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Induced pluripotent stem cells (iPSCs)—a new era of reprogramming
JOURNAL OF
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 415−421 www.jgenetgenomics.org
Induced pluripotent stem cells (iPSCs)—a new era of reprogramming Lan Kang, Zhaohui Kou, Yu Zhang, Shaorong Gao * National Institute of Biological Sciences, NIBS, Beijing 102206, China Received for publication 22 March 2010; revised 9 April 2010; accepted 20 April 2010
Abstract Embryonic stem cells (ESCs) derived from the early embryos possess two important characteristics: self-renewal and pluripotency, which make ESCs ideal seed cells that could be potentially utilized for curing a number of degenerative and genetic diseases clinically. However, ethical concerns and immune rejection after cell transplantation limited the clinical application of ESCs. Fortunately, the recent advances in induced pluripotent stem cell (iPSC) research have clearly shown that differentiated somatic cells from various species could be reprogrammed into pluripotent state by ectopically expressing a combination of several transcription factors, which are highly enriched in ESCs. This ground-breaking achievement could circumvent most of the limitations that ESCs faced. However, it remains challenging if the iPS cell lines, especially the human iPSCs lines, available are fully pluripotent. Therefore, it is prerequisite to establish a molecular standard to distinguish the better quality iPSCs from the inferior ones. Keywords: induced pluripotent stem cells (iPSCs); reprogramming; molecular events
Introduction In mammals, the fertilization unites two most specialized cells (sperm and oocyte) to form a totipotent embryo (zygote). After several rounds of cell division, the first cell fate determination occurs when embryos develop to morulae stage. As the result, some blastomeres differentiate into trophoblast cells and a small proportion of blastomeres remain at pluripotent state and form inner cell mass (ICM) at the blastocyst stage. After implantation, the trophoblast cells will develop into placenta and the ICM will develop into epiblast then into a whole body. Given the appropriate culture conditions, the embryonic stem cell (ESC) lines could be established from the ICM and the resulting ESC lines maintain the most if not all the properties of ICM * Corresponding author. Tel: +86-10-8072 8967; Fax: +86-10-8072 7535. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60060-6
cells (Evans and Kaufman, 1981). ES cells could proliferate infinitely but maintain their identity in vitro and on the other hand, ES cells could differentiate into all different types of cells of the body when appropriately induced. Moreover, ES cells could differentiate into all cell types, including the germ cells, belonging to the three germ layers in the normal embryos. Therefore, ES cells have been widely utilized in the gene targeting studies to investigate the functions of newly discovered genes. Meanwhile, ES cells have been proposed as ideal seed cells for regenerative medicine to cure many degenerative and genetic diseases. However, embryos have to be destroyed and utilized for derivation of new ES cell lines and consequently ethical controversy has to be faced. Furthermore, the other important issue has to be concerned is immune rejection because no customized ES cells are available for specific individuals at present. Therefore, obtaining individually specific pluripotent stem cells will be prerequi-
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site for clinical application using pluripotent stem cells in the future.
Reprogramming of differentiated somatic cells into pluripotent stem cells The most accessible cells in the body are somatic cells, the differentiated cells with unique property. The idea of converting a differentiated somatic cell into pluripotent stem cell or even a totipotent embryo was raised decades ago. However, it was not realized until the born of the cloned sheep “Dolly” in mammals (Wilmut et al., 1997). It was the first evidence indicating that fully differentiated somatic cells could be converted (reprogrammed) into a totipotent embryo by transferring into an enucleated oocyte, which can ultimately develop into a whole animal although this process is very inefficient. Subsequently, therapeutic cloning was proposed to generate histocompatible nuclear transfer embryonic stem (ntES) cells, which could be utilized for regenerative medicine. However, the shortage supply of oocytes, ethical issues and technical challenges impede the progress of therapeutic cloning in human although hundreds of ntES cell lines with fully pluripotency were successfully established in the laboratory mouse. Along with the cloning progress, ES cells were found to be competent to reprogram a differentiated somatic cell into pluripotent state through fusion experiment (Tada et al., 2001). However, the fused cells are tetraploid or multiploid and could not be used clinically. All these evidences provide us very important information that the unknown factors present in either oocytes or ES cells could indeed re-set the cell fate of a differentiated somatic cell. The accumulated knowledge on understanding the gene expression and regulation in ES cells leads to the recent important discovery of induced pluripotent stem cells (iPSCs), a more ideal seed cells closer to clinical application.
