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Induced pluripotent stem cells (iPS) (Fig1.) are those cells in which "pluripotency is induced" by somatic-cell reprogramming which make them behave similar to embryonic stem cells (ES) in morphology, proliferation, growth properties, teratoma formation and expression of ES cell marker genes (Yamanaka et al,2007; IPS essay). The method was first demonstrated by Shinya Yamanaka et al. by producing iPS cell from the mouse embryonic fibroblast (MEF) and adult mouse tail-tip fibroblast by retro-virus mediated transfection of four defined factors (Yamanaka et al,2006). Same was also demonstrated with human fibroblast a year later (Yamanaka et al, 2007). The risk of viral integration into genome and formation of tumours in iPS clones led to the development of other techniques for generation of iPSC in recent years.
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Figure1. Morphology of induced pluripotent stem colony produced from adult human dermal fibroblast using four transcriptional factors by retrovirus vectors. (commentary)
The main advantage of iPS cells over ESC is that it doesn't require human embryo and therefore it brings it out of the ethical consideration. This enables to make patient-specific cell lines. The iPS technology promises to be an effective tool in research, applicable in understanding of disease mechanism, drug screening, patient-specific therapy like juvenile diabetes and spinal cord injury and regenerative medicine (ips essay). The technical difficulty and safety concern for production iPS cells need to be overcome before putting iPS cells to any biomedical application.
Reprogramming of the genome of adult somatic cells can be done by four different techniques (Fig2.). It can be done either by transfer of reprogrammed somatic nuclei into enucleated oocyte or fusion of differentiated cells with ES cells or by nuclear extract of a pluripotent material (Table1.). These techniques led to finding that there are certain factors capable of induction of pluripotent state in differentiated cell (good review). Based on such findings, 24 genes involved in up-regulation of pluripotency in mouse ESC were recognised (good review). Shinya Yamanaka selected only defined and limited set of four transcription factors - octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) and carryout retroviral-mediated introduction into human fibroblast (Fig1.) and then culturing these cells under embryonic stem cell conditions (Yamanka et al,2006; Yamanaka et al, 2007; good review ; ips essay). The coding region of Fbx15 gene expressed in ESC was replaced with neomycin resistance gene in fibroblasts (Yamanaka et al 2006) using constitutive promoter that gives high expression. After transfection, the neomycin-resistant colonies were morphologically identical to ESC (Fig1.) as termed iPS cells by Yamanaka. Thus, direct reprogramming technique formed the classical method for producing iPS cells where the genes of interest are reprogrammed using viral vectors.
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Figure2. Four different strategies to induce reprogramming of genome of the adult somatic cells: Nuclear transfer, Cell fusion, Cell explanation (cell extract) and direct reprogramming by infecting the genome with virus. In direct reprogramming, the somatic cells are infected with the virus which enters the nucleus and enables the expression of cloned genes.
Table1. Various methods for reprogramming somatic cells (Adapted from good review)
Transfer of nucleus from somatic cell to enucleated oocyte and reprogrammed giving rise to whole organism.
Low efficiency. Development of abnormalities in cloned animal. Ethical restrictions.
Fusion of ESC with differentiated cell giving rise to hybrids of differentiated cell.
Cell hybrids lack normal diploid chromosome set.
Permeabilization of differentiated cells and their treatment with a pluripotent cell extract which may give rise to various differentiated cell.
Reprogrammed cell display only some properties of pluripotent cell.
Reprogramming of differentiated cells via transduction of viral vectors expressing Oct4, Sox2, Klf4 and c-Myc genes giving rise to pluripotent state similar to ESC (iPS).
Insertional mutagenesis due to viral integration. Reactivation of transgenes particularly by c-Myc oncogene.
