Induction to the role of pluripotent stem cells

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Somatic cells can be reprogrammed to a embryonic like state by the induction of four transcription factors: Sox2, Oct4, KLf4 and c-myc. The produced cells are called "induced pluripotent stem cells"

The process of inducing pluripotency in somatic cells can be divided into two stages. In the first stage lineage associated genes are repressed and the epigenome of the target cells was reset to an ES cell like ground state. Also an open chromatin state, to allow the transcription of ES cell specific genes, was established in this stage. In the second stage, the interconnected autoregulatory loop and care transctiptional network was reactivated. C-myc and Klf4 are involved by inducing pluripotency in somatic cells. C-myc promotes cell cycle progression and inhibits differentiation. C-myc also facilitates an open chromatin conformation which allow the transcription of ES cell specific genes. Klf4 acts as a cofactor of Oct4 and Sox2 and regulates the activity of endogenous Nanoug through p53 inhibition. Furthermore, the balance between c-myc and Klf4 may also be important by inducing pluripotency.

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In this review, recent developments in the induction of pluripotency in somatic cells are described. Also de role of c-myc and Klf4 by inducing pluripotency and de possibilities and challenges of iPS cells will be discussed.

Introduction

Human embryonic stem (ES) cells are considered to have great potential for biomedical and clinical research. This is primarily because they have the ability to grow indefinitely while maintaining pluripotency and because they are able to differentiate into cells of all three germ layers. They might be used to treat diseases like Parkinson's disease, diabetes, heart failure, sickle cell anemia and spinal cord injury.(1,2,3) Differentiated cells can be reprogrammed to an embryonic-like state by fusion with embryonic stem cells or by transfer of nuclear contents into oocytes.(1) Embryonic like state cells can also be established by long term culture of bone marrow cells and pluripotent stem cells can be generated from adult germ cells.(3) Figure 1 gives an overview of all currently used techniques. All these techniques however, encounter ethical objections related to the destruction of human embryos or oocytes. It is therefore important to find other ways to obtain embryonic-like state cells.

Figure 1: Currently available methods to generate pluripotent stem cells from adult somatic or germ cells(3)

The fact that it is possible to reprogram somatic cells into cells with an embryonic-like state by fusion with ES-cells or by transfer of nuclear contents into oocytes, indicates that unfertilized eggs and ES cells possibly contain factors that can induce pluripotency in somatic cells.(1) Previous research showed that the transcription factors Oct3/4, Sox2 and Nanog play an important role in the maintenance of pluripotency in both embryos en ES cells.(1) Several genes, like E-Ras, c-myc, Krüppel-like-factor 4 (Klf4) and Stat 3, were also found to contribute to the long term maintenance of the ES cell phenotype and the rapid proliferation of ES cells in culture.(1,3) This genes were also frequently up regulated in tumours.(1) In 2006, Takahashi and Yamanaka tested 24 different candidate factors on their ability to induce pluripotency in mouse embryonic fibroblasts (MEF).(1,3) This analysis led to the demonstration that only four factors, Oct 3/4, Sox2, c-myc and Klf4, are necessary to introduce pluripotency in somatic cells. The resulting cells were named "induced pluripotent stem cells" (iPS cells) and where similar to ES cells in morphology, proliferation and teratoma formation.(1,3) In this review, an overview of current developments in research into iPS cells will be given and also the role of the transcription factors KLf4 and c-myc will be discussed.

Development of induced pluripotent stem cells

In the study of Takahashi and Yamanaka combinations of 24 candidate reprogramming genes were retrovirally transduced into mouse fibroblasts. An assay system was developed in which induction of the pluripotent state could be detected as resistance to G418. A neomycin resistance gene was knocked into the Fbx15 locus by homologous recombination. The Fbx15 locus is exclusively expressed in ES cells and resistance to G428, due to reactivation of the neomycin reporter gene, was used as a marker for induced pluripotency.(1,4) Takahashi and Yamanaka demonstrated in their study that there are only four transcription factors, Oct 3/4, Sox2, c-myc and Klf4, necessary for the induction of pluripotency in somatic cells. Induction of only two factors was, in any possible combination, was not able to induce the formation of G418-resistant colonies. A combination of Oct3/4, c-myc and Klf4 was able activate the Fbx15 locus but the morphology of the resulting cells differed from that seen in the cells made by the introduction of four transcription factors.(1) The cells made by the introduction of four transcription factors resembled ES cells in morphology and proliferative characteristics. Moreover, these cells differentiated into all three germ layers (ectoderm, mesoderm and endoderm) in vitro and were able to form teratomas when injected into nude mice.(1,4) Despite the similarities, the Fbx15-selected IPS cells significantly differ from ES cells in gene expression and DNA methylation patterns.(1,3,4) When iPS cells are transplantated into blastocysts, the cells give only rise to chimeric embryos but not to adult of germline competent chimeras.(3) Summarized, the Fbx15-selected iPS cells resemble ES cells in many way but they are clearly not identical to ES cells.(4)

