Current progress and prospects of induced pluripotent stem cells

Transcription

1 Science in China Series C: Life Sciences 2009 SCIENCE IN CHINA PRESS Springer Special Topic Review life.scichina.com Current progress and prospects of induced pluripotent stem cells CHEN LingYi & Liu Lin Key Laboratory of Bioactive Materials of Ministry of Education, College of Life Sciences, Nankai University, Tianjin , China Induced pluripotent stem (ips) cells are derived from somatic cells by ectopic expression of few transcription factors. Like embryonic stem (ES) cells, ips cells are able to self-renew indefinitely and to differentiate into all types of cells in the body. ips cells hold great promise for regenerative medicine, because ips cells circumvent not only immunological rejection but also ethical issues. Since the first report on the derivation of ips cells in 2006, many laboratories all over the world started research on ips cells and have made significant progress. This paper reviews recent progress in ips cell research, including the methods to generate ips cells, the molecular mechanism of reprogramming in the formation of ips cells, and the potential applications of ips cells in cell replacement therapy. Current problems that need to be addressed and the prospects for ips research are also discussed. induced pluripotent stem (ips) cells, somatic cell reprogramming, pluripotency 1 Sources and characterization of pluripotent stem cells Pluripotent stem cells are able to differentiate into all types of cells in the body and to self-renew indefinitely. Thus, pluripotent stem cells have great application potential in regenerative medicine, developmental biology research, and drug development. Pluripotent stem cells should meet the following criteria: (i) Unlimited proliferation in vitro and maintenance of normal diploid karyotype; (ii) expression of pluripotency markers, such as Oct4, Nanog, SSEA1, SSEA4, etc.; (iii) in vitro differentiation potential into cells of three germ layers (ectoderm, mesoderm and endoderm); (iv) capacity to form teratoma (composed of tissues from three germ layers), when injected subcutaneously into immune-deficient mice. A higher standard for pluripotency is that pluripotent stem cells contribute to chimeras and show germline transmission, following injecting into recipient blastocysts and embryo transfer. The most stringent test for pluripotency should be production of live pups merely composed of pluripotent stem cell derivatives through tetraploid embryo com- plementation or 4 8 cell embryo injection [1 3]. However, due to ethical restriction, the last two more rigorous tests cannot be applied to the evaluation of pluripotent stem cells in human. The most well-known pluripotent stem cell is embryonic stem (ES) cells, which are derived from the inner cell mass of the blastocyst [4,5]. Under proper conditions, ES cells can differentiate into more than 200 types of cells, and even are able to form a whole organism [1]. Besides naturally fertilized embryos, in vitro fertilization, parthenogenesis, and somatic cell cloning are alternative sources of blastocysts. Blastocysts from different sources can be used for derivation of ES cells [6,7]. Nevertheless, regardless of types of blastocysts used, isolation and establishment of ES cell lines involve embryo destruction and require oocytes. Thus, destruction of Received March 6, 2009; accepted May 26, 2009 doi: /s Corresponding author ( lingyichen@nankai.edu.cn) Supported by the National Key Basic Research and Development Program of China (Grant No. 2009CB941000), the Ministry of Science and Technology of China, and the transgenic program (Grant No. 2009ZX B), the National Natural Science Foundation of China (Grant No ) and the Ministry of Agriculture of China (Grant No. 2009ZX B). Citation: Chen L Y, Liu L. Current progress and prospects of induced pluripotent stem cells. Sci China Ser C-Life Sci, 2009, 52(7): , doi: /s

2 embryos and derivation of ES cells from primates, especially from human, leads to extensive ethical concerns and debates. Moreover, when transplanted into patients, ES cells or their derivative cells could cause immunological rejection. Both ethical issue and immunological rejection prevent potential application of ES cells in cell replacement therapy. Hence, many studies have been attempted to develop new methods to generate pluripotent stem cells, without involving destruction of embryos or requiring oocytes. Fusion of ES cell and somatic cell can give rise to new pluripotent stem cells. Yet, these pluripotent stem cells are tetraploid, restricting their applications in regenerative medicine and development biology research [8]. Under specific in vitro culture conditions, mouse and human spermatogonial stem cells (SSCs) are spontaneously induced to form ES-like pluripotent cells [9 11]. However, isolation of SSCs involves invasive surgery, and this method is only applicable for males. More importantly, primordial germ cells (PGCs), the progenitor of SSCs, undergo imprint erasure to remove the parental imprints. Subsequently, a male- or female- specific imprint pattern, different from that in adult somatic cells, is re-established during gametogenesis. Hence, ES-like pluripotent cells from in vitro culture of SSCs might cause tumorigenesis due to the unbalanced imprinting status [12], limiting their potential applications in cell replacement therapy. In addition, in vitro culture of PGCs gives rise to embryonic germ (EG) cells [13,14]. Recently, epiblast stem cells (EpiSCs) have been derived from day 4.5 blastocyst [15,16]. Both EG cells and EpiSCs are pluripotent and capable of contributing to chimeric mice. Yet, no germline transmission has been achieved with these two types of cells in the chineras, suggesting that they do not have the most stringent pluripotency. Interestingly, mouse EpiSCs are very similar to human ES cells [15]. Hence, further investigation is needed to assess the pluripotency of human ES cells. 