Direct reprogramming enables the generation of pluripotent stem-cell lines from almost any somatic tissue and mammalian species, thereby avoiding the ethical issues associated with human embryonic stem cells.
Although direct reprogramming is conceptually and technically simple, it is an extremely slow and inefficient process. It is influenced by several variables that affect its efficiency and reproducibility, and the quality of the resulting induced pluripotent stem cells (iPSCs).
Depending on the donor cell type, reprogramming is achieved with different efficiencies and kinetics. These differences are attributed to variations in the endogenous levels of certain reprogramming factors, differentiation status and/or intrinsic epigenetic states that are more amenable to reprogramming.
Different factors are able to promote reprogramming, including genes that are normally expressed in early development, factors that directly or indirectly affect cell proliferation, chromatin remodellers or microRNAs.
At present in the iPSC field, it is still difficult to unambiguously designate a reprogramming strategy that is fitting for all purposes. In each case, one will have to evaluate the most appropriate starting cell types, factors, culture conditions and delivery method.
Reprogramming methods can be divided into two classes, those involving the integration of exogenous genetic material and those involving no genetic modification of the donor cells. Among these methodologies, retroviral delivery of OCT4, SOX2, KLF4 and MYC (the OSKM set) into fibroblasts is still the most widely used.
Integrative reprogramming approaches generate heterogeneous iPSC lines, which could obscure comparative analysis between lines. The use of Cre-deletable vectors has partially solved this problem.
Among non-integrative reprogramming systems, the recently published RNA-based approach seems promising on the basis of the high efficiency it achieves. Although appealing, the high gene dosages of the reprogramming factors resulting from direct messenger RNA delivery may represent an oncogeneic risk owing to higher expression levels of MYC.
A crucial challenge in the iPSC field is to evaluate how these various methodologies affect the quality of iPSCs in terms of transcriptional signatures, epigenetic status, genomic integrity, stability, differentiation and tumorigenic potential. Whole-genome sequencing platforms will probably have an important role in the future in assessing the integrity of the genome of iPSCs and will certainly improve our understanding of the mechanism by which reprogramming occurs in a specific cell type.
Pluripotent stem-cell lines can be obtained through the reprogramming of somatic cells from different tissues and species by ectopic expression of defined factors. In theory, these cells — known as induced pluripotent stem cells (iPSCs) — are suitable for various purposes, including disease modelling, autologous cell therapy, drug or toxicity screening and basic research. Recent methodological improvements are increasing the ease and efficiency of reprogramming, and reducing the genomic modifications required to complete the process. However, depending on the downstream applications, certain technologies have advantages over others. Here, we provide a comprehensive overview of the existing reprogramming approaches with the aim of providing readers with a better understanding of the reprogramming process and a basis for selecting the most suitable method for basic or clinical applications.
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We apologize to those authors whose publications cannot be mentioned here owing to space constraints. F.G. was partially supported by a fellowship from the Swiss National Science Foundation. Work in the laboratory of J.C.I.B. was supported by grants from the G. Harold and Leila Y. Mathers Charitable Foundation, the Cellex Foundation, MICINN and Sanofi-Aventis.
The authors declare no competing financial interests.
Relative efficiencies of reprogramming depending on factors and/or delivery modes in mouse embryonic fibroblasts (MEFs). (PDF 350 kb)
Direct reprogramming of diverse mouse cell types. (PDF 393 kb)
Direct reprogramming of diverse human cell types. (PDF 541 kb)
Disease iPS lines (PDF 316 kb)
- Inner cell mass
(ICM). In mammals, a cluster of pluripotent cells found inside the blastocyst that give rise to all the cells of the body of the embryo proper. Embryonic stem cells, which are derived from ICM cells, are the closest in vitro counterpart of ICM cells.
(Also known as POU5F1). A POU homeodomain transcription factor that has a crucial role in early embryonic development and is necessary for the maintenance of embryonic stem cell pluripotency.
Transcription factor of the SRY-related HMG-box family involved in the regulation of embryonic development and in the determination of cell fate. SOX2 is required to maintain self-renewal of undifferentiated embryonic and neural stem cells.
A member of the Krüppel-like family of zinc finger transcription factors that is involved in cell proliferation, differentiation and survival. KLF4 has both transcriptional activation and repression domains.
(Also known as c-MYC). MYC is among the most frequently dysregulated oncogenes in human cancer. This transcription factor controls the expression of hundreds of target genes, many of which are also oncogenes or tumour suppressors, and have roles in cell proliferation and the cell cycle.
Combination of the OCT4, SOX2, KLF4 and MYC transcription factors, also known as the 'Yamanaka factors'. This was the first combination that was reported to reprogramme somatic cells into a pluripotent state.
