The SOX transcription factors as key players in pluripotent stem cells.

Stem Cells and Development The SOX transcription factors as key players in pluripotent stem cells (doi: 10.1089/scd.2014.0297) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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The SOX transcription factors as key players in pluripotent stem cells

Essam M. Abdelalim, Mohamed M. Emara, and Prasanna R. Kolatkar

Qatar Biomedical Research Institute, Qatar Foundation, Education City, 5825 Doha, Qatar.

Address correspondence to: Essam M. Abdelalim, PhD Qatar Biomedical Research Institute, Qatar Foundation, Education City, Doha 5825, Qatar Tel.: +974-44546432

Fax: +974-44541770

E-mail: [email protected]

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Abstract Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), and induced PSCs (iPSCs) are able to self-renew and differentiate into a multitude of specialized cellular lineages. In these cells, the pluripotential identity is maintained by a group of transcription factors (TFs). Among these factors, SOX TFs play an essential role, not only in regulating pluripotency, but also in mediating self-renewal and differentiation. Some SOX TFs are highly expressed in undifferentiated PSCs, while others are upregulated upon differentiation to promote specific lineage differentiation. Further roles of SOX factors in pluripotency are highlighted through their critical involvement in iPSCs generation. To perform these multiple functions and activities, SOX TFs are strongly associated with a complex regulatory network(s) that involves the binding of SOX factors to variant trans-acting partners to activate or suppress specific genes. Although, SOX2 has attracted special attention as a critical factor in maintaining PSCs characteristics and as an integral component that is required to reprogram somatic cells into pluripotency, new reports widely appreciated that other SOX TFs, such as SOX1, SOX3, or re-engineered SOX7 and SOX17, can compensate for the absence of SOX2 and thus play a fundamental role during the reprogramming process as well as maintaining pluripotency. These findings indicate that the recent progress has greatly expanded our knowledge about the role of SOX factors in PSCs. Thus, in this review we summarize what is currently known about the roles of SOX factors in PSCs as well as their role in somatic cell reprogramming. Also, we intend to provide an update on their relationship with other factors in regulating the characteristics and early differentiation of PSCs. Key Words: ESCs, iPSCs, SOX, Pluripotency, Self-renewal, Reprogramming, Differentiation.

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Introduction Embryonic stem cells (ESCs) [1, 2] and induced pluripotent stem cells (iPSCs) [3, 4] are characterized by their unique ability to self-renew and differentiate into all types of cells. Stem cell fate is a precariously balanced phenomenon that is dependent on a variety of factors including extracellular as well as intracellular molecules. The effector molecules themselves are heterogeneous and include proteins (transcription factors, chromatin modifiers, etc.), miRNAs, as well as a host of other moieties. Among these effector classes are the transcription factors (TFs), which are regulatory proteins that bind to a specific DNA sequence to mediate gene expression. Over the past few decades these factors have attracted a great deal of attention as functional proteins that play a significant role in regulating a large number of cellular processes. These processes include, but are not limited to, gene regulation, cell proliferation, cell survival, and cell development. The central role of TFs in mediating gene expression highlights their direct and/or indirect role in controlling pluripotency and stem cell fate [5-8]. TFs can mediate stem cell fate through different strategies. These include the activation of genes that maintain pluripotency or upregulation of various defined factors that direct stem cell differentiation to specific stem cell lineage [5-7, 9, 10]. Early in cell development, TFs make important decisions to decide if cells will remain as stem cells or if they will differentiate into the different germ layers. Central to this decision making process are several players including Oct4, Klfs, the Sox family, and a host of key factors that decide the balance of pluripotency or differentiation. SRY-related high-mobility-group box (SOX) family proteins comprise group of transcriptional regulators having a highly conserved high mobility group (HMG) domain similar to that of sex determining region Y protein (SRY), which mediates DNA binding [11-14]. Sox proteins are divided into groups SOXA-SOXH based on the degree of amino acid identity within

