iPSC Research Methods

Induced pluripotent stem cells (iPSCs) are a type of stem cell that can maintain self-renewal and have pluripotent differentiation capabilities. In 2006, the research team led by Shinya Yamanaka of Kyoto University in Japan took the lead in reporting the research on iPSC. They introduced the four transcription factors "OCT3/4, SOX2, c-MYC and KLF4" into mouse embryos or skin fibroblasts, and found that they can be induced to transform. The iPSC are very similar to embryonic stem cells in morphology, gene and protein expression, epigenetic modification status, cell doubling ability, embryoid body and teratoma formation ability, differentiation ability, etc.

Soon, several laboratories also published similar research results. At the end of 2007, Thompson laboratory and Yamanaka Shinya laboratory almost simultaneously reported that using iPSC technology can also induce human skin fibroblasts into iPSC. The difference is that Japanese laboratories still use OCT3/4, SOX2, c-MYC, and KLF4, while the Thompson laboratory used the lentiviral vector to introduce the factor combination of OCT4, SOX2, NANOG, and LIN28. These research results have caused a great sensation in the scientific community and the mass media, and as a result, they were listed as the second among the top ten scientific and technological breakthroughs in 2007 by the American "Science" magazine.

In 2008, the research boom of iPSC continued to rise, and several remarkable progress was made. Harvard University's George Daley laboratory used this technology to convert skin cells collected from patients with 10 different genetic diseases into iPSC. These cells will play an important role in establishing disease models and drug screening. Other scientists have also discovered that iPSC can be differentiated under appropriate induction conditions, such as becoming blood cells, and then used to treat diseases. Another Harvard University laboratory found that after the introduction of three transcription factors (Ngn3 (also known as Neurog3), Pdx1 and Mafa) that play an important role in cell development into mouse pancreatic exocrine cells by viruses, these mouse pancreatic exocrine cells can directly transform into stem-like cells and can secrete insulin and effectively lower blood sugar.

Introduction to iPSC Research Methods

The iPSC technology is undoubtedly the most outstanding achievement made by the Stem Cell Research Institute, so it was selected as the top scientific and technological breakthrough in 2009. Unlike classic embryonic stem cell technology and somatic cell nuclear transfer technology, iPSC technology does not use embryonic cells or egg cells, avoiding ethical disputes. Moreover, the patient can use iPSC derived from autologous cells, thus avoiding immune rejection. In all, iPSC technology is greatly promising for clinical cell therapy.

So far, iPSCs have been successfully derived from the skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, amniotic membrane, peripheral blood cells, breast epithelial cells, adipose stem cells, umbilical cord matrix, and placenta.

Fig.1 A step-by-step guide to iPSC technology.

1. iPSC Reprogramming Methods

Through a large number of related technologies, somatic cell reprogramming can be achieved to generate induced pluripotent stem cells (iPS cells). We provide traditional and non-integrated iPSC generation tools for a range of somatic cell types. At present, a combination of multiple transcription factors has successfully induced the production of reprogrammed iPSC cells. Commonly used iPSC reprogramming methods include chemical methods and lentiviral and retroviral transfection.

• Chemical methods: Rapamycin (mTOR inhibitor) and Y27632 (ROCK inhibitor) can be used in combination to reprogram human breast cancer cells (MDA-MB-468) into iPSC. The mouse embryonic fibroblasts (MEFs) which have been transduced to express Oct4 and Klf4 can be reprogrammed into iPSC by the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR99021. BIX 01294, histone methyltransferase inhibitor combined with Oct4 and Klf4 expression can reprogram mouse embryonic fibroblasts (MEF) into iPSC.
• Lentiviral and retroviral transfection: this method can integrate DNA encoding pluripotent genes into the host genome, forcing somatic cell expression and pluripotency. It is well-known that 6-related transcription factors, such as Oct4, Klf4, Sox2, cMyc, etc., can induce iPSC.

2. iPSC Culture

iPSC research requires close attention to culture conditions. Two commonly used iPSC culture systems are feeder-dependent culture system or feeder-free culture system.

• Feeder-dependent culture system uses a layer of feeder cells, which can provide iPSC with growth factors and extracellular matrix proteins needed to maintain its health and expansion. The feeder cells originate from human and murine.
• Feeder-free culture system uses an alternative medium, which provides all the growth factors required for iPSC.

Note：don' t forget to check your iPSC and to make sure they are in a pluripotent state. You can use WB or ICC to analyze the expression of undifferentiated cell markers (e.g., OCT4, SOX2, KLF4, and c-MYC, etc.) of iPSC.

3. iPSC Genetical Modification

Transform your induced pluripotent stem cells to analyze gene and protein expression, study differentiation pathways, and disease pathways. Choose from transfection products based on lipid and electroporation technology and a range of cloning technologies. For example, the most widely used method at present is to introduce mutations that mimic disease occurrence into iPSCs through CRISPR/Cas9, or repair mutations in iPSC disease models, and then differentiate to obtain the required cells for research or treatment. Genetic modification of iPSC through CRISPR/Cas9 is currently a hot topic in iPSC research.

4. iPSC Differentiation Methods

iPSC differentiation requires standardized culture methods to ensure reproducible and reliable results. If your iPSC are expressing undifferentiated cell markers, then you can start to differentiate your iPSCs into the cell type you are interested in. Due to the pluripotency of iPSCs, they may differentiate into any cell type such as nerve cells and cardiomyocytes. This differentiation can be accomplished simply by using different biochemical reagents and growth factor mixtures ("cocktail").

Different target cells require different inducing factors, which are briefly summarized below:

5. iPSC Characterization

Characterization and verification of induced pluripotent stem cells to ensure that they are pluripotent or differentiated into target cell lines is a key step in the research process.

• Firstly, after iPSC differentiates into the target cell type (such as nerve, heart, lung, etc.), it should be checked whether the cells no longer express the above-mentioned undifferentiated cell markers (such as OCT4, KLF4, c-MYC, SOX2, etc.).
• Next, you should also check whether the generated cell type is exactly your target cell type. The differentiation markers of different target cells are listed below:

If you'd like to know more about our stem cell service, please directly send us an e-mail or contact us for your special requests.

References

1. Takahashi, K.; et al. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006, 126(4):663-676.
2. Yu, J.; et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007, 318 (5858):1917-1920.
3. Takahashi, K.; et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007, 131(5):861-872.
4. Park, I. H.; et al. Disease-specific induced pluripotent stem cells. Cell. 2008, 134(5):877-86.
5. Zhou, Q.; et al. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008, 455(7213):627-632.

For Research Use Only. Not For Clinical Use.