The iPS lines obtained by direct reprogramming differed in gene expression profiling when compared to ESCs. Also, they gave rise to unviable chimeras after injection at blastocyst stage. So, instead antibiotic resistance gene Nanog or Oct4 as promoter were used which was able to produce viable chimeras when introduced at blastocysts stage and differentiated into the three germ layers of embryo. But depending upon cell types, the formation of iPS colonies was relatively at low frequency (0.05-0.1%) within the culture. Thereafter, the use of antibiotic resistant genes was eliminated. It was observed that the properties of iPSs did not differ from ESC in absence of selection, in fact the frequency increased. This technology has been successful in producing iPSs from various cells such as hepatocytes, neural stem cells and pancreatic Î²-cells in humans (good review). Since, reprogramming has been done using various factors, experiments have shown production of human iPS using OCT4, SOX2, NANOG and LIN28 and production of mouse iPS using Sox (Sox1,Sox3,Sox15 & Sox18), Myc (1-Myc & n-Myc) and Klf (Klf1,Klf3 & Klf5) have been successful although the efficiency was comparatively lower than the original four set of genes.
Lentivirus as the vector to obtain iPS worked more efficiently than using retrovirus as vectors. It resulted in higher rate of iPS production. However, lentivirus are silenced later and gets reactivated more frequently as the compared to retrovirus. Thus, viral intergration shows adverse effects in the reprogrammed cells. The integrated virus at random genome sites causing mutation and interferes with the regulatory system of that gene. Particularly it is the case with c-Myc gene which gives rise to serious consequences like apoptosis and cell malignancy.
FURTHER IMPROVEMENTS IN iPS METHODOLOGY
Reduce number of reprogramming factors
Reprogramming using viral vectors may result in reactivation of cloned genes. To improve the safety of iPS cultures, it is essential to reduce the set of transcriptional factors used for reprogramming. By elimination of c-Myc, the silencing of retroviral vectors is done more quickly and occurrence of tumour in chimeric mouse also reduces. However, this results in longer reprogramming and low frequency of production of iPS colonies. For culturing the cells in reprogramming where c-Myc is not utilized, the medium is enhanced with Wnt3a protein factor which is involved in pluripotency maintenance. For showing the possibility of reprogramming using one, two or three factors, neural stem cells are used as source for experiment since it endogenously expresses Sox2, c-Myc, Klf4 , Oct4 and also AP and (stage-specific embryonic antigen) SSEA-1 (one factor).
"Reducing the number of factors decreases the chance of retroviral insertional mutagenesis (one factor)". To reduce the viral integration occurring through c-Myc, reprogramming is done using only two factors and endogenous expression of other factors. Some recent experiments done on neural stem cells have demonstrated that reprogramming is possible by utilising only two reprogramming factors. Neural stem cells expresses SOX2 twice more than ESCs and equal amount of C-MYC protein. With various combinations it showed different time required for iPS production. For example, iPSs was produced most rapid using Oct4-Klf4 whereas it took one-two weeks more using Oct4-c-Myc combination.
Such findings opened new doors for producing safe iPS cultures where the set of reprogramming factors can be reduced. After two factors reprogramming, recent experiment have demonstrated that production of iPS cells from neural stem cell is also possible using only one factor (one factor). This is called one-factor induced pluripotent stem cells (1F iPS). The exogenous expression of Oct4 was shown to be sufficient enough for producing iPS from adult mouse. The properties of 1F iPS were found to be similar in vitro as well as in vivo. They were capable of efficiently differentiating into NSCs, cardiomyocytes and germ cells in vitro and formation of teratoma and germ line transmission in vivo (one factor). Previous studies have revealed that there exist about twenty retroviral integrations for all four factors (Aoi et al., 2008; Wernig et al., 2007) (one factor). So while using only 1F iPS, it reduces to five integrations by only Oct4 transgene (one factor). But it stills remains the matter of concern that viral integration cannot be completely eliminated.
