Available online at www.sciencedirect.com
ScienceDirect New insights into the conserved mechanism of pluripotency maintenance Xingliang Zhou, Humberto Contreras-Trujillo and Qi-Long Ying Pluripotent stem cells provide a powerful tool for both basic and translational research. The establishment and maintenance of germline-competent pluripotent stem cells in vitro, however, have only succeeded in the mouse and rat. From in vivo studies on pluripotency during embryogenesis and in vitro studies on existing pluripotent stem cells, several mechanisms have been uncovered for maintenance of both the naı¨ve and the primed pluripotent states. Current clues strongly indicate that such mechanisms are likely conserved among different species. A better understanding of how these mechanisms work together to control cell fate choice will guide future research in both stem cell biology and regenerative medicine. Address Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Corresponding author: Ying, Qi-Long ([email protected])
Current Opinion in Genetics & Development 2015, 34:1–9 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Amander T Clark and Thomas P Zwaka
http://dx.doi.org/10.1016/j.gde.2015.06.002 0959-437X/# 2015 Elsevier Ltd. All rights reserved.
Introduction Mammalian development, from the fertilized egg to the various types of highly specified somatic cells, is accompanied by progressive loss of developmental potential which is termed ‘potency’. During the early cleavage divisions, each cell in the embryo is able to contribute to the formation of a whole embryo [1]. Such developmental potential is termed ‘totipotency’. Upon the formation of the blastocyst, cells in the embryo are separated into two groups, the inner cell mass (ICM) and the trophoblast. Cells in the ICM are able to give rise to all types of cells in the newborn animal, excluding the extraembryonic tissue [2]. Such developmental potential is termed ‘pluripotency’. Pluripotency is not an innate property of the zygote, but emerges in the ICM only after the formation of the pre-implantation blastocyst, and www.sciencedirect.com
persists in the epiblast until the post-implantation egg cylinder stage. Following gastrulation, the majority of cells in the developing embryo undergo further specification and their contribution is limited to the derivatives of their tissue origin. Such developmental potential is termed ‘multipotency’. The only exception for this specification process is the primordial germ cells (PGCs) which can regain pluripotency given appropriate cues and contribute to the germline. Pluripotent stem cells (PSCs) possess the ability to give rise to any type of cell in the body, including derivatives from all three primary germ layers, that is, ectoderm, mesoderm and endoderm, in addition to the germline. PSCs exist transiently during the early stages of embryogenesis in vivo. However, when provided with proper culture conditions, PSCs are capable of indefinite in vitro self-renewal while retaining their developmental potential. Several types of PSCs have been isolated and established from embryos at various developmental stages (Table 1). For example, embryonic stem cells (ESCs) can be established from the ICM of pre-implantation embryos [3,4], epiblast stem cells (EpiSCs) can be established from the epiblast of post-implantation embryos [5,6], and embryonic germ cells (EGCs) can be established from the PGCs at a later developmental stage [7– 10]. Alternatively, PSCs, such as induced pluripotent stem cells (iPSCs), can be generated from somatic cells through reprogramming [11]. Among different types of PSCs, rodent ESCs and iPSCs show robust ability to form any type of cell in the body including gametes and have been utilized to generate genetically modified cell lines and animal models [12,13]. To date, despite that ESC-like cells from various species have been reported, only ESCs derived from mouse and rat possess the ‘naı¨ve’ state pluripotency [12,14,15]. This feature is characterized by the expression of pluripotency markers, for example, Rex1 and Nr0b1, two active X chromosomes in female cells and, most importantly, the ability to reenter embryonic development and give rise to germline-competent chimeric progenies. In contrast to the naı¨ve state pluripotency, mouse EpiSCs and currently available human ESCs [16] represent the ‘primed’ state pluripotency. This state is characterized by the expression of early specification markers, for example, Fgf5 and Brachyury, one active X chromosome in female cells, and most importantly, the inability to incorporate and contribute into developing embryos to form germline-competent chimeras. Therefore, based on Current Opinion in Genetics & Development 2015, 34:1–9
2 Cell reprogramming, regeneration and repair
Table 1 Different types of PSCs derived from developing mouse embryos Embryonic day Developmental stage Tissue of origin Potency in vivo PSC type Potency in vitro
E0 to E2.0
E3.5
E5.25
E7.5 to E13.5
Zygote to early 8-cell embryo Blastomere Totipotent – –
Pre-implantation blastocyst ICM Pluripotent ESCs Pluripotent
Post-implantation egg cylinder Epiblast Pluripotent EpiSCs Pluripotent
Late gastrula to presomite PGCs Unipotent EGCs Pluripotent
the developmental potential and molecular properties (see Table 2 for comparison), it is believed that the primed state PSCs are developmentally more specified than the naı¨ve state PSCs [17]. The conversion from primed state pluripotency back to naı¨ve state pluripotency has been achieved by genetic manipulation in mouse EpiSCs [18,19,20,21]. Recently, several reports [22,23,24,25,26,27,28] have shown that human PSCs with similar properties of mouse ESCs can be established from currently available primed state human ESCs or human fibroblasts. Present clues strongly suggest that the properties of PSCs are commonly shared among different species and the maintenance of pluripotency is governed by a highly conserved mechanism from mouse to human. In this review, we will discuss recent findings about the mechanisms that maintain naı¨ve state and primed state pluripotency in vitro.
