As viable human brain tissue is not available for use in studying disease development and creating therapies for neurological disorders like Huntington’s disease (HD), researchers desperately needed an alternative cell source for this purpose. Embryonic stem cells fit this role but have many disadvantages, especially for treatments, including immune rejection by the recipient. Some of these drawbacks have been overcome by a recent discovery that revolutionized the face of stem cell biology. In 2006, Shinya Yamanaka’s research group at Kyoto University made a groundbreaking announcement: they had discovered that adult cells could be genetically engineered to revert back to apluripotent, stem cell-like state. As iPSC (induced pluripotent stem cell) production rapidly improved, the cells were soon able to compete with traditional fetal, embryonic, and adult stem cells. The primary advantages of iPSCs compared to other stem cells are: a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and b) the lack of ethical implications because cells are harvested from a willing adult without harming them. These patient-specific cells can be used to study diseases in vitro, to test drugs on a human model without endangering anyone, and to hopefully act as tissue replacement for diseased and damaged cells.
Although there are many problems that still must be addressed for iPS technology, such as the tendency for tumors to evolve after iPSC transplantation and the low efficiency of the technology, iPSCs could completely change how diseases are approached in biomedical research. For HD and other neurological disorders, iPSCs could create perfect models for the cells of the central nervous system that are harmed in the diseases.
The Discovery of iPSCs^
The creation of pluripotent cells has been widely studied for decades. In 1976, the first method of fusion of an adult somatic cell with embryonic cells to create pluripotent stem cells was reported. However, fusion with embryonic cells created unstable cells that were rejected by immune systems after transplantation. If the genes that induced pluripotency could be isolated from their parent embryos and injected into somatic cells, these problems could be avoided.
Yamanaka’s research team studied twenty-four genes expressed by embryonic stem cells in an effort to track down these essential genes that induce pluripotency. To detect pluripotency, they looked for cells expressing genes that were traditionally expressed only in embryos. They discovered that the addition of four genes induced a cell into a pluripotent state capable of then becoming many different cell types.
The Four Factors^
Subsequent studies showed that other gene combinations were also successful in reengineering cells into iPSCs, but none were as efficient as the first four. Adding other genes that are expressed in early development was shown to increase reprogramming efficiency, and the specific genes needed varied depending on the cell type that was being forced back to its pluripotent state. As the four factors and their alternatives were largely discovered by trial and error, it is not known how the genes induce pluripotency. Discovering how genes work may point to ways of improving the efficiency of the process and assessing the quality of iPSCs.
The specific genes that induce iPSCs tell scientists a lot about the characteristics of the cells themselves. Pluripotent stem cells are very closely related to tumor cells. Both can survive and proliferate indefinitely, and a test of pluripotency is whether a cell can create a tumor. It is therefore no surprise that two tumor-related genes, c-Myc and Klf4, are needed to create iPSCs. Another requirement of pluripotent stem cells is open and active chromatin structures (for more information on chromosomes, click here and DNA transcription click here). The c-Myc gene codes for proteins that loosen the chromatic structure, stimulating differentiation. Klf4 impedes proliferation. c-Myc and Klf4 in this way regulate the balance between proliferation and differentiation. If only c-Myc and Klf4 are used in the engineering of iPSCs, tumor cells will arise—instead of pluripotent stem cells. Oct3/4 and Sox2 are required to direct cell fate towards a more embryonic stem cells (ESC)-like phenotype. Oct3/4 directs specific differentiation, such as neural and cardiac differentiation, while Sox2 maintains pluripotency. Oct3/4 and Sox2 together ensure that iPSCs are indeed pluripotent stem cells and not tumor cells.
Amounts and Timing of Reprogramming Factors^
The programming of iPSCs depends both on the original cell type being transformed and the levels of each reprogramming factor that is expressed. Expressing Oct3/4 more than the other genes increases efficiency. Increasing the expression of any of the other three genes decreases the efficiency. There is clearly a correlation between gene expression ratio and reprogramming efficiency, but the optimal ratio is likely to vary depending on the cell type being reprogrammed. For instance, when neural progrenitor cells are reprogrammed, they do not require Sox2 as they express this gene sufficiently already. The level of expression of other important genes for maintaining pluripotency also can affect the reprogramming process and the quality of the resulting cells.
The effect gene expression ratio has on reprogramming may explain why efficiency is typically so low (less than 1% of cells are reprogrammed successfully). Reprogramming is a slow process, and so the timing of various events may also exert a great influence over thecell’s success. The minimum time for the full reprogramming of a mouse somatic cell into an iPSC is between eight and twelve days. The timing of the mechanism for cellular reprogramming may also be a reason for low efficiency, as the cells can only proceed if the right molecular events happen in the correct order.
