Induced pluripotent stem cell

Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of specific genes.

Induced Pluripotent Stem Cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many respects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.

iPSCs were first produced in 2006 from mouse cells and in 2007 from human cells. This has been cited as an important advance in stem cell research, as it may allow researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos. Because iPSCs are developed from a patient's own somatic cells, it was believed that treatment of iPSCs would avoid any immunogenic responses; however, Zhao et al. have challenged this assumption.

Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose significant risks that could limit its use in humans. For example, if viruses are used to genomically alter the cells, the expression of cancer-causing genes or oncogenes may potentially be triggered. In February 2008, in ground-breaking findings published in the journal Cell, scientists announced the discovery of a technique that could remove oncogenes after the induction of pluripotency, thereby increasing the potential use of iPS cells in human diseases. In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).

Production of iPSCs
[[Image:Dedifferentiation Methods (2010) - Bischoff, Steven R.tif|thumb|right|350px|Dedifferentiation to totipotency or pluripotency: an overview of methods.Various methods exist to revert adult somatic cells to pluripotency or totipotency. In the case of totipotency, reprogramming is mediated through a mature metaphase II oocyte as in somatic cell nuclear transfer (Wilmut et al., 1997). Recent work has demonstrated the feasibility of enucleated zygotes or early blastomeres chemically arrested during mitosis, such that nuclear envelope break down occurs, to support reprogramming to totipotency in a process called chromosome transfer (Egli and Eggan, 2010).

Direct reprogramming methods support reversion to pluripotency; though, vehicles and biotypes vary considerably in efficiencies (Takahashi and Yamanaka, 2006). Viral-mediated transduction robustly supports dedifferentiation to pluripotency through retroviral or DNA-viral routes but carries the onus of insertional inactivation. Additionally, epigenetic reprogramming by enforced expression of OSKM through DNA routes exists such as plasmid DNA, minicircles, transposons, episomes and DNA mulicistronic construct targeting by homologous recombination has also been demonstrated; however, these methods suffer from the burden to potentially alter the recipient genome by gene insertion (Ho et al., 2010). While protein-mediated transduction supports reprogramming adult cells to pluripotency, the method is cumbersome and requires recombinant protein expression and purification expertise, and reprograms albeit at very low frequencies (Kim et al., 2009). A major obstacle of using RNA for reprogramming is its lability and that single-stranded RNA biotypes trigger innate antiviral defense pathways such as interferon and NF-kB-dependent pathways. In vitro transcribed RNA, containing stabilizing modifications such as 5-methylguanosine capping or substituted ribonucleobases, e.g. pseudouracil, is 35-fold more efficient than viral transduction and has the additional benefit of not altering the somatic genome (Warren et al., 2010).

An overarching goal of reprogramming methods is to replace genes with small molecules to assist in reprogramming. No cocktail has been identified to completely reprogram adult cells to totipotency or pluripotency, but many examples exist that improve the overall efficiency of the process and can supplant one or more genes by direct reprogramming routes (Feng et al., 2009; Zhu et al., 2010).]] iPS cells are typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells (although this technique is becoming less popular since it is known to be prone to cancer formation), such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3/4 (Pou5f1) and Sox2, although it is suggested that other genes enhance the efficiency of induction. After 3–4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.

First generation
Induced pluripotent stem cells were first generated by Shinya Yamanaka's team at Kyoto University, Japan in 2006. Yamanaka used genes that had been identified as particularly important in embryonic stem cells (ESCs), and used retroviruses to transduce mouse fibroblasts with a selection of those genes. Eventually, four key pluripotency genes essential for the production of pluripotent stem cells were isolated; Oct-3/4, SOX2, c-Myc, and Klf4. Cells were isolated by antibiotic selection of Fbx15+ cells. However, this iPS cell line showed DNA methylation errors compared to original patterns in ESC lines and failed to produce viable chimeras if injected into developing embryos.

Second generation in mice
In June 2007, the same group published a breakthrough study along with two other independent research groups from Harvard, MIT, and the University of California, Los Angeles, showing successful reprogramming of mouse fibroblasts into iPS cells and even producing viable chimera. These cell lines were also derived from mouse fibroblasts by retroviral mediated reactivation of the same four endogenous pluripotent factors, but the researchers now selected a different marker for detection. Instead of Fbx15, they used Nanog which is an important gene in ESCs. DNA methylation patterns and production of viable chimeras (and thereby contributing to subsequent germ-line production) indicated that Nanog is a major determinant of cellular pluripotency.