BMP4, mESCs could be maintained without feeder cells in serum-free medium. Alternatively, mESCs self-renewal can also be achieved by combined use of two small molecule inhibitors (2i), CHIR99021 (CHIR) and PD0325901 (PD03) [12]. CHIR and PD03 inhibit glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (Mek), respectively. 2i can robustly maintain naı¨ve state of PSCs in vitro in the absence of LIF, serum or BMP4, and has been widely used to derive germline-competent ESCs/iPSCs from various strains of mice [12,32]. Recently, 2i has been utilized in the derivation of the first authentic rat ESC lines [14,15] and in the generation of the first genetically manipulated rat model using ESC-based gene-targeting technology [33], suggesting that the fundamental mechanisms governing naı¨ve state pluripotency may be conserved across species.
Mechanisms governing the naı¨ve state pluripotency in vitro
The molecular mechanisms underlying ESC self-renewal mediated by LIF and 2i have been extensively studied (Figure 1). LIF activates Janus kinase 1 (Jak1) upon its binding to LIFR/gp130 heterodimer receptors. Jak1 then phosphorylates cytosolic signal transducer and activator of transcription 3 (Stat3) at Tyr705 and Ser727, which leads to the dimerization and nuclear translocation of Stat3. As a transcription factor, nuclear Stat3 is able to activate its target genes, such as Klf4 and c-Myc, among which many possess the ability to promote ESC self-renewal [34].
Mouse ESCs (mESCs) were first derived by culturing ICM cells on a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs), known as ‘feeder cells’, in serum-containing medium [3,4]. MEFs support mESC self-renewal by secreting leukemia inhibitory factor (LIF) [29,30]. The essential factor in serum for LIFmediated mESC self-renewal is bone morphogenetic protein 4 (BMP4) [31]. If cultured with LIF and Table 2 Comparison among different types of pluripotent stem cells Cell type
Mouse ESCs/iPSCs
Mouse EpiSCs
Pluripotent state Colony morphology Culture condition
Naı¨ve Dome LIF/BMP4 or 2i
Primed Flat Activin/FGF
X chromosome inactivation Single-cell dissociation Teratoma Chimera Germline transmission Refs.
XaXa Tolerant Yes Yes Yes [3,4]
XaXi Sensitive Yes No No [5,6]
a
Naı¨ve state human PSCs
Mouse EGCs
Human ESCs/iPSCs
Human EGCs
Naive Dome SCF/FGF/LIF or Serum/LIF or LIF/2i – Sensitive Yes Yes No [7,8,69]
Primed Flat Activin/FGF
Primed Flat SCF/FGF/ forskolin
Naı¨ve Dome 2i and other factors a
XaXi Sensitive Yes No – [16]
– Sensitive Yes – – [9,10]
XaXa Tolerant Yes Yes – [22,23,24,25, 26,27,28]
Culture conditions used to derive and maintain naı¨ve state human PSCs vary among different reports.