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4 the HMG-box (Table 1). Several SOX proteins have been identified and found to act as critical TFs involved in embryonic development and cell fate decision [15, 16]. The SOXA group consists only of SRY, encoded in mammalian Y-chromosomes [17]; SOXB group consists of two sub-groups (SOXB1 and SOXB2); SOXB1 includes SOX1, SOX2, and SOX3 [18], whereas SOXB2 proteins include SOX14 and SOX21 [19]; SOXC includes SOX4, SOX11 and SOX12 [20]; SOXE includes SOX8, SOX9 and SOX10 [21]; and SOXF includes SOX7, SOX17 and SOX18 [22]; SOXD group includes SOX5, SOX6 and SOX13 [23]; SOXG (SOX15) [24] and SOXH (SOX30) [25] proteins are structurally related to SOXB1 and SOXD proteins, respectively (Table 1). All the structures of Sox HMG domains solved to date are highly similar including the bending of the DNA (Fig.1). The importance of TFs in the stem cell field increased significantly as a result of the breakthrough discovery by Shinya Yamanaka, who showed that overexpression of four transcription factors (OCT4, KLF4, SOX2, and C-MYC) enables reprogramming of somatic cells to specific type of cells that have the same characteristics and properties of natural pluripotent stem cells (PSCs) [3, 4]. These cells are called iPSCs and have the potential to selfrenew and differentiate into the three germ layers [26]. iPSCs were initially induced

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transducing mouse [3] or human [4] fibroblasts with retroviral vectors expressing the above mentioned reprogramming factors. Since iPSCs discovery, several other factors have been used to elicit the most efficient approach for iPSCs generation. These factors range from small compounds that mimic TFs [27] to drugs that alter TFs or RNA molecules that overexpress reprogramming factors. Although many factors are important for ESC and iPSC maintenance or generation, the duo of OCT4 and SOX2 stands out as two of the most critical factors. Several

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5 studies previously showed that the knockdowns of these factors result in inducing differentiation and losing pluripotency, hence their importance in stem cell regulation. In this review, we highlight the main role of SOX factors in mediating stem cell fate. We provide an overview of the SOX factors during preimplantation embryo development and their role to orchestrate the operation of self-renewal and differentiation in PSCs as well as their importance in the iPSC generation. Finally, we discuss the relationship between Sox factors and other factors in PSCs.

The role of SOX transcription factors in preimplantation embryos One of the first events that occurs during embryonic development is the formation of the blastocyst, which is a hollow sphere composed of an outer trophectoderm (TE) layer and a population of founder cells called inner cell mass (ICM). The ICM cells have the potential to differentiate into the three germ layers derived tissues (ectoderm, mesoderm, and endoderm) in a prevalent phenomenon known as pluripotency (Fig. 2). This makes the ICM a valuable source to derive ESCs, which are PSCs. Therefore, the genes expressed in the ICM are the same as those expressed in ESCs cultured in vitro [1, 2]. Although many factors have been found to be important for the development of preimplantation embryos, SOX TFs stands out as one of the most critical factors [28-30]. Out of the 30 SOX genes, which have been identified in mammals, the most well known of these factors is SOX2, which is expressed at all developmental stages and has been shown to be essential for early embryogenesis. During the early stages of development, SOX2 is expressed in both ICM and TE, whereas at later stages its expression becomes restricted to the ICM [31, 32]. At the late blastocyst stage, where ICM segregates into primitive endoderm, primitive ectoderm, and epiblast cells, SOX2 expression is only detectable