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A significant improvement was reported less than a year later. Three groups generated iPS cells competent to give rise to adult and germline chimeras by using more stringent selection makers, endogenous Nanog- or Oct4 expression. Nanog and Oct4 were considered to be better markers of complete reprogramming because they are crucial to the pluripotent state, while Fbx15 is dispensable for pluripotency. Unlike the previous produced cells, Nanog- and Oct4- selected iPS cells are highly similar to ES cells in gene expression profiles and in DNA methylation patterns.(3,4)

Before iPS cell technology could live up to its therapeutic promise, it was important to demonstrate that reprogramming by defined factors could also be achieved in human somatic cells.(4) A few months after discovering that IPS cells could be induced from MEF culture by the introduction of four transcription factors, Takahashi & Yamanaka (2006) succeeded in generating iPS cells by introducing the human orthologs of the four transcription factor encoding genes into human fibroblasts.(2) Two other groups also demonstrated that the principle of reprogramming by defined factors could be achieved in human somatic cells. This indicated that the cellular networks governing pluripotency in somatic cells were simular across species.(2,4) In all previous studies, the human iPS cells closely resembled ES cells in many of their characteristics, including gene expression, morphology, patterns of DNA methylation and histone modifications. Human iPS cells were also able to give rise to all three germ layers in vitro. The injection of human iPS cells into blastocysts for the development of a term fetus comes up to practical and ethical concerns. However when human iPS were injected into nude mice, the iPS cells formed mature teratomas.(2)

The process of inducing pluripotency in somatic cells

Although transcription factor-induced reprogramming is one of the most promising ways to produce stem cells, it is still a poorly understood process. The question arose whether restoration of the pluripotent state of somatic cells takes place as a random process or is guided through a set of specific events. Schepers and Copray (2009), described two recent studies in which lentiviral vectors were used to study intermediate stages in the reprogramming process. In both studies it was found that the generation of iPS cells occurs through a predictable succession of events.(4) The observed changes in the cells are shown in table 1.

Table 1: The observed changes in het transfected fibroblasts (4)

Number of days after transduction

Observed changes in transfected fibroblasts

3

Down regulation of fibroblast specific genes such as Thyl and up regulation of the ES cell-specific markers alkaline phosphate and FBx15

5-9

Up regulation of ES cell marker gene SSEA1

10-16

Endogenous expression of pluripotency Oct4, Sox2 and Nanog.

Reactivation of telomerase and reactivation of the silenced X chromosome

> 16

The observed cells became increasingly independent of viral transgene expression.

As shown in table 1, the cells are almost completely independent of viral transgene expression after 16 days of reprogramming. They are however not fully reprogrammed at this time point. When the transgene expression was down regulated, the cells transformed back into their differentiated form. It might be that after prolonged expression of the viral vectors a larger portion of the cells is able to reach a pluripotent state. When this pluripotent state is reached, the viral vectors will be completely silenced.(4)

Whit the previous described observations, Schepers and Copray made the assumption that the process of reprogramming by defined factors consists of two stages. The classification of the two stages is shown in figure 2.

Figure 2: Reprogramming by defined factors viewed as a two state model. In the first stage lineage genes were down regulated and ES cell specific genes up regulated. Also an open chromatin state was established. In the second stage the ES cell transcript(4)

The first stage serves to repress lineage associated genes and to reset the epigenome of the target cell to a ES cell-like ground state. One of the earliest events in this stage is the repression of lineage-associated genes by exogenous Oct4 and Sox2. Concomitant whit the repression of this genes, ES cell specific genes, as Fbx15 and SSEA1 are up regulated. This genes can be activated so early in the reprogramming process because they have a relatively accessible chromatin states, even in differentiated cells. Other ES cell specific genes, such as transcription factors and components of signal transduction pathways, may possibly also be up regulated by exogenous Oct4 and Sox2. The combined effect of up regulation of these ES cell genes and repression of lineage associated genes is presumably already sufficient to produce a "quasi-pluripotent" phenotype. This phenotype however, regresses back to an differentiated state when transgene expression is stopped.(4)