Induced pluripotent stem cells (iPSCs) Through analyzing the gene expression profiles of ESCs, many highly expressed genes in ESCs have been identified. Based on these findings, the landmark experiment was conducted by Yamanaka and his colleague in 2006 (Takahashi and Yamanaka, 2006), in which they utilized
retroviral infection approach and successfully introduced 24 transcription factors that are highly expressed in ESCs into the fibroblast cells derived from the fetal mice. Surprisingly, some ES-like colonies appeared in the culture dish in two weeks post retroviral infection. Moreover, these ES-like cells could be propagated in vitro and resemble ES cells morphologically after many cell passages. Furthermore, the pluripotency of these so called induced pluripotent stem cells (iPSCs) was further characterized. Although no chimeric mice were generated, the iPSCs could indeed form teratomas in the immune deficient mice, which contain three germ layers structure. More importantly, by reducing the factors one by one in the process of retroviral infection, they found ultimately that four transcription factors including Oct4, Sox2, Klf4 and c-Myc were essential for converting the fibroblast cells into iPSCs (c-Myc was found dispensable for iPSCs induction in the latest results (Nakagawa et al., 2008)). This phenomenal experiment opens up a new era of reprogramming through which no ethical controversies and no limitations of cell sources will be worried about. Somatic cells and ES cells are totally different populations in morphology, gene expression, functional activity and so on. So it is surprising that just four or even three genes’ expression lead to this tremendous transformation. Obviously, all these transcription factors play pivotal roles in reprogramming of the differentiated cell fate.
The magic four transcription factors Oct-3/4 is an octamer binding factor which recognizes the octamer motif (ATTTGCAT) (Okamoto et al., 1990; Scholer et al., 1990). It is expressed in unfertilized oocytes, pregastrulation embryos, primordial germ cells and the germ lines but not in other adult organs and it also presents in embryonic stem cells and embryonal carcinoma cells and is down-regulated during differentiation (Scholer et al., 1989; Okamoto et al., 1990; Rosner et al., 1990; Pesce et al., 1998). The absence of Oct-3/4 results in peri-implantation lethality, Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass (ICM) cells are restricted to differentiation along the trophoblast lineage (Nichols et al., 1998). In addition, the precise level of Oct-3/4 is critical for stem cell pluripotency. A less than two-fold increase in expression causes differentiation into primitive endoderm and mesoderm and repression of Oct-3/4 induces loss of
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pluripotency and dedifferentiation to trophectoderm (Niwa et al., 2000). Therefore, Oct4 has been considered as the master regulator of pluripotency in ESCs. Sox2 is a member of SRY-related HMG-box transcription factor family which expressed in early embryo, ICM, epiblast, and germ cells like Oct4, and besides that it became restricted to the prospective neural plate and chorion and associated with the developing central nervous system after gastrulation (Yuan et al., 1995; Wood and Episkopou, 1999). Sox2 homozygous mutant embryos exhibited normal at blastocyst stage but failed to survive shortly after implantation due to epiblast development failure (Avilion et al., 2003) and its disruption in mouse ES cells also led to differentiation (Ivanova et al., 2006). Thus Sox2 is critical for early embryo development and ES cell pluripotency maintenance. Klf4, also named gut-enriched Krüppel-like factor, is a zinc finger-containing transcription factor which contains an essential binding site with G/AG/AGGC/TGC/T sequence (Shields et al., 1996; Shields and Yang, 1998). Klf4 is highly expressed in gastrointestinal tract and correlates with its development (Ton-That et al., 1997), and meanwhile it also functions as a growth suppressor with its expression decreased in tumorigenesis (Dang et al., 2000) and increased during DNA damage (Zhang et al., 2000). Knockout of Klf4 leads to shortly death after birth due to loss of epidermal barrier function (Segre et al., 1999). Interestingly, Krüppel-like factors are also required for the self-renewal of ES