Creation of a Germline Competent Model^
In the first studies of iPSCs, the cells were shown to be similar to ESCs in morphology and proliferation. But the cells were not germline-competent, in other words they were unable to differentiate into cells that expressed genes of the parent cells, and so they could not give rise to adult chimeras when transplanted into blastocysts. As chimeras play key roles in biomedical research, scientists identified iPSCs through a stricter gene marker that only identified iPSCS that were germline competent. It was found that cells that expressed Nanog, a gene closely tied to pluripotency, were germline competent. These cells also were virtually indistinguishable from ESCs in gene expression, and were more stable. The transgenes were better silenced in the Nanog identified cells although 20% of the iPSCs still developed tumors due to the reactivation of c-Myc. Unfortunately this stricter criterion also decreased efficiency to only 0.001-0.03%. While subsequent studies improved this efficiency by varying methods, the fact remains that iPSCs are generated with incredibly low efficiency.
Characteristics of iPSCs^
iPSCs exhibit many characteristics that are related to their pluripotency. They lose proteins that are common to somatic cells and gain proteins common to embryonic cells. They also lose the G1 checkpoint in their cell cycle control mechanism, which embryonic stem cells lack as well. During the reprogramming of somatic cells in the iPS mechanism, the cell cycle structure of stem cells must be reestablished. Another distinguishing characteristic of pluripotent stem cells is their open chromatin structure, as this is needed to maintain pluripotency and to access genes rapidly for differentiation. iPSCs have the open chromatin structure associated with ESCs and other pluripotent cells. Finally, female iPSCs show reactivation of the somatically silenced X chromosome. A very early step of stem cell differentiation is the inactivation of one of the two X chromosomes in female mammals, a random process. By the reactivation of this X chromosome, iPSCs show that they are truly pluripotent and identical to ESCs.
Non-retroviral Methods of iPSC Production^
A huge barrier to the eventual use of iPSC-derived treatments is the use of retroviruses to force the expression of the four key genes, discussed above, and activating their transcription factors. Retroviruses can carry target DNA that is inserted into a host cell’s genome upon injection, making them ideal for incorporating the four genes into target cells. However, this DNA and the rest of the viruses’ genomes remain in the host genome, which can lead to transcription of unwanted genes and greatly increases the risk of tumors. The expression of the four transgenes must be silenced after reprogramming to avoid harmful gene expression. c-Myc, a tumor-promoting gene, especially must be silenced after cellular reprogramming or the risk of tumor development becomes too great for clinical use. These retroviral methods in which the transgenes are still present in the pluripotent cells pose a danger to safety, and also are less closely related to ESCs in gene expression than their non-retroviral alternatives. Methods of reprogramming iPSCs without transgene expression in the reprogrammed cell is therefore essential not only for potential therapies and clinical applications, but also for reliable and accurate in vitro models of diseases. Yet, the low efficiency of alternatives remains a worry. Whether these methods will be viable for human clinical use remains to be seen.
The excision strategy (transient transfection) of iPSC generation allows the transgenes to briefly integrate into the genome but then removes them once reprogramming is achieved. An example of this site/enzyme combination is the loxP site and the Cre enzyme. In a study of Parkinson’s disease (PD), specific iPSCs, this loxP/Cre combination was used to generate the iPSCs. Neural differentiation was then induced on the iPSCs to test whether they could differentiate into dopaminergic neurons, the cells harmed in PD. The differentiation was successful, indicating the transgenes had been excised. However, a loxP site remains in iPSC genome as does some residual viral DNA, so there is still a small potential for insertional mutagenesis. The piggyBac site/enzyme system on the other hand is capable of excising itself completely, not leaving any remnants of external DNA in the iPSC genome. The piggyBac system also was much more efficient than other non-retroviral methods, with comparable efficiency to retroviral methods, but with the added benefits of safety and ease of application.
Adenoviral methods do not pose the same threats as retroviral methods of generating iPSCs. Adenoviruses work like all viruses by hijacking their hosts’ cellular machinery to replicate their own genome and reproduce, but unlike retroviruses they do not incorporate their genome into the host DNA. Because the transgenes are never even incorporated into the host’s genome they do not have to be excised. Instead, the genes are expressed directly from the virus’ genome. iPSCs created by adenoviral methods demonstrated pluripotency, but have extremely low reprogramming efficiency. Viralgenomic material could not be detected in any of the iPSCs, and no tumor formation was reported. This suggests that the use of non-integrating adenoviral methods substantially lowers the threat of tumorgenesis. The successful creation of iPSCs from adenoviral methods proves definitively that safer, non-retroviral methods can also successfully reengineer cells.