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6 in pluripotent epiblast cells and multipotent cells of the extraembryonic ectoderm (ExEn) [31] but not in primitive endoderm cells [33, 34]. The critical role of SOX2 in the development of preimplantation embryo has been provided by the observation that Sox2 deletion in mouse zygote causes early embryonic death. This embryonic lethal phenotype has been found to be specifically due to the inability to form the epiblast without affecting TE formation [31]. These results have been supported by the work done by Keramari and his colleagues [28] who have related the unobserved effect of Sox2 deletion on TE formation to the presence of the maternal Sox2. The maternal form of SOX2 is found in oocytes and preimplantation embryo and persists until the blastocyst stage, suggesting that maternal SOX2 might mask a phenotype in TE during the early development of Sox2-deficient zygote [28]. Moreover, the knockdown of both maternal and zygotic Sox2 results in a complete arrest of embryos at the morula stage and suppression of TE formation [28]. This is compounded with the failure of Sox2-deficient embryos to produce ESCs, from ICM, trophoblast stem cells (TSCs), or TE [31]. Taken together these findings indicate that SOX2 is essential for the segregation of the TE from the ICM lineage during early embryogenesis. Another key member of the SOX family that plays a role in embryonic development is SOX17. Earlier studies have shown that Sox17 transcript starts to express, during mouse development, at embryonic day 6 in the ExEn [35, 36]. However, a recent study recorded an earlier expression of SOX17 at the morula and blastocyst stages, implying the important role of SOX17 in preimplantation embryos [33]. An overall expression of SOX17 is initially observed at the 32-cell stage, whereas at the late blastocyst stage, SOX17 expression becomes limited to the primitive endoderm lineages [33]. Indeed, the expression of SOX17 at this stage co-localizes and coincides well with the expression of the primitive endoderm marker GATA6, which has

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7 previously been shown to be the master regulator of early extraembryonic development [37, 38]. Therefore it is not surprising that SOX17 has been found to be required for the formation of extraembryonic endoderm stem cell lines, called XEN cells [33, 39]. To that end, two Sox transcription factors, SOX2 and SOX17, have been shown to play different essential roles in the early development of the preimplantation embryo. However, the question remains as to whether other SOX transcription factors form a wide regulatory network along with SOX2 and SOX17 to mediate preimplantation embryonic development.

The role of SOX transcription factors in pluripotent stem cells Members of the SOX family exhibit a demarcated phenomenon in governing pluripotency, in which SOX2 is considered to be the most well studied factor. In PSCs, SOX2 appears closely linked to the other two core pluripotent transcription factors OCT4 and NANOG. The three proteins are known to be the key players in a network of intrinsic transcription factors that play a critical role in maintaining the self-renewal and pluripotent identities of PSCs [40] by regulating the expression of pluripotency genes and suppressing genes, which stimulate differentiation [5, 31, 41, 42]. Interestingly, it has been reported that although the NIH3T3 cells (differentiated dermal fibroblast cells) express low levels of OCT4, NANOG, and SOX2, these cells have stemness properties and are able to differentiate into other cell types [43]. This finding suggests that even in the presence of low levels of these crucial pluripotency regulators, the cells can sustain certain level of pluripotency. Previous data highlighted the role of SOX2 in this dynamic regulatory circuit. For instance, a number of research studies showed that SOX2 complexes with OCT4 in a cooperative interaction to maintain ESC pluripotency [44-49] while others showed that Nanog transcription factor is dependent on SOX2 and OCT4 to maintain the ESC

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8 characteristics [41, 48]. The synergistic action between OCT4 and SOX2 extends to include the activation of several ESC-specific enhancers, that contain binding sites for both OCT4 and SOX2, such as FGF4 [44], UTF1 [45], FBX15 [46], NANOG [48], and LEFTY [47]. Remarkably, OCT4 and SOX2 bind to their own enhancers and thus activate an auto-regulatory loop to maintain ESC in a pluripotent state [49-51]. Taken together, these findings support the notion that SOX2, OCT4, and NANOG function predominantly to form a regulatory network that orchestrates pluripotency in PSCs. Within this network the three transcription factors (SOX2, OCT4, and NANOG) regulate the expression of themselves as well as other factors. For example, ectopic expression of SOX2 in embryonal carcinoma cells (ECCs) and ESCs leads to suppression of its own gene as well as Sox2/Oct4 target genes involving Oct4, Nanog and Fgf4 genes [52]. This kind of regulation appears to be dose dependent. Notably, the dose of Sox2 in ESCs appears to be crucial to maintain the undifferentiated state of these cells, as an increase or a decrease in SOX2 protein levels leads to ESCs differentiation. Elevated SOX2 shows different regulatory effects between mouse ESCs and human ESCs. In mouse ESCs, slight elevation in the levels of SOX2 protein (less than two fold increase) reduces the expression of Sox2/Oct4 target genes (Nanog and lefty) and cues ESC differentiation into neuroectoderm, mesoderm, and TE lineages but not the endoderm one [53]. By contrast, the overexpression of SOX2 in stable human ESC lines downregulates OCT4 and NANOG expressions and induces ESC differentiation into TE lineage by regulating the expression of the TE marker CDX2 [54]. Indeed, knockdown experiments in human or mouse ESCs supports the important role of SOX2 expression in this regulatory system. Deletion of Sox2 in mouse ESCs [42, 50] or its knockdown in human ESCs results in suppression of ESC pluripotency and induction of the differentiation into TE [55]. Another study

