Induced pluripotent stem cells

Derivation of the first stable pluripotent hESC lines (Thomson et al., 1998) initiated the wide- spread research into early human development and regenerative medicine. However, the ethical concerns, limited supply of human embryos and obscurity of the genetic background of the cell lines have been problematic for the application of hESCs. (Yamanaka, 2008) For these reasons

19

together with the breakthrough insight that somatic cells can be reprogrammed back to PSCs (Figure 7), the discovery of iPSC technology revolutionized the field of biomedical technology (Takahashi et al., 2007; Yu et al., 2007). iPSCs share the characteristics of hESCs, such as plu- ripotency marker expression and differentiation capacity, as well as a need for supporting matrix.

In addition, the cell culture and differentiation methods are similar for both stem cell types (Takahashi et al., 2007).

Figure 7. Production of hESCs from the inner cell mass of the early stage embryo blastocyst and of iPSCs from the patient’s somatic cells using reprogramming factors. Pluripotent hESCs and iPSCs can be differentiated into various cell types using specific differentiation protocols.

The pioneering work of iPSCs was published in 2006, when the first mouse iPSC lines were generated from mouse embryonic fibroblasts with retroviral transfections (Takahashi and Ya- manaka, 2006). The first human iPSCs (hiPSCs) were discovered in two separate studies (Takahashi et al., 2007; Yu et al., 2007). In these studies, human skin fibroblasts were repro- grammed back to a pluripotent state using combinations of retrovirally transfected transgenes of

20

OCT4 (octamer-binding transcription factor 4), SOX2 (sex determining region Y-box 2), c-MYC (myelocytomatosis viral oncogene homolog) and KLF4 (Kruppel-like factor 4) (Figure 7) (Takahashi et al., 2007) or of lentivirally transfected factors of OCT4, SOX2, NANOG (Nanog homeobox) and LIN28 (lin-28 homolog A) (Yu et al., 2007). These genes are involved in maintaining the self-renewal capacity, undifferentiated state, pluripotency and proliferation of PSCs (Takahashi et al., 2007; Yu et al., 2007). Some variations of these gene combinations have been tested for reprogramming. For example, combinations of all six factors, OCT4, SOX2, c- MYC, KLF4, NANOG and LIN28, have increased the reprogramming efficiency (Liao et al., 2008). Reducing the number of reprogramming factors takes advantage of endogenously ex- pressed genes, decreasing the need for ectopic expression of pluripotency factors (Lai et al., 2011). For example, OCT4 alone has been shown to reprogram human neural stem cells into iPSCs (Kim et al., 2009), and c-MYC has been excluded when reprogramming human fibro- blasts because these cells express c-MYC endogenously (Nakagawa et al., 2008). To date, hiP- SCs have been generated from a wide spectrum of somatic cells, including keratinocytes (Aasen et al., 2008), neural stem cells or progenitor cells (Kim et al., 2009), astrocytes (Ruiz et al., 2010), amniotic cells (Li et al., 2009a), adipose tissue (Sun et al., 2009), cord blood cells (Takenaka et al., 2010), T lymphocytes (Seki et al., 2011), skeletal muscle stem cells (Tan et al., 2011), peripheral blood cells (Loh et al., 2010; Staerk et al., 2010), and human urine-derived cells (Zhou et al., 2011).

Originally developed virus-based iPSC technology (Takahashi et al., 2007; Yu et al., 2007) utilizes vectors that can integrate into the genome and that could have harmful consequences, e.g., cell death, residual expression, reprogramming factor re-activation, immunogenicity, un- controlled transgene silencing, and insertional mutagenesis (Hu, 2014). To overcome these problems, different strategies for iPSC reprogramming have been developed (Figure 8).