Current Opinion in Genetics & Development 2015, 34:1–9
www.sciencedirect.com
Mechanism of pluripotency maintenance Zhou, Contreras-Trujillo and Ying 3
Figure 1
FGF
LIF LIF R
P
gp130
FGF R
Wnt Lrp5/6
FGF R
Frizzled
P Jak
Dvl
P
P SHP2
Socs3 PTP
Grb2
CHIR99021
SOS P p85 P P
Stat3
P
Ras/Raf
APC Ck1α
PI3K
Stat3 P
PD0325901
SUMOylation
Akt
β-Catenin
Erk1/2
PIAS
P P
Gsk3
Mek
P
importin
Stat3
Axin
β-Catenin P P P
P
Stat3
β-Catenin Erk1/2 P
target gene
target gene Stat3
P P
Stat3
Self-renewal Differentiation Klf4 Socs3 c-Myc Cdx2 Gbx2 Tfap2c Tfcp2l1 ... Pim1 Pim3 Rhox5 ...
target gene Tcf/Lef1
TF
Differentiation Gmnn Psmb Ifna14 ...
Self-renewal Differentiation Klf4 Axin2 Esrrb Cdx1 Tfcp2l1 Brachyury ... ...
Current Opinion in Genetics & Development
Signaling pathways involved in the maintenance of naı¨ve state pluripotency in vitro. Naı¨ve state pluripotency can be maintained in vitro via activation of LIF/Stat3 pathway. Alternatively, it can also be maintained via simultaneously activation of Wnt/b-catenin pathway and inhibition of FGF/MAPK pathway by two small molecule inhibitors (2i).
Activation of Stat3 is the principle effect of LIF on mESC self-renewal, as demonstrated by Niwa et al. using a GCSFR/gp130 chimeric receptor which consists of the extracellular domain of the granulocyte-colony stimulating factor (GCSF) receptor fused to the transmembrane and cytoplasmic region of gp130 [35]. Addition of GCSF is sufficient to maintain self-renewal of mESCs overexpressing this chimeric receptor in the absence of LIF. Consistently, Stat3 phosphorylation mutant (Stat3Y705F) impedes mESC self-renewal even in the presence of LIF [35]. The critical role of Stat3 in maintaining naı¨ve pluripotency was further confirmed as activation of Stat3 promotes somatic cell reprogramming and inhibition of Jak1 abolishes iPSC generation [36]. In addition, studies have identified several LIF/Stat3 downstream targets as pluripotency regulators, such as Pim1, Pim3, Pramel7, Rhox5, Gbx2 and Tfcp2l1 [19,20,21]. Overexpression of these Stat3 targets can partially substitute the effect of LIF in mESC self-renewal. It is important to highlight that only knock-down of Tfcp2l1, which is also a Wnt/bcatenin target (see below), abolished LIF/Stat3-mediated mESC self-renewal [21]. These findings have reiterated the diverse effects of LIF on mESC self-renewal. In recent years, several studies have shed light on the detailed relationship between Stat3 activation and www.sciencedirect.com
mESC self-renewal. For example, Huang et al. [37] showed that the phosphorylation of Stat3 at Tyr705 and Ser727 has distinct functions on mESC self-renewal. Phosphorylation at Tyr705 is essential for ESC self-renewal mediated by LIF/Stat3. However, phosphorylation at Ser727 is dispensable for ESC self-renewal. Nonetheless, it can promote optimal ESC proliferation and survival. Interestingly, blocking the phosphorylation at Ser727 did not hamper self-renewal but reinforced it by inhibiting neural differentiation. Tai et al. [38] demonstrated that activation level of Stat3 has to be controlled within an optimal range in order to promote ESC selfrenewal. Activation of LIF/Stat3 signaling induces expression of self-renewal-promoting genes and genes that induce ESC differentiation. On one hand, activation of genes that promote self-renewal has to reach a certain threshold to maintain pluripotency, as hypoactivation of Stat3 failed to prevent mesoderm/endoderm specification of ESCs. On the other hand, activation of the genes that promote differentiation cannot exceed a certain level, as hyperactivation of Stat3 led to trophoblast differentiation and ESC death. Thus, rather than simply turning on and off LIF/Stat3 signal, it is the fine tuning of this signaling pathway that keeps the balance between self-renewal and differentiation and therefore maintains naı¨ve state pluripotency in vitro. Current Opinion in Genetics & Development 2015, 34:1–9