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9 reported that knockdown of SOX2 in human ESCs induces their differentiation into trophectodermal and endodermal lineages [54]. It is worth to note that the discrepancy between these two observations has been explained to be as a result of using two different culturing systems; one with feeder layers [55] and the other with feeder-free culture system [54]. Like SOX2, any variations in OCT4 levels lead to changes in the stem cell fate decision. High expression levels of Oct4 induce the differentiation of the ExEn and mesoderm, while lower levels stimulate TE differentiation [56]. Interestingly, the deletion of Oct4 in mouse ESCs abrogates the expression of SOX2 that switch the cell from pluripotent state to a differentiated state [57]. Altogether, these data suggest that the undifferentiated PSCs should maintain appropriate levels of SOX2 protein to sustain the unique ability to self-renew. Additionally two other SOX TFs, SOX3 and SOX15, have also been reported to be expressed in undifferentiated human ESCs [58] and mouse ESCs [59], respectively. Although both proteins share a high homology with the Sox2 HMG DNA binding domain, they behave in an alternate manner in maintaining pluripotency. A recent report showed that the knockdown of SOX3, which is expressed in low levels in hESCs, has no effect on the pluripotency of these cells [58]. Interestingly, the knockdown of SOX2 in hESCs upregulates the expression of SOX3, and maintain the undifferentiated state, suggesting that SOX3 may compensate the absence of SOX2 to maintain the ESCs phenotype [58]. Moreover, concurrent suppression of SOX2 and SOX3 leads to loss of pluripotency and induction of differentiation [58]. These observations suggest a counter relationship between the levels of SOX2 and SOX3 in which human ESCs that express high levels of SOX2 and low levels of SOX3 have the ability to keep the cells under normal status of self-renewal and differentiation, whereas in the presence of low levels SOX2, and abundant levels of SOX3, hESCs maintain the pluripotency, but not self-renewal (Fig. 3). On

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10 the other hand, mouse ESCs lacking SOX15 maintain normal self-renewal abilities and show no defects during differentiation process [59].

The role of SOX transcription factors in somatic cell reprogramming The discovery of iPSCs technology significantly increased the importance of SOX TFs in pluripotency. This technology is founded on the notion of overexpressing SOX2 along with other three transcription factors (OCT4, KLF4, and C-MYC) to enable the reprogramming of differentiated (somatic) cells to pluripotent cells (iPSCs) that closely resemble ESCs [3, 4]. Although, the generation of these PSCs is based on the presence of the cocktail factors mentioned above, SOX2 has been found to be necessary for the success of this process [3, 4]. The requirement of this activity of SOX2 is even stronger, because although C-MYC and KLF4 can be replaced or removed without loss of the reprogramming efficiency [60-63], the SOX2/OCT4 appears essential for the reprogramming process [63-65]. In addition, it has been previously shown that human iPSCs have been generated from human cord blood using OCT4 and SOX2 alone [66, 67]. Notwithstanding the above findings, it has been found that in the cells, which express appropriate levels of SOX2, such as neural progenitor cells (NPCs), SOX2 overexpression is not required for their reprogramming into iPSCs [68]. Thus, overexpression of SOX2 during the reprogramming of NPCs reduces the reprogramming efficiency [68], which confirms the fact that too high levels of SOX2 can induce the differentiation of pluripotent stem cells [54]. The mechanisms underlying the role of SOX2 during the cellular reprogramming process remain largely elusive. However, some reports shed the light on possible mechanisms. A previous study showed that SOX2/OCT4 increase the efficiency of iPSCs generation by a direct