cells while simultaneous depletion of Klf2, Klf4 and Klf5 leads to ES cell differentiation (Jiang et al., 2008). c-Myc was initially recognized as an oncogene which is highly expressed in tumors caused by chromosome translocation (Crews et al., 1982). c-Myc belongs to Myc transcription factor family which contains a basic helix-loop-helix (bHLH) domain, through which c-Myc could specifically recognize the CACGTG E-box sequence (Blackwell et al., 1990) and bind DNA as a heterodimer with MAX (Blackwood and Eisenman, 1991). c-Myc plays important roles in cell cycle, apoptosis, development, signal transduction, transcriptional and post-transcriptional regulatory mechanisms, non-coding RNAs, stem cell biology and the molecular basis of cancer (Meyer and Penn, 2008). A null c-Myc mutation causes embryonic lethality before 10.5 days of gestation in homozygotes but deletion of c-Myc does not impair cell division in ES cells (Davis et al., 1993). Interestingly, evidence indicates that
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c-Myc plays an important role in mouse ES cell self-renewal and pluripotency through LIF/STAT3 pathway (Cartwright et al., 2005). Many genes have been characterized as important for pluripotency in ESCs. Among these, Oct4, Sox2 and Nanog form a core pluripotency regulatory circuitry. These three transcription factors are found playing critical roles in regulating the expression of pluripotent genes, and simultaneously suppressing the expression of many differentiation related genes. They exert the functions through co-occupying their target genes, and meanwhile they can bind at their own and each other’s promoters to form an interconnected autoregulatory loop. In this autoregulatory circuitry, these three factors function collaboratively to maintain their own expression and maintain the pluripotent state of ESCs (Boyer et al., 2005; Jaenisch and Young, 2008).
Pluripotency of induced pluripotent stem cells (iPSCs) Although the original iPSCs obtained could differentiate in vitro, no chimeric mice could be produced from these cells after injection into the normal embryos. The follow-up studies from both Yamanaka’s lab and Jaenisch’s lab successfully demonstrated that chimeric mice with germline transmission ability could be generated from the iPSCs using an improved selection strategy (Takahashi and Yamanaka, 2006; Okita et al., 2007; Wernig et al., 2007; Kang et al., 2009; Zhao et al., 2009). More importantly, iPSCs could be successfully derived from the differentiated somatic cells simply based on the morphology changes and no genetic selection was needed, which indicated that human somatic cells without genetic modification could be reprogrammed successfully. However, no iPSCs lines generated by then were in accordance with the most stringent criterion for evaluating the pluripotency of ESCs. It is well known that the golden standard for evaluating the pluripotency of ESCs is tetraploid blastocyst complementation assay, through which the pluripotent stem cells with fully pluripotency could autonomously generate a live animal. However, no iPSCs lines have been proven able to autonomously generate a whole animal through complementation with a tetraploid embryo until our group together with the other group’s discoveries were published independently (Kang et al., 2009; Zhao et al., 2009).
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Through these studies, we can conclude that the iPSCs reprogrammed from the somatic cells by defined transcription factors could indeed recapitulate the fully pluripotency, which are functionally equivalent to normal ES cells derived from fertilized embryos. Therefore, iPSCs are promising to be potentially used in regenerative medicine for treating many diseases in the future and indeed many patient-specific iPSCs lines have been established (Dimos et al., 2008; Soldner et al., 2009). However, a molecular standard is required for distinguishing the bona fide iPSCs in human because the tetraploid complementation assay can not be performed to test pluripotency of iPSCs in human. We propose that further digging the molecular signature including the mRNA expression profile, microRNA expression profile, epigenetic modification characteristics of different iPSCs lines through deep sequencing would provide us some clues to screen out the relative better quality iPSCs.