Non-DNA methods of iPSC generation^
Recent studies have implied that perhaps genetic material is not required for iPS cellular reprogramming. The substitution of transgenes with small molecules that promote iPSC generation would be a safe, clinically appropriate way of creating iPSCs, though it remains to be seen if small molecules will be able to completelyreplace genetic methods of iPSC generation or are just useful as supplementary aids to the process. Protein transduction is a different method shown to entirely replace gene delivery. In this method fusion proteins are created, which fuse each of the transgenes to a cell-penetrating peptide sequence that allows it to cross the cellular membrane. Reprogramming without DNA intermediates should eliminate the risk of tumorgenesis and distorted gene expression due to the reactivation of the transgenes.
Issues Facing the Use of iPSCs^
With iPSC research being a hotspot for several years now, many of the problems the technology first faced have been studied and resolved. iPSCs are now germline competent, can be generated from many different types of human and animal somatic tissue, and can be generated in a variety of retrovirus-free methods. This lack of retroviruses ended worries about transgene reactivation and subsequent tumorgenesis. The nature of the transgenes in question made the risk of tumor development particularly prevalent, as two of the genes, c-Myc and Klf4, directly inducing tumorgenesis. Retroviral delivery posed a threat to safety in its increased risk of tumorgenesis and in its tendency to alter gene expression. When other methods were established that did not require retroviruses, these concerns were put to rest, yet these new methods’ efficiencies must be improved and some issues still remain concerning the safety of iPSCs and their abilities to act on par with any other pluripotent cell.
Even without the use of retrovirsues, tumorgenesis is still a large concern for iPSCs, especially if they are ever to be used as cell replacement therapies. Using retroviral methods, twenty percent of iPSCs developed tumors in one study, and though this number has significantly lowered, it must become negligible for iPSCs to be considered for clinical use. It is telling that the assay for pluripotency in stem cells is the ability to form teratomas, or tumors. This test of “stemness” illustrates the precariously close link between stem cells and tumor cells. There are several proposals on how to prevent this tumor formation. The idea to sort cells before transplantation and after differentiation, so that only well-differentiated neural progenitors will be transplanted, is one such proposal. Another proposal is to genetically modify iPSCs so that they will have a suicide gene to self-destruct when tumors are created. Finally, some antioxidants, such as Resveratrol, have been shown to have tumor-suppressing qualities, and could potentially aid in any treatment proposed to prevent tumors (for an article about the potential of Resveratrol for the treatment of HD, click here).
Directed differentiation has been a perennial problem in stem cell biology, and iPSCs bring their own unique characteristics to the dilemma. As with ESCs, iPSCs sometimes have the tendency to not fully differentiate. Also, as with all stem cell research with neurodegenerative diseases, a more efficient and comprehensive method to differentiate cells into neural progenitors and specific neuronal tissue must be discovered, as current methods are imperfect and slow.
Quality Assessment and Variability^
In iPSC research there is a need to establish methods to evaluate the reprogramming process and the final quality of the cells. To create human iPSCs suitable for cell replacement therapies, there must be tests to ensure that all pluripotent cells have differentiated, and that the cells have not been genetically altered during reprogramming or during differentiation. With cells derived from diseased individuals for an autologous treatment, there is naturally the concern that the underlying genetic cause of the disease remains in the iPSCs and will manifest itself in the same way. Some studies have indicated that iPSC lines differ drastically, which makes the reproducibility of any particular phenotype difficult. Analyzing this variability may help discover which somatic tissue is best for generating iPSCs.
A problem that has not been significantly improved upon since the beginnings of iPSC research is the technology’s low efficiency. Some hypothesize that the addition of other factors would greatly aid the reprogramming process, and that reprogramming success depends on specific amounts and ratios of the four factors, which are only achieved by chance in a small percentage of the cells. Modifying the culture conditions is another area of study for increasing efficiency and rate of iPSC production. For cellular transplantation therapies, other questions must also be considered, such as the optimal cell dose and source tissue, and the best way to deliver the cells. There are potential solutions to this problem, though. Induction efficiencies have been improved up to a hundred times initial values by use of different somatic starting cells and the aid of small molecules. Although there are barriers to iPSC production, research in this field is still in its infancy and has made impressive gains for the short time it has been going on. As more studies are conducted on iPSCs, these issues may be resolved and iPSCs may enter a state capable of clinical use.