2 The establishment of ips cells Somatic cells can be reprogrammed by nuclear transfer or cell fusion with ES cells, implying that some factors in oocytes and ES cells are able to initiate the reprogramming process, and to change the epigenetic status of somatic cells, resulting in acquisition of pluripotency and cell proliferation like zygotes. Given these phenomena, can the reprogramming factors be directly applied in somatic cells and reverse the differentiated status to the pluripotent status? This question was positively answered in Takahashi and Yamanaka chose 24 [17] transcription factors highly expressed in early mouse embryos, ES cells and embryonic carcinoma cells. Through screening various combinations of these 24 factors, they found that simultaneous expression of Oct4, Sox2, c-myc, and Klf4 (these four factors are also called Yamanaka factors) efficiently reprogram mouse embryonic fibroblasts (MEF) and mouse tail-tip fibroblasts (TTF) to form colonies that show ES-like colony morphology and proliferate similar to ES cells. These ES-like colonies are named induced pluripotent stem (ips) cells. Further characterization of ips cells revealed that ips cells express ES cell specific marker genes. Subcutaneous transplantation of ips cells into nude mice leads to the formation of teratoma, which is composed of tissues from three germ layers. ips cells also contribute to mouse embryonic development after injection into blastocysts, however, no live chimera mouse is born. This breakthrough on ips cells brought stem cell research to the frontier of life sciences, and drew great interest and attention. The successful derivation of ips cells solves the problems faced by ES cell researchers. First, the generation of ips cells is more convenient and flexible. With ectopic expression of few transcription factors, embryonic, neo-natal, or adult somatic cells are reprogrammed and give rise to pluripotent stem cells. Patient-specific ips cells circumvent immunological rejection after cell engraftment. Secondly, the derivation of ips cells no longer requires early embryos or germ cells, avoiding ethical issues associated with ES cells. However, even though they formed teratoma and contributed to embryonic development, the ips cells in this study failed to produce live chimera animals, not to mention germline transmission, implying limited pluripotency of the original ips cells, unlike ES cells [17]. The major reason was that a neomycin-resistant gene driven by the Fbx15 promoter was used to select ips cells in this study. Yet, Fbx15 gene is not an appropriate pluripotency marker. Despite high expression of Fbx15 in undifferentiated ES cells, Fbx15 is dispensable for self-renewal and pluripotency maintenance of ES cells [18]. Therefore, the ips cells selected by Fbx15 are not authentic pluripotent stem cells. Then better selection strategies with Oct4 or Nanog Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

3 reporter system in replacing Fbx15 selection were applied to obtain ips cells. Both Oct4 and Nanog are involved in self-renewal and pluripotency maintenance [19 21]. Thus, activation of endogenous Oct4 and Nanog gene is more closely related to the pluripotent status. Using Oct4 or Nanog reporters, several groups tried to reprogram somatic cells with Yamanaka factors, and obtained ips cells with pluripotency. The new ips cells are able to generate chimeric mice with high efficiency, to contribute to germ cell development, and to produce ips cell progeny mice through germline transmission [22 24]. Furthermore, after injection into tetraploid blastocysts, ips cells support embryonic development up to the late-gestation stage [23]. These ips cells also share similar epigenetic and transcriptional profiles with ES cells, such as DNA methylation, histone H3 methylation, X chromosome activation, and global gene expression profiles [22 24]. Successful generation of mouse ips cells laid a solid groundwork for deriving human ips cells. Soon after the first report of mouse ips cells, four independent groups reprogrammed human embryonic, neo-natal, and adult fibroblasts, and established human ips cell lines [25 28]. In these studies, except for Thomson group who used lentiviral vectors that express OCT4, SOX2, NANOG, and LIN28 for reprogramming, the other three groups reprogrammed somatic cells with retrovirus vectors expressing OCT4, SOX2, KLF4 and C-MYC [25 28]. Despite different combinations of factors for ips cell generation, the ips cells from these 4 groups shared similar colony morphology, gene expression profile, and differentiation potential. The two groups of reprogramming factors will help us understand the molecular mechanism of somatic cell reprogramming. Moreover, even though Park et al. [27] failed to reprogram healthy adult skin fibroblasts with 4 Yamanaka factors, addition of htert and SV40 large-t antigen to the 4 factors enabled derivation of ips cells from healthy adult skin fibroblasts. More interestingly, htert and SV40 large-t antigen viral DNA were not detectable in the genomic DNA of these ips cells. Hence, further investigations are warranted to elucidate the functions of htert and SV40 large T antigen in the reprogramming of somatic cells into ips cells. Recently, more published papers on human ips cells confirmed that the generation of ips cells is robust and reproducible [29 34]. In addition to mouse and human ips cells, rhesus monkey, rat and pig ips cells have also been established [35 39]. Monkey, rat and pig ips cells have great application potential in research on human diseases through modeling human diseases. Alternatively, cell transplantation with monkey, rat and pig ips cells or their derivatives provide valuable animal experimental data, which is the basis for cell replacement therapy in human patients. Moreover, pig ips cells have great application potential in agricultural practice, such as construction of gene-targeted or transgenic animals. Beyond that, the generation of monkey, rat and pig ips cells proves that the mechanism for pluripotency maintenance is highly conserved among animal species. 