Exploring the molecular events in iPSCs induction Although the induction of iPSCs formation is not challenging technically, the reprogramming mechanisms underlying remain elusive. It turns to be very difficult to understand the detailed molecular mechanism involved in iPSCs induction because only a very small proportion of the cells become pluripotent eventually. But, the inducible strategy utilized by several labs could potentially disclose this mystery.
Inducible system and secondary iPSCs Inducible iPSCs system is based on the combination of the Tet-On gene inducible system with the lentiviral infection system (Brambrink et al., 2008; Stadtfeld et al., 2008a). The reprogrammable mice could be generated from the inducible iPSCs by either tetraploid blastocyst complementation or nuclear transplantation approach (Kang et al., 2009; Kou et al., 2010). Subsequently, all the cells of the mice generated from the inducible iPSCs are homogenous and their genomes contain the exogenous factors whose expression could be re-induced by simply adding doxycycline in the culture medium. Therefore, inducible iPSCs system excludes the variations in efficiency of viral infection that was utilized widely in iPSCs induction. Subsequently, many fundamental questions in repro-
gramming could be potentially addressed using the inducible iPSCs system.
Sequential gene expression in iPSCs induction Once doxycycline was supplemented in the culture medium, the four exogenous transcription factors (Oct4, Sox2, Klf4 and c-Myc) could be re-activated in the somatic cells, which could in turn alter the global gene expression landscape in the cells. Consequently, the cell surface markers specific for stem cells will appear. Therefore, flow cytometry has been successfully utilized for sorting out the stem cell surface marker positive cells at certain time points and then the gene expression profile of these cells could be investigated (Brambrink et al., 2008; Stadtfeld et al., 2008a). Moreover, live cell imaging has been applied in situ tracing the cell surface markers’ sequential expression of the colonies appeared (Chan et al., 2009). These two strategies have been applied in detecting real time sequential gene expression changes notwithstanding the limitation in surface markers.
Reprogramming efficiency Comparison of reprogramming efficiency is the most used assay at present to reveal the effects of gene manipulation, small molecules and differentiation status of different donor cells on reprogramming outcome (Huangfu et al., 2008; Mikkelsen et al., 2008; Eminli et al., 2009; Hong et al., 2009; Kawamura et al., 2009). The efficiency and required time are the most direct used parameters of reprogramming. The factors that could increase the efficiency of reprogramming or could decrease the required time for reprogramming are considered important in this process. Epigenetic modifications such as DNA methylation and histone acetylation are well known to be one of the major gene expression regulation systems. And during the reprogramming, there are numerous pluripotency associated genes to express and differentiation associated genes to shut down, so it’s not surprising to see the molecules inhibiting DNA methylation (AZA) and histone deacetylation (VPA and TSA) highly increase the efficiency of reprogramming (Huangfu et al., 2008; Mikkelsen et al., 2008). Suppression of p53-p21 pathway has been shown able to increase the iPSCs generation (Hong et al., 2009;
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Kawamura et al., 2009). It is possible that iPSCs have a cell cycle much shorter than somatic cells without the checkpoint in G1 phase, and to gain this change, cell cycle regulation factors have to be engaged. And maybe more importantly, the dramatic changes in the cells need the company of rapid cell division. Wnt3a and Nanog which help the pluripotent circuitry establishment also have positive effect on the efficiency of reprogramming (Liao et al., 2008; Marson et al., 2008; Hanna et al., 2009).
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oughly. But we believe that iPSCs create an attainable opportunity to cure many diseases completely.
Acknowledgements This work was supported by the grants from the Ministry of Science and Technology of China (Nos. 2008AA022311, 2010CB944900 and 2008AA1011005). We thank members of our laboratory for helpful comments on this manuscript.
