| 背景 | The CDy1 dye was developed as part of a collaboration including the Agency for Science, Technology and Research (A*STAR) in Singapore. Researchers screened the Diversity-Oriented Fluorescence Library (DOFL) to identify novel small-molecule imaging probes specific for pluripotent stem cells and discovered the fluorescent rosamine compound, CDy1, could selectively stain live ESCs and iPSCs1. The stain could be used to identify and isolate ESCs from a mixture containing MEF feeder cells. Additional characterization of CDy1 revealed that CDy1 can be used to determine iPSC reprogramming at 10 days post retroviral infection of the Oct-4, Sox2, KLF4 and c-Myc transcription factors, while the GFP reporter system did not show expression of GFP until 17 days post retroviral infection, indicating that Stem Cell CDy1 Dye can be used to determine iPSC reprogramming at earlier time points than genetic reporter systems1.Conventional methods used to identify stem cells include characterization by colony morphology, screening for expression of alkaline phosphatase and immunostaining for stem cell markers. Stem Cell CDy1s ability to stain living cells provides a significant benefit over methods such as immunostaining and alkaline phosphatase detection which require cell fixation, thereby rendering the cells unusable for future experiments. Another commonly used staining method, the Aldefluor method, uses BODIPY-aminoacetaldehyde, which is a substrate of aldehyde dehydrogenase, to stain hematopoietic stem cells and some cancer stem cells. A comparison of the CDy1 dye and the Aldefluor stain revealed that Aldefluor does not stain ESCs1. The CDy1 dye has also been shown by subsequent published studies to be selective for other stem cell types, including neural stem cells, as shown by work from the laboratory of Dr. Geoffrey W. Osborne at the University of Queensland2, and cancer stem cells, as demonstrated by research from the laboratory of Dr. Robert Hawley at George Washington University3.The drawbacks and limitations presented by conventional methods makes Active Motifs Stem Cell CDy1 Dye an ideal solution for the identification and isolation of stem cells. With CDy1s ability to stain living cells, there is no longer a need to sacrifice precious stem cell samples. Cells that have been stained will have the same growth rate, morphology and differentiation capability as unstained stem cells. Simply add Stem Cell CDy1 Dye to the culture medium of living cells. Following a 1 hour incubation and 3 hour destaining, cells are washed and prepared for imaging4. The spectral properties of the CDy1 dye enables detection of stained cells using fluorescence microscopy with TRITC or Cy3 filter sets, while a 488 nm laser and a PE-Texas Red filter can be used for analysis with flow cytometry4. Figure 1: CDy1 Staining Coincides with Expression of Stem Cell Markers.Mouse embryonic stem cells (mESCs) grown on a mouse embryonic fibroblast (MEF) feeder layer were stained without fixation with Stem Cell CDy1 dye. CDy1 staining was visualized by fluorescent microscopy. The cells were then fixed in 4% paraformaldehyde, stained with Oct-4 antibody and visualized with FITC-conjugated secondary antibody. The images show that mESC aggregates, but not MEF feeder cells, stain positive for both CDy1 (red) and Oct-4 (green). Scale bar, 100 μm. The images were kindly provided courtesy of Dr. Y-T Chang at the National University of Singapore, Republic of Singapore.References- Im, C.N. et al. (2010) Angew. Chem. Int. Ed. Engl., 49: 7497-7500.
- Vukovic, J. et al. (2013)Stem Cells Dev. doi:10.1089/scd.2012.0660.
- Hawley, T.S. et al.(2013)Am J Hematol. 88, 265-272.
- Kang, N.Y. et al. (2011) Nat. Protoc., 6: 1044-1052.
 Figure 1: FACS analysis of mESCs and MEFs stained by Stem Cell CDy1 Dye.Mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) were stained with the Stem Cell CDy1 Dye and then analyzed by flow cytometry (FACS) using side scatter (SSC) and a PE-Texas-Red channel. CDy1 enables clear differentiation and isolation of the pluripotent mESCs (red) vs. the MEFs (blue). Protocol information for flow cytometry analysis of CDy1 stained mESCs is referenced in Kang N.-Y. et al.(2011)Nat Protoc. Figure 2: Stem Cell CDy1 Dye Selectively Stains Pluripotent Stem Cells.Human embryonic stem cells (hESC), mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSC) were grown on feeder cells and stained without fixation with either DMSO as a negative control or Stem Cell CDy1 dye. The cells were then visualized by fluorescent microscopy. The images show selective staining of all types of embryonic stem cells (hESCs, mESCs and iPSCs) within the mixed cell cultures by the CDy1 dye. Furthermore, punctate staining is observed within the formed stem cell colonies. This staining pattern results from the CDy1 dye selectively staining pluripotent cells, and not differentiated cells, within the colonies as some of the cells have already begun the process of early stage differentiation. The DMSO stain of hESCs is provided as a representative negative control image. These images were kindly provided courtesy of Rhoda Mondeh-Lowor at California State University, San Marcos. Figure 3: Stem Cell CDy1 Dye Selectively Stains iPSCs in Live Mixed Cell Populations.Human induced pluripotent stem cells (hiPSCs) derived from umbilical cord blood were grown on a mouse embryonic fibroblast (MEF) feeder layer and stained without fixation with Stem Cell CDy1 dye. The cells were then visualized using phase contrast and fluorescent (TRITC) filters. The images show selective staining of hiPSC aggregates within the mixed cell culture by the CDy1 dye. These images were kindly provided courtesy of Dr. H. Zaehres at the Max Planck Institute for Molecular Biomedicine in Germany.Figure 4: CDy1 Staining Coincides with Expression of Stem Cell Markers.Mouse embryonic stem cells (mESCs) grown on a mouse embryonic fibroblast (MEF) feeder layer were stained without fixation with Stem Cell CDy1 dye. CDy1 staining was visualized by fluorescent microscopy. The cells were then fixed in 4% paraformaldehyde, stained with Oct4 antibody and visualized with FITC-conjugated secondary antibody. The images show that mESC aggregates, but not MEF feeder cells, stain positive for both CDy1 (red) and Oct4 (green). Scale bar, 100 μm. The images were kindly provided courtesy of Dr. Y-T Chang at the National University of Singapore, Republic of Singapore. Figure 5: CDy1 dye is specific for pluripotent stem cells unlike alkaline phosphatase staining methods.Human embryonic stem cells (hESC), mouse embryonic stem cells (mESC), and induced pluripotent stem cells (iPSC) were grown on feeder cells and stained without fixation with either Stem Cell CDy1 dye (CDy1, red) or alkaline phosphatase (ALP, green) using a competitors live ALP staining kit. The images show that, unlike alkaline phosphatase staining methods, CDy1 is highly specific for pluripotent stem cells and does not produce background signal in other cells, such as feeder or differentiated cells. In contrast, alkaline phosphatase staining only differentially stains pluripotent cells at higher levels but still produces background staining in feeder cells and differentiated cells. These images were kindly provided courtesy of Rhoda Mondeh-Lowor at California State University, San Marcos. |