3 Optimization of ips cell derivation Initially, the prevailing method to establish ips cells is as follows: somatic cells are infected by retroviruses expressing four Yamanaka factors; the infected cells are then cultured in standard mes medium; next, drug selection is applied for the activation of pluripotencyassociated promoters, such as Oct4 and Nanog promoters; once morphologically ES-like colonies are obtained, pluripotency of ips cells is evaluated with various assays [17,19 21]. With this method, the efficiency of ips cell generation is extremely low. Moreover, various ips cell lines show heterogeneous pluripotency. In addition, these ips cells have their own safety problems, such as possible mutations by viral insertions and the oncogenic potential of c-myc, preventing the potential applications of ips cells in clinics. In order to solve the safety issues and to improve the efficiency of ips cell derivation, many studies have been carried out to optimize the method of establishing ips cells from different aspects (Figure 1 and Table 1). 3.1 Selection of ips cells based on colony morphology or silencing of transgenic GFP Among the first few studies of ips cells, the donor somatic cells all carried genetic modifications, so that ips cells could be selected by activation of drug-resistance genes. However, genetic modification through homologous recombination is an inefficient and time-consuming process. It is even harder to perform homologous recombination in human somatic cells. To circumvent the requirements for genetic modification, two independent groups attempted to isolate ips cells based on morphology criteria, but not drug-selection. Again, they trans- 624 Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

4 duced mouse MEF and TTF cells with retroviral vector-carried four Yamanaka factors. After culture of the transduced cells in mes conditions for more than 10 days, large colonies with ES cell colony morphology were picked. With further characterization of the picked colonies, they identified the successfully reprogrammed ips cell lines [40,41]. The most stringent assay, tetraploid embryo complementation, was performed to assess the developmental potential of the ips cells isolated based on colony morphology. The result showed that these ips cells are capable of supporting embryonic development as late as 14 days, further confirming the pluripotency of the ips cells isolated based on morphology criteria [40]. These studies demonstrated that it is feasible to reprogram non-genetic modified somatic cells and isolate ips cells. This allows us to derive human ips cells from unmodified human somatic cells. Despite the fact that ips induction requires expression of Yamanaka factors, ips cells silence the virally expressed genes after the successful reprogramming. Therefore, if viruses co-express GFP gene and Yamanaka factors are used to reprogram somatic cells, silencing of GFP expression can serve as a marker for ips colonies, allowing easy identification of ips cells [42]. 3.2 Reducing the tumorigenicity of ips cells Another important issue concerns about the safety associated with ips cells. Among the four Yamanaka factors, c-myc is a well-known oncogene, and Klf4 has also been implicated in carcinogenesis [43,44]. Therefore, it is necessary to carefully analyze the tumor formation probability of ips cells before applying ips cells in clinics. It has been shown that around 20% of ips cell F1 progeny mice developed tumors, due to the reactivation of the oncogene c-myc [22]. To reduce the tumorigenicity of ips cells, c-myc was omitted from the reprogramming cocktails, and only three factors, Oct4, Sox2 and Klf4 were used to derive ips cells. Even though the lack of c-myc dramatically decreased efficiency in the derivation of ips cells, ips cells with high quality and pluripotency were successfully established. More importantly, no tumor was observed in ips cell F1 progeny mice 100 days after birth without c-myc [45,46]. The reduced derivation efficiency of ips cells due to the lack of c-myc can be compensated by some smallmolecules. Marson and his colleagues found that addi- Figure 1 The schematic illustration of ips cell derivation. Through retrovirus, lentivirus, adenovirus, plasmid transfection, transposon, or protein transduction, specific combination of transcription factors, for example, Yamanaka factors, Oct4, Sox2, Klf4, and c-myc, are ectopically expressed, and induce the reprogramming of somatic cells to pluripotent ips cells. Some small-molecule chemicals, such as DNA methyltransferase inhibitors, histone deacetylase inhibitors, and inhibitors of signaling pathways, promote the generation of ips cells. These small-molecule chemicals can reduce the number of transcription factors required for reprogramming, and enhance the efficiency in the derivation of ips cells. Additional factors or RNAi knock-down of specific genes may promote ips cell formation (see Table 1 for details). Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

5 Table 1 The methods of ips cell derivation Species Yamanaka factors* Other small-molecule Ectopic expression Other factors chemicals and biologically Type of somatic cells O S K M methods active molecules References fibroblasts retrovirus [17,22 24,40,41, 47] fibroblasts inducible lentivirus [48,49] fibroblasts single lentivirus** [50,51] hepatocytes,stomach cells retrovirus [52] fibroblasts, hepatocytes adenovirus [53] fibroblasts plasmid transfection [54,55] fibroblasts PB transposon [56] VPA fibroblasts protein transduction [57] C/EBPα, Pax5 RNAi - B lymphocytes inducible lentivirus [58] Mouse RNAi knock-down fibroblasts, of Pax7, Pax3, 5 -azac Blymphocytes Gata6, and Sox9 inducible lentivirus [59] - fibroblasts retrovirus [45,46] - Wnt3a fibroblasts inducible lentivirus [60] - 5 -azac, VPA fibroblasts retrovirus [61] - - Essrb fibroblasts retrovirus [62] - - NSCs, NPCs retrovirus,inducible lentivirus [63,64] - - BIX NPCs retrovirus [65] - - BIX-01294, BayK8644 fibroblasts retrovirus [66] - - CHIR99021, PD fibroblasts, NSCs retrovirus [67] Human NSCs retrovirus [68] fibroblasts retrovirus [25,28 30] fibroblasts lentivirus [69] fibroblasts inducible lentivirus [33,34] keratinocytes retrovirus [32] keratinocytes single lentivirus** [55] CD34 + blood cell retrovirus [70] fibroblasts plasmid transfection [54] fibroblasts PB transposon [56] c-myc, Klf4 fibroblasts episomal vector [71] fibroblasts protein transduction [72] - - Nanog,Lin28 fibroblasts lentivirus [26] - - Nanog,Lin28 CHIR99021, PD , A fibroblasts lentivirus [36] htert,large T fibroblasts retrovirus [27] p53 RNAi, UTF1 fibroblasts lentivirus [42] - fibroblasts retrovirus [45] - - VPA fibroblasts retrovirus [31] mir-302 skin cancer cell retrovirus [73] Monkey fibroblasts retrovirus [35] Rat - CHIR99021, PD , A liver progenitor cells retrovirus [36] fibroblasts, bone marrow cells lentivirus [37] Pig Fibroblasts retrovirus [38] bone marrow cells lentivirus [39] * O, OCT4; S, SOX2; K, KLF4; M, C-MYC. ** With the combination of 2A peptide sequence and IRES, a single viral vector expresses 4 Yamanaka factors. 626 Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