Non-viral integration (adenovirus)
To avoid the viral integration into the genome, iPS cells were also generated using adenoviruses. Adenoviruses don't integrate in host genome during their life cycle. The reprogramming is done without their integration into genome. After few passages they are removed from the cells. Therefore, the iPS produced is free from any genomic modification. However, there is still little chance of virus getting integrated into the genome. For that appropriate test are carried out to detect viral genome in the DNA. Construction of adenovirus vector is relatively time consuming. Also, the efficiency of the reprogramming is much lower as compared for retroviral delivery. The low efficiency could be occurring because the transgene expression requires eight days. Therefore, makes it difficult to maintain high level of reprogramming factors using adenovirus (many ways).
Another approach to avoid viral integration is to use plasmid vectors for transfection. Mouse iPSC lines from MEF were successfully generated using serial transfection and transient expression of two plasmids, one expressing c-Myc and second expressing Oct4, Klf4, and Sox2 showed no evidence of viral integration into the genome (Okita et al 2008). However, the frequency obtained for reprogramming was extremely low (Table2&3). It also developed tetraploid clones. The experiment was able to achieve only 8% of clones free of transgene integration. Most of them lead to the tumour formation and prenatal death. The efficiency can be improved by 33% using 4 serial transfections and 2 plasmids. Thus, it increases laborious work.
Production of Human iPS cells from fibroblast by single transfection with Epstein-Barr nuclear antigen-1(EBNA1) based episomal vectors free of viral integration and transgene sequences was recently performed(episomal vector). This EBNA1 is suitable for inserting the reprogramming factors into host genome as it can be transfected without viral packaging and can be eliminated from the cells by culturing in the absence of drug selection (episomal). The EBNA1 vectors replicate only once per cell cycle and with help of drug selection it can be established as stable episomes about 1% of the intial transfected cells which is comparatively very low (Fig5). Initially episomal vectors failed to produce hiPSC. Later combination of OCT4, SOX2, NANOG, LIN28, c-Myc, KLF4, and SV40LT, were successful in producing iPS cell colonies from human foreskin fibroblasts using oriP/EBNA1-based vectors. Due to toxicity produced by c-Myc, substantial cell death occurred. Stable episomes were established at different frequency depending on cell types.
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Figure3. Illustrates three different strategy to produce mouse iPS cells- Retroviral or lentiviral transduction, adenoviral transduction and plasmid transfection (many ways).
Table2. Efficiency achieved by use of various combination and number of reprogramming factors (many ways).
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Although adenoviral and plasmid transfection method are transient and improves the safety of iPS cultures by eliminating the risk of inserting mutagenesis, the reprogramming efficiency is not achieved more than techniques using retroviral and lentiviral as vectors (Table3) (piggybac). Therefore, the alternative method of excision strategy was introduced for removing the viral genome after transfection as to get rid of the problem arising from the reactivation of oncogene.
Cre-loxP recombination technique is used for excision of the viral genome in established iPS cell lines to avoid viral integration but leaves behind the residual vector sequences (Fig4.a). In this technique, full length recombinant viral DNA is generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle (crelox 1999). This excision strategy can be applied to lentiviral and adenoviral vector methods for producing iPSC. The factors are connected with 2A peptide linkers. It simplifies and reduces the time consumed by viral vectors but may lead to genomic instability (Fig5.).
PiggyBac (PB), also called sleeping beauty, is an excision strategy using transposons (host-independent factor) to deliver doxycycline-inducible transcription factors (Fig4.b) (piggybac). PB system requires inverted terminal repeats and transiesnt expression of transposase enzyme which will catalyze the excision event. The integration of transposons are simple and has improves safety profile compared to plasmid vectors (Fig5.) (sleeping beauty). This method was able to generate stable iPS cells with characteristic similar to ESCs and succeed in several rigorous differentiation assays (PiggyBac). The natural propensity of this excision strategy for traceless removal of reprogramming factors joined with viral 2A sequences delivered by individual PB insertions from the seamless excision is advantageous (PiggyBac). The vector elimination using PB system in human has not yet been demonstrated and removal of multiple transposon is laborious.