Improvement of iPSCs generation and the perspective of iPSCs application
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GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 415−421 www.jgenetgenomics.org
Induced pluripotent stem cells (iPSCs)—a new era of reprogramming Lan Kang, Zhaohui Kou, Yu Zhang, Shaorong Gao * National Institute of Biological Sciences, NIBS, Beijing 102206, China Received for publication 22 March 2010; revised 9 April 2010; accepted 20 April 2010
Abstract Embryonic stem cells (ESCs) derived from the early embryos possess two important characteristics: self-renewal and pluripotency, which make ESCs ideal seed cells that could be potentially utilized for curing a number of degenerative and genetic diseases clinically. However, ethical concerns and immune rejection after cell transplantation limited the clinical application of ESCs. Fortunately, the recent advances in induced pluripotent stem cell (iPSC) research have clearly shown that differentiated somatic cells from various species could be reprogrammed into pluripotent state by ectopically expressing a combination of several transcription factors, which are highly enriched in ESCs. This ground-breaking achievement could circumvent most of the limitations that ESCs faced. However, it remains challenging if the iPS cell lines, especially the human iPSCs lines, available are fully pluripotent. Therefore, it is prerequisite to establish a molecular standard to distinguish the better quality iPSCs from the inferior ones. Keywords: induced pluripotent stem cells (iPSCs); reprogramming; molecular events
Introduction In mammals, the fertilization unites two most specialized cells (sperm and oocyte) to form a totipotent embryo (zygote). After several rounds of cell division, the first cell fate determination occurs when embryos develop to morulae stage. As the result, some blastomeres differentiate into trophoblast cells and a small proportion of blastomeres remain at pluripotent state and form inner cell mass (ICM) at the blastocyst stage. After implantation, the trophoblast cells will develop into placenta and the ICM will develop into epiblast then into a whole body. Given the appropriate culture conditions, the embryonic stem cell (ESC) lines could be established from the ICM and the resulting ESC lines maintain the most if not all the properties of ICM * Corresponding author. Tel: +86-10-8072 8967; Fax: +86-10-8072 7535. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60060-6
cells (Evans and Kaufman, 1981). ES cells could proliferate infinitely but maintain their identity in vitro and on the other hand, ES cells could differentiate into all different types of cells of the body when appropriately induced. Moreover, ES cells could differentiate into all cell types, including the germ cells, belonging to the three germ layers in the normal embryos. Therefore, ES cells have been widely utilized in the gene targeting studies to investigate the functions of newly discovered genes. Meanwhile, ES cells have been proposed as ideal seed cells for regenerative medicine to cure many degenerative and genetic diseases. However, embryos have to be destroyed and utilized for derivation of new ES cell lines and consequently ethical controversy has to be faced. Furthermore, the other important issue has to be concerned is immune rejection because no customized ES cells are available for specific individuals at present. Therefore, obtaining individually specific pluripotent stem cells will be prerequi-
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site for clinical application using pluripotent stem cells in the future.
Reprogramming of differentiated somatic cells into pluripotent stem cells The most accessible cells in the body are somatic cells, the differentiated cells with unique property. The idea of converting a differentiated somatic cell into pluripotent stem cell or even a totipotent embryo was raised decades ago. However, it was not realized until the born of the cloned sheep “Dolly” in mammals (Wilmut et al., 1997). It was the first evidence indicating that fully differentiated somatic cells could be converted (reprogrammed) into a totipotent embryo by transferring into an enucleated oocyte, which can ultimately develop into a whole animal although this process is very inefficient. Subsequently, therapeutic cloning was proposed to generate histocompatible nuclear transfer embryonic stem (ntES) cells, which could be utilized for regenerative medicine. However, the shortage supply of oocytes, ethical issues and technical challenges impede the progress of therapeutic cloning in human although hundreds of ntES cell lines with fully pluripotency were successfully established in the laboratory mouse. Along with the cloning progress, ES cells were found to be competent to reprogram a differentiated somatic cell into pluripotent state through fusion experiment (Tada et al., 2001). However, the fused cells are tetraploid or multiploid and could not be used clinically. All these evidences provide us very important information that the unknown factors present in either oocytes or ES cells could indeed re-set the cell fate of a differentiated somatic cell. The accumulated knowledge on understanding the gene expression and regulation in ES cells leads to the recent important discovery of induced pluripotent stem cells (iPSCs), a more ideal seed cells closer to clinical application.