4 Cell reprogramming, regeneration and repair
In addition to LIF/Stat3 signaling pathway, Wnt/b-catenin has also been identified as a crucial element in the maintenance of naı¨ve state pluripotency in vitro [12]. In the absence of Wnt ligand, cytosolic b-catenin is continuously phosphorylated by Ck1a/Gsk3 and further degraded in a proteasome-dependent manner. Upon Wnt ligand or CHIR stimulation, Gsk3 is functionally inhibited, which leads to the cytosolic stabilization and nuclear translocation of b-catenin. Nuclear b-catenin then interacts with Tcf/Lef1 family transcription factors to activate downstream targets [39]. The effect of Wnt/b-catenin pathway in the maintenance of naı¨ve pluripotent state was originally observed as the addition of Gsk3 inhibitor enhances mESC self-renewal in LIF/serum condition [40]. This observation has been fortified with the emergence of 2i [12], as CHIR is able to rescue the insufficiency of PD03 to maintain mESC self-renewal in the absence of serum. The fact that CHIR failed to maintain b-catenin / mESC self-renewal [41] confirmed the central role of Wnt/b-catenin in 2i-mediated ESC self-renewal. Recent studies have shown that Tcf3 is a transcription repressor of several crucial pluripotency factors [42]. Upon b-catenin binding, the repressive effect of Tcf3 is alleviated and the expression levels of core pluripotency factors, such as Oct4 and Nanog, are increased. Moreover, recent reports also identified several critical pluripotency genes, for example, Klf2, Esrrb and Tfcp2l1, as direct downstream targets of Wnt/b-catenin signaling in mESCs [20,21,43]. It is important to note that Tfcp2l1 is identified as the downstream target of both LIF/Stat3 and Wnt/b-catenin signaling pathways, which indicates its critical role as the intersection of these two major pathways that regulate naı¨ve state pluripotency. It should also be noted that activation of Wnt/b-catenin signaling pathway alone, by either Wnt3a or Gsk3 inhibitors, can only maintain short term (
ScienceDirect New insights into the conserved mechanism of pluripotency maintenance Xingliang Zhou, Humberto Contreras-Trujillo and Qi-Long Ying Pluripotent stem cells provide a powerful tool for both basic and translational research. The establishment and maintenance of germline-competent pluripotent stem cells in vitro, however, have only succeeded in the mouse and rat. From in vivo studies on pluripotency during embryogenesis and in vitro studies on existing pluripotent stem cells, several mechanisms have been uncovered for maintenance of both the naı¨ve and the primed pluripotent states. Current clues strongly indicate that such mechanisms are likely conserved among different species. A better understanding of how these mechanisms work together to control cell fate choice will guide future research in both stem cell biology and regenerative medicine. Address Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Corresponding author: Ying, Qi-Long ([email protected])
Current Opinion in Genetics & Development 2015, 34:1–9 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Amander T Clark and Thomas P Zwaka
http://dx.doi.org/10.1016/j.gde.2015.06.002 0959-437X/# 2015 Elsevier Ltd. All rights reserved.
