Induced
pluripotent stem cells (induced pluripotent stem cells, iPS cells) is
rearranged by gene approach, "induced" normal cell embryo back to the
original state, like embryonic stem cells
can be differentiated, such cells are called "iPS". Yamanaka iPS cell
research groups in Japan in 2006 named, they screened through multiple
Oct-4, Sox2, Klf4 and c-Myc four retrovirus-mediated gene transfer by
murine fibroblasts, to reprogram The result is a similar embryonic stem cells
(ES) cells with pluripotent stem cells, stem cells and named such iPS
cells [1]. The findings were "Nature" and "Science" magazine in 2007 the
first and the second largest scientific progress. Because of different
iPS and ES, no damage embryos, thus avoiding the obstacle ES research
and development of ethical issues, and because of the specificity with
individual sources, which does not involve the immune rejection, and
therefore have a broad and important basic research and clinical
applications value. Currently iPS related research has become a hot spot,
many of the world from every angle in the laboratory to explore the
mechanisms and applications, and the establishment of multi-party
validation of iPS cells repeatability. But in general, iPS research is
in its infancy, and how successfully established iPS cells still exist
many problems, this article will combine an overview of relevant
research in recent years, for at this stage there are two main problems
as well as safety-iPS cells induction efficiency do an analysis.
A security issue
The past two years, the world's attention to the reason for iPS cells showed explosive growth, mainly due to its huge potential clinical treatment prospects for establishing a secure and efficient iPS cells to treat disease is one of the ultimate goal.
Yamanaka first established iPS cells, ES cells, these cells express specific marker genes can also be immunodeficient mice teratoma, a tumor tissue from three germ layers; also chimera, but did not get viable chimeric mice [1]. This is mainly due to his use of retroviral vectors inserted mutation in mice genetically modified cells as well as four factors is oncogene c-Myc, and Klf4 also have some ability to cause cancer [2,3]. In fact there are also studies prove: Virus-mediated activation of c-Myc increased again iPS chimeric mice tumors originated chance [4,5].
In order to reduce the tumorigenicity of iPS cells, researchers have constructed various improvements from iPS cells, there are mainly two ways: First deprecated oncogene; firstly, to seek a safe gene transfer methods.
2007 Thomson team used Oct-4, Sox2, Nanog and Lin28 4 genes to reconstruct human somatic cells to iPS cells [6], first proved without oncogene c-Myc and Klf4 can also be successfully induced. After, Nakagawa group [5] and Wernig group [7] of these three factors have been successful attempts. Although not cause induction of c-Myc iPS cell efficiency is significantly reduced, but the pluripotency of iPS cells obtained and better quality, and the resulting offspring mice born iPS 100 days did not form tumors. Recently many studies have shown that only two genes can induce iPS cells successfully, but there must be a small molecule compounds involved. Feng group [8] with Essrb replace c-Myc and Klf4 these two potential carcinogen, and Oct-4 and Sox2 together to be effective in the fetal rat fibroblast reprogramming of iPS cells; Huangfu D group [9] find a histone deacetylase inhibitor (VPA, valproic acid) not only will be only two Oct-4 and Sox2 transcription factors transfected fibroblasts induced into iPS cells, and can be re-programmed rate up to 1%. Even Kim et al [10] reported that only one factor Oct4 can be adult mouse neural stem cells (neural stem cells, NSCs) reprogrammed into iPS cells because NSCs expressing Sox2, and Klf4.
So far, the transcription factor can be obtained gene into somatic cells iPS cells mainly in the following ways: retrovirus [1], lentiviral [6], adenovirus [11], transposons [12] and plasmids [ 13]. Retroviral and lentiviral-mediated transgene way that viral vector integrated into the host genome, to achieve transgene stably expressed, but the two transgenic methods easily activated oncogene; through adenovirus transcription factor genes into somatic cells, transient expression of these transcription factor, which gene is generally not integrated into the viral vector in the host genome, but the first, the method for the induction of somatic cells compared to the efficiency of iPS cells retroviruses and lentiviruses much lower [11] Moreover fact, adenovirus vectors may also be integrated into the genome, although this possibility is very small, this is not the best way; through transposons and plasmids principle mediated expression mediated through its four factors, inducing and the establishment of iPS cells, iPS cells, and the transient expression of these transposase enzyme is induced by removal of exogenous factors, such iPS cells will no longer express exogenous inducing factor, genomic DNA sequences does not cause any change in the security improved. But in the process of establishment of iPS cell transit transposon insertion on the safety of iPS cells long-term effects have yet to be further to verify.