Induced pluripotent stem cells (iPSCs) Through analyzing the gene expression profiles of ESCs, many highly expressed genes in ESCs have been identified. Based on these findings, the landmark experiment was conducted by Yamanaka and his colleague in 2006 (Takahashi and Yamanaka, 2006), in which they utilized
retroviral infection approach and successfully introduced 24 transcription factors that are highly expressed in ESCs into the fibroblast cells derived from the fetal mice. Surprisingly, some ES-like colonies appeared in the culture dish in two weeks post retroviral infection. Moreover, these ES-like cells could be propagated in vitro and resemble ES cells morphologically after many cell passages. Furthermore, the pluripotency of these so called induced pluripotent stem cells (iPSCs) was further characterized. Although no chimeric mice were generated, the iPSCs could indeed form teratomas in the immune deficient mice, which contain three germ layers structure. More importantly, by reducing the factors one by one in the process of retroviral infection, they found ultimately that four transcription factors including Oct4, Sox2, Klf4 and c-Myc were essential for converting the fibroblast cells into iPSCs (c-Myc was found dispensable for iPSCs induction in the latest results (Nakagawa et al., 2008)). This phenomenal experiment opens up a new era of reprogramming through which no ethical controversies and no limitations of cell sources will be worried about. Somatic cells and ES cells are totally different populations in morphology, gene expression, functional activity and so on. So it is surprising that just four or even three genes’ expression lead to this tremendous transformation. Obviously, all these transcription factors play pivotal roles in reprogramming of the differentiated cell fate.
The magic four transcription factors Oct-3/4 is an octamer binding factor which recognizes the octamer motif (ATTTGCAT) (Okamoto et al., 1990; Scholer et al., 1990). It is expressed in unfertilized oocytes, pregastrulation embryos, primordial germ cells and the germ lines but not in other adult organs and it also presents in embryonic stem cells and embryonal carcinoma cells and is down-regulated during differentiation (Scholer et al., 1989; Okamoto et al., 1990; Rosner et al., 1990; Pesce et al., 1998). The absence of Oct-3/4 results in peri-implantation lethality, Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass (ICM) cells are restricted to differentiation along the trophoblast lineage (Nichols et al., 1998). In addition, the precise level of Oct-3/4 is critical for stem cell pluripotency. A less than two-fold increase in expression causes differentiation into primitive endoderm and mesoderm and repression of Oct-3/4 induces loss of
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pluripotency and dedifferentiation to trophectoderm (Niwa et al., 2000). Therefore, Oct4 has been considered as the master regulator of pluripotency in ESCs. Sox2 is a member of SRY-related HMG-box transcription factor family which expressed in early embryo, ICM, epiblast, and germ cells like Oct4, and besides that it became restricted to the prospective neural plate and chorion and associated with the developing central nervous system after gastrulation (Yuan et al., 1995; Wood and Episkopou, 1999). Sox2 homozygous mutant embryos exhibited normal at blastocyst stage but failed to survive shortly after implantation due to epiblast development failure (Avilion et al., 2003) and its disruption in mouse ES cells also led to differentiation (Ivanova et al., 2006). Thus Sox2 is critical for early embryo development and ES cell pluripotency maintenance. Klf4, also named gut-enriched Krüppel-like factor, is a zinc finger-containing transcription factor which contains an essential binding site with G/AG/AGGC/TGC/T sequence (Shields et al., 1996; Shields and Yang, 1998). Klf4 is highly expressed in gastrointestinal tract and correlates with its development (Ton-That et al., 1997), and meanwhile it also functions as a growth suppressor with its expression decreased in tumorigenesis (Dang et al., 2000) and increased during DNA damage (Zhang et al., 2000). Knockout of Klf4 leads to shortly death after birth due to loss of epidermal barrier function (Segre et al., 1999). Interestingly, Krüppel-like factors are also required for the self-renewal of ES