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11 activation of the miR-200 family [53, 69]. Another recent study reported that during the early stage of reprogramming process, transient SOX2-mediated inhibition of mammalian target of rapamycin (mTOR) enhances the reprogramming efficiency through a temporary increase in the autophagy process, which is required for iPSC generation [70]. In this study, SOX2 has been found to inhibit mTOR by recruiting the NuRD complex to the mTOR promoter, resulting in transcriptional suppression of mTOR [70]. Several other members of the SOX family have been tested for their ability to generate iPSCs [62]. Remarkably, it has been shown that SOX1 and SOX3, which are closely related factors to SOX2, can replace SOX2 during the reprogramming process [62]. Similarly, the replacement of SOX2 by SOX15 or SOX18, during reprogramming could generate iPSCs, but not as efficiently as members of SOXB1 subfamily. In contrast, neither SOX7 nor SOX17 permit reprogramming when they substituted SOX2 within the reprogramming factors mixture [62]. Interestingly, it has been found that a single amino acid replacement in the DNA binding/OCT interaction domains in the Sox17 gene can change its function, and the reengineered SOX17 (SOX17EK) is able to replace SOX2 during reprogramming process [71]. Furthermore, when the conserved glutamate in either SOX7 or SOX17 is mutated to lysine in the corresponding position in SOX2 HMG domain, both proteins are converted into efficient reprogramming factors and produced 10-50 times more colonies containing fully reprogrammed pluripotent cells than those observed with SOX2 [64, 72, 73]. On the other hand, no pluripotent colonies have been detected when similar mutations are introduced to other family members; SOX4, SOX5, SOX6, SOX8, SOX9, SOX11, SOX13, and SOX18. However, attaching the C-terminus of SOX17 to the pointmutated HMG domains of SOX18 and SOX4 (SOX18EKC17 and SOX4-EKC17) enable them to generate iPSCs, but with very low efficiency [73]. Collectively these data illustrate the core

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12 role of SOX1B family members in somatic cell reprogramming and their high potential in efficient iPSCs generation. This, however, does not exclude the possibility that other SOX factors may play an important role in somatic cell reprogramming. Yet, such a possible role remains unclear although the recent comparison of the HMG domain sequence from Sox family members that demonstrated a phylogenetic evidence for the close relationship between SOX14, SOX15, and SOX21, to SOX1B family [74]. Thus other SOX factors require further investigation.

The role of SOX factors in the differentiation of pluripotent stem cells Some SOX factors, such as SOX2, SOX7, and SOX17, play an important regulatory role during the differentiation of PSCs. SOX7 and its closely related group member, SOX17, are involved in determining the endodermal fate [35, 75, 76] and are known to be upregulated during the differentiation process [36, 76]. In mouse ECs, SOX17 expression is detected in both ExEn and definitive endoderm (DE), whereas SOX7 is not expressed except in the ExEn [35]. Consistent with this observation, SOX7 has been found to be required for stimulating the master regulators of ExEn differentiation, GATA4 and GATA6, and in turn ExEn differentiation [75], whereas SOX17 has been shown to function in the differentiation of both DE [35] and ExEn [33, 36]. Beside these studies that have been done in mouse models, the important role of SOX7 and SOX17 in endoderm differentiation has been also highlighted in the developmental studies of Xenopus Sox7 (Xsox7) [77] and Sox17 (Xsox17) [78-80] as well as those done on Sox17 zebrafish (Zsox17) [81]. In human ESCs, overexpression of SOX17 promotes differentiation to the DE phenotype, while SOX7 overexpression induces cell differentiation to the ExEn [76]. The ExEn derived as a