Integrity can not completely avoid the use of viruses or transposons, to establish a more secure way to create iPS cells do? Fortunately, this year, almost the same time, Zhou H group [14] and Kim D group [15] in "Cell Stem Cell", introduced a new method, by direct protein-mediated, respectively, of the mouse and human fibroblasts induced by a iPS cells. That will be designed to pass through the membrane short peptide chain is connected to the reprogramming factor protein, such a fusion protein to penetrate the cell membrane into the cell interior, and execute functions of reprogramming. Unlike Zhou H group in the test small molecule compounds added VPA (valproic acid) to improve the efficiency of reprogramming, and Kim D team did not add any substance. Because this protein induces direct way does not involve any genetic modification, so for now, this is the most secure method. To sum up, through continuous exploration, iPS cells is gradually increased security, but these methods increase security, while induction of iPS cells are making efficiency is greatly reduced.
A security issue
The past two years, the world's attention to the reason for iPS cells showed explosive growth, mainly due to its huge potential clinical treatment prospects for establishing a secure and efficient iPS cells to treat disease is one of the ultimate goal.
Yamanaka first established iPS cells, ES cells, these cells express specific marker genes can also be immunodeficient mice teratoma, a tumor tissue from three germ layers; also chimera, but did not get viable chimeric mice [1]. This is mainly due to his use of retroviral vectors inserted mutation in mice genetically modified cells as well as four factors is oncogene c-Myc, and Klf4 also have some ability to cause cancer [2,3]. In fact there are also studies prove: Virus-mediated activation of c-Myc increased again iPS chimeric mice tumors originated chance [4,5].
In order to reduce the tumorigenicity of iPS cells, researchers have constructed various improvements from iPS cells, there are mainly two ways: First deprecated oncogene; firstly, to seek a safe gene transfer methods.
2007 Thomson team used Oct-4, Sox2, Nanog and Lin28 4 genes to reconstruct human somatic cells to iPS cells [6], first proved without oncogene c-Myc and Klf4 can also be successfully induced. After, Nakagawa group [5] and Wernig group [7] of these three factors have been successful attempts. Although not cause induction of c-Myc iPS cell efficiency is significantly reduced, but the pluripotency of iPS cells obtained and better quality, and the resulting offspring mice born iPS 100 days did not form tumors. Recently many studies have shown that only two genes can induce iPS cells successfully, but there must be a small molecule compounds involved. Feng group [8] with Essrb replace c-Myc and Klf4 these two potential carcinogen, and Oct-4 and Sox2 together to be effective in the fetal rat fibroblast reprogramming of iPS cells; Huangfu D group [9] find a histone deacetylase inhibitor (VPA, valproic acid) not only will be only two Oct-4 and Sox2 transcription factors transfected fibroblasts induced into iPS cells, and can be re-programmed rate up to 1%. Even Kim et al [10] reported that only one factor Oct4 can be adult mouse neural stem cells (neural stem cells, NSCs) reprogrammed into iPS cells because NSCs expressing Sox2, and Klf4.
So far, the transcription factor can be obtained gene into somatic cells iPS cells mainly in the following ways: retrovirus [1], lentiviral [6], adenovirus [11], transposons [12] and plasmids [ 13]. Retroviral and lentiviral-mediated transgene way that viral vector integrated into the host genome, to achieve transgene stably expressed, but the two transgenic methods easily activated oncogene; through adenovirus transcription factor genes into somatic cells, transient expression of these transcription factor, which gene is generally not integrated into the viral vector in the host genome, but the first, the method for the induction of somatic cells compared to the efficiency of iPS cells retroviruses and lentiviruses much lower [11] Moreover fact, adenovirus vectors may also be integrated into the genome, although this possibility is very small, this is not the best way; through transposons and plasmids principle mediated expression mediated through its four factors, inducing and the establishment of iPS cells, iPS cells, and the transient expression of these transposase enzyme is induced by removal of exogenous factors, such iPS cells will no longer express exogenous inducing factor, genomic DNA sequences does not cause any change in the security improved. But in the process of establishment of iPS cell transit transposon insertion on the safety of iPS cells long-term effects have yet to be further to verify.