cells while simultaneous depletion of Klf2, Klf4 and Klf5 leads to ES cell differentiation (Jiang et al., 2008). c-Myc was initially recognized as an oncogene which is highly expressed in tumors caused by chromosome translocation (Crews et al., 1982). c-Myc belongs to Myc transcription factor family which contains a basic helix-loop-helix (bHLH) domain, through which c-Myc could specifically recognize the CACGTG E-box sequence (Blackwell et al., 1990) and bind DNA as a heterodimer with MAX (Blackwood and Eisenman, 1991). c-Myc plays important roles in cell cycle, apoptosis, development, signal transduction, transcriptional and post-transcriptional regulatory mechanisms, non-coding RNAs, stem cell biology and the molecular basis of cancer (Meyer and Penn, 2008). A null c-Myc mutation causes embryonic lethality before 10.5 days of gestation in homozygotes but deletion of c-Myc does not impair cell division in ES cells (Davis et al., 1993). Interestingly, evidence indicates that
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c-Myc plays an important role in mouse ES cell self-renewal and pluripotency through LIF/STAT3 pathway (Cartwright et al., 2005). Many genes have been characterized as important for pluripotency in ESCs. Among these, Oct4, Sox2 and Nanog form a core pluripotency regulatory circuitry. These three transcription factors are found playing critical roles in regulating the expression of pluripotent genes, and simultaneously suppressing the expression of many differentiation related genes. They exert the functions through co-occupying their target genes, and meanwhile they can bind at their own and each other’s promoters to form an interconnected autoregulatory loop. In this autoregulatory circuitry, these three factors function collaboratively to maintain their own expression and maintain the pluripotent state of ESCs (Boyer et al., 2005; Jaenisch and Young, 2008).
Pluripotency of induced pluripotent stem cells (iPSCs) Although the original iPSCs obtained could differentiate in vitro, no chimeric mice could be produced from these cells after injection into the normal embryos. The follow-up studies from both Yamanaka’s lab and Jaenisch’s lab successfully demonstrated that chimeric mice with germline transmission ability could be generated from the iPSCs using an improved selection strategy (Takahashi and Yamanaka, 2006; Okita et al., 2007; Wernig et al., 2007; Kang et al., 2009; Zhao et al., 2009). More importantly, iPSCs could be successfully derived from the differentiated somatic cells simply based on the morphology changes and no genetic selection was needed, which indicated that human somatic cells without genetic modification could be reprogrammed successfully. However, no iPSCs lines generated by then were in accordance with the most stringent criterion for evaluating the pluripotency of ESCs. It is well known that the golden standard for evaluating the pluripotency of ESCs is tetraploid blastocyst complementation assay, through which the pluripotent stem cells with fully pluripotency could autonomously generate a live animal. However, no iPSCs lines have been proven able to autonomously generate a whole animal through complementation with a tetraploid embryo until our group together with the other group’s discoveries were published independently (Kang et al., 2009; Zhao et al., 2009).
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Through these studies, we can conclude that the iPSCs reprogrammed from the somatic cells by defined transcription factors could indeed recapitulate the fully pluripotency, which are functionally equivalent to normal ES cells derived from fertilized embryos. Therefore, iPSCs are promising to be potentially used in regenerative medicine for treating many diseases in the future and indeed many patient-specific iPSCs lines have been established (Dimos et al., 2008; Soldner et al., 2009). However, a molecular standard is required for distinguishing the bona fide iPSCs in human because the tetraploid complementation assay can not be performed to test pluripotency of iPSCs in human. We propose that further digging the molecular signature including the mRNA expression profile, microRNA expression profile, epigenetic modification characteristics of different iPSCs lines through deep sequencing would provide us some clues to screen out the relative better quality iPSCs.