6 tion of soluble Wnt3a into mes medium improves reprogramming efficiency with only three factors, Oct4, Sox2 and Klf4 [60]. A histone deacetylase inhibitor valproic acid (VPA) also enhances the efficiency of ips cell generation in combination with Oct4, Sox2 and Klf4 [61]. Alternatively, Essrb, together with Oct4 and Sox2, can efficiently reprogram MEF into ips cells, thus replacing two potent oncogenic factors, c-myc and Klf4 [62]. While several studies focused on the oncogenic effect of reprogramming factors, Aoi et al. [52] addressed the tumorigenicity of ips cells from a different aspect by looking at the donor cell types for generation of ips cells. ips cells were first derived from adult mouse hepatocytes and gastric epithelial cells as well as MEF cells, and then ips cell F1 progeny mice were obtained through germline transmission. The authors found that the tumor formation rate and the death rate of the F1 mice with the stomach cell or hepatocyte origin are lower than those of the F1 mice derived from MEForigin ips cells, indicating that donor cell types influence tendency in tumor formation of ips cells. The mechanism of the donor cell effect on ips mouse tumorigenicity and death rate remains elusive. Yet, these data suggest that appropriate types of donor somatic cells have to be carefully chosen, and factors with oncogenic potential should be avoided in order to ensure the safety of ips cells in therapeutic applications. 3.3 Reduction of viral integration sites to enhance the safety of ips cells Except for the tumorigenicity of c-myc, retrovirus and lentivirus mediating the transduction of reprogramming factors also have safety issues. Because both retrovirus and lentivirus are integrating viruses, they insert viral sequences together with exogenous genes into genomic DNA, and might cause mutations by insertion. The use of retroviruses and lentiviruses during ips cell generation should be restricted, or even avoided, to minimize the chance of insertion mutation. In order to reduce the number of viruses, some studies took advantage of key transcriptional factors for pluripotency expressed in donor cells. For example, Sox2 is expressed in neural stem cells (NSCs) and neural progenitor cells (NPCs), so that NSCs and NPCs can be reprogrammed to form ips cells with only Oct4 and Klf4 [63,64]. Recently, Kim et al. [68] showed that just one factor Oct4 is sufficient to reprogram adult mouse NSCs, most likely because of the endogenous expression of Sox2 and Klf4 in NSCs. Alter- natively, four Yamanaka factors can be expressed from a single viral vector through the combination of 2A peptide sequence and IRES technology, hence reducing viral integration sites and insertion mutation rate in ips cells [50,51]. Another strategy is to use small-molecules to replace reprogramming factors. Several groups screened small-molecule libraries, and identified some chemicals facilitating the production of ips cells. An inhibitor of the G9a histone methyltransferase, BIX is able to substitute Oct4, and reprogram NPCs to ips cells together with the rest three Yamanaka factors. In addition, BIX promotes the reprogramming efficiency of NPCs in the presence of Oct4 and Klf4 [65]. The combination of BIX and BayK8644 (a L-channel calcium agonist) enables reprogramming MEF to ips cells with Oct4 and Klf4 [66]. If VPA, a histone deacetylase inhibitor, is added into cell culture during reprogramming, ips cells can be efficiently induced from human fibroblasts even with two factors OCT4 and SOX2, thus avoiding two oncogenes C-MYC and KLF4 [31]. To circumvent the reactivation of c-myc and reduce the tumorigenicity of ips cells, foreign genes could be excised in established ips cells. Recently, Cre/LoxP recombination was applied to remove exogenous reprogramming factors after establishment of ips cells, resulting in factor-free ips cells [54,74]. However, a part of the vector backbone remains at the integration site after Cre-mediated factor deletion. Therefore, Cre-mediated factor deletion cannot completely avoid insertion mutations. To minimize the risk of insertion mutations, two groups reprogrammed somatic cells with piggybac (PB) transposon, and the integrated exogenous genes were removed by transient expression of transposase in the resultant ips cells. These ips cells are free of exogenous genes, and PB transposons are seamlessly removed from their integration sites. Compared to Cre-mediated excision, the PB transposon system appears to be a safer method to derive ips cells [54,56]. Nevertheless, the safety issue of the PB transposon system, especially whether the transposon insertions during ips cell formation have any long-term effect on the resulting ips cells, remains to be determined. The safest method of generating ips cells is not to use integrating viruses or transposons at all. The reprogramming factors are transiently expressed to induce formation of ips cells. Establishment of ips cells requires continuous expression of Yamanaka factors for around days [48,49]. Thus, the duration of tran- Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