- Cord blood
The fraction of blood remaining in the placenta and the umbilical cord after childbirth. Cord blood is a rich source of haematopoietic stem cells, which have been used extensively for transplantation in the treatment of diseases such as leukaemia and other cancers.
- CD133+ cells
Cells expressing the CD133 antigen, a 97 kDa glycoprotein composed of five transmembrane domains. This cell-surface marker is expressed by immature haematopoietic stem/progenitor cells but not their mature counterparts.
A homeobox transcription factor expressed in undifferentiated cells, including fetal gonads (ovary and testis), inner cell mass and embryonic stem cells. NANOG expression in the inner cell mass prevents this from differentiating into extra-embryonic endoderm and trophectoderm.
- Alkaline phosphatase
A hydrolase enzyme responsible for dephosphorylating molecules such as nucleotides, proteins and alkaloids under alkaline conditions. It is often used as marker of pluripotency.
A tumour suppressor that responds to diverse cellular stresses by regulating genes involved in cell-cycle arrest, apoptosis, senescence, DNA repair and changes in metabolism. Downregulation of p53 improves reprogramming efficiency.
- Moloney murine leukaemia virus
(MMLV). A retrovirus composed of an ssRNA genome replicating through a DNA intermediate that integrates into the host genome. MMLV infects only actively dividing cells.
Delivery of nucleic acids (plasmid DNA, linear DNA or RNA) into cells by a non-viral method. Common transfection methods include calcium phosphate treatment, electroporation, nucelofection and the use of cationic lipid vehicles.
A genus of retroviruses with long incubation periods that cause chronic, progressive and usually fatal diseases, such as HIV in humans. They are the only retroviruses that are able to replicate in non-dividing cells.
An inducible promoter system based on the tetracycline operon, which is present in a variety of vectors. In Tet-OFF vectors, gene expression is turned on when tetracycline or doxycycline is removed from the culture medium, whereas Tet-ON systems are induced only when doxycycline is added.
Cre is a 38-kDa type I topoisomerase protein from bacteriophage P1 that catalyses site-specific intramolecular (excision or inversion) and intermolecular (integration) recombination between loxP sites. The loxP site consists of two 13bp inverted repeats separated by an 8bp asymmetric spacer region.
A transcription unit made up of several open reading frames, resulting in the translation of separate proteins. Internal ribosome entry site or 2A-peptide sequences allow such multigene expression constructs to be engineered.
(PB). A TTAA-specific transposon, originally described in the order Lepidoptera. This mobile genetic element stably transfers exogenous DNA into a variety of cells. The PB system is composed of a donor plasmid, co-transfected with a helper plasmid expressing the transposase. Once integrated, PBs can be precisely deleted upon remobilization by the transposase.
The FLP recombination system, derived from the 2μ plasmid of Saccharomyces cerevisiae, mediates site-specific intramolecular (excision or inversion) and intermolecular (integration) recombination between FRT sites. The FRT site consists of two 13bp inverted repeats separated by an 8-bp asymmetric spacer.
- Adenoviral vector
A vector based on adenoviruses, which are medium-sized viruses with a double-stranded linear DNA genome. Recombinant adenoviral vectors allow transient, high-level expression of exogenous genes without integrating into the host genome.
- Sendai viral vector
A vector based on a negative sense, ssRNA paramyxovirus. F-deficient Sendai viral vectors replicate in the form of negative-sense ssRNA in the cytoplasm of infected cells, allowing the transfer of foreign genetic material.
An extrachromosomal DNA element that can replicate within a cell independently of the chromosome. Commonly used episomal vectors (also referred to as plasmids) contain an origin of replication and an antibiotic resistance cassette, allowing propagation in bacteria.
Human LIN28 is a cytoplasmic RNA-binding protein containing an amino-terminal cold-shock domain and two carboxy-terminal CCHC zinc finger domains. It is expressed in various undifferentiated embryonic cell types, as well as adult cardiac and skeletal muscle cells. The expression of LIN28 is regulated by microRNAs.
The PhiC31 (ΦC31) integrase from bacteriophage PhiC31 is a serine-type site-specific recombinase that mediates the recombination between the heterotypic target sites attB and attP. Unlike Cre or FLP, this system allows irreversible deletion, inversion or integration between its target sites.
- B18R protein
A vaccinia virus decoy receptor for type I interferons. In some cell types it increases cell viability after RNA transfection.
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González, F., Boué, S. & Belmonte, J. Methods for making induced pluripotent stem cells: reprogramming à la carte. Nat Rev Genet 12, 231–242 (2011). https://doi.org/10.1038/nrg2937
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