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One of the most important functions of the first stage is to establish a permissive chromatin state. This state is likely the result of the transcription factors Klf4 and c-myc. Trough this open chromatin state transcription of ES specific genes is possible. 4) Exogenous Oct 4 and Sox2 may also activate histon modification enzymes to aid in the general unfolding of chromatin. Transcription of pluripotency genes like, Oct4, Sox2 and Nanog will also be promoted though multiple rounds of cellular division. This leads to the progressive loss of DNA methylation marks at promoters of pluripotency genes.(4) When all of this was completed the second stage of induced reprogramming was reached. In this stage, the interconnected autoregulatory loop and care transcriptional network will be reactivated. Exogenous Oct4 and Sox2 are, though the unfolding of chromatin, able to target and activate endogenous Oct4, Sox2 and Nanog loci. Expression of these pluripotency regulators will be self maintained through the autoregulatory loop.(4) Finally the pluripotent state becomes completely dependent on the endogenous autoregulatory circuit and a new induced pluripotent stem cell is formed.

Secondary reprogramming of Human Fibroblasts

Reprogramming of somatic cells to a pluripotent state has, so far, been achieved in mouse and human cells by viral transduction of the four transcription factors, Oct4, Sox2, c-myc and Klf4, into the genome of the host cell. This process is however, highly inefficient whit only 0.001 to 0.1% of the infected cells eventually becoming reprogrammed to a pluripotent state.(5) In 2008, Hockemeyer et all, developed a Doxycycline (DOX)-inducible "secondary" reprogramming system to efficiently generate iPS cells without additional viral infections.(5) In this experiment, DOX-inducible lentiviral vectors, which carry the mouse or the human cDNAs encoding the four transcription factors, were used to transduce either four (Oct4, Sox2, c-myc and Klf4.) or three (Oct4, Sox2 and Klf4) reprogramming factors into human fibroblasts. Thereafter, the cells were infected with a lentivirus, transducing the reverse tetracycline transactivator. This process was repeated several times. The infected cells were cultured in the presence of DOX and the detected iPS cell lines all showed a morphology characteristic for human ES cells. They were also able to express the pluripotency markers Oct4, Sox2 and Nanog. The cells made in this process were named primary iPS cell.(5)

The primary iPS cells were differentiated in the absence of DOX into secondary fibroblasts. These secondary cells acquired a homogenous fibroblast-like morphology. The secondary cells were then plated in the presence or absence of DOX under human ES cell culture conditions. After 20 to 25 days, the cells treated with DOX gave rise to secondary iPS cells. No human ES cell-like colonies appeared in any of the secondary fibroblast lines cultured in the absence of DOX. This indicates that derivation of secondary iPS cells was dependent on DOX induced transgene expression. The

efficiency of this new reprogramming process was 0.26 to 2% which is a great improvement over the old technique.(5)

The role of the transcription factors c-myc and Klf4.

ES cells and other pluripotent stem cells resemble tumour cells in many aspects. ES cells are, like tumour cells, also immortal and proliferate rapidly. When transplantated into immune-deficient mice ES-cells start to form tumours. In a sense, induced pluripotent stem cells are reversibly transformed cells. Taking the above data into account, it is not hard to imagine that the introduction of pluripotency in somatic cells is partly caused by two tumor associated gene products, c-myc and Klf4.(3) In the next part c-myc and Klf4 induced pluripotency in somatic cells will be discussed.

c-myc

The c-myc proto-oncogene is a multidomain transcription factor with a lot of functions in cellular processes such as cell growth, proliferation, loss of differentiation and apoptosis. Global analyses showed that c-myc is involved in the transcriptional regulation of 10% of the genome.(4) C-myc is part of the basic helix-loop-helix-leucine zipper (bHLH-LZ) family. This family is able to both activate and repress gene expression. Activation occurs via dimerization of c-myc with its bHLH-LZ partner MAX or by direct binding of c-myc to the DNA sequence CACGTA. This DNA sequence is called the E-box.(6) The 150 amino acid N-terminal region of c-myc is required for transactivation of target genes.(7) The C terminus of myc contains the basic bHLH-LZ motif for dimerization with is partner MAX and is also involved in transactivation through binding to CBP and p300, which have histone acetylase activities.(3)