7 siently expressed reprogramming factors has to be at least 10 days. This might be a challenging issue. Stadtfeld et al. [53] used adenoviruses to transiently express the four Yamanaka factors and established ips cells without exogenous DNA insertion. Yet, the efficiency of ips cell derivation is extremely low. Unexpectedly, a large proportion of ips cell lines produced with adenoviruses are tetraploid. How adenoviruses induce the tetraploid state in ips cells needs further investigation. Alternatively, Okita et al. [55] applied liposome-mediated transfection of non-viral expression vectors to induce ips cells. To achieve prolonged expression of the transcription factors, multiple transfections were performed. Again, ips cell lines were isolated at low efficiency, similar to the adenovirus-mediated method. Moreover, some ips cell lines derived with this method had plasmid DNA integration. Non-integration episomal vectors were applied in human ips cell induction, but again with low efficiency. After ips cells were obtained, episomal vectors are gradually lost in the absence of drug selection, and vector and transgene-free human ips cells can be isolated by subcloning [71]. A more intriguing method is to deliver the reprogramming proteins, instead of genes, into somatic cells to induce ips cells. The protein transduction can be mediated by cell-penetrating peptides. Thus far, both mouse and human protein-induced ips cells have been generated [57,72]. Yet, the efficiency of ips cell induction is greatly reduced with the protein transduction method, and can be expectedly improved in combination with other methods, i. e. using small chemical molecules. From the safety perspective of ips cells in clinical applications, induction of ips cells with proteins is the safest method so far, since no genetic modification is involved during reprogramming. Other methods, such as adenovirus, plasmids, and episomal vector mediated reprogramming, also improve the safety to a certain extent. However, all these methods with improved safety are associated with reduced reprogramming efficiency. Thus, efforts to improve reprogramming efficiency are necessary. 3.4 Enhancing the efficiency of ips cell derivation The efficiency of ips cell generation is low, especially when reducing the number of reprogramming factors, or not using retrovirus/lentivirus. In the initial study, the efficiency of ips cell derivation was only 0.02% [17]. The low efficiency might be due to low efficiency in viral transduction or reprogramming itself. Viral transduction efficiency is below 100% so that the fraction of cells infected by all four factors is even lower. A doxycycline-inducible lentiviral system was constructed to improve reprogramming efficiency. Upon doxycycline treatment, all cells express four Yamanaka factors, eliminating the effect of low viral transduction efficiency. Even so, generation of ips cells is still inefficient, maximum 2%, implying that somatic cell reprogramming is a stochastic process, and that not every cell expressing all four Yamanaka factors is induced to form ips cells [33,34,58]. The low efficiency of ips cell generation not only affects clinical applications of ips cells, but also makes it difficult to study the mechanisms of ips cell formation. In order to enhance reprogramming efficiency, many studies were conducted to look for new reprogramming factors or small-molecules. As mentioned above, BIX-01294, an inhibitor of the G9a histone methyltransferase, improves the reprogramming effect of Oct4 and Klf4 on NPCs [65]. DNA methyltransferase and histone deacetylase inhibitors, such as 5-aza-cytidine (AZA) and VPA, also enhance derivation rates of ips cells [31,59,61]. When purified recombinant proteins were used to reprogram MEF cells, ips cells can be derived only in the presence of VPA [57]. Recently, knockdown of p53 and over-expression of UTF1, in combination with four Yamanaka factors, or even in the absence of c-myc, increase the efficiency of ips cell production by 100 folds [42]. Alternatively, rates of deriving ips cells can be improved by selection of donor cell types. When induced with retrovirally expressed four Yamanaka factors, the ips cell derivation rate from juvenile human primary keratinocytes is at least 100-fold higher than that from human fibroblasts. Furthermore, reprogramming of keratinocytes is twofold faster than reprogramming of fibroblasts. Taking advantage of high reprogramming efficiency of keratinocytes, ips cells can be established with keratinocytes isolated from single adult human hair [32]. Donor cells from single hair is enough for generation of ips cells, avoiding complicated and invasive surgical procedures and facilitating the derivation of patient-specific ips cell lines. In summary, for the safety of ips cells, the number of exogenous transcription factors has to be reduced, and integrating virus should be avoided. However, these methods significantly decrease the efficiency of ips cell derivation. With ensured ips cell safety, there are at least 628 Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

8 two strategies to improve rates of generating ips cell: one is small-molecules, such as DNA methyltransferase inhibitors and histone deacetylase inhibitors; and the other is the use of appropriate donor cell types, such as keratinocytes and adult stem cells. These two methods are not exclusive. Combination of these two strategies might lead to highly efficient reprogramming. 4 The molecular mechanism underlying generation of ips cells To efficiently establish safe ips cell lines, it is necessary to understand the molecular mechanism underlying reprogramming during ips cell derivation. 4.1 The dynamics of pluripotency marker activation during ips cell induction The expression dynamics of pluripotency markers was first investigated to understand the mechanism of ips cell formation. With the doxycycline-inducible lentiviral system, Stadtfeld el al. [48] and Brambrink et al. [49] studied how mouse MEF cells are reprogrammed by Yamanaka factors. They found that the fibroblast-specific gene Thy1 is first down-regulated, accompanied by up-regulation of alkaline phosphatase and a ES cell surface marker SSEA1, at the initial phase of reprogramming, while the activation of endogenous Nanog, Sox2 and Oct4 can only be detected at the late stage of reprogramming. The expression of telomerase and the reactivation of X chromosome are also late events of reprogramming [48]. In addition, induction of ips cells requires continuously expression of exogenous Yamanaka factors for at least days [48,49]. 