Origin Cells Used^
Another potential way to improve iPSC generation efficiency is to establish the best somatic cells type to reprogram for the cleanest, easiest reprogramming. Many different tissue types have been reprogrammed, including fibroblasts, neural progenitor cell, and stomach epithelial (stomach lining) cells. Certain cell types are much more efficient and rapid than others. There is also the probability that subtly varying iPSCs are generated from different types of starting tissue, some of which may prove to be useful for research or replacement purposes.
An interesting “type” of somatic cell was used in studies of secondary iPSCs. iPSCS were initially generated and then implanted into blastocysts to create chimeric animals. Somatic cells from these chimeras were then removed and iPSCs were generated from these cells, creating secondary iPSCs. These secondary iPSCs were generated more efficiently. The differentiation status of thecells to be reprogrammed also affects efficiency, as adult progenitor cells are reprogrammed at three hundred times the efficiency of completely differentiated somatic cells.
An interesting possibility for the reprogramming methods of iPSCs is the potential for transdifferentiation. It may not always be necessary to reprogram cells all the way back to their most primitive pluripotent stem cell state, and instead reprogram one type of adult somatic tissue directly into a different type, bypassing the lengthy processes of complete reprogramming and subsequent differentiation. For example, in theory fibroblasts that can be easily and safely obtained from a patient’s skin could be converted into neurons or heart muscle cells without ever passing through a pluripotent stage. This would have advantages not only in the conservation of time and resources but also for safety, as transdifferentiation does not pose the risk of tumorgenesis as the cells never are pluripotent. Unfortunately, the technology for such processes is very difficult. To reprogram cells directly into a different cell type, the qualities and characteristics of the desired cell type must be comprehensively understood. For iPSCs the desired cell type was embryonic stem cells, which were very well researched and characterized, but for many types of cell of interest, including cells of the central nervous system, there are still many unanswered questions about the target cell population. Excitingly, the Wernig lab at Stanford has recently created induced neurons (iN) directly from mouse fibroblasts.
A potential use of iPSCs for cellular therapy that can be applied much more quickly than actual replacement of damaged tissue is the transplant of pluripotent cells as support cells rather than replacement neurons. These cells offer neuroprotection by preventing inflammation and producing neurotrophic factors (for the therapeutic use of neurotrophic factors in HD, click here). In various studies, the transplantation of iPSCs has significantly improved host neuronal survival and function. This bystander mechanism of therapy is of huge immediate potential in iPSCs, and Dr. Nolta’s lab recently submitted a request for a clinical study of the same mechanism using mesenchymal stem cells to the FDA. For a detailed study of the use of iPSCs for this purpose click here.
Stem cell biology has been an area of great interest and intense debate since its inception, and iPSC technology has furthered this research and created hope for potential therapeutic applications. While there are still many barriers to the clinical use of stem cells, iPSCs may help elucidate the nature of both pluripotent stem cells and of many disease pathologies to reach an eventual concrete connection between the two. With their potential for autologous cell replacement and disease modeling in vitro iPSCs are the future of stem cell research, and as such they are key players in the battle against HD.
For Further Reading^
Abeliovich, Asa and Claudia A. Doege. “Reprogramming Therapeutics: iPS Cell Prospects for Neurodegenerative Disease.” Neuron. 12 Feb, 2009, 61 (3): 337-39.
Short, approachable article reviewing two studies deriving iPSCs from patients with neurological disorders.
Cox, Jesse L. and Angie Rizzino. “Induced pluripotent stem cells: what lies beyond the paradigm shift.” Experimental Biology and Medicine. Feb 2010, 235 (2): 148-58.
Very detailed, mostly accessible review of the state of iPS research and the discoveries to date, as well as what iPS cells mean for stem cell biology and modern medical approaches. Perfect thorough introduction to iPS technology.
Crook, Jeremy Micah, and Nao Rei Kobayashi. “Human stem cells for modeling neurological disorders: Accelerating the drug discovery pipeline.” Journal of Cellular Biochemistry. 105 (6): 1361-66.
Accessible, interesting article that argues the greatest potential for iPSCs is to test potential drugs for neurological diseases in vitro and find problems early on in the drug development, saving time and resources.
Gunaseeli, I., et al. “Induced Pluripotent Stem Cells as a Model for Accelerated Patient- and Disease-specific Drug Discovery.” Current Medicinal Chemistry. 2010, 17: 759-766.
Readable review on the future of iPS cells, comparing them with other stem cells and elucidating their pontential drawbacks. Good summary of the landmark discoveries in iPS technology to date.