Unfortunately, one of the four genes used (namely, c-Myc) is oncogenic, and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported that one can create iPSCs even without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.

Two-fathered mice
Reproductive scientists in University of Texas MD Anderson Cancer Center have created mice with nuclear DNA (nDNA) solely from two fathers, using iPS technology. Foetal fibroblasts from one father (XY) were cultivated and one percent of the resultant cells had spontaneously lost a Y-chromosome; like an individual with Turner Syndrome (X0). These cells were injected in female blastocysts (XX), which gestated in surrogate mothers to form female chimeras (X0/XX). When these mated with male mice (XY). Some of the offspring had nDNA from the original father and also from the mated male but not from the female blastocysts or the surrogate mother. Both male and female two-fathered mice were viable.

Human induced pluripotent stem cells
In November 2007, a milestone was achieved by creating iPSCs from adult human cells; two independent research teams' studies were released - one in Science by James Thomson at University of Wisconsin–Madison and another in Cell by Shinya Yamanaka and colleagues at Kyoto University, Japan. With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system. Thomson and colleagues used OCT4, SOX2, NANOG, and a different gene LIN28 using a lentiviral system.

Limitations of the transcription factor approach
Although the traditional method using transcription factors such as Oct3/4, Sox2, c-Myc, etc. pioneered by Yamanaka and Thompson was good proof of concept that somatic cells can be reprogrammed to iPS cells, there are still many key challenges for this method to overcome:
 * 1) Throughput: the throughput of successfully reprogrammed cells has been incredibly low. For example, the rate at which somatic cells were reprogrammed into iPS cells in the Yamanaka mouse study was .01-.1%. The low efficiency rate may reflect the need for precise timing, balance, and absolute levels of expression of the reprogramming genes. It may also suggest a need for rare genetic and/or epigenetic changes in the original somatic cell population or in the prolonged culture.
 * 2) Genomic Insertion: genomic integration of the transcription factors limits the utility of the transcription factor approach because of the risk of mutations being inserted into the target cell’s genome. A common strategy for avoiding genomic insertion has been to use a different vector for input.  plasmids,  adenovirus es, and  transposon  vectors have all been explored, but these often come with the tradeoff of lower throughput.
 * 3) Tumors: another main challenge was mentioned above—some of the reprogramming factors are oncogenes that bring on a potential tumor risk. Inactivation or deletion of the tumor suppressor p53, which is the master regulator of cancer, significantly increases reprogramming efficiency. Thus there seems to be a tradeoff between reprogramming efficiency and tumor generation.
 * 4) Incomplete reprogramming: reprogramming also faces the challenge of completeness. This is particularly challenging because the genome-wide epigenetic code must be reformatted to that of the target cell type in order to fully reprogram a cell. However, three separate groups were able to find mouse embryonic fibroblast (MEF)-derived iPS cells that could be injected into tetraploid blastocysts and resulted in the live birth of mice derived entirely from iPS cells, thus ending the debate over the equivalence of embryonic stem cells (ESCs) and iPS with regard to pluripotency. However, some of the other techniques profiled below demonstrated limited or incomplete equivalent to ESCs.

The table at right summarizes the key strategies and techniques used to develop iPS cells over the past half-decade. Rows of similar colors represents studies that used similar strategies for reprogramming.

Mimicking transcription factors with small compounds
One of the main strategies for avoiding problems (1) and (2) has been to use small compounds that can mimic the effects of transcription factors. These molecule compounds can compensate for a reprogramming factor that does not effectively target the genome or fails at reprogramming for another reason; thus they raise reprogramming efficiency. They also avoid the problem of genomic integration, which in some cases contributes to tumor genesis. Key studies using such strategy were conducted in 2008. Melton et al. studied the effects of histone deacetylase (HDAC) inhibitor valproic acid. They found that it increased reprogramming efficiency 100-fold (compared to Yamanaka’s traditional transcription factor method). The researchers proposed that this compound was mimicking the signaling that is usually caused by the transcription factor c-Myc. A similar type of compensation mechanism was proposed to mimic the effects of Sox2. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of calcium channels in the plasma membrane in order to increase reprogramming efficiency. It is foreseeable that such experiments will continue to find small compounds that improve efficiency rates. Ultimately, the goal is to discover a cocktail of reprogramming factors and compounds that efficiently and reliably reprogram somatic cells to iPS cells.

