Induced pluripotent stem cells (iPSCs) have opened up the possibility for therapeutics research. They can be acquired by reprogramming the differentiated cells of human beings and animals, thereby eliminating the need to use embryonic stem cells in clinics and research.
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iPSCs play an important role in cell replacement therapy, toxicology, biomedicine, and pharmacology, yet how safe is the technology that is based on iPSC? The answer depends on the methods used in the production of iPSCs. It is essential that methods developed for culturing human iPSC do not use animal cells. The popularity that iPSCs have gained all over the world necessitates setting up a method that is authentic for producing iPSCs.
iPSCs can be produced from adult somatic cells (e.g., fibroblasts, peripheral blood cells) by using specific reprogramming factors, which in turn have to be introduced into the cells using appropriate methods. There are three main steps in the generation of iPSCs, namely (1) setting up and establishing an initial somatic cell culture, (2) introducing iPSCs, and (3) description of iPSCs and its development.
First, the somatic cells, such as peripheral blood cells and fibroblasts are isolated and grown as a pure culture. The next is to introduce the reprogramming factors into these cells. This is done either by using integrating systems or by using non-integrating methods.
In the first method, retroviral, lentiviral (or inducible lentiviral) plasmids that integrate with the host cell DNA are introduced into the host cells. In the second method, plasmid DNA, synthetic mRNA, recombinant proteins, transgenes and viral vectors (sendai virus, adenovirus) that do not integrate with the host DNA are introduced into the host cells.
To induce expression of the reprogramming factors, the transfected cells must be incubated on a feeder layer of cells (these may be fibroblasts or keratinocytes) and suitable media conditions. iPSC generation happens over time, when the expression of the reprogramming factors is steady.
Finally, the presence of iPSCs is confirmed by their morphological and physicochemical characteristics. Reprogrammed cultures may appear as flat, sharp-edged cells that are closely-packed and mitotically active.
As cells cannot be characterized by morphology alone, they are characterized further by their expressed cell surface proteins (e.g., alkaline phosphatase, SSEA-4) or by the expression of the introduced transcription factors (e.g., Sox2, Nanong, Oct4). These markers also confirm iPSC generation from the physicochemical angle.
There are three main steps in generating iPSCs, namely (1) setting up and establishing an initial cell culture, (2) induction of iPSCs, and (3) characterization and expansion of iPSCs.
First, the source cells are isolated and grown as a pure culture. The next is to introduce the reprogramming factors into these cells.
This is done either by using integrating systems or by using nonintegrating methods. In the first method, retroviral, lentiviral (or inducible lentiviral) plasmids that integrate with the host cell DNA are introduced into the host cells. In the second method, plasmid DNA, transgenes, recombinant proteins, synthetic mRNA, and viral vectors that do not integrate with the host DNA are introduced into the host cells.
For expression of the reprogramming factors, the transfected cells are incubated on a feeder layer of cells, which under appropriate conditions, serve as an extracellular matrix and provide nourishment to the host cells. iPSC generation happens over time, when the expression of the reprogramming factors is steady.
Finally, the presence of iPSCs is confirmed by their morphological and physicochemical characteristics. After this, they are grown in sufficient quantities (expanded).
Factors involved in production of iPSCs
Generating iPSCs involves introducing reprogramming factors into isolated somatic cells. This introduction can be done by one of two approached, which either use integrating viral vector systems or non-integrating systems. The term integrating viral vector systems implies the use of viruses and their integration into the somatic cell genome. Lentivirus and retrovirus have generally been used for this purpose. Although high efficiencies have been obtained using this method, there is also an associated risk of cancer. For this reason, other approaches are also being investigated.
The non-integrating methods do not involve integration of the vector genome with that of the host genome. The use of certain viral vectors, proteins, plasmid DNA and RNA have also been explored in these methods, with varying degrees of success. For example, introduction of reprogramming factors was reproducible with the Epstein-bar derived plasmid oriP/EBNA1, but with a low efficiency.
Cell-penetrating peptides have been used to fuse with host cells and directly deliver the reprogramming factors into the host cell. Self-replicating RNA replicons expressing Yamanaka’s factors have also been used for transfection into fibroblasts; the process yielded cells with all the properties indicative of pluripotent stem cells.
It is believed that the reprogramming factors have individual roles as well as complementary roles, all of which become operational and transform the somatic cells into iPSCs.
Reviewed by Afsaneh Khetrapal BSc (Hons)