Hailei Yao

Epimorphin Regulates Bile Duct Formation via Effects on Mitosis Orientation in Rat Liver Epithelial Stem-Like Cells

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3α and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, β1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPβ.

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem‐like cells (WB‐F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co‐culture of EPM‐overexpressed mesenchymal cells (PT67EPM) with WB‐F344 cells. Here, we showed that EPM could promote WB‐F344 cells differentiation into bile duct‐like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin‐1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA‐WB‐F344 cell line (WBRhoA‐DN) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up‐regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM‐induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase‐reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPβ. Taken together, these data demonstrated that EPM regulates bile duct formation of WB‐F344 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis. J. Cell. Physiol. 226: 2807–2816, 2011.

Neuronal restrictive silencing factor silencing induces human amniotic fluid-derived stem cells differentiation into insulin-producing cells.

Islet cell replacement represents the most promising approach for the treatment of type I diabetes. However, it is limited by a shortage of pancreas donors. Here, we report that human amniotic fluid-derived stem cells (hAFSCs) can be induced to differentiate into functional insulin-producing cells by knocking down neuronal restrictive silencing factor (NRSF). In this study, lentiviral vectors were used to deliver small interference NRSF (siNRSF) RNA into hAFSCs. After infection with lentivirus containing siNRSF, hAFSCs were successfully induced to differentiate into insulin-producing cells. The differentiated siNRSF-hAFSCs expressed genes specific for islet cells, such as Pdx1, Hnf4α, Isl-1, Nkx6.1, Insulin, and Glut2. These cells also produced and released C-peptide in a glucose-responsive manner. These findings indicated that hAFSCs could be induced to differentiate into insulin-producing β-like cells by NRSF silencing.

Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture

Cell-cell contacts and interactions between pancreatic β-cells and/or other cell populations within islets are essential for cell survival, insulin secretion, and functional synchronization. Three-dimensional (3D) culture systems supply the ideal microenvironment for islet-like cluster formation and functional maintenance. However, the underlying mechanisms remain unclear. In this study, mouse insulinoma 6 (MIN6) cells were cultured in a rotating 3D culture system to form islet-like aggregates. Glucose-stimulated insulin secretion (GSIS) and the RhoA/ROCK pathway were investigated. In the 3D-cultured MIN6 cells, more endocrine-specific genes were up-regulated, and GSIS was increased to a greater extent than in cells grown in monolayers. RhoA/ROCK inactivation led to F-actin remodeling in the MIN6 cell aggregates and greater insulin exocytosis. The gap junction protein, connexin 36 (Cx36), was up-regulated in MIN6 cell aggregates and RhoA/ROCK-inactivated monolayer cells. GSIS dramatically decreased when Cx36 was knocked down by short interfering RNA and could not be reversed by RhoA/ROCK inactivation. Thus, the RhoA/ROCK signaling pathway is involved in insulin release through the up-regulation of Cx36 expression in 3D-cultured MIN6 cells.

RhoA promotes differentiation of WB-F344 cells into the biliary lineage

When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells in which RhoA expression was upregulated. To further investigate the role of RhoA in WB cell differentiation initiated by Matrigel treatment, we constructed constitutively active RhoA-expressing vectors and stably transfected them into WB-F344 cells. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopically active RhoA expression were found to form bile-duct-like structures even without Matrigel treatment. Besides, ROCK inhibitor Y27632 treatment eliminated luminal morphogenesis. F-actin cytoplasmic staining further verified that the RhoA-ROCK signal pathway was involved in differentiation of WB cells into the biliary lineage. In conclusion, our results suggested that the RhoA-ROCK-stress fibre system plays an obligatory role in Matrigel-induced WB-F344 cell luminal morphogenesis and further differentiation.

Legume Lectin FRIL Preserves Neural Progenitor Cells in Suspension Culture In Vitro

In vitro maintenance of stem cells is crucial for many clinical applications. Stem cell preservation factor FRIL (Flt3 receptor-interacting lectin) is a plant lectin extracted from Dolichos Lablab and has been found preserve hematopoietic stem cells in vitro for a month in our previous studies. To investigate whether FRIL can preserve neural progenitor cells (NPCs), it was supplemented into serum-free suspension culture media. FRIL made NPC grow slowly, induced cell adhesion, and delayed neurospheres formation. However, FRIL did not initiate NPC differentiation according to immunofluorescence and semiquantitive RT-PCR results. In conclusion, FRIL could also preserve neural progenitor cells in vitro by inhibiting both cell proliferation and differentiation.

Wnt3a is involved in the early stage of miPSC and mESC haemopoietic differentiation

The Wnt/β‐catenin signalling pathway is important in regulating not only self‐renewal of haemopoietic progenitors and stem cells but also haemopoietic differentiation of ESCs (embryonic stem cells). However, it is still not clear how it affects haemopoietic differentiation. We have used a co‐culture system for haemopoietic differentiation of mouse ESCs and iPSCs (induced pluripotent stem cells) in which the Wnt3a gene‐modified OP9 cell line is used as stromal cells. The number of both Flk1+ and CD41+ cells generated from both co‐cultured mouse ESCs and mouse iPSCs increased significantly, which suggest that Wnt3a is involved in the early stages of haemopoietic differentiation of mouse ESCs and mouse iPSCs in vitro.