R‐spondin 1 and noggin facilitate expansion of resident stem cells from non‐damaged gallbladders

Pioneering studies within the last few years have allowed the in vitro expansion of tissue‐specific adult stem cells from a variety of endoderm‐derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R‐spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5‐positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non‐regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R‐spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder‐derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride‐treated mice. Human gallbladders were also a source of organoid‐forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.     

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R‐spondin

R‐spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R‐spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R‐spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R‐spondin‐induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt‐inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R‐spondin, can be strongly suppressed by R‐spondin, suggesting that R‐spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R‐spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.     

Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas

1. Download : Download high-res image (231KB)
2. Download : Download full-size image

     

Analysis of gene expressions of embryonic stem‐derived Pdx1‐expressing cells: Implications of genes involved in pancreas differentiation

We have recently reported the method by which embryonic stem (ES) cells were induced into Pdx1‐expressing cells. To gain insights into the ES cell‐derived Pdx1‐expressing cells, we examined gene expression profiles of the cells by microarray experiments. Microarray analyses followed by a comparison with the data of the cells in developing pancreatic and adult islet suggested that the ES cell‐derived Pdx1‐positive cells were immature pancreatic progenitor cells with endodermal characteristics. The analyses of the genes upregulated in the ES cell‐derived Pdx1‐positive cells would give us knowledge on early pancreatic development. Here, we first listed the genes and found that these contained not only those known to be expressed in the endoderm or pancreatic progenitor cells, but also those known to be involved in left–right axis formation. Second, we examined the gene expression patterns and found that several genes were expressed in the ventral foregut lip at the anterior intestinal portal in E8.5 embryo. Given that the Pdx1/GFP‐expressing cells are first observed in the same region at the anterior intestinal portal, these results suggest that the pancreatic progenitor cells first give rise at the ventral endoderm prior to the formation of dorsal and ventral pancreatic buds.     

MiR‐30‐5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β‐catenin signaling axis

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell‐like characteristics (stemness) and chemoresistance. MiR‐30‐5p has been shown to function as a tumor suppressor by targeting the Wnt/β‐catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR‐30‐5p exerts anti‐oncogenic effects in CRC by regulating the USP22/Wnt/β‐catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR‐30‐5p family expression. After identifying the 3’UTR of USP22 as a potential binding site of miR‐30‐5p, we constructed a luciferase reporter containing the potential miR‐30‐5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR‐30‐5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR‐30‐5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR‐30‐5p is normally decreased in CD133 + CRC cells, miR‐30‐5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR‐30‐5p expression is normally reduced in 5‐fluorouracil (5‐FU) resistant CRC cells, whereas miR‐30‐5p overexpression in 5‐FU resistant cells reduces sphere formation and cell viability. Inhibition of miR‐30‐5p reversed the process. Finally, we determined that miR‐30‐5p attenuates the expression of Wnt/β‐catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β‐catenin protein levels in CRC stem cells.     

Knock‐down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC‐derived dendritic cells

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β‐N‐acetyl‐hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay‐Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock‐down immunogenic DCs (i‐DCs) from CD34⁺‐haemopoietic stem cells (CD34⁺‐HSCs), thus mimicking the Tay‐Sachs (HEXA?/?) and Sandhoff (HEXB?/?) cells. We showed that the absence of β‐N‐acetyl‐hexosaminidase activity does not alter the differentiation of i‐DCs from HSCs, but it is critical for the activation of CD4⁺T cells because knock‐down of HEXA or HEXB gene causes a loss of function of i‐DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β‐N‐acetyl‐hexosaminidase A isoenzyme within this mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Wnt/β‐catenin signalling induces MLL to create epigenetic changes in salivary gland tumours

We show that activation of Wnt/β‐catenin and attenuation of Bmp signals, by combined gain‐ and loss‐of‐function mutations of β‐catenin and Bmpr1a, respectively, results in rapidly growing, aggressive squamous cell carcinomas (SCC) in the salivary glands of mice. Tumours contain transplantable and hyperproliferative tumour propagating cells, which can be enriched by fluorescence activated cell sorting (FACS). Single mutations stimulate stem cells, but tumours are not formed. We show that β‐catenin, CBP and Mll promote self‐renewal and H3K4 tri‐methylation in tumour propagating cells. Blocking β‐catenin–CBP interaction with the small molecule ICG‐001 and small‐interfering RNAs against β‐catenin, CBP or Mll abrogate hyperproliferation and H3K4 tri‐methylation, and induce differentiation of cultured tumour propagating cells into acini‐like structures. ICG‐001 decreases H3K4me3 at promoters of stem cell‐associated genes in vitro and reduces tumour growth in vivo. Remarkably, high Wnt/β‐catenin and low Bmp signalling also characterize human salivary gland SCC and head and neck SCC in general. Our work defines mechanisms by which β‐catenin signals remodel chromatin and control induction and maintenance of tumour propagating cells. Further, it supports new strategies for the therapy of solid tumours.     

Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes

BackgroundMegakaryocytes (MKs), a rare population of bone marrow cells, are responsible for the production of platelets. Sick neonates are predisposed to developing thrombocytopenia (platelet count <150 × 109/L) and neonates are affected by several megakaryocyte disorders as compared to adults.HypothesisMicroRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant hematopoiesis, but their role in regulation of biological differences between adult and neonatal megakaryopoiesis is unknown.MethodsTo study this, we cultured human cord blood (CB) and peripheral blood (PB) derived CD34⁺ cells in the presence of thrombopoietin for 14 days and collected cultures expressing >90% CD41⁺ by flow cytometry and studied 88 miRNAs involved in stem cell development and differentiation. miRNA validation studies were performed in Dami cell line.ResultsOut of 88 miRNAs involved in stem cell development, let-7b was the only miRNA down regulated (∼10-fold) in neonates compared to adult-MKs. Let-7b has not been previously described in MKs, however reduced expression of let-7b was found in several human cancers, suggesting that it functions as a tumor suppressor. Our results showed the inhibitory effect of let-7b on wnt signaling pathway by regulating Fzd4 (frizzled family receptor 4) and thereby regulating proliferation as well as differentiation. Let-7b down regulation induced mitochondrial biogenesis and its markers PGC-1α and NRF1 during megakaryocyte development.ConclusionsOur findings for the first time unveil the novel role of let-7b/Fzd4 axis through wnt signaling by regulating mitochondrial biogenesis during megakaryocyte development.     

PTEN-mediated AKT/β-catenin signaling enhances the proliferation and expansion of Lgr5+ hepatocytes

Rationale: Compelling evidence suggests that Lgr5+ hepatocytes repair liver damage by promoting the regeneration of hepatocytes and ductal cells in the case of liver injury. The PTEN-mediated AKT/β-catenin signaling plays a key role in the regulation of innate immune regulation in the liver. However, the signaling pathways that control Lgr5+ hepatocyte proliferation in the liver remain unclear.Methods: In order to assess the involvement of PTEN-mediated AKT/β-catenin signaling in the expansion of Lgr5+ hepatocytes upon liver injuries, the Lgr5-CreER; Rosa-mTmG lineage tracing system was used to target Lgr5+ hepatocytes.Results: The tracing of Lgr5+ hepatocytes showed that PTEN deletion and β-catenin activation significantly promoted the proliferation of Lgr5+ hepatocytes. In converse, the simultaneous inhibition of PTEN and β-catenin limited Lgr5+ hepatocyte proliferation in the liver. Our findings provide an insight into understanding how PTEN-mediated AKT/β-catenin signaling regulates the proliferation of Lgr5+ hepatocytes.Conclusion: The outcomes can improve the application potential of Lgr5+ hepatocytes in the treatment of liver injury diseases and provide a new treatment option for liver cancer.     

Development of the duct system during exocrine pancreas differentiation in the grass snake Natrix natrix (Lepidosauria,Serpentes)

We analyzed the development of the pancreatic ducts in grass snake Natrix natrix L. embryos with special focus on the three‐dimensional (3D)‐structure of the duct network, ultrastructural differentiation of ducts with attention to cell types and lumen formation. Our results indicated that the system of ducts in the embryonic pancreas of the grass snake can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different from that in other vertebrates, which was related to the specific topography of the snake's internal organs. The process of duct remodeling in Natrix embryos began when the duct walls started to change from multilayered to single‐layered and ended together with tube formation. It began in the dorsal pancreatic bud and proceeded toward the caudal direction. The lumen of pancreatic ducts differentiated by cavitation because a population of centrally located cells was cleared through cell death resembling anoikis. During embryonic development in the pancreatic duct walls of the grass snake four types of cells were present, that is, principal, endocrine, goblet, and basal cells, which is different from other vertebrate species. The principal cells were electron‐dense, contained indented nuclei with abundant heterochromatin, microvilli and cilia, and were connected by interdigitations of lateral membranes and junctional complexes. The endocrine cells were electron‐translucent and some of them included endocrine granules. The goblet cells were filled with large granules with nonhomogeneous, moderately electron‐dense material. The basal cells were small, electron‐dense, and did not reach the duct lumen.     

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983–1002, 2016     

iPSC‐derived mesenchymal stem cells exert SCF‐dependent recovery of cigarette smoke‐induced apoptosis/proliferation imbalance in airway cells

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.