A chemical probe that labels human pluripotent stem cells.

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

[1]  H. Nakauchi,et al.  The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype , 2001, Nature Medicine.

[2]  N. Greenfield,et al.  Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes. , 1977, Biochimica et biophysica acta.

[3]  S. Kane,et al.  Alteration of substrate specificity by mutations at the His61 position in predicted transmembrane domain 1 of human MDR1/P-glycoprotein. , 1997, Biochemistry.

[4]  D. Williams,et al.  Chloromethyl-X-rosamine (MitoTracker Red) photosensitises mitochondria and induces apoptosis in intact human cells. , 1999, Journal of cell science.

[5]  K. Ueda ABC Proteins Protect the Human Body and Maintain Optimal Health , 2011, Bioscience, biotechnology, and biochemistry.

[6]  C. Gilissen,et al.  Hematopoietic stem cells exhibit a specific ABC transporter gene expression profile clearly distinct from other stem cells , 2010, BMC pharmacology.

[7]  B. Mannervik,et al.  Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases. , 2002, Analytical Biochemistry.

[8]  Jun Li,et al.  A fluorescent rosamine compound selectively stains pluripotent stem cells. , 2010, Angewandte Chemie.

[9]  M. Murakami,et al.  The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells , 2003, Cell.

[10]  M. Uhlén,et al.  Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs , 2009, Drug Metabolism and Disposition.

[11]  H. Schöler,et al.  Oct‐4: Gatekeeper in the Beginnings of Mammalian Development , 2001, Stem cells.

[12]  Peter J. Donovan,et al.  Derivation of pluripotent stem cells from cultured human primordial germ cells , 1998 .

[13]  M. Mann,et al.  Analysis of proteins and proteomes by mass spectrometry. , 2001, Annual review of biochemistry.

[14]  P. Heytler uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones. I. Some characteristics of m-Cl-CCP action on mitochondria and chloroplasts. , 1963, Biochemistry.

[15]  Norio Nakatsuji,et al.  Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. , 2006, Biochemical and biophysical research communications.

[16]  Rita Padányi,et al.  High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. , 2008, Biochimica et biophysica acta.

[17]  K. Moitra,et al.  Evolution of ABC transporters by gene duplication and their role in human disease , 2011, Biological chemistry.

[18]  D. Sahoo,et al.  An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells , 2011, Nature Biotechnology.

[19]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[20]  R. Benz,et al.  The kinetic mechanism by which CCCP (carbonyl cyanidem-Chlorophenylhydrazone) transports protons across membranes , 2005, The Journal of Membrane Biology.

[21]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[22]  P. Twentyman,et al.  On the relationship between the probenecid‐sensitive transport of daunorubicin or calcein and the glutathione status of cells overexpressing the multidrug resistance‐associated protein (MRP) , 1995, International journal of cancer.

[23]  A. Schnerch,et al.  Distinguishing Between Mouse and Human Pluripotent Stem Cell Regulation: The Best Laid Plans of Mice and Men , 2010, Stem cells.

[24]  H. Nakauchi,et al.  Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. , 2008, Blood.

[25]  Peter W. J. Rigby,et al.  A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo , 1990, Nature.

[26]  S. Yamanaka,et al.  Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts , 2013, Proceedings of the National Academy of Sciences.

[27]  C. Eaves,et al.  ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. , 2004, Blood.

[28]  T. Amachi,et al.  Nonequivalent Nucleotide Trapping in the Two Nucleotide Binding Folds of the Human Multidrug Resistance Protein MRP1* , 2000, The Journal of Biological Chemistry.

[29]  J. Thomson,et al.  Preimplantation Human Embryos and Embryonic Stem Cells Show Comparable Expression of Stage‐Specific Embryonic Antigens , 2002, Stem cells.

[30]  I. Tamai,et al.  Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells. , 1990, The Journal of biological chemistry.

[31]  Gerd Schmitz,et al.  Real-time reverse transcription-PCR expression profiling of the complete human ATP-binding cassette transporter superfamily in various tissues. , 2003, Clinical chemistry.

[32]  Xue-Jun Li,et al.  Lack of ABCG2 Expression and Side Population Properties in Human Pluripotent Stem Cells , 2009, Stem cells.

[33]  J. Thomson,et al.  Isolation of a primate embryonic stem cell line. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Uesugi,et al.  A mitochondrial surface-specific fluorescent probe activated by bioconversion. , 2011, Angewandte Chemie.

[35]  K. Niakan,et al.  BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages , 2011, Cell stem cell.

[36]  Norio Nakatsuji,et al.  A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. , 2012, Cell reports.

[37]  T. Graf Faculty Opinions recommendation of Induction of pluripotent stem cells from adult human fibroblasts by defined factors. , 2007 .

[38]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[39]  Tetsuhiro Kikuchi,et al.  Small‐molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells , 2011, Journal of neuroscience research.

[40]  H. Green,et al.  An established pre-adipose cell line and its differentiation in culture. , 1974, Cell.

[41]  J. Schellens,et al.  Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. , 2002, Molecular cancer therapeutics.

[42]  I. Holland,et al.  ABC transporters: bacterial exporters-revisited five years on. , 1999, Biochimica et biophysica acta.

[43]  H. Okumura,et al.  Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function , 2001, International journal of cancer.

[44]  I. Pastan,et al.  Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Nichols,et al.  Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells , 2003, Cell.

[46]  Janet Rossant,et al.  Interaction between Oct3/4 and Cdx2 Determines Trophectoderm Differentiation , 2005, Cell.

[47]  Hanmin Wang,et al.  Cardiac induction of embryonic stem cells by a small molecule inhibitor of Wnt/β-catenin signaling. , 2011, ACS chemical biology.

[48]  G. Stein,et al.  Differential regulation of H4 histone gene expression in 3T3‐L1 pre‐adipocytes during arrest of proliferation following contact inhibition or differentiation and its modulation by TGFβ1 , 1992 .

[49]  Y. Bergman,et al.  A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells , 1995, Molecular and cellular biology.

[50]  Jun-Seok Lee,et al.  Combinatorial rosamine library and application to in vivo glutathione probe. , 2007, Journal of the American Chemical Society.

[51]  Paul A. Clemons,et al.  The binding of fluorophores to proteins depends on the cellular environment. , 2011, Angewandte Chemie.