The generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the enforced expression of a few embryonic transcription factors. This discovery has raised fundamental questions about the mechanisms by which transcription factors influence the epigenetic conformation and differentiation potential of cells during reprogramming and normal development. In addition, iPSC technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders with autologous cells. In this review, we summarize the progress that has been made in the iPSC field over the last 4 years, with an emphasis on understanding the mechanisms of cellular reprogramming and its potential applications in cell therapy.

The third principle that contributed to the discovery of induced pluripotency was the observation that lineage-associated transcription factors—which help to establish and maintain cellular identity during development by driving the expression of cell type-specific genes while suppressing lineage-inappropriate genes—can change cell fate when ectopically expressed in certain heterologous cells. This idea was first demonstrated by the formation of myofibers in fibroblast cell lines transduced with retroviral vectors expressing the skeletal muscle factor MyoD ( Davis et al. 1987 ). Subsequently, Graf and colleagues ( Xie et al. 2004 ; Laiosa et al. 2006 ) discovered that primary B and T cells could be converted efficiently into functional macrophages upon overexpression of the myeloid transcription factor C/EBPα. More recently, researchers have identified sets of transcription factors that induce the conversion of pancreatic acinar cells into insulin-producing β cells by overexpressing the pancreatic factors MafA, Pdx1, and Ngn3 ( Zhou et al. 2008 ); the conversion of fibroblasts into neurons by the activation of the neural factors Ascl1, Brn2, and Myt1l ( Vierbuchen et al. 2010 ); and the conversion of fibroblasts into cardiomyocytes by the cardiac factors Gata4, Mef2c, and Tbx5 ( Ieda et al. 2010 ). Of note, these experiments proved that lineage conversions are not restricted to cell types within the same lineage or germ layer, since fibroblasts are mesodermal in origin, whereas neurons are derived from ectoderm. Some of the early transdifferentiation experiments provided the intellectual framework for a more systematic search for transcription factors that could induce the conversion of differentiated cells to a pluripotent state, which is discussed below.

While SCNT is a powerful tool to probe the developmental potential of a cell, it is technically challenging and not well suited for genetic and biochemical studies. Thus, another major advance toward isolating iPSCs was the establishment of immortal pluripotent cell lines from teratocarcinomas, tumors of germ cell origin. These cell lines were called embryonal carcinoma cells (ECCs) ( Stevens and Little 1954 ; Kleinsmith and Pierce 1964 ) and could be clonally expanded in culture while retaining pluripotency ( Finch and Ephrussi 1967 ; Kahan and Ephrussi 1970 ). Importantly, when ECCs were fused with somatic cells, such as thymocytes, the resulting hybrid cells acquired biochemical and developmental properties of ECCs and extinguished features of the somatic fusion partner ( Miller and Ruddle 1976 , 1977 ). The dominance of the pluripotent state over the somatic state in hybrids suggested that soluble trans-acting factors must exist in ECCs that can confer a pluripotent state upon somatic cells, and that these factors should be identifiable.

During mammalian development, cells gradually lose potential and become progressively differentiated to fulfill the specialized functions of somatic tissues. For example, only zygotes and blastomeres of early morulas retain the ability to give rise to all embryonic and extraembryonic tissues ( Kelly 1977 ), and are therefore called “totipotent,” while cells of the inner cell mass (ICM) of the blastocyst can give rise to all embryonic but not all extraembryonic tissues, and are hence called “pluripotent.” Cells residing in adult tissues, such as adult stem cells, can only give rise to cell types within their lineage and are called either “multipotent” or “unipotent,” depending on the number of developmental options they have. Upon terminal differentiation, cells entirely lose their developmental potential.

The discovery of induced pluripotency represents the synthesis of scientific principles and technologies that have been developed over the last six decades. These are (1) the demonstration by somatic cell nuclear transfer (SCNT) that differentiated cells retain the same genetic information as early embryonic cells; (2) the development of techniques that allowed researchers to derive, culture, and study pluripotent cell lines; and (3) the observation that transcription factors are key determinants of cell fate whose enforced expression can switch one mature cell type into another. In this section, we briefly summarize these three areas of research and the influence they had on the generation of induced pluripotent stem cells (iPSCs).

For mouse iPSCs, the expression status of the imprinted Gtl2 gene has been described recently as a refined marker that allows for the prospective identification of clones that support the development of tetraploid embryo complementation mice and therefore appear developmentally indistinguishable from ESCs ( L Liu et al. 2010 ; Stadtfeld et al. 2010b). Whether the human homolog MEG-3 or any other gene has similar predictive value in human iPSCs remains to be tested.

For human iPSCs, expression of surface markers such as TRA-1-81 has been shown to enrich for reprogrammed cells ( Lowry et al. 2008 ). A more stringent approach to identify faithfully reprogrammed human iPSCs without the use of drug selection combines the detection of surface markers with that of “indicator retroviruses” expressing fluorescent proteins, which become silenced upon acquisition of pluripotency ( Chan et al. 2009 ).