Integrity can not completely avoid the use of viruses or transposons, to establish a more secure way to create iPS cells do? Fortunately, this year, almost the same time, Zhou H group [14] and Kim D group [15] in "Cell Stem Cell", introduced a new method, by direct protein-mediated, respectively, of the mouse and human fibroblasts induced by a iPS cells. That will be designed to pass through the membrane short peptide chain is connected to the reprogramming factor protein, such a fusion protein to penetrate the cell membrane into the cell interior, and execute functions of reprogramming. Unlike Zhou H group in the test small molecule compounds added VPA (valproic acid) to improve the efficiency of reprogramming, and Kim D team did not add any substance. Because this protein induces direct way does not involve any genetic modification, so for now, this is the most secure method. To sum up, through continuous exploration, iPS cells is gradually increased security, but these methods increase security, while induction of iPS cells are making efficiency is greatly reduced.
2-induced efficiency
Stage, iPS cells obtained inefficient, about the very few to a few thousandths, to effectively obtain a greater number of iPS cells, the researchers tried a variety of methods. On the whole, with the efficiency of iPS cells obtained are closely related to the following main points.
2.1 of small molecules in a growing number of studies show that by the combination of different transcription factors or associated with the use of small molecule compounds, can greatly improve the murine and human cell reprogramming of iPS cells, the efficiency of these small molecular compounds in promote cell reprogramming plays an important role. When added to the culture medium or small molecule compounds BIX PD0325901 with CHIR99021, can significantly improve the efficiency of reprogramming [16]. G9a histone methyltransferase inhibitor BIX-01294 was found to be replaced first Oct-4, and the remaining three NPCs reprogramming factors induce the formation of iPS cells together. In addition, BIX-01294 can also improve Oct-4 and Klf4 two factor reprogramming of NPCs efficiency [16]. In addition, Park et al [17] in only four reprogramming factors induce healthy adult skin fibroblasts did not form iPS cells after four factors based on adding hTERT and SV40 large T antigen, can be from healthy adult human fibroblasts induces the formation of iPS cells. Mice mature B cells need to import myeloid transcription factor C / EBPα can be transformed into iPS cells [18], and when added to the culture medium 5-AZA may also mature B cells induced into iPS cells [19]. Recently Hyenjong Hong et al [20] found that by blocking called "p53" gene path, skin cells can be transformed into iPS cells, the success rate to about 10%, the conversion rate is about hundred times the original. However, they cautioned that: "p53" inhibit cancer cells, preventing tumor growth, so by blocking the "p53" Path iPS cells to improve conversion rates, they should also pay attention to the potential risks.
2.2 somatic cells derived from different tissues in inducing efficiency of different Aasen et al [21] Studies have shown that parts from the hair root sheath cuticle germinal epidermal stem-like cells as donor cells, the same retrovirus-mediated expression through Oct-4 , Sox2, Klf4 and c-Myc, successfully induced iPS cell formation, and after detection of mRNA levels and found that induction efficiency compared with fibroblasts, increased approximately 100 times, and iPS clones appear much shortened to 10 days, occurring at a rate also increased nearly twice as much.
In addition to two, the signal transduction pathway, such as the Wnt pathway [22]; epigenetic modification of [9] can improve the efficiency of iPS cell formation.
Reprogramming somatic cells resulting iPS cells inefficiencies reason is mainly because the molecular mechanisms underlying the present stage of its research is unclear. As a dedifferentiated cells by means of iPS cells reprogrammed a slow and complex process, there are many molecules involved. And Oct-4, Sox2, Klf4 and c-Myc four basic factors in the formation of iPS cells in the mechanism is not clear. Furthermore, even by the most versatile ways to induce viral transfection, the transfection efficiency is not 100%, while the proportion of cells expressing four factors on the lower. In addition, these four factors induce iPS cells also belong to the Almighty to get random event, and the event is dependent on the somatic cells Oct-4 transcription factors such as dry leakage occurs randomly randomness of expression and epigenetic changes [23, 24]. Imagine if we can choose the right reprogramming adult cells as the starting cell, adding the appropriate small molecule compounds to catalyze, to put this random event becomes inevitable event, bound to high-efficiency induction of iPS cell formation.