Haruhisa, Inoue. “Neurodegenerative disease-specific induced pluripotent stem cell research.” Experimental Cell Research. 2010.
General overview of use of iPS cells specific to neurological diseases for modeling diseases in vitro and eventually using as a cellular replacement therapy. Good, non-technical overview of the various potential pathways of iPS technology.
Hung, Chia-Wei, et al. “Stem Cell-Based Neuroprotective and Neurorestorative Strategies.” International Journal of Molecular Science. 2010, 11(5): 2039–2055.
Overview of various neurological diseases and the potential of stem cell therapeutics, either using adult neural stem cells or iPS stem cells. Experiment descriptions are fairly technical, but the review’s reflections and discussion are accessible and interesting.
Laowtammathron, Chuti, et al. “Monkey hybrid stem cells develop cellular features of Huntington’s disease.” BioMed Center Cell Biology. 2010, 11 (12).
Detailed article on the establishment of pluripotent HD monkey model cell line and its use in the study of Huntington’s.
Marchetto, Maria C.N., et al. “Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases.” Human Molecular Genetics. 2010, 19 (1).
Fairly technical review describing the use of iPSCs for modeling neurological disorders.
Niclis, J.C., et al. “Human embryonic stem cell models of Huntington’s Disease.” Reproductive Biomedicine Online. July 2009, 19 (1): 106-13.
Detailed, technical article on the use of human embryonic stem cell lines for HD.
O’Malley, James. “New strategies to generate induced pluripotent stem cells”. Current Opinions in Biotechnology. Oct. 2009: 20 (5): 516-21.
Longer technical article on the various strategies to generate iPS cells without using potentially dangerous viral vectors.
Okita, Keisuke, et al. “Generation of germline-competent induced pluripotent stem cells.” Nature. 19 Jul, 2007, 448(7151):313-17.
Fairly technical article about an early study in iPS research, where cells were selected for Nanog expression rather than the less pertinent gene Fbx15. This higher caliber of selected cells were germline-competent.
Okita, Keisuke, et al. “Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors.” Science. 7 Nov, 2008, 322 (5903): 949-53.
Technical article about the advancements in finding non-viral, clinically applicable methods of creating iPS cells.
Orlacchio, A., et al. “Stem Cells: An Overview of the Current Status of Therapies for Central and Peripheral Nervous System Diseases.” Current Medicinal Chemistry. 2010, 17: 595-608.
Technical review on the various types of stem cells used in the studies of neurological diseases and the progress made to date with these cells.
Park, In-Hyun, et al. “Disease-Specific Induced Pluripotent Stem Cells.” Cell. 2008, 134 (5): 877-86.
Fairly accessible article on the creation of iPS cells with genetic defects, as tools for studying the symptoms and experimenting with treatments of various diseases.
Robbins, Reisha D., et al. “Inducible pluripotent stem cells: not quite ready for prime time?” Current Opinion in Organ Transplantation. 15 (1): 61-57.
Clear review of the barriers facing clinical use of iPSCs, accessible and realistic.
Soldner, Frank, et al. “Parkinson’s Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors.” Cell. 6 Mar, 2009, 136 (5): 964-77.
Technical article about first successful derivation of iPS cells from a patient with a neurodegenerative disease without using viral vectors. Relevant to HD research as a protocol that will likely be followed for subsequent creation of neurodegenerative iPSC lines for in vitro study.
Stradtfeld, Matthias, et al. “Induced Pluripotent Stem Cells Generated Without Viral Integration.” Science. 7 Nov, 2008, 322 (5903): 945-49.
Technical article outlining a method for creating iPS cells using excisable adenoviruses, rather. than retroviruses that have the potential to harm the cells.
Takahashi, Kazutoshi, et al. “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors” Cell. 30 Nov, 2007, 131(5): 861-72.
Landmark article in the discovery of induced pluripotent stem cells and the factors that create them. Short, but fairly technical.
Yamanaka, Shinya. “Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors.” Cell Proliferation. Feb, 2008, 41 (Suppl. 1):51-6
Short review, less technical summary of first iPS discovery by Yamanaka. Perfect for quick overview of the basics of iPS cell generation.
Yamanaka, Shinya. “Strategies and New Developments in the Generation of Patient-Specific Pluripotent Stem Cells.” Cell: Stem Cell. 7 June 2007, 1(1): 39-49.
Comprehensive review of various methods for creating pluripotent stem cells with a detailed introduction to iPSC methods. Fairly accessible, and very thorough.
A. Lanctot 2011