Reprogramming has been studied extensively for decades. Nuclear transfer into an oocyte gives somatic cells pluripotency to produce cloned animals. For example, Dr J. Gurdon and his colleagues showed that frog somatic cell nuclei can be reprogrammed after transfer into enucleated oocytes, and they develop into feeding tadpoles [1
]. Reprogramming in vertebrates was also proven by the creation of cloned animals from sheep [2
] and mice [3
]. In addition to oocytes, human [4
] and mouse embryonic stem (ES) [5
] cells also can reprogramme somatic cells into an ES cell-like state after cell fusion. These results demonstrate that terminally differentiated cells can revert to a state of pluripotency in response to external stimulation.
The accumulated understanding of the mechanisms underlying pluripotency in ES cells led to attempts to revert somatic cells into a pluripotent state using defined factors. Twenty-four candidate factors were transduced into mouse embryonic fibroblasts (MEFs) by retroviral delivery and this identified four factors that can convert fibroblasts into induced pluripotent stem (iPS) cells [6
]. iPS cells have been generated from mouse [6
], rat [7
], monkey [9
], pig [10
], dog [11
], rabbit [12
] and human [13
]. Most of the iPS cells are derived using the Oct3/4
reprogramming factors. The original iPS cell induction system used retroviral vectors, which integrate transgenes into the host genome. The insertion of tumorigenic genes, like c-Myc
, and activation of proto-oncogenes by LTR increase the risk of tumour formation [15
Mouse iPS cells were generated using a plasmid vector in 2008, showing that iPS cells can be induced by the transient expression of reprogramming factors [17
]. The goals of those experiments were to increase transfection efficiency in primary cells and to maintain transgene expression long enough (a few weeks) for iPS cell induction. Three essential reprogramming factors (Oct3/4
) were connected in a single plasmid using the 2A sequence, which enables expression of multiple proteins from a single RNA transcript. The stoichiometric balance of these core transcription factors is thought to be important for iPS cell induction, and therefore all six possible orders of the factors in the retrovirus system were examined to determine the most effective arrangement. The three factors were then placed into a plasmid vector with a constitutively active CAG promoter, which yielded high expression [18
]. This vector ensures co-expression of the three core factors in all of the transfected cells. In addition, another expression vector for c-Myc
was constructed. The transfection of the plasmids into MEFs was repeated multiple times to achieve the sustained expression required for iPS cell generation. After four weeks we obtained iPS cell colonies, albeit at a very low frequency. As expected, iPS cell clones in which transgenes had been integrated into the host genome were frequently observed. However, no transgene integration was detected in approximately one-third of the established mouse iPS cell clones. The integration-free iPS cell clones had the potential to differentiate into various cell types of the three germ layers. Furthermore, they were able to form chimeric mice when transplanted into blastocysts, which were competent for germline transmission.
The frequency of iPS cell generation by plasmids, however, was very inefficient. The estimated efficiency is less than 0.0002%, which is at least 1000-fold lower than that of viral induction. The fact that reprogramming efficiency of human fibroblasts with retrovirus is approximately 10-fold lower than that of mouse fibroblasts suggested that the generation of integration-free human iPS cells would be extremely inefficient using the same method. Subsequently, several methods for integration-free human iPS cell generation have been reported. The approach can be divided into four groups based on delivery methods of the reprogramming factors: (i) virus [13
], (ii) DNA [21
], (iii) RNA [25
], and (iv) protein [26
] (). We calculated induction efficiency of the methods from the best result reported in each article. Because of the differences in their experimental settings, it is hard to compare their efficiency correctly. However, as predicted, non-integration methods are extremely inefficient in general. Notably, recent reports showed significant improvement of non-integration method. Sendai virus is a minus strand RNA virus. Fusaki et al
] infected Sendai virus vectors encoding reprogramming factor into human fibroblasts and obtained iPS cells. Because Sendai virus replicates its genome in the cytoplasm of infected cells, this vector system can stably express reprogramming factors and achieve high reprogramming efficiency. The established iPS cells, on the other hand, tended to carry the virus genome even after long-term culture. To obtain viral-free cells, an additional approach was needed such as the elimination of virus-containing cells through negative selection against virus antigen hemagglutinin–neuraminidase or using a temperature sensitive mutant. Direct delivery of synthetic mRNA also generated iPS cells at high efficiency [25
]. The mRNA sustained high and relatively long expression of encoding reprogramming factors by using modified ribonucleotides. However, reprogramming via modified RNAs is technically difficult, sensitive to reagents and requires labour-intensive procedures. Therefore, further improvements in reprogramming methods are absolutely required for reproducible generation of integration-free human iPS cells. A summary of several topics associated with iPS cell generation and a discussion of the future in reprogramming methods for medical and other applications are herein provided.
iPS induction methods in human fibroblasts.