Induced pluripotent stem cells (iPS) are somatic cells that can be reprogrammed by expressing a combination of embryonic transcription factors. Like embryonic stem cells, iPS cells can differentiate into all three germ cell layers: ectoderm, mesoderm, and endoderm. The reprogrammed cells can be used to generate stem cells for diseases, drug development, and personalized regenerative stem cell therapy.
The discovery of induced pluripotent stem (iPS) cells
Although the actual discovery of pluripotent stem cells was made in 2006, several studies paved the way for this breakthrough. Sir John Gurdon demonstrated the first example of reprogramming when he generated tadpoles from enucleated unfertilized egg cells of frogs transplanted with the nucleus from the epithelial somatic cells of tadpoles. This method – known as somatic cell nuclear transfer – was performed in 1962 and gave rise to the idea of cloning.
Sir Ian Wilmut and his colleagues used the principle of somatic cell nuclear transfer to clone Dolly the sheep thirty-five years after Sir John Gurdon’s experiments. These two experiments showed that the nucleus of differentiated somatic cells contains all the genetic information necessary to create an entire organism ‘from scratch’.
The mouse embryonic stem cell lines and human embryonic stem cell lines were created in 1981 and 1998, respectively. These cell lines were created from pre-implantation embryos and had the propensity to create any cell in the body. In another landmark discovery, Takashi Tada and his colleagues showed reprogramming of somatic cells by fusing adult somatic cells with embryonic stem cells, creating in turn cells that could express pluripotency-related genes in 2001.
Induced pluripotent stem cells
All these discoveries paved the way for Shinya Yamanaka and Kazutoshi Takahashi when they developed first induced pluripotent stem cells in 2006. They employed a different method of reprogramming where they used a retrovirus to deliver four reprogramming transcription factors: Octamer-binding transcription factor-3/4 (Oct 3/4), Kruppel Like Factor-4 (Klf4), sex-determining region Y-box 2 (SOX2) and c-Myc. These four factors are also called “OSKM” factors. The same method was used by Yamanaka and his team to subsequently develop human induced pluripotent stem cells (hiPSCs).
Producing iPS cells for use in research and medicine
Armed by the knowledge of previous studies which demonstrated that unfertilized eggs and embryonic stem cells contain factors that can bestow pluripotency to somatic cells, Yamanaka and his team hypothesized that factors which aid in maintaining embryonic stem cell identity can also have critical roles to induce pluripotency in somatic cells.
Transcription factors – such as Oct3/4, Sox2 and Nanog – had been shown to have roles in maintaining pluripotency in early embryos and embryonic stem cells. Also, genes such as Stat3, E-Ras, c-Myc, Klf4, and b-catenin had been shown to be involved in the long-term maintenance of embryonic stem cell phenotype and proliferation of embryonic stem cells in culture conditions. Thus, in 2006 Yamanaka and his colleagues selected these factors to test their efficacyin inducing pluripotency in somatic cells.
Twenty-four selected candidate genes were tested in an assay where the induction of pluripotent state was detected as resistance to the antibiotic G418 (an aminoglycoside antimicrobial related to gentamycin). The candidate genes were introduced into the mouse embryonic fibroblasts, and the transduced cells were cultured in feeder cells kept in embryonic stem cell medium containing high concentrations of the aforementioned antibiotic.
In short, the presence of all 24 candidate genes created antibiotic-resistance colonies. These resistant colonies also possessed embryonic stem cell-like properties, including morphology and proliferation features. Further analysis revealed that these colonies also harbored embryonic stem cell markers, such as Oct3/4, Nanog, E-Ras, Cripto, Dax1, Zfp296 and Fgf. Therefore, this experiment revealed that combining these 24 candidate genes could induce embryonic stem cell marker genes.
However, to investigate which of these candidate genes were indispensable for this conversion process, the research team observed the effect of withdrawing individual factors from the pool of candidate genes. They found that removing four factors – Oct3/4, Klf4, Sox2, and c-Myc – led to either no colonies or flatter, less embryonic stem cell-like colonies. These results suggest that the aforementioned four factors have an important role to generate induced pluripotent stem cells from mouse embryonic fibroblasts.
What are the advantages and disadvantages of iPS cells?
Induced pluripotent stem cells are not derived from embryonic stem cells, and this negates the ethical concerns present in the field regarding the utilization of embryonic stem cells. As they can be obtained from somatic cells of the same donor who will receive the transplant, histocompatibility issues are drastically reduced.
This method has helped to develop our understanding regarding the “reprogramming” process and the information is transferable to in vivotherapies aimed at reprogramming damaged or diseased cells. Thus, patient-specific drugs can be developed to test them without endangering other animals. This also provides a “human model” for testing various drugs candidates, in contrast to animal models that necessitate more resources and time and are less efficient.
The main issue is the use of retroviruses to generate iPSCs as they are associated with cancer. More specifically, retroviruses can insert their DNA anywhere in the genome and subsequently trigger cancer-causing gene expression. Also, as mentioned before, the c-Myc (one of the genes used in reprogramming) is a known oncogene, and its overexpression can cause cancer.
Also, in certain non-dividing cell types (such as PBMCs or elderly skin fibroblasts) the reprogramming rate of somatic cells to iPSCs is very low (less than 0.02 %). There is also a need to assess the quality and variability of the reprogramming process. For example, iPSCs often have a tendency not to fully differentiate. Hence, there is a need to pursue a quantitative assessment of the final quality of cells and screen for any genetic or epigenetic alterations during the reprogramming process.
CRISPR/Cas9 gene-editing of iPS cells
Gene editing of iPS cells has shown great potential for exploring the molecular/cellular mechanisms that underpin various neurodegenerative, immunological, hematological, metabolic, and cardiac diseases. At present, CRISPR/Cas9 is being used in research to generate disease models and restore the normal functions of cells. This was a significant help to scientists to uncover the genetic cause for a myriad of conditions.
CRISPR/Cas9 has also been used to knockout various gene mutations, including DNMT3B (the cause of muscular dystrophy 2 amongst others), COL7A1 (dystrophic epidermolysis bullosa), and ABCA1 (familial HDL deficiency and Tangier disease).
In one study, scientists deleted CGG repeats known to cause Fragile X syndrome in FMR1gene, curing the condition. Similarly, Kim et al. used CRISPR to correct a point mutation in iPSC cells derived from patients who had a mutation in the Ataxia Telangiectasia Mutated (ATM) gene.