One important function of c-myc is its ability to promote cell cycle progression. C-myc promotes G1-S progression through both gene activation and repression. RNA polymerase ΙΙΙ is activated by c-myc for example. This polymerase is involved in the generation of transfer RNA and 5S ribosomal RNA required for protein synthesis in growing cells. It is activated by c-myc via binding to TFΙΙΙB. C-myc induces also cyclin E-CDK2 activity early in the G1 phase of the cell cycle. This activation is regarded as an essential event in myc induced G1-S progression.(7)

As noted before, c-myc also represses genes to promote the cell cycle progression. Cyclin dependent kinase (CDK) inhibitors P15 and P21 are for example inhibited through c-myc. The myc-MAX heterodimer interactes with positively acting transcription factors such as MIZ-1 and SP1. The interaction of myc-MAX with MIZ-1 blocks the association of MIZ-1 with its own coactivator p300. This results in the down-regulation of P15 and P17 what allows CDK to continue whit preparing the cell for replication.(7)

C-myc also stimulates proliferation and is responsible for the loss of differentiation of cells. Numerous studies demonstrated that the MYC/MAX/MAD network plays an important role in regulating cell proliferation an differentiation. In general, expression of different members of the MAD/MXΙΙ protein family coincides with down-regulation of c-myc expression means that cells begin to exit the cell cycle and acquire a terminally differentiated phenotype.(7)

Curiously, c-myc also have some apoptotic activity despite his proto-oncogen character. Generally assumed is that the induction of cell cycle entry sensitizes the cell to apoptosis. Cell proliferative and apoptotic pathways are this way coupled. However, the apoptotic pathways are suppressed so long as appropriate survival factors deliver anti-apoptotic signals. (7)

C-myc also is a key component of active chromatin and is associated whit several histone acetyltransferase (HAT) complexes. consequently, as described before, it is thought that an important reprogramming function of c-myc is to facilitate an open chromatin conformation in the first stage of the reprogramming process through global histone acetylation, thereby providing Oct4 and Sox2 access to their target loci.(3,4) The overall hypothesis is that c-myc induces pluripotenty in somatic cells by its ability to promote cell cycle progression and proliferation, by its inhibiting effect on the differentiation and by its ability to facilitate an open chromatin conformation in the first stage of the reprogramming process.

In addition, recent studies show that c-myc also enhances the DNA synthesis by causing increased replication origin activity. This function of c-myc is independent of its transcription factor activity. Summarized, c-myc induced DNA replication and cell cycle progression may provide dividing cells with opportunities to reset their epigenome aiding in the reprogramming to pluripotency.(4) Finally, it has also been observed that c-myc directly up regulates expression of TERT. This gene encodes the enzymatic subunit of telomerase. Somatic cells that have been completely reprogrammed to a pluripotent state show elevated telomerase activity similar to ES cells and this may be a consequence of c-myc activity.(4)

Klf4

Klf4 belongs to the relatively large family of Sp-1 like transcription factors. This family has more than 20 members. A hallmark of this protein family is the presence of a DNA binding motif that contains several C2H2 zinc fingers. The zinc finger domains of Klf4 can bind to CACCC elements and to GC rich sequences of DNA in the regulatory sequences of target genes. The highly conserved linker sequence Krüppel interconnects the zinc finger domains. Apart from the shared DNA binding domains, Klfs contain various other domains involved in transcriptional activation or repression and protein-protein interaction.(8,9)

Klf4 is also known as gut-enriched Krüppel-like-factor. Recent studies imply that KLf4 is mainly expressed in adult tissues that have a high rate of cell turnover like epithelial cells of the GI tract and skin. Klf4 is also highly expressed in non-dividing cells.

In the human body, Klf4 is known to play important roles in cellular processes, including development, proliferation, differentiation and apoptosis. A major mechanism by which KLf4 regulates these diverse processes is by its capacity to act as a sequence-specific transcription factor. Klf4 is a transcription factor that can both activate and repress genes that are involved in cell-cycle regulation and differentiation. Curiously Klf4 can function both as a tumor-suppressor and a proto-oncogene.(9,10) In 2005, the molecular mechanisms underlying this dual function of KLf4 was partially declared by Rowland et all. They showed that ectopic expression of Klf4 suppresses cell proliferation. However, the inactivation of only one of Klf4s target genes, P21, is sufficient change this suppressing effect of Klf4. In p21 null cells, Klf4 promotes cell proliferation by down regulation of p53. Therefore p21 may function as a switch that determines the outcome of klf4 signaling. (3)(9)