To improve the overall low efficiencies of generating iPSCs with most nonintegrating approaches, screens for chemical compounds that promote reprogramming have been performed. This led to the identification of a number of molecules that significantly increase reprogramming efficiencies in the context of Oct4, Klf4, Sox2, and c-Myc overexpression (for review, see Desponts and Ding 2010 ; Li and Ding 2010 ). Notably, some of these molecules can also replace individual reprogramming factors, raising the possiblility of deriving iPSCs solely with chemicals ( Desponts and Ding 2010 ; Li and Ding 2010 ). However, it should be noted that chemical substitution of a reprogramming factor is, in most cases, associated with a significant decrease in the number of iPSC clones generated, indicating that no single chemical compound is able to entirely replace the function of a transcription factor. Another potential caveat of chemical reprogramming approaches is the introduction of genetic or epigenetic abnormalities into resultant iPSCs, especially since many of the reported compounds are potent modulators of DNA and chromatin modifications.

Successful reprogramming has been achieved recently without the use of viral or plasmid vectors at all. Specifically, iPSCs have been derived from both mouse and human fibroblasts by delivering the reprogramming factors as purified recombinant proteins ( Zhou et al. 2009 ) or as whole-cell extracts isolated from either ESCs ( Cho et al. 2010 ) or genetically engineered HEK293 cells ( D Kim et al. 2009 ). While the use of purified proteins represents an attractive approach for the generation of transgene-free iPSCs, its efficiency is extremely low and, in the recombinant protein approach, required the addition of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) to the culture media. A more efficient and safer way of producing integration-free iPSCs may be the introduction of modified RNA molecules encoding for the reprogramming factors into somatic cells, which has been validated recently ( Warren et al. 2010 ).

Reprogramming efficiencies with current nonintegrating methods are several orders of magnitude lower (∼0.001%) than those achieved with integrating vectors (0.1%–1%) ( Table 3 ), most likely because factor expression is not maintained for a sufficient length of time to allow complete epigenetic remodeling. To avoid this issue, several laboratories have developed integration-dependent gene delivery vectors with incorporated loxP sites that can be subsequently excised from the host genome by transient expression of Cre recombinase ( Kaji et al. 2009 ; Soldner et al. 2009 ). This approach enables the efficient generation of iPSCs from different cell types, especially if polycistronic vectors are used ( Chang et al. 2009 ; Sommer et al. 2010 ). It remains to be seen, however, whether short vector sequences, which inevitably remain in the host cell DNA after excision, affect cellular function. Transgene-free iPSCs can also be generated with piggyBac transposons, mobile genetic elements that can be introduced into and removed from the host genome by transient expression of transposase ( Woltjen et al. 2009 ; Yusa et al. 2009 ). The low error rate of this process allows for a seamless excision, but requires characterization of integration sites in iPSCs before and after transposon removal. It also remains unclear if transposase expression can induce nonspecific genomic alterations in iPSCs ( Stadtfeld and Hochedlinger 2009 ).

Approaches to derive iPSCs free of transgenic sequences are aimed at circumventing the potentially harmful effects of leaky transgene expression and insertional mutagenesis. This is particularly important when considering iPSC technology in a therapeutic setting. Techniques to generate integration-free iPSCs can be subdivided into three categories: (1) those that use vectors that do not integrate into the host cell genome, (2) those that use integrating vectors that can be subsequently removed from the genome, and (3) those that do not use nucleic acid-based vectors at all ( Table 3 ).

In a modification of the conventional secondary system, mouse strains lacking individual reprogramming transgenes have been generated as a screening platform for the identification of small molecules that can substitute for a given reprogramming factor ( Markoulaki et al. 2009 ). Because lentiviral transgenes, however, often exhibit heterogeneous expression patterns in secondary cells, several primary iPSC clones need to be screened to identify the ones that efficiently reactivate the factors, a process that can be quite cumbersome. The recent development of “reprogrammable” mouse strains, which contain a single inducible polycistronic transgene in a defined genomic position, has solved this issue, and also enables the breeding of animals into desired mutant backgrounds for mechanistic studies ( Carey et al. 2010 ; Stadtfeld et al. 2010a).

Inducible vector systems have been employed to generate so-called “secondary” reprogramming systems, which do not rely on direct factor delivery into target cells. These systems entail differentiating “primary” iPSC clones, generated with doxycyline-inducible lentiviral vectors or transposons, into genetically homogeneous somatic cells using either in vitro differentiation (for human cells) ( Hockemeyer et al. 2008 ; Maherali et al. 2008 ) or blastocyst injection (for mice) ( Wernig et al. 2008a ; Woltjen et al. 2009 ). These somatic cells are then cultured in doxycycline-containing media, thus triggering the formation of “secondary” iPSCs at efficiencies that depend on the specific cell type used but are generally several orders of magnitude higher than the efficiencies obtained after primary infection. Secondary systems therefore (1) allow for the reprogramming of large quantities of genetically homogeneous cells for biochemical studies and cells that are difficult to culture or transduce, and (2) facilitate the comparison of genetically matched iPSCs derived from different somatic cell types.

iPSC derivation is ethically and legally less problematic and technically more feasible than SCNT. In order to use iPSCs as efficient research tools and ultimately translate this technology into clinical applications, suitable techniques of factor delivery and efficient identification of faithfully reprogrammed cells are crucial. Thus, recent advances in the area of iPSC generation and identification are discussed in the following section.

Mechanisms underlying iPSC formation

In the following section, we introduce models that have been developed to explain the low efficiency of reprogramming at a cellular level. We then discuss key molecular events that may act as barriers during the reprogramming process, and speculate on the role of the individual reprogramming factors as well as on supporting and antagonizing factors during epigenetic remodeling. This is followed by a discussion of different pluripotent states that have been identified recently and that can be interconverted by some of the same transcription factors. Finally, we address the question of whether iPSCs are molecularly and functionally equivalent to fertilization-derived ESCs.