Figure4. Excision strategy by a) Cre-loxP recombination and b) Piggy-Bac transposition. a)(grey bar- lentiviral vector, white triangle -loxP sites and black line - genome of fibroblast) the expression of Cre eliminate the factor and leaves the external DNA of loxP site integrated after reprogramming. b)(grey bar -PB transposons and white rectangles- terminal repeats) PB Transposase eliminates the transposon completely (O'malley et al., 2009)
To fully obviate the need of viral vector in reprogramming of somatic cells for generation of iPS cells, another approach used is based on protein products of the genes that will help in insertion of transgene (good review). The recombinant OCT4 and SOX2 proteins was obtained and then fused with TAT domain (which are arginine rich domains) that allows the transfer of protein from the culture medium into the cell (Good review) (Zhou et al., 2009). They specifically bind to DNA in landing sites of the corresponding reprogramming factors (good review). However, the method of direct protein transduction is very slow, inefficient and requires optimization (Table3). This technique is cost-effective since there is large availability of proteins. The cell-free protein synthesis (CFPS) produces factors from E.coli based methods. Thus, it surpasses the folding step and eliminates toxicity and aggregation occurring from the in vivo production of reprogramming factors (Belmonte et al., 2009).
Table3. Reprogramming efficiency and safety advantages of different reprogramming strategies.
To enhance the efficiency of reprogramming, transcriptional factors are supplemented with some small molecules/chemicals. Compounds or substances which are capable of altering the chromatin structure are used for catalyzing the reprogramming which includes DNA methyl-transferase inhibitor 5-aza-cytidine (AZA), histone deacetylase (HDAC), chemical that inhibit Tgf-b signalling and deacetylase inhibitor valporic acid (VPA)(small molecules; epigenetic reprogramming) (Table4). VPA was found to replace the c-Myc gene in iPS generation and increased the reprogramming efficiency by 100-fold (good review). Whereas histone methyl-transferase inhibitor G9a BIX-01294 produces iPSs with three factors without the Oct4 gene, but the reprogramming rate of somatic cells is extremely low in this case (good review). Small molecules efficiently reprogram cells and generate stable iPSC lines. Use of small molecules would make the reprogramming independent of the protein transduction and reduce the laborious work cause by it. Small molecules will also eradicate safety concerns of the transgenic approaches (Kaji et al., 2009; Kim et al., 2009; Okita et al., 2008)(small molecule). Use of small molecules allows utilization of reduce number of transcriptional factors.
Very recent experiments also put forth the hypothesis that reprogramming might be possible by only use of chemicals agent replacing the vectors for gene delivery (good review). High level of efficiency will be achieved is doubtful and so far it has not been achieved on human cell lines. They can only enhance the expression of genes for pluripotency and might not repress genes of differentiation. Therefore, small molecules may not be sufficient enough to completely replace the reprogramming factors (good review).
Table 4. Function of various small molecules in reprogramming of iPSC
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Figure5. Pros and Cons of different reprogramming strategies (nature piggy)
MAINTENANCE OF iPS CULTURE
iPS cells are co-cultured with mouse embryonic fibroblast (MEF) feeder cells. Recent studies have revealed that, feeder cells does not accelerate or initiate the reprogramming factors or increase the frequency of iPS colonies (MEF). Their main role remains to provide the feeder conditions and microenvironment that improve the growth of primary iPS colonies. Addition of bFGF, WntsandBMP4 and activin A (bFA) to medium helps sustaining the pluripotency of iPS. For regenerative medicine, human iPS cells are needed free of animal feeders (MEF). For efficient long term pluripotency maintenance of human and mouse iPS cells, it is co-culture with mitomycin-C-inactivated. The disadvantage includes destruction of embryo for preparation of feeder layer and the process is time consuming. The factors generated by MEF play crucial role in iPSC proliferation and maintenance. For example, Oct4, Sox2 and Nanog actively transcribed genes such as Lefty2, STAT3 and FGF2, etc. The resulting lower expression of iPSC could be affected by the downstream factors secreted by MEF. Experiments have shown that bFGF and activin A promotes iPSC proliferation in absence of feeder cells. Recent studies have also shown that hypoxic condition enhances the efficiency for production of mouse and human iPSC. It promotes the survival of iPSC and increases the GFP-positive colonies from 2/3-factor transduced MEF.