Introduction Mammalian development, from the fertilized egg to the various types of highly specified somatic cells, is accompanied by progressive loss of developmental potential which is termed ‘potency’. During the early cleavage divisions, each cell in the embryo is able to contribute to the formation of a whole embryo [1]. Such developmental potential is termed ‘totipotency’. Upon the formation of the blastocyst, cells in the embryo are separated into two groups, the inner cell mass (ICM) and the trophoblast. Cells in the ICM are able to give rise to all types of cells in the newborn animal, excluding the extraembryonic tissue [2]. Such developmental potential is termed ‘pluripotency’. Pluripotency is not an innate property of the zygote, but emerges in the ICM only after the formation of the pre-implantation blastocyst, and www.sciencedirect.com
persists in the epiblast until the post-implantation egg cylinder stage. Following gastrulation, the majority of cells in the developing embryo undergo further specification and their contribution is limited to the derivatives of their tissue origin. Such developmental potential is termed ‘multipotency’. The only exception for this specification process is the primordial germ cells (PGCs) which can regain pluripotency given appropriate cues and contribute to the germline. Pluripotent stem cells (PSCs) possess the ability to give rise to any type of cell in the body, including derivatives from all three primary germ layers, that is, ectoderm, mesoderm and endoderm, in addition to the germline. PSCs exist transiently during the early stages of embryogenesis in vivo. However, when provided with proper culture conditions, PSCs are capable of indefinite in vitro self-renewal while retaining their developmental potential. Several types of PSCs have been isolated and established from embryos at various developmental stages (Table 1). For example, embryonic stem cells (ESCs) can be established from the ICM of pre-implantation embryos [3,4], epiblast stem cells (EpiSCs) can be established from the epiblast of post-implantation embryos [5,6], and embryonic germ cells (EGCs) can be established from the PGCs at a later developmental stage [7– 10]. Alternatively, PSCs, such as induced pluripotent stem cells (iPSCs), can be generated from somatic cells through reprogramming [11]. Among different types of PSCs, rodent ESCs and iPSCs show robust ability to form any type of cell in the body including gametes and have been utilized to generate genetically modified cell lines and animal models [12,13]. To date, despite that ESC-like cells from various species have been reported, only ESCs derived from mouse and rat possess the ‘naı¨ve’ state pluripotency [12,14,15]. This feature is characterized by the expression of pluripotency markers, for example, Rex1 and Nr0b1, two active X chromosomes in female cells and, most importantly, the ability to reenter embryonic development and give rise to germline-competent chimeric progenies. In contrast to the naı¨ve state pluripotency, mouse EpiSCs and currently available human ESCs [16] represent the ‘primed’ state pluripotency. This state is characterized by the expression of early specification markers, for example, Fgf5 and Brachyury, one active X chromosome in female cells, and most importantly, the inability to incorporate and contribute into developing embryos to form germline-competent chimeras. Therefore, based on Current Opinion in Genetics & Development 2015, 34:1–9
2 Cell reprogramming, regeneration and repair
Table 1 Different types of PSCs derived from developing mouse embryos Embryonic day Developmental stage Tissue of origin Potency in vivo PSC type Potency in vitro
E0 to E2.0
E3.5
E5.25
E7.5 to E13.5
Zygote to early 8-cell embryo Blastomere Totipotent – –
Pre-implantation blastocyst ICM Pluripotent ESCs Pluripotent
Post-implantation egg cylinder Epiblast Pluripotent EpiSCs Pluripotent
Late gastrula to presomite PGCs Unipotent EGCs Pluripotent
the developmental potential and molecular properties (see Table 2 for comparison), it is believed that the primed state PSCs are developmentally more specified than the naı¨ve state PSCs [17]. The conversion from primed state pluripotency back to naı¨ve state pluripotency has been achieved by genetic manipulation in mouse EpiSCs [18,19,20,21]. Recently, several reports [22,23,24,25,26,27,28] have shown that human PSCs with similar properties of mouse ESCs can be established from currently available primed state human ESCs or human fibroblasts. Present clues strongly suggest that the properties of PSCs are commonly shared among different species and the maintenance of pluripotency is governed by a highly conserved mechanism from mouse to human. In this review, we will discuss recent findings about the mechanisms that maintain naı¨ve state and primed state pluripotency in vitro.
BMP4, mESCs could be maintained without feeder cells in serum-free medium. Alternatively, mESCs self-renewal can also be achieved by combined use of two small molecule inhibitors (2i), CHIR99021 (CHIR) and PD0325901 (PD03) [12]. CHIR and PD03 inhibit glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (Mek), respectively. 2i can robustly maintain naı¨ve state of PSCs in vitro in the absence of LIF, serum or BMP4, and has been widely used to derive germline-competent ESCs/iPSCs from various strains of mice [12,32]. Recently, 2i has been utilized in the derivation of the first authentic rat ESC lines [14,15] and in the generation of the first genetically manipulated rat model using ESC-based gene-targeting technology [33], suggesting that the fundamental mechanisms governing naı¨ve state pluripotency may be conserved across species.
