Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

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Generation of ventralized human thalamic organoids with thalamic reticular nucleus

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Effects of sodium butyrate on the differentiation of pancreatic and hepatic progenitor cells from mouse embryonic stem cells

Recently significant progress has been made in differentiating embryonic stem (ES) cells toward pancreatic cells. However, little is known about the generation and identification of pancreatic progenitor cells from ES cells. Here we explored the influence of sodium butyrate on pancreatic progenitor differentiation, and investigated the different effects of sodium butyrate on pancreatic and hepatic progenitor formation. Our results indicated that different concentration and exposure time of sodium butyrate led to different differentiating trends of ES cells. A relatively lower concentration of sodium butyrate with shorter exposure time induced more pancreatic progenitor cell formation. When stimulated by a higher concentration and longer exposure time of sodium butyrate, ES cells differentiated toward hepatic progenitor cells rather than pancreatic progenitor cells. These progenitor cells could further mature into pancreatic and hepatic cells with the supplement of exogenous inducing factors. The resulting pancreatic cells expressed specific markers such as insulin and C‐peptide, and were capable of insulin secretion in response to glucose stimulation. The differentiated hepatocytes were characterized by the expression of a number of liver‐associated genes and proteins, and had the capability of glycogen storage. Thus, the current study demonstrated that sodium butyrate played different roles in inducing ES cells toward pancreatic or hepatic progenitor cells. These progenitor cells could be further induced into mature pancreatic cells and hepatocytes. This finding may facilitate the understanding of pancreatic and hepatic cell differentiation from ES cells, and provide a potential source of transplantable cells for cell‐replacement therapies. J. Cell. Biochem. 109: 236–244, 2010. © 2009 Wiley‐Liss, Inc.     

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells

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Blood vessel formation in cerebral organoids formed from human embryonic stem cells

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

外源性Wnt3a持续作用对小鼠胚胎干细胞Wnt／β-catenin信号通路的调控作用

目的观察Wnt/β-catenin信号通路是否在体外以外源性Wnt3a持续作用小鼠胚胎干细胞后被激活,并进一步调控该通路下游基因的表达。方法应用外源性Wnt3a持续作用ES-E14TG2a小鼠胚胎干细胞21d,通过细胞免疫荧光及Western Blotting检测细胞内β-catenin蛋白,以观察该蛋白的胞内积聚情况;同时QRT-PCR检测WNT下游靶标基因的表达量,采用完全随机F检验并用LSD法进行两两比较,来确定经典WNT／β-catenin信号通路是否被激活。结果ES-E14TG2a小鼠胚胎干细胞经Wnt3a连续培养21d后,β-catenin蛋白的细胞荧光明显较强,而对照组中的荧光强度较弱,说明细胞内β-catenin蛋白没有被降解而是在胞内大量积累;Western Blotting检测结果显示Wnt3a连续培养21d后ES-E14TG2a细胞内β-catenin蛋白条带明显比空白对照的蛋白条带粗;ES—E14TG2a细胞经wnt3a培养后Pitx2、Frizzled、Sox17的表达量均持续上升,Pitx2在培养7d、14d、21d分别为4．17±0．20、7．27±0．35、8．59±0．21（F=222．757,P=0．000）;Frizzled在培养7d、14d、21d分别为1．01±0．06、2．93±0．22、5．44±0．30（F=302．703,P=0．000）;Sox17在培养7d、14d、21d分别为8．45±0．41、18．35±0．17、34．93±0．16（F=7217．083,P=0．000）;Oct4培养到7d、14d的表达量持续增加分别为1．22±0．21、1．56±0．04,而连续培养21d后Oct4基因的表达量下降为1．15±0．07（F=8．827,P=0．016）。结论Wnt3a持续作用可激活Wnt／β-catenin信号通路,并调控下游基因的表达。     

Identification of microRNAs expressed highly in pancreatic islet‐like cell clusters differentiated from human embryonic stem cells

Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self‐renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES‐T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet‐like cell clusters derived from T3 cells), which expressed pancreatic islet cell‐specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES‐T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein‐coding mRNAs. The T3pi showed very high expression of microRNAs, miR‐186, miR‐199a and miR‐339, which down‐regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics.     

Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing

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Generation and characterisation of rabbit monoclonal antibodies against the native cell surface antigens of embryonic stem cells

Embryonic stem (ES) cells are potent resources for cell therapy, and monoclonal antibodies (mAbs) against native cell surface markers of ES cells could be useful tools for therapeutic applications. Here, we report the development of a feasible approach, which could be used in mass production, for experimentally producing rabbit mAbs against native cell surface antigens on the cell surface. Two of the 14mAbs, which were selected at random, could be bound to the cell surface antigens of mES cells. The immunocytochemistry (ICC) and Western blot results showed that mAb 39 recognises conformational epitopes. The target antigen of mAb 39 was then successfully purified using an improved immunoprecipitation approach in which mAb was bounded to intact mES cells before the cells were lysed. The LC-LTQ mass spectrum analysis showed that the target antigen of mAb 39 was Glut3. This result was further confirmed by Western blot using commercially available antibodies against Glut3. Further experiments showed that mAb 39 exhibited an antiproliferative effect on mES cells. We also found that Glut3 was differentially expressed among the mES cell population as detected by flow cytometry.     

Establishment of a novel embryonic stem cell line by a modified procedure

To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is diffucult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouseSry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.Abbreviations DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - FIAU 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil - LIF leukemia inhibitory factor - NEAA non-essential amino acids     

Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.     

The TRβ1 is essential in mediating T3 action on Akt pathway in human pancreatic insulinoma cells

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor β1 has been hypothesized. The serine‐threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic β‐cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non‐genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRβ1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor β1. We showed that the thyroid receptor β1 mediates the T3 regulation of the cdk4·cyc D1·p21^CIP1·p27^KIP1 complex formation and activity. In addition TRβ1 is essential for the T3 upregulation of the Akt targets β‐catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the β1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway. J. Cell. Biochem. 106: 835–848, 2009. © 2009 Wiley‐Liss, Inc.