The cell density also plays an important role in the efficiency of the generation of iPSC. The density of passages has to be maintained every time. High-density growth of cells will results in death as there is limitation of space, nutrients and reagents. For the appropriate efficieny and growth of iPSC, the cells are passaged with density depending upon the area of flask and required confluency.
APPLICATIONS OF iPS TECHNOLOGY AND ITS CHALLENGES
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Figure6. In vitro and In vivo application of iPS cells (commentary)
There are several advantages of the iPS technology including analysis of the disease mechanism by obtaining iPS lineages, developing patient-specific cell based therapies, robust and high-throughput drug testing, studying early developmental processes in vitro cell culture and development of non viral gene therapy. (epigenetic reprogramming 1) The progress of iPS technology will greatly have impact on the research of regulation for stem cell differentiation. Also, it will enhance the understanding of signalling interaction which is critical for developing artificial tissue and organ (epigenetic reprogramming 1). Before all this possible application is put to use, there lies a series of technical challenges and hence, understanding the recent advancement in iPS technology is essential (Table 5).
Table 5. Potential Obstacles of different methods for generating reprogramming factor-free iPS cells (technical challenge).
The technical challenges faced includes developing of reprogramming factor-free hiPSCs (table5), gene-targeting strategies to produce differentiation gene markers, disease-specific phenotypes in vitro and in vivo for establishing disease-specific lines. So far, 10 disease specific iPSC lines have been generated including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and Parkinson's disease and various genetic diseases (table6).
Table6. Patient disease-specific iPSC lines.
In vivo applications of iPS cell technology is possible as it overcomes restrictions associated with human embryos and also immune rejection caused by ESC after transplantation. Another concern is the teratoma formation. Use of terminal differentiated cells might reduce the teratoma formation as few undifferentiated cells can lead to teratoma formation. Production of Î²-cells from terminally differentiated cells has been demonstrated to reduce the risk of teratoma formation. Also, the methods doesn't indicate that reprogramming is fully complete or no. Partial or aberrant reprogramming will increase the risk generating genetic dysfunction. Leaky expression of transgene may also result in same. There lie several obstacles before iPSC technology is successfully applied in regenerative medicine for humans (Fig7. &Table6). So far, iPSC is produced from murine model with sickle cell anaemia and Î²-thalassemia.
Figure7. Application of iPSC technology in regenerative medicine.
In vitro application of iPSC technology includes disease modelling and drug screening. Hepatocytes produce using iPSC with various cytochrome p450 helps in detecting liver toxicity of new drugs. Generation of cardiacomyocytes is useful in testing drugs for long QT syndrome. Such disease model helps in understating the underlying pathogenesis. Study of drugs in vitro can produce novel drugs applicable in vivo. The transgenic mouse with certain mutations helps in studying genetic disease like ALS whose motor neuron have been produced by iPSC. The technical challenge is faced in recapitulating disease and to mimic the epigenetic changes occurring in particular environment over time in cells for producing patient-specific iPSC. For certain kind of genetic disease where more than one cell type is involved, multiple cell types needs to be produced from iPSC.
The iPSC technology which includes reprogramming strategies like retoviral vector, protein transduction, plasmid transfection, excision strategy gives scope for further development of the techniques to increase the efficiency for generating hiPSC. Overcoming the use of transgene, tumour formation and toxicity will enable the application the iPSC technology in regenerative medicine and as a research tool. The promises hold by this tecnnology is yet far from clinic but better understanding of the technical challenges will help it improve further so as to remove the technical limits in clinical applications of iPSC. More research is needed to improve safety and low efficiency issues in iPS colonies.