Transgene reprogramming can be classified into three groups: DNA-based reprogramming, RNA-based reprogramming and protein transduction. Chemical reprogramming with small molecules has also been studied with or without transgene reprogramming (Bayart and Cohen- Haguenauer, 2013; Hu, 2014); for example, the small molecule tranylcypromine and a specific glycogen synthase kinase 3 (GSK-3) inhibitor (CHIR99021), together with OCT4 and KLF4 transgenes (Li et al., 2009c), have been used in chemical reprogramming.

DNA-based technologies are the most widely used methods of reprogramming, and they uti- lize three major forms: virus particles, transposons, and plasmids. Viruses can be retroviruses

21

(Takahashi et al., 2007), lentiviruses (Yu et al., 2007) or adenoviruses (Stadtfeld et al., 2008), although adenoviruses have not been shown to reprogram human cells. (Hu, 2014) Transposons are mobile genetic elements that can move from one position to another within the genome through an excision/insertion mechanism. For example, a piggyback transposon-based non-viral vector that can induce stable genomic integration and persistent gene expression in mammalian cells quite efficiently has been exploited in iPSC production. (Kaji et al., 2009) Reprogramming plasmids can be linearized to enhance integration into the genome for efficient reprogramming.

Non-integrating plasmids include conventional and episomal plasmids, as well as minicircle DNA grouped into plasmids. (Hu, 2014)

Sendai virus is an RNA virus that enables efficient reprogramming of somatic cells and that is already exploited widely in iPSC derivation. It is non-integrating and can therefore be repli- cated in the host cell cytoplasm without entering the nucleus. (Fusaki et al., 2009) Another non- integrating RNA strategy for iPSC generation is to deliver synthetic messenger RNA (mRNA) encoding reprogramming factors directly into the cells. RNA replicons have also been delivered into cells in the form of packaged virions or as synthetic RNA genomes to avoid the integration of transgenes into the genome. One problem with RNA replicons and Sendai viruses is that they can be converted into complementary DNA (cDNA) by possible reverse transcriptases in the human genome; therefore, they can potentially integrate into the genome. (Hu, 2014) Mi- croRNAs (miRNAs), short RNA molecules that bind to complementary sequences on messen- ger RNA and block expression of a gene, have also been used as a reprogramming method. This technique is quite efficient but requires repeated transfection of the miRNAs into the cells (Anokye-Danso et al., 2011; Miyoshi et al., 2011). One method for the generation of transgene- free iPSCs is protein transduction, where reprogramming can be done with mammalian or bac- terial recombinant proteins. (Hu, 2014)

iPSCs may hold great promise for regenerative medicine due to the possibility of generating autologic transplantable cells with this technology. iPSC lines generated with methods causing transgene integrations are not applicable for clinical use because of potentially harmful genetic modification. (Ebert et al., 2012) Some studies have also reported substantial differences in genetic or epigenetic profiles of iPSCs versus hESCs, which must be further investigated before therapeutic application of iPSCs in humans (Kim et al., 2010b). Due to these problems, many methods of reduction or complete elimination of transgene integration into genomes have been studied widely. These methods include the use of a polycistron to reduce the number of integra-

22

tions or the use of the Cre-LoxP system to excise the transgenes (such as lentiviral vectors) from the reprogrammed genome. In addition, transposon transposition of transgenes into the repro- grammed genomes and subsequent excision of transgenes from the reprogrammed genomes have been studied. Repeated transfections of cells with nonreplicating plasmids, the use of non- integrating and replicating episomal plasmids and direct RNA delivery of reprogramming fac- tors (synthetic mRNA, RNA virus, RNA replicon, or miRNA) have also been investigated, and protein transduction and chemical reprogramming methods to eliminate transgene integration into the genome have been studied (Figure 8). (Hu, 2014)

Figure 8. Transgene genome integrating (red) and non-integrating (blue) strategies for iPSC reprogramming and transgene integration reduction methods. The efficiency of each method is categorized as very high (++), high (+), low (-) or very low (--). The figure is modified from (Hu, 2014).

23