Once neurons enter the post-mitotic G0 phase during central nervous system (CNS) development, they lose their proliferative potential. When neurons re-enter the cell cycle during pathological situations such as neurodegeneration, they undergo cell death after S phase progression. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are highly coordinated. In this review, the coordination of cell cycle control and neuronal differentiation during development are discussed, focusing on regulation by the Rb family of tumor suppressors (including p107 and p130), and the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors. Based on recent findings suggesting roles for these families in regulating neurogenesis and neuronal differentiation, I propose that the Rb family is essential for daughter cells of neuronal progenitors to enter the post-mitotic G0 phase without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family regulates the timing of neuronal progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina. Rb's lack of involvement in regulating the initiation of neuronal differentiation may explain why Rb family-deficient retinoblastomas characteristically exhibit neuronal features.
One of the most striking properties of the central nervous system (CNS) is the tremendous diversity of neurons, which display distinct morphologies (Cajal 1909–1911) and gene expression profiles (Gong et al. 2003; Lein et al. 2007). These diverse populations of neurons are generated from a pool of undifferentiated multi-potent progenitor cells during a specific developmental stage in embryogenesis (Turner & Cepko 1987; Holt et al. 1988). During CNS development, different types of neurons in each part of the CNS are generated at specific times. For example, in the cerebral cortex, deep layer neurons that extend axons to subcortical targets are generated at early stages, while more superficial layer neurons that extend axons intracortically are generated at later developmental stages (Angevine & Sidman 1961). Thus, the cell cycle exit of progenitor cells must be temporally coordinated with the initiation of neuronal differentiation to control the number and ratio of different neuronal subtypes in the CNS. Once progenitor cells exit the cell cycle, their daughter neurons enter the post-mitotic G0 phase for terminal differentiation and lose their proliferative potential. This inability of differentiated neurons to undergo proliferation is one of the major reasons brain tissue cannot regenerate following injury. When mature neurons re-enter the S phase in pathological situations such as neurodegeneration, they undergo cell death (Herrup & Yang 2007). Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are tightly controlled.
Our understanding of the molecular mechanisms involved in cell cycle regulation and neuronal differentiation has markedly increased in recent years. For example, the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors was shown to regulate the timing of neuronal progenitor cell cycle exit, thereby contributing to the generation of different neuronal subtypes in the correct ratios (Dehay & Kennedy 2007). On the other hand, the tumor suppressor Rb and its family members (p107 and p130) were found to play a central role in cell cycle regulation during cell differentiation (Burkhart & Sage 2008). Both the Cip/Kip and Rb families regulate neuronal migration, a critical aspect of neuronal differentiation (Frank & Tsai 2009). However, the coordinated regulation of the cell cycle and neuronal differentiation, and the consequences of uncoupled proliferation and differentiation during neuronal development are not well understood. Here, recent reports describing the mechanisms by which cells enter the quiescent, senescent, and post-mitotic G0 phases, the coordination of cell cycle control and neuronal differentiation during development, and the consequences of uncoupled proliferation and differentiation leading to the development of retinoblastoma are reviewed. Based on these findings, I propose that the Rb family is essential for regulating the entrance and maintenance of the daughter neurons of progenitors in the post-mitotic G0 phase, without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family controls the timing of progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina.
Role of the Rb family in regulating cell entrance into the quiescent G0 phase
RB1 was the first tumor suppressor gene cloned from patients with retinoblastoma (Friend et al. 1986). The Rb protein product suppresses the expression of genes important for S phase progression by binding to the E2F transcription factor (Chellappan et al. 1991; Hiebert et al. 1992; Weintraub et al. 1992). During the G1 to S phase progression, Rb is phosphorylated and dissociates from E2F, which induces the expression of S phase-related genes (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989). The Rb family members p107 and p130 also bind to E2F and function to control proliferation. p107 and p130 are expressed during different cell cycle phases, while Rb is expressed throughout the cell cycle. p107 is found in an E2F complex at the late G1 and S phases (Devoto et al. 1992; Shirodkar et al. 1992), while p130 and E2F form a complex during the quiescent G0 and early G1 phases (Cobrinik et al. 1993; Smith et al. 1996).