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13 result of SOX7 overexpression shows a gene profile similar to that of mouse XEN cells [39, 76]. Interestingly, during pancreatic beta cell differentiation, continued SOX17 expression stimulates the expression of PDX1 [76], which is a pancreatic progenitor marker [82]. Although the overexpression of SOX7 and SOX17 in human ESCs does not influence the expression of OCT4 and NANOG, during differentiation these genes are rapidly downregulated [76]. Conversely, the overexpression of SOX17, in mouse ESCs, inhibits pluripotency genes and induces differentiation of ESCs into extraembryonic cells. Furthermore, mouse ESCs lacking Sox17 maintain the expression of the pluripotency factors, such as OCT4, NANOG and SOX2, and interfere with the capability of these cells to differentiate into extraembryonic lineages [33]. Altogether, these findings suggest different roles of SOX17 between mouse and human ESCs, which may reflect intrinsic differences between both ESC types. This difference comes along with a previous study, which showed that mouse and human ESCs differ in the conditions required for the induction of DE SOX17-positive cells [83]. In contrast to SOX7 and SOX17, SOX2 is downregulated during normal differentiation process [42] where it works jointly with OCT4 to suppress the differentiation of either neuroectodermal or mesendodermal layers [84]. Any alterations in levels of either protein will lead to cell differentiation. It has been previously shown that the elevation of SOX2 protein levels stimulates the differentiation of ESCs into neuroectoderm, mesoderm, and TE [53], while high levels of OCT4 direct the differentiation into mesoderm and endoderm [56]. Of note, suppression of either OCT4 or SOX2 triggers the differentiation of ESCs into TE [42, 50, 56]. Thus, the upregulation of OCT4 and SOX2 affects the fate of ESCs differently. Taken together, these findings suggest that SOX2 and OCT4 protein levels should be tightly controlled since

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14 aberrantly high protein levels of SOX2 or OCT4 switch these proteins from an activator to a repressor of ESC self-renewal. It has been shown that SOX2 activates the differentiation to neuroectoderm by inhibiting regulators that are found to be essential for the mesendodermal differentiation [58, 84]. For example, constitutive expression of SOX2 specifically inhibits brachyury, one of the main mesendodermal regulators, and drives the cells toward the neuroectoderm fate [84]. Interestingly, such inhibition is independent of OCT4, which is found to be necessary for the differentiation of cells into the mesendodermal lineage, by suppressing one of the SOXB1 family members, SOX1, that is also known to be a marker for neuroectoderm differentiation [84]. On the other hand, in the absence of OCT4 under undifferentiated culture conditions, SOX2 overexpression triggers SOX1, indicating the importance of SOX2 for NE differentiation. Perhaps the strongest support for the role of SOX2 in neuroectoderm differentiation comes from the finding of Takemoto and his colleagues [85], who demonstrated that in the absence of TBX6, a promoting factor for mesoderm differentiation, SOX2 started to be expressed in the paraxial mesoderm of the mutant mouse embryos and converts the differentiation of this compartment into the neuroectodermal lineage. Moreover, when TBX6 suppresses SOX2 enhancer, N1, SOX2 is repressed and the differentiation is committed towards the mesodermal lineage. Congruent with the above findings, overexpression of SOX2 is sufficient to suppress the differentiation of mesendoderm even under mesendoderm-enhancing culture conditions [58]. In mouse ESCs, it has been found that SOX3 and SOX11 have the opposite effect on the genes that expressed in neural precursor cell (NPC) and neurons [86]. Overexpression of SOX3 activates the genes expressed in the NPCs, while the overexpression of SOX11 activates genes expressed in the neurons. Also, in ESC-derived NPCs, Sox3 binds genes, which are subsequently activated upon neuronal differentiation through their

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15 binding to Sox11 [86]. Furthermore, SOX21, another SOX factor, has been found to induce ESC differentiation if it is overexpressed in ESCs [87]. In this respect, it is logical to assume that SOX factors are differentially regulated to serve a role in determination and differentiation of a specific stem cell fate.

