Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell's EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.

Endodermal stem/progenitor cells have diverse potential applications in research and regenerative medicine, so a readily available source could have widespread uses. Here we describe derivation of human induced endodermal progenitor cells (hiEndoPCs) from gastrointestinal epithelial cells using a cocktail of defined small molecules along with support from tissue-specific mesenchymal feeders. The hiEndoPCs show clonal expansion in culture and give rise to hepatocytes, pancreatic endocrine cells, and intestinal epithelial cells when treated with defined soluble molecules directing differentiation. The hiEndoPC-derived hepatocytes are able to rescue liver failure in FahRag2 mice after transplantation, and, unlike hESCs, transplanted hiEndoPCs do not give rise to teratomas. Since human gastric epithelial cells are readily available from donors of many ages, this conversion strategy can generate clonally expandable cell populations with a variety of potential applications, including personalized drug screening and therapeutic strategies for liver failure and diabetes.

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.

No abstract

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.

Background Generation of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood. Methods We established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell–cell and cell–extracellular matrix interactions which are fundamentally different between 2D and 3D cultures. Results The expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression. Conclusion We developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.

Background Ex vivo production of induced megakaryocytes (MKs) and platelets from stem cells is an alternative approach for supplying transfusible platelets. However, it is difficult to generate large numbers of MKs and platelets from hematopoietic stem cells and progenitor cells (HSPCs). Methods To optimize the differentiation efficiency of megakaryocytic cells from HSPCs, we first employed a platelet factor 4 (PF4)-promoter reporter and high-throughput screening strategy to screen for small molecules. We also investigated the effects and possible mechanisms of candidate small molecules on megakaryocytic differentiation of human HSPCs. Results The small molecule Ricolinostat remarkably promoted the expression of PF4-promoter reporter in the megakaryocytic cell line. Notably, Ricolinostat significantly enhanced the cell fate commitment of MK progenitors (MkPs) from cord blood HSPCs and promoted the proliferation of MkPs based on cell surface marker detection, colony-forming unit-MK assay, and quantitative real-time PCR analyses. MkPs generated from Ricolinostat-induced HSPCs differentiated into mature MKs and platelets. Mechanistically, we found that Ricolinostat enhanced MkP fate mainly by inhibiting the secretion of IL-8 and decreasing the expression of the IL-8 receptor CXCR2. Conclusion The addition of Ricolinostat to the culture medium promoted MkP differentiation from HSPCs and enhanced the proliferation of MkPs mainly by suppressing the IL-8/CXCR2 pathway. Our results can help the development of manufacturing protocols for the efficient generation of MKs and platelets from stem cells in vitro.

BackgroundBi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases.MethodsIn this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium.ResultsIn vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression.ConclusionsHere we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0703-2) contains supplementary material, which is available to authorized users.