Exploring the molecular events in iPSCs induction Although the induction of iPSCs formation is not challenging technically, the reprogramming mechanisms underlying remain elusive. It turns to be very difficult to understand the detailed molecular mechanism involved in iPSCs induction because only a very small proportion of the cells become pluripotent eventually. But, the inducible strategy utilized by several labs could potentially disclose this mystery.
Inducible system and secondary iPSCs Inducible iPSCs system is based on the combination of the Tet-On gene inducible system with the lentiviral infection system (Brambrink et al., 2008; Stadtfeld et al., 2008a). The reprogrammable mice could be generated from the inducible iPSCs by either tetraploid blastocyst complementation or nuclear transplantation approach (Kang et al., 2009; Kou et al., 2010). Subsequently, all the cells of the mice generated from the inducible iPSCs are homogenous and their genomes contain the exogenous factors whose expression could be re-induced by simply adding doxycycline in the culture medium. Therefore, inducible iPSCs system excludes the variations in efficiency of viral infection that was utilized widely in iPSCs induction. Subsequently, many fundamental questions in repro-
gramming could be potentially addressed using the inducible iPSCs system.
Sequential gene expression in iPSCs induction Once doxycycline was supplemented in the culture medium, the four exogenous transcription factors (Oct4, Sox2, Klf4 and c-Myc) could be re-activated in the somatic cells, which could in turn alter the global gene expression landscape in the cells. Consequently, the cell surface markers specific for stem cells will appear. Therefore, flow cytometry has been successfully utilized for sorting out the stem cell surface marker positive cells at certain time points and then the gene expression profile of these cells could be investigated (Brambrink et al., 2008; Stadtfeld et al., 2008a). Moreover, live cell imaging has been applied in situ tracing the cell surface markers’ sequential expression of the colonies appeared (Chan et al., 2009). These two strategies have been applied in detecting real time sequential gene expression changes notwithstanding the limitation in surface markers.
Reprogramming efficiency Comparison of reprogramming efficiency is the most used assay at present to reveal the effects of gene manipulation, small molecules and differentiation status of different donor cells on reprogramming outcome (Huangfu et al., 2008; Mikkelsen et al., 2008; Eminli et al., 2009; Hong et al., 2009; Kawamura et al., 2009). The efficiency and required time are the most direct used parameters of reprogramming. The factors that could increase the efficiency of reprogramming or could decrease the required time for reprogramming are considered important in this process. Epigenetic modifications such as DNA methylation and histone acetylation are well known to be one of the major gene expression regulation systems. And during the reprogramming, there are numerous pluripotency associated genes to express and differentiation associated genes to shut down, so it’s not surprising to see the molecules inhibiting DNA methylation (AZA) and histone deacetylation (VPA and TSA) highly increase the efficiency of reprogramming (Huangfu et al., 2008; Mikkelsen et al., 2008). Suppression of p53-p21 pathway has been shown able to increase the iPSCs generation (Hong et al., 2009;
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Kawamura et al., 2009). It is possible that iPSCs have a cell cycle much shorter than somatic cells without the checkpoint in G1 phase, and to gain this change, cell cycle regulation factors have to be engaged. And maybe more importantly, the dramatic changes in the cells need the company of rapid cell division. Wnt3a and Nanog which help the pluripotent circuitry establishment also have positive effect on the efficiency of reprogramming (Liao et al., 2008; Marson et al., 2008; Hanna et al., 2009).
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oughly. But we believe that iPSCs create an attainable opportunity to cure many diseases completely.
Acknowledgements This work was supported by the grants from the Ministry of Science and Technology of China (Nos. 2008AA022311, 2010CB944900 and 2008AA1011005). We thank members of our laboratory for helpful comments on this manuscript.
Improvement of iPSCs generation and the perspective of iPSCs application
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