Relationship between SOX transcription factors and other factors in pluripotent stem cells SOX proteins perform their gene regulatory functions by interacting with partner trans-acting factors to form a regulatory network of transcription factors, chromatin remodeling factors, and co-activators that tightly controls pluripotency and differentiation of PSCs [88, 89]. This complex formation occurs when the functional binding site of SOX protein is associated with another partner protein binding site, which is essential for SOX-dependent transcriptional regulation [10]. Of this network, SOX2 was shown to interact with about 70 different proteins such as OCT4, NANOG, ESRRB, KLF4, SALL1, and SALL4 (Fig. 4A,B) [90-94]. It has been previously shown that SOX2, OCT4, and NANOG form autoregulatory and feedback loops, which control their own expression and thus control the expression of other regulatory factors to keep the balanced status between self-renewal and differentiation (Fig. 4A,B) [5, 7]. More importantly, this autoregulatory circuit has been found to be conserved in mouse as well as human ESCs and is controlled by OCT4/SOX2 interaction [5]. Although SOX2 is the most well known member of SOX family to complex with OCT4, other members of this family have also been reported to partner with OCT4 to determine stem cell fate. In ESCs, genome-wide TF binding experiments showed that there is extensive overlap in the target genes of Sox2, Oct4, and Nanog [5, 95]. Such 15


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16 strong genomic overlap was studied using a variety of methods but the most conclusive have been the recent Chromatin Immunoprecipitation sequencing (ChIP-seq) experiments [95, 96]. ChIP-Chip analysis of hESCs showed that ~350 genes (out of ~18,000 genes interrogated) bind the three TFs OCT4, NANOG, and SOX2 [5]. However, ChIP-seq analysis of mESCs showed that 600 genes bind Oct4, Nanog, and Sox2 [95]. In addition, other complimentary experiments using genetic knockdowns as well as purified proteins have been used to show both cis and trans interactions involved in this cooperative mechanism. OCT4 (containing the POU5F1 domain) is known to bind the classical octamer motif (ATTTGCAT) and binds adjacent to SOX2 (HMG domain) motif (AACAAAG) predominantly during pluripotent state. Remarkably, the gap between the 2 binding motifs has been shown to be critical for accommodating different Sox family members to interact with OCT4 [97]. Indeed, individual motifs for binding OCT4 and SOX family members are essentially the same, however, it is the spacing between the motifs, which leads to co-assembly of different SOX family members with OCT4 and thereby lead to different types of cell fate outcomes [67]. In addition to these findings, a single-cell gene expression profiling of mouse blastocyst revealed a marked coexpression of SOX2, SOX17, and OCT4. More importantly, such defined expression has been observed in the ICM at an early developmental stage before lineage segregation [58, 98], suggesting an intriguing possibility that SOX2 can compete with SOX17 for OCT4 binding and thus affect stem cell fate. In agreement with this possible scenario, stable expression of SOX17 in ESC lines switches the binding preference of OCT4 from SOX2 to SOX17 [72]. Such an interesting swap in binding activity is recognized by a single base pair difference that distinguishes OCT4/SOX2 canonical motif binding from the opposing OCT4/SOX17 compressed motif binding (Fig. 5). The cooperative binding of OCT4 and SOX2 is dependent on the presence of a one base pair space between the

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17 two binding motifs, whereas this interspanning base pair is absent in OCT4 and SOX17 binding mode. This interchangeable interaction mediates a developmental switch, where the cell divert from pluripotent state, in case of OCT4-SOX2 interaction, to the endodermal differentiation fate when OCT4 and SOX17 complex together [64, 71, 72]. The different SOX family members binding with diverse interfaces likely lead to recruitment of additional cell fate determining factors and thereby lead to specific cell developmental pathways. The precarious cell fate balance not only hinges on a single base pair difference in the cis regulatory element but also is affected by the presence of a single key SOX residue at the protein-protein interaction interface, which has been found to be crucial for partnering POU5F1 domain on the proper DNA element [11, 71, 99]. Importantly, the type of this single residue differs between SOX2 and SOX17 as shown by amino acid sequence alignment analysis. Sox2 harbors a basic lysine residue, which is highly conserved among other members of SOXB1 subfamily, whereas a conserved glutamate residue within subfamily SOXF is the candidate residue of SOX17 [71]. Recent biochemical studies using purified components as well as genomic and cell biology approaches have shown that mutating this particular residue can in fact impart the properties of the SOX containing this residue [72, 73]. Point mutation of the basic lysine amino acid, in SOX2/OCT4 interface, to acidic glutamate, significantly weakened Sox2 interaction with Oct4 DNA in mobility shift assays upon the canonical element and abolished its partnering ability as confirmed by Chip-seq analysis. In contrast, when the conserved glutamate in SOX17 was mutated to a lysine residue the intensity of SOX17/OCT4 complex noticeably increased upon the canonical cis element. Given these results, it is perhaps not surprising that SOX17 was converted into a potent induced pluripotent stem cell TF and SOX2 conversely was transformed into an endodermal TF. Likewise, similar mutations within SOX7 and SOX18, other

