技术资料

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct,in the absence of proliferation and multipotent progenitor generation,or indirect,by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state,induced by brief exposure to reprogramming factors,followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells,including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies. View Publication

Green tea is a popular drink consumed daily by millions of people around the world. Previous studies have shown that some polyphenol compounds from green tea possess anticancer activities. However,systemic evaluation was limited. In this study,we determined the cancer chemopreventive potentials of 10 representative polyphenols (caffeic acid,CA; gallic acid,GA; catechin,C; epicatechin,EC; gallocatechin,GC; catechin gallate,CG; gallocatechin gallate,GCG; epicatechin gallate,ECG; epigallocatechin,EGC; and epigallocatechin gallate,EGCG),and explored their structure-activity relationship. The effect of the 10 polyphenol compounds on the proliferation of HCT-116 and SW-480 human colorectal cancer cells was evaluated using an MTS assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with propidium iodide (PI)/RNase or annexin V/PI. Among the 10 polyphenols,EGCG showed the most potent antiproliferative effects,and significantly induced cell cycle arrest in the G1 phase and cell apoptosis. When the relationship between chemical structure and anticancer activity was examined,C and EC did not show antiproliferative effects,and GA showed some antiproliferative effects. When C and EC esterified with GA to produce CG and ECG,the antiproliferative effects were increased significantly. A similar relationship was found between EGC and EGCG. The gallic acid group significantly enhanced catechin's anticancer potential. This property could be utilized in future semi-synthesis of flavonoid derivatives to develop novel anticancer agents. View Publication

Differences in global levels of histone acetylation occur in normal and cancer cells,although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases,histones are globally deacetylated by histone deacetylases (HDACs),and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs),preventing further reductions in pH(i). Conversely,global histone acetylation increases as pH(i) rises,such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i),particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus,acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors. View Publication

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability,spheroid formation,apoptosis,DNA-binding and transcriptional activities,and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability,spheroid formation,and Gli-DNA binding and transcriptional activities,and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4,TRAIL-R2/DR5 and Fas,and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail,Slug and Zeb1. In addition,GANT-61 inhibited pluripotency maintaining factors Nanog,Oct4,Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability,spheroid formation,apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore,GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression,and suppression of Gli1,Gli2,Bcl-2,CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs,and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs. View Publication

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs,heterogeneity,and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs,but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques,such as coculture,physical manipulation,sorting,or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First,epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days,iPSCs showed upregulation of mesodermal genes (MSX2,NCAM,HOXA2) and downregulation of pluripotency genes (OCT4,LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes,reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype,expressed high levels of vimentin and N-cadherin,and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs,whereas adipogenic differentiation was limited,as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture,providing a robust,clinically applicable,and efficient system for generating MSCs from human iPSCs. View Publication

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date,most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-β (TGFβ) signaling pathways,and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however,the role of Wnt/β-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β-catenin signaling in mouse embryonic stem cells,we identified a compound,ID-8,that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8,we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements,serum,serum replacement,or albumin. Using this culture system,we have shown that several human pluripotent cell lines maintained pluripotency (textgreater20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds. View Publication

The interaction between HER2 and aryl hydrocarbon receptor (AhR) signaling cascades during mammosphere formation of MCF-7 cells was studied. A newly established clonal MCF-7 cell line (HER2-5),stably overexpressing HER2,showed significantly enhanced levels of AhR mRNA and protein compared with MCF-7 cells. AhR was required for the HER2-mediated induction of interleukin-6 mRNA and for mammosphere formation in HER2-5 and MCF-7 cells. Mammosphere forming efficiency was suppressed by an AhR antagonist in a dose-dependent manner,as well as by knockdown of AhR. Taken together,these results indicate that AhR enhances mammosphere formation by human HER2-overexpressing breast cancer cells. View Publication

Regenerative medicine is in need of solid,large animal models as a link between rodents and humans to evaluate the functionality,immunogenicity,and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model,genetically close to humans and readily used worldwide in clinical research. Until now,only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore,we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV,SB431542,PD0325901,and ascorbic acid via a novel,excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4,KLF4,SOX2,C-MYC). Endogenous pluripotency markers like OCT3/4,KLF4,SOX2,C-MYC,LIN28,NANOG,and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors,megakaryocytes,adipocytes,chondrocytes,and osteogenic cells. Thus,all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations. View Publication