Mechanisms governing the naı¨ve state pluripotency in vitro
The molecular mechanisms underlying ESC self-renewal mediated by LIF and 2i have been extensively studied (Figure 1). LIF activates Janus kinase 1 (Jak1) upon its binding to LIFR/gp130 heterodimer receptors. Jak1 then phosphorylates cytosolic signal transducer and activator of transcription 3 (Stat3) at Tyr705 and Ser727, which leads to the dimerization and nuclear translocation of Stat3. As a transcription factor, nuclear Stat3 is able to activate its target genes, such as Klf4 and c-Myc, among which many possess the ability to promote ESC self-renewal [34].
Mouse ESCs (mESCs) were first derived by culturing ICM cells on a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs), known as ‘feeder cells’, in serum-containing medium [3,4]. MEFs support mESC self-renewal by secreting leukemia inhibitory factor (LIF) [29,30]. The essential factor in serum for LIFmediated mESC self-renewal is bone morphogenetic protein 4 (BMP4) [31]. If cultured with LIF and Table 2 Comparison among different types of pluripotent stem cells Cell type
Mouse ESCs/iPSCs
Mouse EpiSCs
Pluripotent state Colony morphology Culture condition
Naı¨ve Dome LIF/BMP4 or 2i
Primed Flat Activin/FGF
X chromosome inactivation Single-cell dissociation Teratoma Chimera Germline transmission Refs.
XaXa Tolerant Yes Yes Yes [3,4]
XaXi Sensitive Yes No No [5,6]
a
Naı¨ve state human PSCs
Mouse EGCs
Human ESCs/iPSCs
Human EGCs
Naive Dome SCF/FGF/LIF or Serum/LIF or LIF/2i – Sensitive Yes Yes No [7,8,69]
Primed Flat Activin/FGF
Primed Flat SCF/FGF/ forskolin
Naı¨ve Dome 2i and other factors a
XaXi Sensitive Yes No – [16]
– Sensitive Yes – – [9,10]
XaXa Tolerant Yes Yes – [22,23,24,25, 26,27,28]
Culture conditions used to derive and maintain naı¨ve state human PSCs vary among different reports.
Current Opinion in Genetics & Development 2015, 34:1–9
www.sciencedirect.com
Mechanism of pluripotency maintenance Zhou, Contreras-Trujillo and Ying 3
Figure 1
FGF
LIF LIF R
P
gp130
FGF R
Wnt Lrp5/6
FGF R
Frizzled
P Jak
Dvl
P
P SHP2
Socs3 PTP
Grb2
CHIR99021
SOS P p85 P P
Stat3
P
Ras/Raf
APC Ck1α
PI3K
Stat3 P
PD0325901
SUMOylation
Akt
β-Catenin
Erk1/2
PIAS
P P
Gsk3
Mek
P
importin
Stat3
Axin
β-Catenin P P P
P
Stat3
β-Catenin Erk1/2 P
target gene
target gene Stat3
P P
Stat3
Self-renewal Differentiation Klf4 Socs3 c-Myc Cdx2 Gbx2 Tfap2c Tfcp2l1 ... Pim1 Pim3 Rhox5 ...
target gene Tcf/Lef1
TF
Differentiation Gmnn Psmb Ifna14 ...
Self-renewal Differentiation Klf4 Axin2 Esrrb Cdx1 Tfcp2l1 Brachyury ... ...
Current Opinion in Genetics & Development
Signaling pathways involved in the maintenance of naı¨ve state pluripotency in vitro. Naı¨ve state pluripotency can be maintained in vitro via activation of LIF/Stat3 pathway. Alternatively, it can also be maintained via simultaneously activation of Wnt/b-catenin pathway and inhibition of FGF/MAPK pathway by two small molecule inhibitors (2i).
