8], and pancreatic cells [9] under the appropriate culture conditions. Ese characteristics of ES cells make them a valuable model system for differentiation study and cell-based regeneration therapies. E farnesoid X receptor (FXR, NR1H4), may modulate the differentiation into myocyte [13] during myogenesis of tissue-speci c stem cells. In liver activated FXR induces liver regeneration by a homeostatic mechanism [17] and affects vascular remodeling [18]. In the intestine, it protects the tissue from bacterialinduced mucosal injury by bile acids [19]. It is known that the FXR activators inhibit cell proliferation, trigger differentiation, and induce apoptosis. Studies on the effects of activated FXR on proliferation or differentiation of ES cells are scarce
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