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The reprogramming kinetics & efficiencies

To better evaluate the reprogramming systems, investigators are seeking for various methods and markers to determine the efficiency in the reprogramming process. Epithelial characteristics and activation of some ESC markers are acquired in somatic cells after initiation of reprogramming through MET transition, which is deemed as a critical but nonessential step for reprogramming. Later, pluripotency-related genes are activated, and markers of AP, SSEA1, NANOG and the surface marker TRA-1-60 gradually turn to be expressed in the different reprogramming stages [8–۱۱]. Cell surface markers of CD44 and ICAM1 can be used to indicate the gradual reprogramming process including mesenchymal state, epidermal state, early pluripotent state and late pluripotent state [12]. The ratio between the number of original cells receiving the set of TFs and the number of genuine iPSC colonies and the kinetics of reprogramming are important for the successful reprogramming, while they are hard to be measured. Besides, the donor cell type and defined culture conditions will undoubtedly influence the reprogramming efficiencies and kinetics. Compared with fibroblasts, human primary keratinocytes can be reprogrammed 100-times more efficiently than MEFs [13]. And the intrinsic epigenetic states in specific donor cells contribute to the higher efficiency, fewer TFs and the quality of the resulting iPSCs [14]. For example, neural stem cells with endogenous expression of Sox2 can be reprogrammed in the absence of Sox2 or with Oct4 alone [15,16]. Concurrently, an increase in proliferation rate and a decrease in cell size are molecularly accompanied with the sequential transition [17]. Telomerase reverse transcriptase and the SV40 large T antigen, which have positive effects on proliferation, can also increase the quantity of resulting iPSCs [18]. Small molecules and miRNA which are able to regulate the cell cycle may take effect to increase the number of fully reprogrammed colonies [19,20]. Intriguingly, hypoxic conditions [21], growth factors secreted by feeder cells [22] and additions in culture medium [23] can absolutely improve the reprogramming efficiency. The kinetics are regulated by multiple factors, consequently there is no golden standard for accurate evaluation about the reprogramming for various reprogramming conditions.

Viral vector approaches for reprogramming

During the reprogramming process, induction silencing occurs gradually but viral genes are expressed constitutively. Despite the possibility of making safe iPSCs, nonintegrating viruses display a rather low gene transfer capacity and thus repeated infections are often required for many cell types. Consequently, retroviruses [24] and lentiviruses [25] are still the widely applicable delivery systems.

Retrovirus

As the most common choice in studies, retroviruses from replication-defective vectors can infect their target cells and deliver their viral payload but avoid cell lysis and death by inhibiting the lytic pathway. The infectivity of retroviruses is limited to dividing cells, thus the cell type for reprogramming is under restrictions. Retrovirus-mediated iPSCs stained positive for alkaline phosphatase, showed renewed expression of pluripotency genes, exhibited ultrastructural features including massive glycogen granules in the cytoplasm [26] and formed teratomas in vivo [27]. Recently a polycistronic cassette encoding four TFs separated by 2A peptides was tested in a retrovirus under an LTR or EF1α promoter, and the efficiency was much higher (up to 0.6%) than any other vectors [28]. In brief, the insertional mutagenesis, residual expression and reactivation of TFs, as well as titer loss during viral concentration and storage inhibited the infection of species and cell types resulting in reprogramming limitations.

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