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Quality control of iPS Cells

Overview of Test Results

検査結果の概要

The iPS cell is called the pluripotent stem cell, which is a type of cell that has the ability to change into any cell type of the body (pluripotency). These pluripotent cells express the protein called “undifferentiation markers”; therefore, investigation of the undifferentiation markers in different ways is necessary to identify iPS cell. In addition, in vitro expansion of iPS cells infrequently accompanies abnormalities in the chromosome (deletion and/or duplication); therefore, the presence or absence of chromosome abnormality in the expanded iPS cells is necessary to be confirmed with karyotypic analysis.


Test for Undifferentiation Marker

Pluripotent cells express the proteins called “undifferentiation markers”, such as Oct3/4, Nanog, TRA1-60, and TRA1-81. To confirm whether the growing iPS cells show pluripotency, expression of the undifferentiation markers described above will be determined by qPCR assay and the immunostaining method, or the cell surface antigens, such as Tra-60 and Tra1-81, will be confirmed by FACS analysis.

Test for Undifferentiation Marker
Test for Undifferentiation Marker

Test for Undifferentiation Marker

Pluripotent cells express the proteins called “undifferentiation markers”, such as Oct3/4, Nanog, TRA1-60, and TRA1-81. To confirm whether the growing iPS cells show pluripotency, expression of the undifferentiation markers described above will be determined by qPCR assay and the immunostaining method, or the cell surface antigens, such as Tra-60 and Tra1-81, will be confirmed by FACS analysis.

Undifferentiated test with rBC2LCN

rBC2LCN has a high specificity to sugar chains present on the cell surface of undifferentiated human ES cells and human iPS cells, which can be used for detecting undifferentiated iPS cells. rBC2LCN can also be used to monitor for an increase or decrease of human ES/iPS cells in the culture condition


Karyotype Test

The repeated iPS cell passages (a process that transplants grown cells to new dishes) infrequently cause abnormalities in the chromosome (deletion and/or duplication); therefore, the presence or absence of chromosome abnormality in the grown iPS cells will be confirmed with the karyotypic analysis. The occurrence of chromosome abnormality may lead to an overgrowth of iPS cells or inhibition of the differentiation into specific cells; therefore, it is preferable to conduct the test in each case when the important stock is created.

Karyotype Test
Karyotype Test

Examples of Chromosome Test Results

Cells: 253G4(P40) / Differential staining: G / No. of cells analyzed: 50 / Karyotype analysis results: 46, XX[20]


Triploblastic Differentiation Test

To confirm that iPS cells show pluripotency, the tissue called the “embryoid body” is induced in vitro by reproducing the developmental process and the presence of the triploblastic tissues (endoderm, mesoderm, ectoderm) in the tissue will be confirmed. The ectoderm marker (PAX6, MAP2), mesoderm marker (T, MSX1), and endoderm marker (SOX17, AFP) will be confirmed by qPCR and immunostaining.

Triploblastic Differentiation  Test
Triploblastic Differentiation  Test
Triploblastic Differentiation  Test

The adherent culture will be conducted for the embryoid body formed from 201B7 cells, and the triploblastic differentiation will be confirmed by the immunostaining of each marker. Left: confirmation of endoderm differentiation by AFP staining, Center: confirmation of mesoderm differentiation by α-SMA staining, Right: confirmation of ectoderm differentiation by MAP2 staining.