Serum-starved cells enter the quiescent G0 phase prior to the late G1 restriction point (Pardee 1974; Fig. 1A). When quiescent G0 cells receive growth stimulation, a few hours later they re-enter the late G1 phase and proliferate (Zetterberg & Larsson 1985; Fig. 1A). p130 functions to maintain the quiescent G0 phase by an epigenetic mechanism involving the recruitment of chromatin-modifying enzymes that suppress E2F-target genes (Takahashi et al. 2000; Rayman et al. 2002). When cells arrest at the quiescent G0 phase, p130 forms a complex with E2F4 and the histone deacetylase HDAC1, associated with the low levels of histone H3 and H4 acetylation (Fig. 1B). When cells advance to the late G1 phase, the p130-E2F4-HDAC1 complex is replaced by E2F1 and E2F3, associated with the high levels of histone acetylation. Thus, Rb family members suppress E2F-dependent transcription in both phosphorylation- and chromatin modification-dependent manners (Fig. 2A). Rb also suppresses cell cycle progression in E2F-independent manners, involving Cdk inhibition (Fig. 2B). In one mechanism, Rb binds to S-phase kinase-associated protein (Skp2), the F-box protein of the E3 ubiquitin ligase complex (Ji et al. 2004). This interaction prevents the degradation of p27, a Cip/Kip family member, via the ubiquitin-proteasome pathway. In a second mechanism, Rb interacts with both Skp2 and anaphase-promoting complex/cyclosome (APC/C; Binne et al. 2007). This interaction leads to Skp2 ubiquitylation and degradation, thereby stabilizing p27.
Although the Rb family members use different mechanisms to prevent S phase progression, redundancy among these family members is likely to mask their individual functions. Therefore, Rb−/−; p107−/−; p130−/− (Rb-TKO) cells are ideal for elucidating Rb family functions in suppressing S phase progression. The mouse embryonic fibroblasts (MEFs) derived from Rb-TKO embryonic stem (ES) cells are resistant to G1 arrest triggers such as contact inhibition and serum starvation (Dannenberg et al. 2000; Sage et al. 2000). Thus, use of the triple knockout showed that the Rb family is essential for inducing cultured MEFs to enter the quiescent G0 phase.
Role of the Rb family in regulating cell entrance into the senescent or post-mitotic G0 phase
The quiescent G0 phase is different from the senescent and post-mitotic G0 phases with respect to the terminal differentiation of cells and their responses to growth factor stimulation. In contrast to quiescent G0 phase cells, senescent and post-mitotic G0 phase cells are incapable of proliferating after growth factor stimulation (Fig. 1A). Senescent cells can be distinguished from post-mitotic cells by the expression of senescence-associated β-galactosidase (SA-βgal; Dimri et al. 1995) and the presence of senescence-associated DNA-damage foci (SDFs; d'Adda di Fagagna et al. 2003; Takai et al. 2003) and senescence-associated heterochromatic foci (SAHFs; Narita et al. 2003). SAHFs are characterized by the presence of heterochromatin-associated histone modifications such as tri-methylated lysine 9 at histone H3 (H3K9Me3) and the methyl-lysine binding protein HP1 (Fig. 1B). Consistent with biochemical data showing that Rb associates with Suv39h1, the H3K9 histone methylase, and HP1 (Nielsen et al. 2001), the loss of Rb prevents SAHF formation and inhibits cells from entering the senescent G0 phase (Chicas et al. 2010). In contrast to quiescent cells, senescent cells are characterized by the association of Rb, rather than p107 or p130, with the E2F-target genes important for S phase progression. Moreover, the inactivation of Rb in senescent MEFs causes them to re-enter the cell cycle and proliferate (Sage et al. 2003). Thus, among the Rb family members, Rb is uniquely required for cells to enter and remain in the senescent G0 phase.