4.2 Transcriptional regulation and epigenetic regulation in the formation of ips cells The most widely used Yamanaka factors are all transcription factors [17]. Even in the four factors used by Thomson group, three of them (OCT4, NANOG and SOX2) are also transcription factors [26]. Therefore, the information about the down-stream targets and regulatory effects of these reprogramming factors should shed lights on the mechanism of ips cell induction. Several groups studied the genome-wide binding sites of these reprogramming factors as well as other transcription factors involved in pluripotency. Oct4, Nanog and Sox2 appear to cross-regulate each other, and form a circuitry regulating the pluripotent state in ES cells. The regulatory circuitry maintains the expression of genes necessary for pluripotency maintenance, and suppresses the expression of genes associated with differentiation [75 80]. This explains why ectopically expressed Yamanaka factors can activate endogenous ES cell marker genes, and reprogram somatic cells. Sridharan and his colleagues further analyzed the genome-wide binding profiles of the four Yamanaka factors in ips, ES and partially reprogrammed cells [81]. They found that the binding profiles of the four Yamanaka factors show significant overlap in ips and ES cells, but not in partially reprogrammed cells. Two groups of genes are of interest: the first group of genes are co-bound by c-myc and any of the other three factors in ES cells; the second group of genes encode pluripotency regulators and are occupied only by Oct4, Sox2, and Klf4, but not c-myc in ES cells. In partially reprogrammed cells, genes in the first group show an ES cell-like binding and expression pattern, while genes in the second group are not occupied by these factors, and are not transcriptionally activated. The histone methylation status of genes in the second group in partially reprogrammed cells is at an intermediate state between fibroblasts and ES/iPS cells. The reason for failure of Oct4, Sox2 and Klf4 to occupy their targets in partially reprogrammed cells might be that binding of Oct4, Sox2 and Klf4 needs other factors which are absent in partially reprogrammed cells, such as Nanog. Moreover, c-myc plays important roles in suppression of fibroblast specific genes and activation of the embryonic metabolic program during the initial stage of somatic cell reprogramming, suggesting various roles of the four factors at different stages of reprogramming [81]. However, low efficiency of reprogramming cannot be explained by transcriptional regulation. Not every cell expressing four Yamanaka factors is programmed into ips cells. Also, small-molecules inhibiting epigenetic modifying enzymes promote the ips cell generation, suggesting that reprogramming of epigenetic information is a major component in ips cell formation [31,61,65]. After systematically comparing the epigenetic properties of ips, ES and MEF cells, Maherali et al. [24] and Mikkelsen et al. [59] found that ips and ES cells have strong similarity in DNA methylation and methylation of histone H3 lysine 4 (H3K4) and lysine 27 (H3K27), but distinct from the donor MEF cells. In addition, the originally silenced X chromosome is reactivated in female ips cells. All these data demonstrate that the epi- Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

9 genetic information in somatic cells has to be reprogrammed to establish a new pluripotent cell specific epigenetic program during ips cell formation. Mikkelsen et al. also isolated partially reprogrammed cell lines, which show an intermediate epigenetic state between ips cells and MEF cells [59]. Single clone of partially reprogrammed cell can give rise to two populations of cells, expressing or not expressing the Oct4-GFP reporter gene. Treatment with DNA methyltransferase inhibitor AZA could facilitate the conversion from partially reprogrammed cells to fully reprogrammed ips cells. Taken together, epigenetic reprogramming during ips cell induction is a stochastic and inefficient process. If the epigenetic reprogramming can be controlled, the efficiency of deriving ips cells will be improved. MicroRNAs also play a certain role in somatic cell reprogramming through their effects on transcriptional and epigenetic regulations. LIN28, one of the four reprogramming factors used by Thomson group, blocks the maturation of the Let7 family of micrornas in ES cells, thus suppressing the function of these micror- NAs [82]. LIN28 might facilitate ips cell formation by suppressing the biosynthesis of micrornas related to differentiation and then affecting the expression of the micrornas targets. Alternatively, other micrornas might activate pluripotency associated genes, and promote generation of ips cells. For example, ectopic expression of mir-302s induces ips cell formation from human melanoma Colo and prostate cancer PC3 cells [73]. mir-291-3p, mir-294 and mir-295 promote the reprogramming efficiency by Oct4, Sox2 and Klf4, but not by these factors plus c-myc. The promoters of these three micrornas are bound by c-myc, suggesting that they are downstream effectors of c-myc in reprogramming [83]. 4.3 Reprogramming of telomerase and telomeres in ips cells Telomeres are repetitive sequences (TTAGGG)n at the ends of chromosomes, which are bound by telomere-associated proteins. The major function of telomeres is to protect the ends of chromosomes and to maintain genomic stability. Telomere length is usually maintained by telomerase. Telomerase and telomeres also contribute to ips cell induction and pluripotency maintenance. Compared to donor somatic cells, ips cells exhibit higher telomerase activity [48]. Correspondingly, the length of telomeres in ips cells is increased [84]. Moreover, the epigenetic modifications at telomeres are similar between ips and ES cells, as evidenced by low trimethylation of H3K9 and H4K20. The transcriptional activities in telomeric regions are also enhanced in ips and ES cells. Even though ips cells can be derived from telomerase deficient (Terc / ) MEF, these Terc / ips cells show defective telomere elongation and cannot produce live chimeras, suggesting that the pluripotency of telomerase deficient ips cells is compromised. Furthermore, the efficiency of reprogramming is reversely correlated with increased generation of telomerase deficient mice with shorter telomeres [84]. 