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1.
Development ; 142(24): 4253-65, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26493401

ABSTRACT

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line.


Subject(s)
Biomarkers/metabolism , Cell Differentiation , Endoderm/cytology , Hepatocytes/cytology , Pluripotent Stem Cells/cytology , Animals , Antibodies/metabolism , Cell Line , Cell Separation , Ectoderm/cytology , Hepatocytes/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Kinetics , Mesoderm/cytology , Mice, Inbred BALB C , Pancreas/embryology , Pluripotent Stem Cells/metabolism , Staining and Labeling
2.
Development ; 140(15): 3285-96, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23861064

ABSTRACT

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.


Subject(s)
Cyclic AMP/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Activins/metabolism , Cell Aggregation , Cell Culture Techniques , Cell Differentiation , Endoderm/cytology , Endoderm/metabolism , Humans , Nodal Protein/metabolism , Signal Transduction , Transcriptome
3.
Cell Stem Cell ; 8(2): 228-40, 2011 Feb 04.
Article in English | MEDLINE | ID: mdl-21295278

ABSTRACT

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression, we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly, we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts.


Subject(s)
Activins/metabolism , Bone Morphogenetic Proteins/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Nodal Protein/metabolism , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation/physiology , Flow Cytometry , Humans , Mice , Pluripotent Stem Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction
4.
Drug Chem Toxicol ; 32(3): 186-90, 2009.
Article in English | MEDLINE | ID: mdl-19538014

ABSTRACT

HESA-A is a natural compound of herbal-marine origin with cytotoxic and antitumor effects. The anticancer effects of HESA-A has been the subject of both in vivo and in vitro studies. This study was to investigate the mechanism of HESA-A teratogenicity. We assessed the HESA-A-induced apoptosis in mouse fetus in vitro by using the vital staining and TUNNEL methods. HESA-A, in lower doses, had no significant effect on apoptosis but, in higher doses of 20 and 40 muL, increased cell death. A dose of 100 muL induced the cell death with both apoptosis and necrosis mechanisms. HESA-A changed the cell-death pattern; in moderate doses of the drug, the apoptosis-to-necrosis ratio was more than 1, and in higher doses, this ratio was less than 1.


Subject(s)
Anticarcinogenic Agents/toxicity , Apoptosis/physiology , Embryo, Mammalian/drug effects , Plant Preparations/toxicity , Teratogens/toxicity , Animals , Anticarcinogenic Agents/chemistry , Apoptosis/drug effects , Dose-Response Relationship, Drug , Embryo Culture Techniques , Embryo, Mammalian/pathology , Female , In Situ Nick-End Labeling , Indicators and Reagents/chemistry , Male , Metals, Heavy/analysis , Mice , Mice, Inbred BALB C , Necrosis/chemically induced , Neutral Red/chemistry , Plant Preparations/chemistry , Pregnancy
5.
J Biol Chem ; 283(31): 21371-81, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18544539

ABSTRACT

The c-Myc transcription factor is commonly dysregulated in cancer. c-Myc also sensitizes cells to apoptosis induced by a variety of toxic events. c-Myc turnover is rapid and mediated by the proteasome and intracellular calpains. Therefore, c-Myc accumulation could contribute to cell death associated with protease inhibitors. We investigated the response of c-Myc-positive and c-Myc-negative rat fibroblast cells to proteasome and calpain inhibitors. Apoptosis induced by the proteasome inhibitor, epoxomycin, was c-Myc-independent, whereas apoptosis induced by the calpain inhibitor, PD150606, or by knockdown of calpain small subunit 1 (CPNS1) was strongly dependent on c-Myc. HL60 cells knocked down for c-Myc expression exhibited reduced calpain activity and decreased sensitivity to PD150606 but not epoxomycin. Calpain inhibitor- or CPNS1 knockdown-induced apoptosis in c-Myc-positive fibroblasts was associated with cell detachment and could be prevented by plating cells on fibronectin, suggesting an anoikis phenomenon. c-Myc stimulated calpain activity by suppressing calpastatin expression, the endogenous calpain inhibitor. Knockdown of calpastatin in c-Myc-negative cells led to a restoration of calpain activity, enhanced cell growth, cell cycle redistribution, anchorage independence, and tumorigenicity in immunodeficient mice. Taken together, these results indicate that c-Myc regulates calpain activity through calpastatin; apoptosis induced by calpain inhibition is dependent on c-Myc, and calpastatin knockdown promotes transformation in c-Myc-negative cells.


Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/chemistry , Proto-Oncogene Proteins c-myc/metabolism , Acrylates/pharmacology , Animals , Apoptosis , Cell Adhesion , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Fibronectins/metabolism , HL-60 Cells , Humans , RNA, Small Interfering/metabolism , Rats
6.
Mol Cancer Ther ; 7(3): 483-91, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18347136

ABSTRACT

Econazole is an azole antifungal with anticancer activity that blocks Ca(2+) influx and stimulates endoplasmic reticulum (ER) Ca(2+) release through the generation of mitochondrial reactive oxygen species (ROS), resulting in sustained depletion of ER Ca(2+) stores, protein synthesis inhibition, and cell death. c-Myc, a commonly activated oncogene, also promotes apoptosis in response to growth factor withdrawal and a variety of chemotherapeutic agents. We have investigated the role of c-myc in regulating sensitivity to econazole. Here, we show that c-myc-negative cells are profoundly resistant to econazole. c-Myc-negative rat fibroblasts failed to generate mitochondrial ROS in response to econazole and consequently failed to deplete the ER of Ca(2+). HL60 cells knocked down for c-myc expression also displayed decreased ROS generation and decreased econazole sensitivity. Addition of H(2)O(2) restored sensitivity to econazole in both c-myc-negative rat fibroblasts and c-myc knocked-down HL60 cells, supporting a role for ROS in cell death induction. c-Myc-negative cells and HL60 cells knocked down for c-myc have reduced mitochondrial content compared with c-myc-positive cells. The hypoxia sensor, hypoxia-inducible factor-1alpha (HIF-1alpha), interacts antagonistically with c-myc and also regulates mitochondrial biogenesis. Knockdown of HIF-1alpha in c-myc-negative cells increased mitochondrial content restored ROS generation in response to econazole and increased sensitivity to the drug. Taken together, these results show that c-myc and HIF-1alpha regulate sensitivity to econazole by modulating the ability of the drug to generate mitochondrial ROS.


Subject(s)
Econazole/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Mitochondria/physiology , Proto-Oncogene Proteins c-myc/physiology , Animals , Calcium/metabolism , Endoplasmic Reticulum/metabolism , HL-60 Cells , Humans , Rats , Reactive Oxygen Species/metabolism
7.
Cytometry A ; 71(7): 475-85, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17458879

ABSTRACT

BACKGROUND: Calpains are intracellular, calcium-sensitive, neutral cysteine proteases that play crucial roles in many physiological and pathological processes. Calpain regulation is complex and activity is poorly correlated with calpain protein levels. Therefore a full understanding of calpain function requires robust methods for measuring activity. METHODS: We describe and characterize a flow cytometric method for measuring calpain activity in live cells. This method uses the BOC-LM-CMAC reagent that readily diffuses into cells where it reacts with free thiols to enhance retention. RESULTS: We show that the reagent is cleaved specifically by calpains and follows saturation kinetics. We use the assay to measure calpain activation following PDGF stimulation of rat fibroblasts. We also show that the calpain inhibitor PD150606 inhibits calpain with a K(i) of 12.5 muM and show that Mek inhibitors PD89059 and U0126 also suppress calpain activity. We also show that the assay can measure calpain activity in subpopulations of cells present in unfractionated cord blood or in HL60 human myelomonocytic leukemia cells. CONCLUSION: Taken together, these experiments demonstrate that this assay is a reliable and useful method for measuring calpain activity in multiple cell types.


Subject(s)
Calpain/metabolism , Flow Cytometry/methods , Acrylates/pharmacology , Animals , Calpain/analysis , Calpain/antagonists & inhibitors , Cells, Cultured , Coumarins/pharmacology , Dipeptides/pharmacology , Enzyme Activation/drug effects , Fetal Blood/cytology , Fetal Blood/enzymology , Fetal Blood/metabolism , HL-60 Cells , Humans , Kinetics , Models, Biological , Platelet-Derived Growth Factor/pharmacology , Rats , Substrate Specificity , Temperature
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