The role of Rb family members in regulating the entrance and maintenance of cells in the post-mitotic G0 phase, leading to terminal differentiation is context-dependent (Burkhart & Sage 2008; Indovina et al. 2013). During skeletal muscle differentiation, the loss of Rb in undifferentiated cells leads to S phase progression both in vitro and in vivo but does not affect the initiation of differentiation, suggesting that Rb is required for cells to enter the post-mitotic G0 phase (Gu et al. 1993; Schneider et al. 1994; Novitch et al. 1996; Zacksenhaus et al. 1996). However, Rb-deficient myotubes fail to fully mature. Rb interacts with the basic helix-loop-helix (bHLH) muscle transcription factor MyoD and may control terminal differentiation via this factor (Gu et al. 1993), suggesting that Rb regulates the entrance of cells into the post-mitotic G0 phase as well as their advancing differentiation. In contrast, the inactivation of Rb in post-mitotic myocytes and myotubes does not lead to S phase progression (Camarda et al. 2004; Pajcini et al. 2010). Additional signals such as Cyclin D and Cdk4 overexpression and Arf suppression are required for the S phase progression of Rb-deficient post-mitotic myocytes and myotubes. Although the roles of p107 and p130 in maintaining the post-mitotic state of skeletal muscle myotubes have not yet been characterized, the inactivation of both Rb and p130 leads post-mitotic cardiac myocytes to enter the S phase (Sdek et al. 2011), suggesting that the Rb family is required for maintaining cardiac myocytes and possibly other cell types in the post-mitotic G0 phase.
Cip/Kip family regulation of neural progenitor cell cycle exit and the initiation of neuronal differentiation
Unlike myocyte differentiation, the timing of progenitor cell cycle exit affects the total neuron number, because neurogenesis is limited to a specific developmental stage. If more progenitor cells exit the cell cycle at early developmental stages, fewer progenitor cells are produced, and therefore fewer neurons are generated in total. In contrast, if fewer progenitor cells exit the cell cycle at the early stages, more progenitor cells are produced and therefore more neurons are generated in total. Since glial cells proliferate even after development (Ge et al. 2012), the number of progenitor cells to produce glial cells at the later gliogenic stages may not affect the total number of glial cells. In fact, neurogenesis peaks later in the development of the larger primate cerebral cortex than in that of a smaller rodent (Finlay et al. 1998). The timing of progenitor cell cycle exit also affects the ratio of specific neuronal subtypes, because of the birth-date-dependent neurogenesis of each subtype. In the retina, cone genesis peaks earlier than rod genesis (Young 1985). As cone and rod photoreceptors are responsible for color and night vision, respectively, it was hypothesized that among closely related species, more retinal progenitor cells exit the cell cycle at the early stages in diurnal species than in nocturnal ones. In fact, the retinal progenitor cells in the diurnal capuchin monkey exit the cell cycle earlier and produce more early-born cone and ganglion cells than do those in the nocturnal owl monkey (Dyer et al. 2009). Thus, the timing of progenitor cell exit from the cell cycle and initiation of differentiation impacts the total neuron number and the ratio of neuronal subtypes. Deregulated timing may alter neuronal numbers and neuron subtype ratios, which may affect neuronal functions. Thus, genetic alterations that result in timing differences may contribute to evolution at the species level and may cause neuronal defects at the individual organism level.
The timing of progenitor cell cycle exit and neuronal differentiation initiation is regulated by the Cip/Kip family. Although the Cip/Kip family member p21 directly regulates cell cycle exit during skeletal muscle differentiation (Guo et al. 1995; Halevy et al. 1995; Parker et al. 1995; Skapek et al. 1995; Zhang et al. 1999), in the CNS, p21 indirectly regulates the cell cycle exit of adult neural stem cells by negatively regulating the pluripotency factor, Sox2 (Kippin et al. 2005; Marques-Torrejon et al. 2013). In contrast, the other Cip/Kip family members, p27 and p57, regulate the cell cycle exit of cortical progenitor cells (Goto et al. 2004; Tarui et al. 2005; Tury et al. 2011; Mairet-Coello et al. 2012). In primates, more upper layer neurons (late-born neurons) are generated in the primary than in the secondary visual cortex (Rockel et al. 1980), and a recent report indicates that the Cip/Kip family contributes to these differences (Lukaszewicz et al. 2005). The G1 phase is shorter, p27 expression is lower, and Cyclin E expression is higher in the progenitors of the primary visual cortex at the developmental stages when the upper layer progenitors expand to generate a sufficient number of upper layer neurons. p27 also contributes to neuronal differentiation not only by suppressing Cdk activity but also by regulating RhoA-mediated neuronal migration (Kawauchi et al. 2006; Nguyen et al. 2006). p57 also regulates both cell cycle arrest and neuronal migration (Itoh et al. 2007; Tury et al. 2011; Mairet-Coello et al. 2012). Thus, the Cip/Kip family coordinates the timing of progenitor cell cycle exit, the initiation of neuronal differentiation, and neuronal migration by multiple signaling pathways. Although recent studies have revealed that the p21-Cdk and p21-Sox2 pathways coordinate cell cycle exit and the initiation of differentiation in adult neural stem cells, future studies are required to elucidate the molecular mechanism(s) by which the Cip/Kip family coordinates these activities in cortical progenitors.