4.4 Signal transduction in somatic cell reprogramming Except for transcriptional and epigenetic regulation in the nucleus, signal transduction on the cell surface and in the cell also plays a critical role in somatic cell reprogramming. For example, Wnt signaling pathway is coupled directly with the core transcriptional regulation circuitry of pluripotency. Addition of soluble Wnt3a into the culture medium activates Wnt signal pathway and allows highly efficient production of ips cell with only three factors Oct4, Sox2 and Klf4 [60]. Simultaneous inhibition of mitogen-activated protein kinase (MAPK) signaling and glycogen synthase kinase-3 (GSK3), promotes ips cell formation from brain-derived NSCs. Furthermore, Sox2 and c-myc are dispensable in the presence of these inhibitors, and ips cells can be induced from NSCs with Oct4 and Klf4 [67]. These signal transduction events might facilitate ips derivation through activating of endogenous reprogramming factors or pluripotency associated genes. Further investigations are necessary to understand how signal transduction pathways regulate somatic cell reprogramming. In summary, induction of ips cell is to reprogram the epigenetic and expression profiles by few transcription factors, and to reverse the differentiated state of somatic cells into a pluripotent embryonic cell-like state (Figure 2). First, c-myc promotes the suppression of genes specific for differentiated cells; Oct4, Sox2 and Klf4 cooperate to activate genes associated with pluripotency; meanwhile, the epigenetic information, including histone H3K4 and H3K27 methylation, X chromosome activation, and telomere elongation, is reprogrammed into a pluripotent state. In addition, micrornas and signaling transduction pathways facilitate ips cell formation by their regulatory effects on epigenetic and 630 Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

10 transcriptional regulation. 5 Applications of ips cells Because of their pluripotency and therapeutic potential in cell replacement therapy, ips cells become the most active field of biomedical research (Figure 3A). After establishment of ips cells, the ultimate goal of stem cell researchers and clinicians is to treat patients with patient-specific ips or ips derivative cells. To prove the therapeutic potential of ips cells, Hanna et al. treated sickle cell anemia mice with ips derived hematopoietic progenitors (HPs) [47]. They used a hu- manized sickle cell anemia mouse model, in which α and β-globin genes were replaced by human α, Aγ and β S (sickle) genes. First, ips cells were derived from TTF cells of the sick mouse. The human β S -globin gene was then replaced by human wild type β A -globin through homologous recombination. Next, HPs were derived from in vitro differentiation of corrected ips cells, and purified HPs were transplanted into irradiated hβ S /hβ S male mice. Compared with untreated control mice, transplantation of HPs derived from corrected ips cells significantly suppresses the symptoms of sickle cell anemia, as shown by the reduced degree of polychroma- Figure 2 The molecular mechanism of ips cell generation. During the process of somatic cell reprogramming, somatic cell-specific genes, such as Thy1 in fibroblasts, are first suppressed (the height of triangles in the figure represents the gene expression level at various time points). Next, some pluripotent specific phenotypes appear, for example, expression of alkaline phosphatase and SSEA1. The endogenous pluripotency transcription factors, Oct4, Sox2, and Nanog, are activated at the late stage during formation of ips cells. Activation of X chromosomes (red and green mark the active and silence X chromosomes, respectively), increased telomerase activity, and elongation of telomeres are late events in ips cell induction. Red diamonds and green trapezoids indicate trimethylation of histone H3K4 and H3K27, respectively. c-myc plays important roles in initiation of reprogramming, suppressing somatic cell-specific genes and activating the embryonic metabolic program, while Oct4, Sox2, and Klf4 are involved in activating pluripotency associated genes. If pluripotency associated genes, especially the late activated genes, are not transcriptionally and epigenetically activated, cells are then arrested at a partially reprogrammed state. Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

11 sia, decrease of anisocytosis and poikolocytosis, and increased red blood cell counts [47]. In another study, endothelial progenitor cells derived from ips cells were engrafted into the liver of hemophilia A mice, and showed obvious therapeutic effects, preventing mouse death from bleeding [85]. Although these two experiments were performed in mice, they prove the feasibility of applying ips cells in the combined gene therapy and cell replacement therapy, and provide a valuable animal experimental principle to support similar therapy in patients. Recently, Raya et al. [86] reprogrammed genetically corrected Fanconi anaemia fibroblast cells and obtained corrected Fanconi-anaemia-specific ips cells that can give rise to phenotypically normal hematopoietic progenitors of the myeloid and erythroid lineages. This study further demonstrates the therapeutic potential of ips cells. Most of ips cell lines were established with virus-mediated expression of reprogramming factors. Genomic DNA of these ips cells likely contains viral DNA insertion. Therefore, these ips cells are not suitable for treatment of patients. However, during the final steps of erythropoiesis, mature erythrocytes lose their nuclei and genomic DNA through nuclear condensation and enucleation. If ips cells differentiate into mature erythrocytes, viral DNA as well as the genomic DNA will be eliminated from the cells. Hence, mature erythrocytes derived from ips cells are safe for treatment of patients. A highly efficient system that enables differentiation of human ES cells into functional oxygen-carrying erythrocytes on a large scale has been established [87]. If this system is applied to patient-specific ips cells, it will offer an unlimited resource of patient-specific erythrocytes. It is of great clinical value for treatment of patients with certain blood diseases, such as sickle cell anemia and hemophilia. It might also help providing erythrocytes for patients with the rare blood type. In addition to applications in blood diseases, ips cells can be applied to treatment of neural and cardiovascular diseases. ips cells can be efficiently differentiated in vitro into neural precursor cells, which give rise to neuronal and glial cell types in culture. After engraftment into the fetal mouse brain, these ips