In short, the current situation, to improve the safety of iPS cells seem to induce iPS cells efficiently generate mutually contradictory, but I believe that with the deepening of research, iPS cell reprogramming and pluripotency molecular mechanism will gradually clear . Efficiently and safely establish iPS cell lines to a more adequate basis for early studies and clinical applications, and ultimately for the patient to bring the gospel of great significance.
Stage, iPS cells obtained inefficient, about the very few to a few thousandths, to effectively obtain a greater number of iPS cells, the researchers tried a variety of methods. On the whole, with the efficiency of iPS cells obtained are closely related to the following main points.
2.1 of small molecules in a growing number of studies show that by the combination of different transcription factors or associated with the use of small molecule compounds, can greatly improve the murine and human cell reprogramming of iPS cells, the efficiency of these small molecular compounds in promote cell reprogramming plays an important role. When added to the culture medium or small molecule compounds BIX PD0325901 with CHIR99021, can significantly improve the efficiency of reprogramming [16]. G9a histone methyltransferase inhibitor BIX-01294 was found to be replaced first Oct-4, and the remaining three NPCs reprogramming factors induce the formation of iPS cells together. In addition, BIX-01294 can also improve Oct-4 and Klf4 two factor reprogramming of NPCs efficiency [16]. In addition, Park et al [17] in only four reprogramming factors induce healthy adult skin fibroblasts did not form iPS cells after four factors based on adding hTERT and SV40 large T antigen, can be from healthy adult human fibroblasts induces the formation of iPS cells. Mice mature B cells need to import myeloid transcription factor C / EBPα can be transformed into iPS cells [18], and when added to the culture medium 5-AZA may also mature B cells induced into iPS cells [19]. Recently Hyenjong Hong et al [20] found that by blocking called "p53" gene path, skin cells can be transformed into iPS cells, the success rate to about 10%, the conversion rate is about hundred times the original. However, they cautioned that: "p53" inhibit cancer cells, preventing tumor growth, so by blocking the "p53" Path iPS cells to improve conversion rates, they should also pay attention to the potential risks.
2.2 somatic cells derived from different tissues in inducing efficiency of different Aasen et al [21] Studies have shown that parts from the hair root sheath cuticle germinal epidermal stem-like cells as donor cells, the same retrovirus-mediated expression through Oct-4 , Sox2, Klf4 and c-Myc, successfully induced iPS cell formation, and after detection of mRNA levels and found that induction efficiency compared with fibroblasts, increased approximately 100 times, and iPS clones appear much shortened to 10 days, occurring at a rate also increased nearly twice as much.
In addition to two, the signal transduction pathway, such as the Wnt pathway [22]; epigenetic modification of [9] can improve the efficiency of iPS cell formation.
Reprogramming somatic cells resulting iPS cells inefficiencies reason is mainly because the molecular mechanisms underlying the present stage of its research is unclear. As a dedifferentiated cells by means of iPS cells reprogrammed a slow and complex process, there are many molecules involved. And Oct-4, Sox2, Klf4 and c-Myc four basic factors in the formation of iPS cells in the mechanism is not clear. Furthermore, even by the most versatile ways to induce viral transfection, the transfection efficiency is not 100%, while the proportion of cells expressing four factors on the lower. In addition, these four factors induce iPS cells also belong to the Almighty to get random event, and the event is dependent on the somatic cells Oct-4 transcription factors such as dry leakage occurs randomly randomness of expression and epigenetic changes [23, 24]. Imagine if we can choose the right reprogramming adult cells as the starting cell, adding the appropriate small molecule compounds to catalyze, to put this random event becomes inevitable event, bound to high-efficiency induction of iPS cell formation.
In short, the current situation, to improve the safety of iPS cells seem to induce iPS cells efficiently generate mutually contradictory, but I believe that with the deepening of research, iPS cell reprogramming and pluripotency molecular mechanism will gradually clear . Efficiently and safely establish iPS cell lines to a more adequate basis for early studies and clinical applications, and ultimately for the patient to bring the gospel of great significance.
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