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Differentiation Induction

“Differentiation induction” is the creation of desired cells from iPS cells, i.e. pluripotent stem cells. There is a wide range of variation in differentiated cells; therefore, there is also a wide range of variation in the differentiation induction method. All differentiated cells, however, are developed from three germ layers called the “ectoderm”, “mesoderm”, and “endoderm”. As a first step, the differentiation into the “germ layer” that develops the desired cells is induced to obtain the differentiated cells from iPS cells. Here, the method for inducing differentiation into these three germ layers, i.e. “ectoderm”, “mesoderm”, and “endoderm”, is explained. The point of differentiation induction is the concentration of the “growth factor” and timing.

Differentiation into the Ectoderm

Representative tissues/organs derived from the ectoderm

Method for inducing differentiation into the neural ectoderm

Reference
Chambers SM. et al.,Nat Biotechnol. 27, 275-280(2009)

[Neural ectoderm] Induction of dopaminergic neuron


Differentiation into the Mesoderm

Representative tissues/organs derived from the mesoderm

Reference
Sumi, T. et al., Development 135, 2969?2979 (2008).

[Mesoderm] Induction of cartilage

The iPS cells culturing with feeder-free method are used for differentiation. Add WNT and ACTIVIN to the culture medium and then differentiate for 3 days.

Remove WNT and ACTIVIN, and differentiate into the mesoderm by the addition of BMP4.


Differentiation into the Endoderm

Representative tissues/organs derived from the endoderm

Endoderm

Reference
Development 59, 2094?2101 (2010).
Nature 470, 105?109 (2011).

[Endoderm] Induction of hepatocyte


Induction to Oligodendrocyte (Professor Okano’s Method)

1. Article: Kuroiwa-Numasawa et al., Stem Cell Reports, 2015

2. Features of the differentiation induction method

The differentiation induction in the embryoid body (EB) state and subsequent repeated neurosphere (NS) formation after cell separation allow high-efficiency differentiation induction to oligodendrocyte within about 55-99 days. The mechanism of Pelizaeus-Merzbacher Disease, an incurable disease of the nervous system, was elucidated by this differentiation induction method in Prof. Okano’s laboratory at Keio University.

3. Scheme of the differentiation induction method

*:DSB: Dorsomorphin, SB431542, BIO

4. Practical example of the differentiation induction method

5. Point of operation

The point of operation is the creation of EB and NS with a uniform size (40 μm – 100 μm) using various devices for sphere formation.


Induction to Dopaminergic Neuron (Professor. Takahashi’s Method)

1. Article: Doi et al., Stem Cell Reports, 2014

Patent number: Publication number 2013-501502 Method for inducing differentiation into neural progenitor cells from pluripotent stem cells

2. Features of the differentiation induction method

Start with iPS cells cultured with StemFit®AK02N and iMatrix-511. The differentiation into dopamine-producing nerve cells is induced in combination with adherent culture and suspension culture while reacting with each growth factor and low molecular weight compound. The cells suitable for cell transplantation can be purified by sorting out dopamine-producing nerve cells during differentiation induction; therefore, this is the differentiation induction method to provide dopamine-producing cells most efficiently.

3. Scheme of the differentiation induction method

4. Practical example of the differentiation induction method

5. Point of the differentiation induction method

1. The cell line with repeated passage may show decreased induction efficiency. After initiation, use the cells with the constant passage number.
2. Pay attention to the manufacturer and condition of the CORIN antibody when FACS is used. We are using the same antibody (KAN Research Institute, Inc.) as the original method from Dr. Takahashi’s laboratory.


Induction to Cardiomyocyte (Dr. Yoshida’s Method)

1.Article: Miki et al., Cell Stem Cell, 16, 699

Patent number: Patent application 2013-102375, Efficient myocardial cell induction method
Patent number: Patent application 2014-058926, Cardiomyocyte sorting method

2.Features of the differentiation induction method

This method is for inducing differentiation of iPS cells into cardiomyocyte with the suspension culture system.
Start with iPS cells in the single-cell state and induce to cardiomyocyte while reacting with each growth factor in the suspension culture system.
The mature cardiomyocyte can be obtained with long-term suspension culture.

3.Scheme of the differentiation induction method

4.Extracellular potential measurement results of cardiomyocyte cluster

5.Point of the differentiation induction method

Induction efficiency may vary depending on the culture method of iPS cells and the iPS cell line used; therefore, it is necessary to seek the optimal condition for each case by changing the cell number at the induction initiation and the processing days of Step 1 (BMP4, bFGF, Activin A).


Differentiation Induction of Pancreatic Cells (Dr. Kume’s Method)

1. Article: Ryutgaro Nakashima et al., Genes to Cells, 20, 1028-1045
Hussain Md. Shahjalal et al., J. Mol. Cell Biol., 6, 394-408

Patent number: Patent application 2014-104019

Method for differentiation induction of insulin-producing cells

2.Features of the differentiation induction method

– The differentiation of insulin-producing cells is induced for 19 days by changing the types of differentiation-inducing factors and their combinations in a stepwise fashion with 5 steps.
– The differentiation of insulin-producing cells can also be efficiently induced with the Xeno-free culture system.

3. Scheme of the differentiation induction method

4. Results of the differentiation induction

(1) Results of immunostaining

(2) Results of C-peptide ELISA

5. Point of the differentiation induction method

Differentiation efficiency varies with the iPS cell line, cell density, and the type of matrix used for cell adhesion; therefore, the optimal condition has to be taken into account.