Rb family proteins regulate progenitor cell cycle exit without affecting neuronal differentiation initiation
In contrast to the Cip/Kip family, the Rb family does not regulate the initiation of differentiation in most cases. For example, when Rb is inactivated in mouse otocysts, the progenitors of hair and supporting cells in the utricle, Rb-deficient hair cells are terminally differentiated but continue to proliferate (Sage et al. 2005). Terminally differentiated neurons can also proliferate when the Rb family is inactivated in retinal progenitor cells. Retinal horizontal interneurons generated from Rb−/−; p107+/−; p130−/− (p107-single) mouse retinal progenitor cells re-enter the cell cycle, proliferate, and form retinoblastomas, while maintaining their differentiated state, including the ability to form neurites and synaptic connections (Ajioka et al. 2007). In contrast, p27-deficient retinal progenitor cells extend their retinogenesis stages (Levine et al. 2000). Importantly, the G2/M-phase marker phosphohistone-H3-positive neurons in the retina and the brain are detected when both p27 and the Ink4 family Cdk inhibitor p19 are deleted (Cunningham et al. 2002; Zindy et al. 1999). Since the Ink4 but not the Cip/Kip family suppresses the activity of Cdk4 (Sherr & Roberts 1999), which uniquely phosphorylates Rb at Ser780 (Kitagawa et al. 1996), Ink4-CyclinD/Cdk4-Rb axis may be partly involved in suppressing uncoupled differentiation and proliferation. Thus, some Rb family-deficient cells continue to proliferate while undergoing differentiation, two processes that are usually mutually exclusive.
Rb also regulates neuronal differentiation and maturation in a context-dependent manner. For example, Rb, but not p107 or p130, regulates the differentiation and maturation of rod photoreceptor and starburst amacrine cells in the mouse retina (Zhang et al. 2004a; Johnson et al. 2006; Chen et al. 2007; Table 1). The differentiation and maturation defects in Rb-deficient retina are not due to the deregulation of the timing of the cell cycle exit of retinal progenitor cells. In the mouse retina, one allele each of Rb and p107 is sufficient to regulate the proper cell cycle progression of retinal progenitor cells (Donovan et al. 2006). However, Rb−/−; p107−/− retinal progenitor cells do not exit the cell cycle, but initiate differentiation into the various types of retinal neurons (Chen et al. 2004; Macpherson et al. 2004). As a consequence, differentiating rod and cone photoreceptors, ganglion cells, and bipolar cells undergo cell death, while differentiating amacrine, horizontal, and Müller glia cells proliferate and form retinoblastomas (Table 1). Most of the cell cycle defects are rescued by the additional inactivation of E2f1 (Chen et al. 2007; Table 1). However, the differentiation defect of starburst amacrine cells is not rescued in the Rb−/−; E2f1−/− retina, but is rescued in the Rb−/−; E2f3a−/− retina, suggesting that Rb regulates starburst amacrine cell differentiation in a cell cycle-independent manner (Table 1). The defect of starburst amacrine cell differentiation may not be due to that of the initiation of differentiation because the ratio of starburst amacrine cell marker-positive cells in Rb-deficient retina decreases as development progresses.
Table 1. Summary of Rb family-mutant mouse strains
The retinal cells of Pax6-Cre; Rb Lox/Lox mice deregulate their cell cycle even in the presence of p107 but not develop retinoblastoma.