Alternate vectors
Another key strategy for avoiding problems such as tumor genesis and low throughput has been to use alternate forms of vectors: adenovirus,  plasmids, and naked DNA and/or protein compounds. In 2008, Hochedlinger et al. used an adenovirus  to transport the requisite four transcription factors into the DNA of skin and liver cells of mice, resulting in cells identical to ESCs. The adenovirus  is unique from other vectors like viruses and retroviruses because it does not incorporate any of its own genes into the targeted host and avoids the potential for insertional mutagenesis. More recently (in 2009), Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells . Another advantage of using adenovirus es is that they only need to present for a brief amount of time in order for effective reprogramming to take place. Also in 2008, Yamanaka et al. once again made a huge contribution to the field of iPS cells with the finding that they could transfer the four necessary genes with a plasmid. The Yamanaka group successfully reprogrammed mouse cells by transfection with two plasmid constructs carrying the reprogramming factors; the first plasmid expressed c-Myc, while the second expressed the other three factors (Oct4, Klf4, and Sox2). Although the plasmid methods avoid viruses, they still require cancer-promoting genes to accomplish reprogramming. The other main issue with these methods is that they tend to be much less efficient compared to retroviral methods. Furthermore, transfected plasmids  have been shown to integrate into the host genome and therefore they still pose the risk of insertional mutagenesis. Because non-retroviral approaches have demonstrated such low efficiency levels, researchers have attempted to effectively rescue the technique with what is known as the piggyBac transposon  system. The lifecycle of this system is shown below. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving any footprint mutations in the host cell genome. As you can see in the figure, the piggyBac transposon  system involves the re-excision of exogenous genes, and this is what prevents this method from causing issues like insertional mutagenesis



Drug-Like chemicals, recombinant proteins
In 2009, Ding and colleagues demonstrated a successful alternative to transcription factor reprogramming through the use of drug-like chemicals. This was the first method in human cells that was mechanism-specific for the reprogramming process. Ding tackled the problem of genomic insertion by using purified proteins to transform adult cells into embryonic-like cells. Once his team successfully demonstrated this, they tackled the efficiency problem. Ding’s overall strategy involved biomimicry. He studied the naturally occurring process of MET (mesenchymal to epithelial cell transition), in which fibroblasts are pushed to a stem-cell like state. Ding first looked for drug-like molecules that inhibited compounds known to be involved in the MET process; these compounds included TGFb (transforming growth factor beta) and MEK (mitogen-activated protein kinase). Ding identified the most active molecules and then studied their effects on iPS creation when used singly or in combination. He concluded that there are two chemicals—ALK5 inhibitor SB431412 and MEK inhibitor PD0325901, which when used in combination are highly effective at promoting the transformation from fibroblast to iPS cell. Although this two-chemical technique bested the efficiency of the classical genetic method by 100fold, Ding sought to do better. He continued with the use of the biomimicry strategy, enlisting another natural pathway—the cell survival pathway. After screening several compounds that target this pathway, the team focused on a new compound called Thiazovivin. Using this protein with the two previous chemicals, the team beat the efficiency of the classic method by 200 fold. Furthermore, this method took only two weeks to complete reprogramming while the classic method took four weeks. Another advantage of Ding’s method was that it overcame the genetic insertion problem because the drug-like molecules were based on natural biological processes.

RNA molecules
Studies by Blelloch et al. in 2009 demonstrated that expression of ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhances the efficiency of induced pluripotency by acting downstream of c-Myc . More recently (in April 2011), Morrisey et al. demonstrated another method using microRNA that improved the efficiency of reprogramming to a rate similar to that demonstrated by Ding. MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Morrisey’s team worked on microRNAs in lung development, and hypothesized that their microRNAs perhaps blocked expression of repressors of Yamanaka’s four transcription factors.