Activation of Stat3 is the principle effect of LIF on mESC self-renewal, as demonstrated by Niwa et al. using a GCSFR/gp130 chimeric receptor which consists of the extracellular domain of the granulocyte-colony stimulating factor (GCSF) receptor fused to the transmembrane and cytoplasmic region of gp130 [35]. Addition of GCSF is sufficient to maintain self-renewal of mESCs overexpressing this chimeric receptor in the absence of LIF. Consistently, Stat3 phosphorylation mutant (Stat3Y705F) impedes mESC self-renewal even in the presence of LIF [35]. The critical role of Stat3 in maintaining naı¨ve pluripotency was further confirmed as activation of Stat3 promotes somatic cell reprogramming and inhibition of Jak1 abolishes iPSC generation [36]. In addition, studies have identified several LIF/Stat3 downstream targets as pluripotency regulators, such as Pim1, Pim3, Pramel7, Rhox5, Gbx2 and Tfcp2l1 [19,20,21]. Overexpression of these Stat3 targets can partially substitute the effect of LIF in mESC self-renewal. It is important to highlight that only knock-down of Tfcp2l1, which is also a Wnt/bcatenin target (see below), abolished LIF/Stat3-mediated mESC self-renewal [21]. These findings have reiterated the diverse effects of LIF on mESC self-renewal. In recent years, several studies have shed light on the detailed relationship between Stat3 activation and www.sciencedirect.com
mESC self-renewal. For example, Huang et al. [37] showed that the phosphorylation of Stat3 at Tyr705 and Ser727 has distinct functions on mESC self-renewal. Phosphorylation at Tyr705 is essential for ESC self-renewal mediated by LIF/Stat3. However, phosphorylation at Ser727 is dispensable for ESC self-renewal. Nonetheless, it can promote optimal ESC proliferation and survival. Interestingly, blocking the phosphorylation at Ser727 did not hamper self-renewal but reinforced it by inhibiting neural differentiation. Tai et al. [38] demonstrated that activation level of Stat3 has to be controlled within an optimal range in order to promote ESC selfrenewal. Activation of LIF/Stat3 signaling induces expression of self-renewal-promoting genes and genes that induce ESC differentiation. On one hand, activation of genes that promote self-renewal has to reach a certain threshold to maintain pluripotency, as hypoactivation of Stat3 failed to prevent mesoderm/endoderm specification of ESCs. On the other hand, activation of the genes that promote differentiation cannot exceed a certain level, as hyperactivation of Stat3 led to trophoblast differentiation and ESC death. Thus, rather than simply turning on and off LIF/Stat3 signal, it is the fine tuning of this signaling pathway that keeps the balance between self-renewal and differentiation and therefore maintains naı¨ve state pluripotency in vitro. Current Opinion in Genetics & Development 2015, 34:1–9
4 Cell reprogramming, regeneration and repair
In addition to LIF/Stat3 signaling pathway, Wnt/b-catenin has also been identified as a crucial element in the maintenance of naı¨ve state pluripotency in vitro [12]. In the absence of Wnt ligand, cytosolic b-catenin is continuously phosphorylated by Ck1a/Gsk3 and further degraded in a proteasome-dependent manner. Upon Wnt ligand or CHIR stimulation, Gsk3 is functionally inhibited, which leads to the cytosolic stabilization and nuclear translocation of b-catenin. Nuclear b-catenin then interacts with Tcf/Lef1 family transcription factors to activate downstream targets [39]. The effect of Wnt/b-catenin pathway in the maintenance of naı¨ve pluripotent state was originally observed as the addition of Gsk3 inhibitor enhances mESC self-renewal in LIF/serum condition [40]. This observation has been fortified with the emergence of 2i [12], as CHIR is able to rescue the insufficiency of PD03 to maintain mESC self-renewal in the absence of serum. The fact that CHIR failed to maintain b-catenin / mESC self-renewal [41] confirmed the central role of Wnt/b-catenin in 2i-mediated ESC self-renewal. Recent studies have shown that Tcf3 is a transcription repressor of several crucial pluripotency factors [42]. Upon b-catenin binding, the repressive effect of Tcf3 is alleviated and the expression levels of core pluripotency factors, such as Oct4 and Nanog, are increased. Moreover, recent reports also identified several critical pluripotency genes, for example, Klf2, Esrrb and Tfcp2l1, as direct downstream targets of Wnt/b-catenin signaling in mESCs [20,21,43]. It is important to note that Tfcp2l1 is identified as the downstream target of both LIF/Stat3 and Wnt/b-catenin signaling pathways, which indicates its critical role as the intersection of these two major pathways that regulate naı¨ve state pluripotency. It should also be noted that activation of Wnt/b-catenin signaling pathway alone, by either Wnt3a or Gsk3 inhibitors, can only maintain short term (