Rb also regulates neuronal migration in a cell cycle-independent manner. Differentiating cortical inhibitory neurons generated at the ganglionic eminence migrate tangentially to the cerebral cortex, and Rb-E2F3, but not Rb-E2F1, regulates this migration (Ferguson et al. 2005; McClellan et al. 2007; Table 1). E2F3 binds to the promoter region of the netrin receptor neogenin to activate its expression, while the binding of Rb suppresses neogenin gene expression. Since neogenin overexpression suppresses cell migration from the medial ganglionic eminence, the Rb-E2F3-neogenin pathway is important for regulating the migration of cortical inhibitory neurons (Andrusiak et al. 2011). Recent reports revealed that the Rb family also plays a role in regulating the migration of cortical excitatory neurons (Oshikawa et al. 2013; Svoboda et al. 2013). Although the molecular mechanisms of this regulation have not yet been identified, they are likely to differ from those underlying the regulation of inhibitory neurons, because (i) Rb inactivation alone inhibits the migration of inhibitory neurons while the additional inactivation of p107 and p130 is required to inhibit the migration of excitatory neurons and (ii) neogenin is not upregulated in the absence of Rb family members in excitatory neurons as it is in inhibitory neurons. Thus, the Rb family is essential for promoting cell entry into the post-mitotic G0 phase in general, and regulates neuronal differentiation in a context-specific manner.
Rb family members were also recently shown to be involved in the maintenance of post-mitotic neurons. We recently developed a technique for the conditional inactivation of all Rb family members just after cells enter the post-mitotic G0 phase, by the ex vivo electroporation of a Cre-expressing plasmid with a neuron-specific pMAP2 promoter (Oshikawa et al. 2013; Table 1). When the Rb family is inactivated following cell entry into the post-mitotic G0 phase, differentiating neurons undergo S phase progression, but not cell division. In contrast, Rb family inactivation in cortical progenitor cells leads to differentiating neurons that divide and proliferate. Thus, once differentiating cortical excitatory neurons enter the post-mitotic G0 phase, they are prevented from undergoing cell division even after the inactivation of all Rb family members.
Epigenetic regulation appears to be involved in maintaining cells in the post-mitotic G0 phase. During the neurogenesis of cortical excitatory neurons, H4K20 becomes tri-methylated (Fig. 1B). In mouse MEFs, the Rb family is required for H4K20 tri-methylation (Gonzalo et al. 2005). Rb family members bind to the histone methyl transferases Suv420h1 and Suv420h2, resulting in the transition of mono- to tri-methylated H4K20. Consistent with these findings, H4K20 is not tri-methylated in post-mitotic neurons generated from Rb-TKO progenitor cells, while the tri-methylated status of H4K20 is maintained in post-mitotic neurons in which Rb family members have been conditionally inactivated. Future studies are required to elucidate the biological roles of H4K20 tri-methylation in post-mitotic differentiating neurons.
In mature neurons in the CNS, the Rb pathway is believed to prevent cell cycle re-entry, which can result in cell death associated with the progression of neuronal degeneration (Heintz 1993; Herrup & Yang 2007). In fact, when Rb is inactivated in mature cortical neurons using the CamKCreERT2 transgenic mouse, Rb-deficient mature neurons undergo cell death (Andrusiak et al. 2012; Table 1). Although this study did not address whether Rb-deficient mature neurons undergo S phase progression or whether the redundancy of p107 and p130 compensates for Rb to prevent S phase progression, Rb family inactivation (using the MAP2 promoter system) induced cell death after S phase progression (IA; unpubl. data). Thus, Rb family-deficient progenitors initiate neuronal differentiation without exiting the cell cycle, resulting in proliferation, while the acute inactivation of Rb family in post-mitotic neurons leads to cell death. In contrast to senescent cells, post-mitotic neurons, at least cortical excitatory neurons, do not proliferate after S phase progression, even after Rb family inactivation.
