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This paper is a comparative analysis of the four ways to reprogram somatic cells back into their pluripotent embryonic-like state. Cell fusion, extract-based reprogramming, direct reprogramming, and somatic cell nuclear transfer (SCNT) are the four different methods known to date. Each method offers advantages and disadvantages varying from difficulty of induction to the range of use. Induced pluripotent stem (iPS) cells have a wide range of use in both the research and medical fields. Most articles used for this paper discuss only one method each for reprogramming somatic cells at a time, which can lead to a variety of interpretations regarding the best of the four. The hypothesis that somatic cell nuclear transfer (SCNT) is the best method of reprogramming somatic cells back to a pluripotent embryonic-like state is supported by the articles reviewed.
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Methods of Reprogramming Somatic Cells to a Pluripotent Embryonic-like State
At one point during early embryonic development, cells have a pluripotency characteristic (Byrne, 2008). A pluripotent cell is one that has the potential to form most any cell type in the body. Through histone modifications and DNA methylation governed by specific epigenic codes, these pluripotent cells are programmed into the various cell types of the body. The reverse of this programming is done in order to generate the pluripotent stem cells from adult tissue instead of obtaining them from embryos. There is much debate over the ethics of obtaining stem cells from embryos, and this is why there is a large focus on ways to produce induced pluripotent stem (iPS) cells (Byrne, 2008).
There are four ways to reprogram somatic cells back into their pluripotent embryonic-like state; cell fusion, extract-based reprogramming, direct reprogramming, and somatic cell nuclear transfer (Byrne, 2008). Reprogramming via cell fusion is complete by the reactivation of pluripotency markers such as OCT4, SOX2, and NANOG in the differentiated somatic cell only after having an embryonic stem cell fused. In the extract-based reprogramming method, a crude cell extract is transferred with its associated reprogramming factors into the somatic cells. The direct reprogramming method transfers the reprogramming factors into the somatic cells through a virus-mediated transduction pathway. In the somatic cell nuclear transfer (SCNT) method of reprogramming somatic cells back to a pluripotent embryonic-like state, an enucleated metaphase II oocyte is injected with a differentiated nucleus. This produces a pluripotent
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stem cell that is not genetically modified and is isogenic to the donor. The ability of
having these stem cells be isogenic to their donor eliminates the problems the others have with long-term transplantation since the cells are not rejected by the patientâ€™s immune system (Byrne, 2008). For this reason, a hypothesis that somatic cell nuclear transfer (SCNT) is the best method of reprogramming somatic cells back to a pluripotent embryonic-like state seems most likely.
In a recent report written by Yu and associates, cell fusion was conducted on IMR90 fetal fibroblasts with an emphasis on OCT4, SOX2, NANOG, and LIN28 (Yu et al., 2007). Demethylation patterns of the iPS(IMR90) clones were similar to that of human embryonic stem cells and different to that of the parental IMR90 cells. The reprogramming of these human somatic cells through cell fusion proves very useful since it allows the iPS cells the potential to differentiate into any of the three germ layers of the body. The main problem associated with this method of reprogramming is the viral vectors used at the insertion site. Viral vectors have been known to cause mutations when used for gene insertion. However, these iPS cells are still important in testing new drugs, and additional research is being conducted to use non-viral vectors (Yu et al., 2007).
In another study, the transcription factors OCT4, SOX2, C-MYC, and KLF4 were used to induce fibroblasts to iPS cells (Wernig, 2007). This is another reprogramming method that is accomplished through cell fusion. Although these scientists used some different transcription factors to induce the cells, the same transduction vectors were used to introduce the cells to the target tissue (Wernig, 2007).
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Nuclear and cytoplasmic extracts from undifferentiated pluripotent cells have a regulatory component that that is capable of reprogramming somatic cells (Collas and Taranger, 2006). These cell extracts reprogram somatic nuclear function by enhancing the stem cell genes, modifying histones, and reducing somatic cell specific markers. A problem associated with the extract-based reprogramming method is that the reprogrammed cells do not obtain a fully pluripotent transcriptional state (Collas and Taranger, 2006).
The direct reprogramming method involves the integration of genetic reprogramming factors into multiple locations in the somatic cellâ€™s genome (Meissner, Wernig, and Jaenisch 2007). The reprogramming proteins that get released change the somatic cell nucleus back to a pluripotent state. A downside to this method is that when the genetic integration occurs, the inactivation of other genes has the potential to eventually reactivate and have negative effects. The reprogrammed pluripotent stem cells from this method still has use in the research field (Meissner et al., 2007).
In the SCNT method of reprogramming somatic cells, the DNA is stripped from the cell nucleus and is then injected into a metaphase II oocyte (Byrne, 2008). The cytoplasm in the oocyte epigenetically reprograms the nucleus into a fully pluripotent cell that is isogenic to the donor. The results may be ideal for both transplantation therapy and research, but the efficiency of this process is very low. Since the efficiency is so low, and human oocytes are in short supply, this SCNT technology is not as well studied as the other three methods (Byrne, 2008).
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All of the research studies used for this paper suggest that cell fusion, extract-based reprogramming, and direct reprogramming are excellent modes of reprogramming somatic cells back to a pluripotent embryonic-like state, but the products are not worthy of long-term cell therapy. The limitations of the aforementioned three methods do not make them any less important; it just means that in vitro studies prove more useful. In vivo studies using the SCNT method of reprogramming have less negative effects than the other three methods (Byrne, 2008).
There are a few obstacles in the path to creating successful pluripotent stem cells for patient-based transplantation purposes. The ethical stigma associated with the use of embryonic stem cells from human embryos is a major barrier that has emplaced laws governing the use of these cells (Takahashi, 2007). Another problem arises from the issue of not having an appropriate transduction vector to introduce the new cells without having the potential to do more harm with mutations (Byrne, 2008). In a study done by Kealy et al. (2009), both viral and nonviral transduction vectors were used to transfer a protein reporter gene to the endothelial progenitor cells.
The use of a successful nonviral transduction vector would dramatically increase the range of in vitro studies (Kealy et al., 2009). Although nonviral transduction vectors such as plasmid/liposome DNA vectors have been used, viral vectors have a higher level of expression. Vector related cytotoxicity is one of the main problems associated with gene therapy, and is a problem for both viral and nonviral vectors (Kealy et al., 2009).
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