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[Chemical Probe for human iPS/ES cells]Kyoto Probe 1 (KP-1)

  • Able to distinguish human iPS cells and/or human ES cells from differentiated cells
  • Usable for flow cytometry or live cell imaging
  • Staining while culturing

λex 515 nm

λem 529 nm

Φ 0.45

Fig.1. Fluorescent feature of KP-1.
Fig.1. Fluorescent feature of KP-1.

The feature of KP-1.

1. Able to distinguish human iPS/ES cells from differentiated cells

Fig.2. (A、B) Dyeing iPS colony, formed on the feeder cells, with KP-1. (A: bright field image, B: fluorescent image).

Fig.2. (A、B) Staining iPS colony, formed on the feeder cells, with KP-1. (A: bright field image, B: fluorescent image).

Mechanism of human iPS/ES cell specific staining by KP-1

Mechanism of human iPS/ES cell specific staining by KP-1

KP-1 has cell permeability and localizes in the mitochondria in the human pluripotent stem cells regardless of mitochondrial membrane potential. After the differentiation of the cells, KP-1 is eliminated from the cells by the ABC transporters which do not work in the pluripotent stem cells, and the cells become non-fluorescent (Cell Rep. 2014).

Fig.3.

Fig.3. (C、F) Staining colony of partially differentiated human iPS cells with KP-1. (C: bright field image, F: fluorescent image). Surrounding region of the colony, in which cells were not differentiated, were specifically stained. (D、G) Staining a colony of human ES cells with KP-1. (D: bright field image, G: fluorescent image). (E、H) Dyeing colony of partially differentiated human ES cells with KP-1. (E: bright field image, H: fluorescent image). Differentiated cells were not stained.

2. Usable for flow cytometry or live cell imaging

Fig.4. FACS analysis of human iPS cells

Fig.4. FACS analysis of human iPS cells
(Left panel) When the mixture of human iPS cells and feeder cells were analyzed, most SSEA-4, pluripotent stem cell marker, positive cells were also positive to KP-1. SSEA-4 is pluripotent stem cell. (Center and Right panel) 99.18% of human iPS cells were stained by KP-1.

3. Isolation of human iPS cells by flow cytometry.

Fig.5. Flow cytometry to various somatic cells with KP-1.

Fig.5. Flow cytometry to various somatic cells with KP-1.
When analyzed by flow cytometry with KP-1 probe, human iPS cells are able to be isolated from somatic cells except for nerve cell.

 

[Detail] Mechanism of recognition to distinguish undifferentiated cells and differentiated cells
 KP-1 localizes at mitochondria in the human stem cells, indicating that the difference of the fluorescent intensity derives from mitochondria. It had been reported that expression level of ABC transporters were varied before and after the differentiation. Therefore, members of ABC transporters that support the transport of chemicals were carefully investigated, then mRNA of ABCB1and ABCG2 were identified to be increased after the induction of the differentiation. Fluorescence of KP-1 in the cell expressing ABCB1 and ABCG2 were measured. The model cell expressing excess of ABCB1and ABCG2 was constructed by the genetic modification from KB3-1 cell in which expression level of ABC transporters was initially low. The fluorescence of KP-1 in these cells was not observed. However, after the addition of cyclosporin A (CsA) or Fumitremorgin C (FTC), inhibitors of ABCB1 and ABCG2 respectively, fluorescence of KP-1 became to be observed. These results indicate that KP-1 is substrate of ABCB1 and ABCG2, and is discharged from the inside of the cells. Next, human ES cells and differentiated cells were stained by KP-1, and analyzed by flow cytometer. Fluorescent intensity in the human ES cells were measured to be about 100 times brighter than that in the differentiated cells. However, increase in the fluorescence in the differentiated cells was observed after the treatment of CsA or FTC. These results suggest that fluorescence from KP-1 depends on the activities of ABCB1 and ABCG2 on the cell membrane. (See the figure on “Mechanism of human iPS/ES cell specific staining by KP-1” section.)

Reference
Ting-Fang Kuo, Di Mao, Nao Hirata, Bilon Khambu, Yasuhisa Kimura, Eihachiro Kawase, Hiroki Shimogawa, Makoto Ojika, Norio Nakatsuji, Kazumitsu Ueda, and Motonari Uesugi.
Selective Elimination of Human Pluripotent Stem Cells by a Marine Natural Product Derivative J. Am. Chem. Soc., 2014, 136 (28), pp 979-9801
Hirata N, Nakagawa M, Fujibayashi Y, Yamauchi K, Murata A, Minami I, Tomioka M, Kondo T, Kuo TF, Endo H, Inoue H, Sato S, Ando S, Kawazoe Y, Aiba K, Nagata K, Kawase E, Chang YT, Suemori H, Eto K, Nakauchi H, Yamanaka S, Nakatsuji N, Ueda K, Uesugi M. Cell Rep. 2014 6:1165-1174.
Price
Code No. Product Size Price Protocol MSDS
GC7001-01 Kyoto Probe 1 (KP-1) 10μg×5 $598 Download
377KB
Download
97KB
GC7001-02 Kyoto Probe 1 (KP-1) 10μg×10 $798

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