The mechanism by which klf4 induces pluripotency in somatic cells is still not clear. Several possible roles of Klf4 induced reprogramming have been suggested. The first role of Klf4 in reprogramming may be as a cofactor of Oct4 and Sox2. In differentiated cells, Oct4 and Sox2 together are unable to induce expression of ES cell-specific genes. In the presence of KLf4 expression of these genes does occur. Furthermore, Klf4 plays a role in the activation of endogenous Nanog. Klf4 functions in this case through p53. P53 has been reported to repress Nanog expression during differentiation. Klf4 inhibits p53 actively so endogenous Nanog could be activated. Similar to c-myc, Klf4 is also associates with the p300 HAT complex. So klf4 also facilitate an open chromatin conformation in the first stage of the reprogramming process, thereby providing Oct4 and Sox2 access to their target loci.(4)

Finally, the balance between Klf4 and c-myc may also play a critical role in the transformation process in iPS cells. Expression of c-myc induces p53-dependent apoptosis in primary fibroblasts. Klf4 prevent this process by suppressing p53. Klf4, in turn, activates p21 and suppresses proliferation. C-myc inhibits this anti-proliferation function of Klf4 by suppressing p21. The above examples show that c-myc and Klf4 both influence each other. It is therefore plausible that the balance between those two is important by inducing pluripotency.(3,4)

Conclusion

Cells whit an embryonic-like state can be formed out of somatic cells by the introduction of four transcription factors, Oct 3/4, Sox2, c-myc and Klf4. The produced cells are called "induced pluripotent stem cells". IPS cells resemble ES cells in both morphology and proliferative characteristics. Moreover, these cells are able to give rise to all three germ layers in vitro and they form teratomas when injected into mouse.

The process of inducing pluripotency in somatic cells can be divided into two stages. The first stage serves to repress lineage associated genes and to reset the epigenome of the target cell to a ES cell-like ground state. An open chromatin state, to allow transcription of all ES cell specific genes, will also be established in this stage. In the second stage, the interconnected autoregulatory loop and care transcriptional network will be reactivated.

The transcription factors c-myc and Klf4 are important by inducing pluripotency in somatic cells. C-myc is, for example, able to induce pluripotency by its ability to promote cell cycle progression and proliferation. C-myc also inhibits differentiation and is able to facilitate an open chromatin conformation needed for the transcription of ES cell specific genes. Increased telomerase activity similar to the activity in ES cells and increased replication activity may also be a consequence of c-myc activity. The mechanism by which Klf4 induces pluripotency in somatic cells is still not clear, but for sure is that Klf4 promotes pluripotency in many ways. Klf4 acts as a cofactor of Oct4 and Sox2 and regulates the activity of endogenous Nanog through p53 inhibition. Similar to c-myc, Klf4 facilitates an open chromatin conformation in the first stage of the reprogramming process. C-myc and klf4 have also major influence on each other. Therefore it is not inconceivable that the balance between those two is also important by the induction of pluripotency.

IPS cells are considered to have great potential for biomedical and clinical research. Several diseases have been put forward which possibly may be treated with iPS cells. however, before iPS cells can be applied to treat human diseases, several problems need to be solved first. Therefore it will still take some time before the real potential of iPS cells is made clear.

Discussion

The process by which a somatic cell can be transformed back into a stem cell is increasingly clarified. This allows more and more thinking about the possibilities of induced pluripotent stem cells. As noted before, iPS cells can be of great value for biomedical and clinical research. However there are also some obstacles which should be taken in account. In the next part the advantages and challenges of iPS cells will be discussed.

Advantages of iPS cells.

IPS cells can be derived from differentiated cells like skin fibroblasts or B lymphocytes, without the need for human embryos or oocytes. This means that they do not have the ethical and political baggage of human ES cells. The methods used for generating iPS cells are also relatively simple and achievable by most laboratories using standard techniques and equipment. This way induced pluripotent stem cells have many potential clinical applications.(2)

The actual clinical significance of iPS cells was demonstrated by a number of different groups. Hanna et all (2007) proof the principle for the use of iPS cells in cell transplantation therapy in mouse. Transgenic mice were engineered to suffer from human sickle cell anemia. These mice were successfully treated with hematopoietic progenitor cells that were produced from autologous iPS cells. IPS cells where first created by reprogramming of tail-tip fibroblasts. The mutant allele was then corrected with an intact wild type β-globin gene (HBB) via homologous recombination. Next the gene corrected iPS cells were successfully differentiated into hematopoietic progenitors. When the in vitro produced hematopoietic progenitor cells were transplanted back into the diseased host mice, animals were rescued from all hematological and systemic symptoms associated with sickle cell anemia. (2,4) An overview of the used method is given in figure 3.