Uncoupled proliferation and neuronal differentiation in retinoblastoma formation
Recent advances in whole genome sequencing have revealed that RB1 is the only known tumor suppressor gene that is mutated in human retinoblastoma (Zhang et al. 2012). The p53 pathway is another tumor suppressor pathway that impacts retinoblastoma development. RB1 inactivation upregulates MDMX, the E3 ubiquitin ligase that recognizes and degrades p53, resulting in p53 pathway inactivation in the RB1-deficient human retina (Laurie et al. 2006). In contrast to the human retina, in the mouse Rb-deficient retina, p107 is upregulated and compensates for Rb functions in cell cycle regulation (Donovan et al. 2006). The mouse Rb−/−; p107−/−, Rb−/−; p130−/−, p107-single, and Rb-TKO retinae develop retinoblastoma (Chen et al. 2004; Macpherson et al. 2004; Zhang et al. 2004b; Ajioka et al. 2007; McEvoy et al. 2011; Table 1). In p107-single mice, differentiated horizontal interneurons proliferate and develop retinoblastomas (Ajioka et al. 2007). At the later stages of tumor development, p107-single retinoblastomas lose their differentiated properties and form metastatic retinoblastomas. In contrast, the retinoblastomas from Rb−/−; p107−/− mice exhibit immature neuron-like features even at the early developmental stages (Chen et al. 2004; Macpherson et al. 2004; Zhang et al. 2004b). Importantly, the knock-in mice expressing mutant p27 protein that cannot bind to Cyclins and Cdks (p27 CK− mice) develop retinoblastoma associated with the amplification of retinal progenitor cells (Besson et al. 2007; Table 1).
Interestingly, both mouse and human late-stage retinoblastomas deficient in the Rb family and RB1, respectively, co-express the cell-type-specific genes for amacrine and horizontal interneurons, rod and cone photoreceptors, and retinal progenitor cells, which are usually mutually exclusive (McEvoy et al. 2011; Fig. 3). A subsequent analysis of epigenetically deregulated genes in retinoblastoma identified many of the same pathways that are co-expressed in these hybrid tumor cells. These data suggest that RB1 inactivation can lead to epigenetic changes in key cancer and retinal differentiation pathways and contribute to the expression of this hybrid developmental phenotype in retinoblastoma.
The proto-oncogene, spleen tyrosine kinase (SYK), is highly expressed in retinoblastoma and is required for cell survival in this tumor (Zhang et al. 2012). The SYK promoter region exhibits transcriptional activation landmarks such as H3K4Me3 and acetylated H3K9 and H3K14 (Fig. 3), suggesting that epigenetic changes in RB1-deficient human retinoblastoma are important for tumor development. On the other hand, another report indicates that human retinoblastomas exhibit characteristic features of differentiating cone photoreceptor cells (Xu et al. 2009). MDM2, another p53 E3 ubiquitin ligase, is upregulated by the cone-specific transcription factor RXRγ in humans. A recent gene expression analysis revealed that human retinoblastomas are composed of two subgroups (Kapatai et al. 2013): group 1 tumors express genes associated with multiple retinal cell types and group 2 tumors express genes associated with differentiating cone cells. Future studies are required to understand the mechanisms by which these two retinoblastoma subtypes develop after RB1 loss. Rb-dependent epigenetic changes that induce uncoupled proliferation and differentiation along with p53 pathway inactivation may play an important role in retinoblastoma development.
Conclusions and perspectives
Recent studies have demonstrated that the Rb family regulates the entrance and maintenance of cells in the post-mitotic G0 phase during neuronal development. Importantly, once neurons enter the post-mitotic phase, cell cycle regulators ensure that proliferation and differentiation are mutually exclusive paths, and prevent cell division even after Rb family inactivation. In contrast, Rb family-deficient neuronal progenitor cells initiate differentiation without exiting the cell cycle at least in the progenitor cells of the cerebral cortex and the retina (Fig. 4), resulting in tumor formation in the retina. On the other hand, Cip/Kip family-deficient neuronal progenitor cells tend to proliferate and suppress the initiation of differentiation, resulting in increased or decreased total cell numbers and altered ratios of neuronal subtypes at least in the progenitor cells of the cerebral cortex and the retina (Fig. 4). Future studies are required to understand the mechanism by which post-mitotic neurons are prevented from undergoing cell division. The elucidation of these mechanisms may lead to the development of new regenerative strategies for recovering lost neurons in neurodegenerative diseases.
This work was supported by the Industrial Technological Research Grant Program in 2009 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Canon Foundation, and the Terumo Life Science Foundation.