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Application Notes

Importance of Accurate Cell Counting in Differentiation of Nerve Cells: iPS PORTAL, Inc.


  • “Human induced pluripotent stem cells (iPSCs) can provide a promising source of mid-brain dopaminergic neurons for cell replacement therapy used to treat Parkinson’s disease. Prior to the clinical application of human iPSCs, it is critical to eliminate tumorigenic or inappropriate cells and to enrich the dopaminergic progenitor cells. This can be achieved with cell sorting using a cell surface antigen, CORIN. Cell densities at passage and the initiation of differentiation are determined by a protocol, indicating the importance of accurate cell counting. Precise counting of iPSC cultures by traditional hemocytometer is difficult because of colony formation. Using the Olympus Cell Counter model R1, cells are properly identified regardless of whether they are single cells or colonized, enabling researchers to conduct experiments with high reproducibility and speed. Furthermore, a microscope with high resolution such as Olympus FV3000 is useful for identification of iPSCs and iPSC-derived cells via immunostaining of cell surface markers or transcription factors. We anticipate that the lineup of Olympus products will prove valuable to many aspects of iPSC research.”
  • Counting of cells (density)
    Cell counter
  • Observation of cell morphology
    Phase-contrast microscope
  • Detection of CORIN+ signal
    Confocal laser scanning microscope

  • iPS Portal Inc provides many solutions for drug discovery and regenerative medicine through its Nobel Prize winning state-of-art iPS cell technologies. Olympus Corporation collaborates with iPS Portal Inc through the Instrument Evaluation Services.

  • For research use only.

Differentiation of iPSC-Derived Nerve Cells Depends on Initial Cell Density.

Experimental Results

  • Positive Control group
     

    5 x 106 cells/well
  • Initiation of Differentiation group
    Differentiation was started and cells were counted and placed into positive and negative control groups based upon concentration.

    Cell counter
  • Negative Control group
     

    1 x106 cells/well
  • Normal cell morphology was observed
  • On Differentiation Culture Day 2
    Phase-contrast images were obtained on differentiation culture day 2. In the positive control group (differentiation was started at a density of 5 x 106 cells/well), normal cell morphology was observed. While, in the group where differentiation was started at a density of 1 x 106 cells/ well, cell morphology was distinctly different from the control group.

    Phase-contrast microscope
  • Abnormal cell morphology was observed
  • CORIN+ signal (green) with reasonable intensity and CORIN+ cell ratio was detected
  • On Differentiation Culture Day 12
    Immunostaining was performed by the use of anti-CORIN antibody. In the positive control group, normal CORIN positive (CORIN+) signal with reasonable signal intensity and the percentage of CORIN+ cells was observed. While, in the group where differentiation was started at a density of 1 x 106 cells/well, signal intensity and the percentage of CORIN+ cells were lower than those of control group, although CORIN+ signal was observed.

    Confocal laser scanning microscope
  • CORIN+ signal was lower than that of control group

Conclusion

After day 12 of differentiation, sorted CORIN+ cells form spheroids maturating toward dopaminergic neurons. The efficiency of inducing differentiation to dopaminergic neurons depends on the density of the sorted CORIN+ cell at initiation, indicating the importance of an accurate cell count.
Sample preparation, image acquisition and data are courtesy of iPS PORTAL Inc.
Banner image: Human iPSC colony
Image data are courtesy of Isao Asaka, Ph.D. Center for iPS Cell Research and Application. Dept. of Fundamental Cell Technology, Kyoto University.

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