Figure 3: iPS cells in the treatment of sickle cell disease(2).

Hanne and colleagues discovered that iPS cells technology may also be of great value for the treatment of Parkinson disease. They found that reprogrammed fibroblast could efficiently differentiate into neuronal and glial cell types in vitro. These cells were also able to integrate functionally into various brain regions when transplanted into the fetal mouse brain.(4,8) More studies have reported the derivation of therapeutically relevant cell types from iPS cells, including human insulin-secreting cells and mouse and human functional cardiomyocytes.(4)

A number of recent studies demonstrated that iPS cells can also be generated from patients. This cells are probably of more use than ES cells for the study and treatment of human diseases. For example, iPS cells can be used for de development of disease models for diseases that lack adequate in vitro or animal models, like disorders affecting the brain and the heart. Ultimately they can be used for transfer of gene corrected autologous progenitors. These disease or mutation specific cell lines offer an opportunity to map out the developmental course of complex medical conditions, such as diabetes and Parkinson's disease, in a manner not possible through animal research alone or by observation of patients. In the long run, patient-specific iPS cell lines may be suitably for cellular therapy, given that they are derived from the patient to be treated, thus minimizing the risk of immune rejection.(2)

Challenges of iPS cells

Although iPS cells have many potential clinical applications, there are still several hurdles to overcome before the full potential of iPS cells can be realized. The first issue amongst these is hurdles is the fact that most iPS cells have, to date, been generated by transduction of somatic cells with retroviruses of lentiviruses. This viruses integrate randomly into the host genome and are silenced during iPS generation. However there is potential for reactivation of these viral transgenes, which include potent oncogenes as c-myc and klf4. Reactivation of c-myc can lead to tumour formation and also to the inhibition of iPS cell differentiation and maturation, leading to a greater risk of immature teratoma formation.(2)

As a major step towards solving this issue, several studies have demonstrated that mouse and human iPS cells can be derived without c-myc, although its absence significantly reduced the reprogramming efficiency. Recent studies show that reprogramming can be achieved in some cells with Oct4 alone. As an alternative c-myc can also be replaced by n-myc, a less tumorigenic member of the Myc family. Using this factor gives an equally high yield in the formation of iPS cells, but unfortunately it has not been investigated whether the use of n-myc results in reduced tumor formation.(2,4)

Another safety issue with the use of retroviruses and lentivirusses relates to the fact that transgene integration leads to mutations within the host genome. The integrated provirus can alter expression of neighboring host genes, leading to oncogensis. Alternative methods that promote reprogramming without integration have actively been sought to solve this problem.(2) In one study researchers were able to produce iPS cells from adult mouse hepatocytes using non-integrating adenoviral vectors.(2,4) They demonstrated that this method allows transient expression of the exogenous reprogramming factors without the use of integrating viruses. In another study, researchers were able produce iPS cells by transfecting mouse fibroblasts with plasmids containing the four transcription factors.(4) This two studies show that there are opportunities to produce iPS cells without the use of integrating viruses, which can solve previously the described problems.

A second key issue by the production iPS cells is the low efficiency of the process. Less that 1% of the cells that have incorporated the four retroviruses really become iPS cells.(1,3) This efficiency can be increased slightly by using a secondary reprogramming system but even than the efficiency is less than 2%.(5) A possible explanation for this low efficiency might be that iPS cells in fact originate from tissue stem cells which coexist in the fibroblast culture. However, previous research showed that the four transcription factors were not able to induce pluripotency in bone marrow stroma cells in a higher rate than normal, despite the fact that bone marrow cells should be more enriched with stem cells that normal fibroblasts.(1)

Another possibility might be that, in addition to the four factors, another factor also need to be activated by retroviral insertion. Candidates for such factors include the polycomb proteins, which play a critical role in the maintenance of pluripotenty. The identification of the missing factor may enable more efficient generation of iPS cells.(3) Alternatively, it is also possible that the levels of the four factors may have narrow ranges. Likely, only a small portion of cells is able to express all four factors at the right levels and is therefore able to acquire ES cell like properties. For example, excess Oct4 is detrimental to pluripotency. In addition, the balance between c-myc and Klf4 may also be a crucial factor by inducing pluripotency.(3)