DataSheet_1_HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway.pdf

ObjectivesNormal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs).MethodsThe 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated.ResultsA highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2.ConclusionOur results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.</p

Effects of ethanol on cell proliferation.

<p>(A, B) Flow cytometry to assess the amount of DNA in the G1, S, and G2 phases during the cell cycle at day 8 after differentiation in the presence or absence of ethanol at 100 mM. (C) qPCR was performed to evaluate the expression of cyclin D1 at days 4 and 8 after differentiation in the presence or absence of ethanol at 100 mM (*p<0.05 vs. no EtOH). (D) Western blot analysis was used to determine protein expression of albumin (ALB), and cyclin D1 at day 8 after differentiation in the presence or absence of ethanol at 100 mM. GAPGH was used as housekeeping gene control.</p

Effects of U0126, Erk inhibitor, on the differentiation of hESC towards hepatocytes.

<p>(A) Western blot analysis was employed to determine the effect of ethanol on the MAPK signaling pathway at day 8 after differentiation in the presence or absence of ethanol at 100 mM. (B) qPCR was used to determine the expression of albumin (ALB), CYP3A4 (3A4), β-catenin (β-cat), and cyclin D1 (D1) at day 8 after differentiation in the presence or absence of U0126 at 10 µM (*p<0.05 vs. no inhibitor; #p<0.005 vs. no inhibitor). (C) qPCR and ELISA were employed to evaluate the relative expression levels of albumin (ALB) and secreted ALB into the medium in the same samples in the presence or absence of U0126 at 10 µM. (p<0.05 vs. no inhibitor). (D–G) Western blot analysis was performed to determine protein expression of albumin (ALB), phosphorylated Erk (p-ERK), phosphorylated GSK-3β at the Ser9 residue (p-GSK3β), and cyclin D1 cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of U0126 at 10 µM. GAPDH and α-Tubulin were control housekeeping genes in cytoplasm and nucleus. (H) Western blot analysis was used to evaluate the expression of albumin (ALB), and cyclin D1 by supplementing high dose FGF-4 (F) or HGF (H) at day 8 after differentiation in the presence or absence of U0126 at 10 µM.</p

Effects of ethanol on the differentiation of hESC towards hepatocytes.

<p>(A) Schematic illustration of the differentiation protocol and the time frame of ethanol treatment. (B) Immunostaining of albumin in the hESC-derived hepatocytes (hEH) at day 24 exposed to the indicated doses of ethanol (x100). (C) Cell viability was assessed at day 24 by MTT assay. (D, E) qPCR was performed to determine the expression of liver genes, ALB and ASGPR1, C/EBPα, and HNF1α at day 24 exposed to the indicated doses of ethanol. (F) Secreted ALB levels in the culture media by hEH were assessed by ELISA at day 24 with or without ethanol treatment. (G) Dynamic expression changes of metabolizing enzymes, CYP3A4, CYP7A1, and UTG1A6 in hEH were evaluated by qPCR at different time points after differentiation in the presence or absence of ethanol at 100 mM. (*p<0.05 vs. no EtOH; #p<0.005 vs. no EtOH).</p

Expression of ethanol metabolizing genes during hepatocyte differentiation of hESC.

<p>qRT-PCR was employed to evaluate the dynamic expression of alcohol dehydrogenases (ADHs), ADH1A, ADH1B and ADH4, and by aldehyde dehydrogenases (ALDH), ALDH2 during the hepatocyte differentiation from ESC-derived hepatic progenitor cells.</p

Effects of IWR-1-endo, a WNT1 inhibitor, on the differentiation of hESC towards hepatocytes.

<p>(A) The entire view of the expression changes of 84 genes representing 10 pathways by PCR Array analysis at day 10 after differentiation in the presence or absence of ethanol at 100 mM. The names of the 84 genes and their expression fold changes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112698#pone.0112698.s001" target="_blank">Figure S1</a>. (B) qPCR was performed to evaluate the expression of Wnt1 at 8 after differentiation in the presence or absence of ethanol at 100 mM (p<0.05 vs. no inhibitor). (C) Western blot analysis was used to determine protein expression of Wnt1, phosphorylated GSK-3β at the Ser9 residue, and TCF1 in cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of ethanol at 100 mM. GAPGH and α-Tubulin were used to as housekeeping gene controls in the cytoplasm and nucleus respectively. (D) qPCR was used to measure the expression of albumin (ALB), alpha fetoprotein (AFP), CYP3A4 (3A4), and TCF1 at day 8 after differentiation in the presence or absence of IWR-1-endo at 0.2 µM (#p<0.005 vs. no inhibitor). (E–G) Western blot analysis was employed to determine protein expression of albumin (ALB), and TCF1 in cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of IWR-1-endo, at 0.2 µM. GAPDH and α-Tubulin as control housekeeping genes in cytoplasm and nucleus.</p

Hepatoma SK Hep-1 Cells Exhibit Characteristics of Oncogenic Mesenchymal Stem Cells with Highly Metastatic Capacity - Figure 7

<p>In vitro invasion assay A–C, the invasion of stained adipose-derived msenchymal stem cells (A), bone marrow-derived mesenchymal stem cells (B), and SK cells (C), were evaluated from transwells at 24 hours after culturing under low serum. D, based on the standard curve, the number of invading cells was quantitated. Magnifications: 100× (A–D).</p

Differentiation of three cell types into adipocytes and osteoblasts.

<p>A, SK cells, adipocyte-derived MSC (MSC-ad), and bone marrow-derived MSC (MSC-BM) were used for the differentiation. B, the three cell types were differentiated into adipocytes determined by Oil Red R staining. C, the three cell types were differentiated into osteoblasts determined by Alizarin Red S staining. D, alkaline phosphatase staining was performed after the osteoblast differentiation. Magnifications: 100X</p

Blocking the non-canonical Wnt pathway can inhibit the specification of WB-F344 cells into myfibroblasts.

<p>(a) mRNA expression of Wnt 5b. (b) Immunostaining of α-SMA and F-actin. (c) The fluorescent intensity of α-SMA. (d) The fluorescent intensity of F-actin. (e) α-SMA was determined by qRT-PCR. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Non-canonical Wnt signaling pathway was activated during the specification of WB-F344 cells into myfibroblasts.

<p>(a) Immunostaining of α-SMA and F-actin. (b) The fluorescent intensity of α-SMA and F-actin. (c) Expression levels of α-SMA, Col I and Col IV. (d) Wnt5a, Wnt5b, frizzled2 and β-catenin were determined by qRT-PCR. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p