Genes of induction
The generation of iPS cells is crucially dependent on the genes used for the induction.

Oct-3/4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.


 * Oct-3/4 (Pou5f1): Oct-3/4 is one of the family of octamer ("Oct") transcription factors, and plays a crucial role in maintaining pluripotency. The absence of Oct-3/4 in Oct-3/4+ cells, such as blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation, and presence of Oct-3/4 thus gives rise to the pluripotency and differentiation potential of embryonic stem cells. Various other genes in the "Oct" family, including Oct-3/4's close relatives, Oct1 and Oct6, fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process.
 * Sox family: The Sox family of genes is associated with maintaining pluripotency similar to Oct-3/4, although it is associated with multipotent and unipotent stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells. While Sox2 was the initial gene used for induction by Yamanaka et al., Jaenisch et al., and Thomson et al., other genes in the Sox family have been found to work as well in the induction process. Sox1 yields iPS cells with a similar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 also generate iPS cells, although with decreased efficiency.
 * Klf family: Klf4 of the Klf family of genes was initially identified by Yamanaka et al. and confirmed by Jaenisch et al. as a factor for the generation of mouse iPS cells and was demonstrated by Yamanaka et al. as a factor for generation of human iPS cells. However, Thomson et al. reported that Klf4 was unnecessary for generation of human iPS cells and in fact failed to generate human iPS cells. Klf2 and Klf4 were found to be factors capable of generating iPS cells, and related genes Klf1 and Klf5 did as well, although with reduced efficiency.
 * Myc family: The Myc family of genes are proto-oncogenes implicated in cancer. Yamanaka et al. and Jaenisch et al. demonstrated that c-myc is a factor implicated in the generation of mouse iPS cells and Yamanaka et al. demonstrated it was a factor implicated in the generation of human iPS cells. However, Thomson et al., Yamanaka et al., and unpublished work by Johns Hopkins University reported that c-myc was unnecessary for generation of human iPS cells. Usage of the "myc" family of genes in induction of iPS cells is troubling for the eventuality of iPS cells as clinical therapies, as 25% of mice transplanted with c-myc-induced iPS cells developed lethal teratomas. N-myc and L-myc have been identified to induce instead of c-myc with similar efficiency.
 * Nanog: In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is necessary in promoting pluripotency. Therefore, it was surprising when Yamanaka et al. reported that Nanog was unnecessary for induction although Thomson et al. has reported it is possible to generate iPS cells with Nanog as one of the factors.
 * LIN28: LIN28 is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and proliferation. Thomson et al. demonstrated it is a factor in iPSC generation, although it is unnecessary.

An open future
The task of producing iPS cells continues to be challenging due to the five problems mentioned above. A key tradeoff to overcome is that between efficiency and genomic integration. Most methods that do not rely on the integration of transgenes are inefficient, while those that do rely on the integration of transgenes face the problems of incomplete reprogramming and tumor genesis. Of course there are a vast number of techniques and methods that have been attempted. Another large set of strategies is to perform a proteomic characterization of iPS cells. The Wu group at Stanford University has made significant headway with this strategy. Further studies and new strategies should help us find optimal solutions to the five main challenges. An interesting experiment might attempt to combine the good of these strategies into an ultimately effective technique for reprogramming cells to iPS cells.