The study of the regulatory signaling hierarchies of human heart development is limited by a lack of model systems that can reproduce the precise developmental events that occur during human embryogenesis. The advent of human pluripotent stem cell (hPSC) technology and robust cardiac differentiation methods affords a unique opportunity to monitor the full course of cardiac induction in vitro. Here,we show that stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a monolayer-based differentiation protocol that used transforming growth factor β superfamily ligands to generate cardiomyocytes. However,insulin did not repress cardiomyocyte differentiation in a defined protocol that used small molecule regulators of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation,we determined that the inhibitory effects by insulin required Wnt/β-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/β-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus,insulin and Wnt/β-catenin signaling pathways,as a network,coordinate to influence hPSC differentiation to cardiomyocytes,with the Wnt/β-catenin pathway dominant to the insulin pathway. Our study contributes to the understanding of the regulatory hierarchies of human cardiomyocyte differentiation and has implications for modeling human heart development. View Publication

Progestins play a deleterious role in the onset of breast cancer,yet their influence on existing breast cancer and tumor progression is not well understood. In luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancer,progestins induce a fraction of cells to express cytokeratin 5 (CK5),a marker of basal epithelial and progenitor cells in the normal breast. CK5(+) cells lose expression of ER and PR and are relatively quiescent,increasing their resistance to endocrine and chemotherapy compared to intratumoral CK5(-)ER(+)PR(+) cells. Characterization of live CK5(+) cells has been hampered by a lack of means for their direct isolation. Here,we describe optical (GFP) and bioluminescent (luciferase) reporter models to quantitate and isolate CK5(+) cells in luminal breast cancer cell lines utilizing the human KRT5 gene promoter and a viral vector approach. Using this system,we confirmed that the induction of GFP(+)/CK5(+) cells is specific to progestins,is dependent on PR,can be blocked by antiprogestins,and does not occur with other steroid hormones. Progestin-induced,fluorescence-activated cell sorting-isolated CK5(+) cells had lower ER and PR mRNA,were slower cycling,and were relatively more invasive and sphere forming than their CK5(-) counterparts in vitro. Repeated progestin treatment and selection of GFP(+) cells enriched for a persistent population of CK5(+) cells,suggesting that this transition can be semi-permanent. These data support that in PR(+) breast cancers,progestins induce a subpopulation of CK5(+)ER(-)PR(-) cells with enhanced progenitor properties and have implications for treatment resistance and recurrence in luminal breast cancer. View Publication

Nuclear reprogramming of adult somatic tissue enables embryo-independent generation of autologous,patient-specific induced pluripotent stem (iPS) cells. Exploiting this emergent regenerative platform for individualized medicine applications requires the establishment of bioequivalence criteria across derived pluripotent lines and lineage-specified derivatives. Here,from individual patients with type 1 diabetes (T1D) multiple human iPS clones were produced and prospectively screened using a battery of developmental markers to assess respective differentiation propensity and proficiency in yielding functional insulin (INS)-producing progeny. Global gene expression profiles,pluripotency expression patterns,and the capacity to differentiate into SOX17- and FOXA2-positive definitive endoderm (DE)-like cells were comparable among individual iPS clones. However,notable intrapatient variation was evident upon further guided differentiation into HNF4α- and HNF1β-expressing primitive gut tube,and INS- and glucagon (GCG)-expressing islet-like cells. Differential dynamics of pluripotency-associated genes and pancreatic lineage-specifying genes underlined clonal variance. Successful generation of glucose-responsive INS-producing cells required silencing of stemness programs as well as the induction of stage-specific pancreatic transcription factors. Thus,comprehensive fingerprinting of individual clones is mandatory to secure homogenous pools amenable for diagnostic and therapeutic applications of iPS cells from patients with T1D. View Publication

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels,under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm,mesoderm,and endoderm. In this chapter,we describe techniques for generation of EBs from human pluripotent stem cells. Once formed,the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research,drug discovery/development,and cellular transplantation therapy. View Publication