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| 1 | Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines显示文摘BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines. | Samah Abdulaali Alharbi Dmitry A Ovchinnikov Ernst Wolvetang | 2021 | World Journal of Gastroenterology2021，27，15： | 2 |
| 2 | Mitochondrial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang EJ Johnson KL Krauer K | | 0，，1： | 1 |
| 3 | Reactive oxygen species and their contribution to pathology in down syndrome显示文摘 | de Haan JB Wolvetang EJ Cristiano F | 1997 | Adv Pharmacol1997，38，： | 1 |
| 4 | Adipoeyte differentiation in human embryonie stem cells transduced with Oct4 shRNA lentivirus显示文摘 | Hannan NR Wolvetang El | 2009 | Stem Cells Dev2009，18，4： | 1 |
| 5 | Mitochon- drial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang E J Johnson K L Krauer K | 1994 | FEBS Letters1994，339，12： | 1 |
| 6 | Reactive oxygen species and their contribution to pathology in Down syndrome显示文摘 | de Haan JB Wolvetang EJ Cristiano F | 1997 | Adv Phar-macol1997，38，： | 1 |
| 7 | Mitochondrial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang E J Krauer K | 1994 | Febs Letter1994，339，12： | 1 |
| 8 | Mechanisms of long non- coding RNAs in mammalian nervous system development, plastici- ty, disease, and evolution显示文摘 | Briggs JA Wolvetang E J Mattick JS | 2015 | Neuron2015，88，5： | 1 |
| 9 | Mitochondrial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang EJ Johnson KL KrauerK | 1994 | FEBS Lett1994，339，12： | 1 |
| 10 | Mitochondrial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang EJ Johnson KL Krauer K | 1994 | FEBS Lett1994，339，12： | 1 |
| 11 | Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro显示文摘 | Thouas GA Trounson AO Wolvetang EJ | 2004 | Biol Reprod2004，76，6： | 1 |
| 12 | Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry显示文摘AIM:The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS:Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays andembryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy. RESULTS:Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripo-tency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to singlecell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival. CONCLUSION:The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments. | Dmitry A Ovchinnikov Jennifer P Turner Drew M Titmarsh Nilay Y Thakar Dong Choon Sin Justin J Cooper-White Ernst J Wolvetang | 2012 | World Journal of Stem Cells2012，4，7： | 1 |
| 13 | Defined high protein content surfaces for stem cell culture显示文摘 | Michael R. Doran Jessica E. Frith Andrew B.J. Prowse Jane Fitzpatrick Ernst J. Wolvetang Trent P. Munro Peter P. Gray Justin J. Cooper-White | 2010 | Biomaterials2010，，19： | 1 |
| 14 | Mitochondria/dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro 显示文摘 | Thouas G A Trounson A O Wolvetang E J | 2004 | Biol Reprod2004，71，： | 1 |
| 15 | Mitochondrial respiratory chain inhibitors induce apoptosis显示文摘 | Wolvetang EJ Johnson KL KrauerK | 1994 | FEBS Lett1994，339，12： | 1 |
| 16 | Mitoehondrial dysfunction in mouse ooeytes results in preimplantation embryo ar- rest in vitro显示文摘 | Thouas GA Trounson AO Wolvetang E J | 2004 | Biol Reprod2004，71，6： | 1 |
| 17 | 3-Methyladenine, an inhibitor ofautophagy, has multiple effects on metabolism 显示文摘 | Caro LH Plomp PJ Wolvetang EJ | 1988 | EurJ Biochem1988，175，2： | 1 |
| 18 | Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro显示文摘 | Thouas G A Trounson A O Wolvetang E J | 2004 | Biology of Reproduction2004，71，6： | 1 |
| 19 | Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin显示文摘 | Wolvetang E J Larm J A Moutsoulas P | 1997 | Cell Growth Differentiation1997，7，10： | 1 |
| 20 | Brief Report: Complement C5a Promotes Human Embryonic Stem Cell Pluripotency in the Absence of FGF2显示文摘 | Owen A. Hawksworth Liam G. Coulthard Stephen M. Taylor Ernst J. Wolvetang Trent M. Woodruff | 2014 | Stem Cells2014，，12： | 1 |
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