Identity
The generated iPSCs were remarkably similar to naturally-isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally-isolated pluripotent stem cells:
 * Cellular biological properties
 * Morphology: iPSCs were morphologically similar to ESCs. Each cell had round shape, large nucleolus and scant cytoplasm.  Colonies of iPSCs were also similar to that of ESCs.  Human iPSCs formed sharp-edged, flat, tightly-packed colonies similar to hESCs and mouse iPSCs formed the colonies similar to mESCs, less flat and more aggregated colonies than that of hESCs.
 * Growth properties: Doubling time and mitotic activity are cornerstones of ESCs, as stem cells must self-renew as part of their definition. iPSCs were mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs.
 * Stem cell markers: iPSCs expressed cell surface antigenic markers expressed on ESCs. Human iPSCs expressed    the markers specific to hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.
 * Stem Cell Genes: iPSCs expressed genes expressed in undifferentiated ESCs, including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
 * Telomerase activity: Telomerases are necessary to sustain cell division unrestricted by the Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase activity and express hTERT (human telomerase reverse transcriptase), a necessary component in the telomerase protein complex.
 * Pluripotency: iPSCs were capable of differentiation in a fashion similar to ESCs into fully differentiated tissues.
 * Neural differentiation: iPSCs were differentiated into neurons, expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of catecholamine-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into dopaminergic neurons. Stem cell-associated genes were downregulated after differentiation.
 * Cardiac differentiation: iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Cardiomyocytes expressed TnTc, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes were downregulated after differentiation.
 * Teratoma formation: iPSCs injected into immunodeficient mice spontaneously formed teratomas after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the three germ layers endoderm, mesoderm and ectoderm; this is unlike other tumors, which typically are of only one cell type. Teratoma formation is a landmark test for pluripotency.
 * Embryoid body: hESCs in culture spontaneously form ball-like embryo-like structures termed “embryoid bodies”, which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPSCs also form embryoid bodies and have peripheral differentiated cells.
 * Chimeric mice: hESCs naturally reside within the inner cell mass (embryoblast) of blastocysts, and in the embryoblast, differentiate into the embryo while the blastocyst’s shell (trophoblast) differentiates into extraembryonic tissues. The hollow trophoblast is unable to form a living embryo, and thus it is necessary for the embryonic stem cells within the embryoblast to differentiate and form the embryo. iPSCs were injected by micropipette into a trophoblast, and the blastocyst was transferred to recipient females. Chimeric living mouse pups were created: mice with iPSC derivatives incorporated all across their bodies with 10%-90% chimerism.
 * Tetraploid complementation: iPS cells from mouse fetal fibroblasts injected into tetraploid blastocysts (which themselves can only form extra-embryonic tissues) can form whole, non-chimeric, fertile mice, although with low success rate.
 * Epigenetic reprogramming
 * Promoter demethylation: Methylation is the transfer of a methyl group to a DNA base, typically the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine sequence). Widespread methylation of a gene interferes with expression by preventing the activity of expression proteins or recruiting enzymes that interfere with expression. Thus, methylation of a gene effectively silences it by preventing transcription. Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity and the active promotion and expression of pluripotency-associated genes in iPSCs.
 * DNA methylation globally: Human iPS cells are highly similar to ES cells in their pattern of which cytosins are methylated, more than to any other cell type. However, on the order of a thousand sites show differences in several iPS cell lines. Half of these resemble the somatic cell line the iPS cells were derived from, the rest are iPSC-specific. Tens of regions which are megabases in size have also been found where iPS cells are not reprogrammed to the ES cell state.
 * Histone demethylation: Histones are compacting proteins that are structurally localized to DNA sequences that can effect their activity through various chromatin-related modifications. H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated, indicating the expression of Oct-3/4, Sox2, and Nanog.

Safety for Regenerative Medicine

 * The major concern with the potential clinical application of iPSCs is their propensity to form tumors. Much the same as ESC, iPSCs readily form teratoma when injected into immunodeficient mice. Teratoma formation is considered a major obstacle to stem-cell based regenerative medicine by the FDA.


 * Since iPSCs can only be produced with high efficiency at this time using modifications, they are generally predicted to be less safe and more tumorigenic than hESC. All the genes that have been shown to promote iPSC formation have also been linked to cancer in one way or another. Some of the genes are known oncogenes, including the members of the Myc family. While omitting Myc allows for IPSC formation, the efficiency is reduced up to 100 fold.


 * A non-genetic method of producing iPSCs has been demonstrated using recombinant proteins, but its efficiency was quite low. However, refinements to this methodology yielding higher efficiency may lead to production of safer iPSCs. Other approaches such as using adenovirus or plasmids are generally thought to be safer than retroviral methods.


 * An important area for future studies in the iPSC field is directly testing iPSC tumorigenicity using methods that mimic the approaches that would be used for regenerative medicine therapies. Such studies are crucial since iPSCs not only form teratoma, but also mice derived from iPSCs have a high incidence of death from malignant cancer. A recent paper came out in the journal Stem Cells indicating that iPS cells are far more tumorigenic than ESC, supporting the notion that iPS cell safety is a serious concern.