cell-derived NPCs migrate into various brain regions and differentiate into neurons and glia [88]. This study demonstrated the therapeutic potential of ips cells in neural diseases. Meanwhile, both mouse and human ips cells have been differentiated into many types of cardiovascular cells, including arterial, venous and lymphatic endothelial cells as well as cardiomyocytes, proving the therapeutic potential of ips cells in cardiovascular diseases [89,90]. ips cells can also be applied to the treatment of infertile patients. Primordial germ cells (PGCs) can be generated through in vitro differentiation of human ips and ES Figure 3 Applications of ips cells. A, The application of ips cells in the combined gene therapy and cell replacement therapy. First, somatic cells, such as fibroblasts and keratinocytes, are isolated from patients. Next, ips cells are induced by ectopically expression of Oct4, Sox2, Klf4, and c-myc. Following correction of defective genes by homologous recombination, ips cells are differentiated in vitro into the desired type of cells, for example, hematopoietic cells and neural cells. At last, these cells are purified and transplanted into patients to cure the disease. B, The application of ips cells in drug screening. A screening system based on patient-specific ips cells is used to screen chemical library. Chemicals that inhibit or change the defective phenotype of the patient-specific ips cells are potential drugs. 632 Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no

12 cells. These in vitro differentiated PGCs show similarity to in vivo PGCs in the gene expression profile. Moreover, the in vitro derived PGCs from ips cells have initiated imprint erasure [91]. If these PGCs can be further differentiated into sperms and oocytes, it will be valuable for infertile patients. ips cells are not only valuable for cell replacement therapy, but also useful for disease modeling in vitro, facilitating studies of mechanisms underlying disease development, drug screening, and development of new therapeutic strategy (Figure 3B). Like ES cells, ips cells support in vitro hematopoiesis. Therefore, in vitro hematopoiesis of patient-specific ips cells allows us to investigate the mechanism of disease development [92]. Some human ips cell lines have been established from patients with neural diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [30,69]. These ALS and SMA patient-specific ips cells can be directed to differentiate into motor neurons [30,69]. The motor neurons originated from ALS and SMA patient-specific ips cells provide in vitro disease models to study these two neural diseases. Once defects of the ALS and SMA motor neurons are well understood, we could correct the defective genes in patient-specific ips cells, obtain functional motor neurons from corrected ips cells, and treat patients with the corrected motor neurons. More ips cell lines have been derived from patients with a variety of genetic diseases, including Parkinson disease, Huntington disease, Down syndrome/trisomy 21, and so on [29,74]. ips cells can also be generated from human blood cells, allowing the generation of patient specific ips cells for diseases in which the disease-causing mutations are restricted to hematopoietic cells [70]. These patient-specific ips cells allow in vitro studies of both normal and pathologic human tissue development, therefore facilitating disease investigation and drug discovery. 6 Prospects of ips cells ips cells can be readily generated from patients themselves, thus solving immunological rejection problem in cell transplantation. As derivation of ips cells does not require embryos or oocytes, no ethical issues would restrict application of ips cells. These two advantages of ips cells move us closer to the realization of autologous cell replacement therapy. However, due to safety concerns, ips cells derived with current technology are not qualified for clinical application. Integrating viruses and reprogramming factors with oncogenic potential should be avoided to provide ips cells with therapeutic quality. In addition, enhancing the efficiencies of ips cell derivation, enriching homogenous populations of ips cells, directed in vitro differentiation, and purification of differentiated cells, all will benefit clinical application of ips cells. Recently, the U.S. Food and Drug Administration approved the first clinical trial of human ES cells, which authorize the biotech company Geron to treat spinal-cord injury patients with human ES cell derived neurons [93]. Even though this is the first step toward applying ES cells in treatment of patients, and there is still a long way to go to achieve the ultimate goal, cell replacement therapy, it is critical that the most important first step has been taken. In the near future, we expect more clinical trials with ips and ES cells, translating basic research into clinical applications. Thus far, no ES cell line has been isolated from domestic farm animals, such as bovine, swine, and sheep. Generation of ips cells from farm animals can be an alternative source of pluripotent cells, and benefit agricultural practice and biomedical research. Pig ips cells have been established [38,39]. Yet, neither chimera pig nor transgenic pig has been produced with pig ips cells. Therefore, efforts are needed to apply pig ips cells in gene-targeted or transgenic animal production, as well as to derive ips cells from other large farm animals. 1 Nagy A, Gócza E, Diaz E M, et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development, 1990, 110: Poueymirou W T, Auerbach W, Frendewey D, et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol, 2007, 25: Huang J, Deng K, Wu H, et al. Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation. Stem Cells, 2008, 26: EvansM J, Kaufman M H. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981, 292: Martin G R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA, 1981, 78: Kim K, Lerou P, Yabuuchi A, et al. Histocompatible embryonic stem cells by parthenogenesis. Science, 2007, 315: Munsie M J, Michalska A E, O'Brien C M, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr Biol, 2000, 10: Cowan C A, Atienza J, Melton D A, et al. Nuclear reprogramming of Chen Lingyi et al. Sci China Ser C-Life Sci Jul vol. 52 no