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18 SOXF subfamily members, lead to loss of both proteins differentiation ability to endoderm and create new versions of SOX proteins that maintain the pluripotent state of cells. Consistently with the above findings, the binding of β-catenin to SOX17 C-terminal activation domain, which is known to be critical for endoderm formation, has also been shown to increase the efficiency of producing iPSCs when using mutated SOX17 [72]. Thus the interplay between the HMG domain as well as the C-terminal activation domain is critical for both partnering of proper POU5F1 domain and the subsequent level of activation. Other factors, which are important for the PSC maintainenace, are additionally regulated by SOX proteins. For example, in ESCs, targets of SOX2 are co-occupied by TFs Smad1 and Smad3 (Smad2/3), the downstream effectors of Transforming growth factor (TGF-β), which is crucial for ESC self-renewal [95, 100]. Also, in hESCs, it has been found that SOX2 is essential for transcription of tropomyosin-related kinase C (TRKC) and that the deficiency of SOX2 in undifferentiated hESCs downregulates TRKC expression [101]. These findings suggest that SOX2 regulates the maintenance of PSC state by involving several mechanisms.

Relationship between SOX factors and miRNAs in pluripotent stem cells In addition to its protein interaction network, mentioned above, SOX2 along with other pluripotent stem cell factors establish another circuit of associations with microRNAs (miRNAs). These miRNA-pluripotent stem cell factors interactions activate the expression of specific miRNAs families or clusters to help to maintain stem cell pluripotency. It has been well documented that the three top pluripotency regulatory proteins (SOX2, OCT4, and NANOG) bind to the promoter regions of pluripotent stem cell-specific miRNA clusters including the mouse miR-290-295, the human miR-371-373, and the conserved miR-302-367 and miR-106a18


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19 363 clusters [102-104]. In hESCs, OCT4 and SOX2 have been found to bind to the conserved promoter region of the miR-302 in a ChIP assay. Interestingly, SOX2 binds to miR-302 in a region juxtaposed to the OCT4 binding site, in a way similar to their binding to target genes, confirming the synergistic regulatory mechanism of the two proteins. Indeed, both proteins have been found to be necessary for the expression and transcriptional activation of miR-302 [103]. This is likely to reflect the involvement of SOX2 and OCT4 in cell cycle mechanism to maintain pluripotency, because miR-302 was found to suppress the expression of a key cell cycle regulatory factor, cyclin D1, in hESCs [8, 103]. Another microRNA cluster to consider for a committed binding to SOX2 and OCT4 and could play a crucial role in pluripotency is miR-200 family. Members of this family are dramatically upregulated in undifferentiated ESCs and neuronal progenitor cells as compared to differentiated stages [105, 106]. During the undifferentiated state, high levels of mouse SOX2 directly activate the promoter of miR-200c/141 gene cluster to express miR-200c, which in-turn inhibits mouse SOX2 expression by a direct binding to the Sox2 3’URT. These series of interactions create a negative feedback loop that regulates SOX2 levels and might eventually lead to Sox2 silencing, and may be other pluripotent stem cell factors, and thus promote cells towards differentiation [106]. Furthermore the ability of SOX2 and OCT4 to bind to the promoter regions of specific miR-200 family clusters and directly activate miR-200s expression has been shown in a recent study using a dual-luciferase reporter assay. The transcriptional activation of miR-200 by SOX2 or OCT4 is accompanied by a promotion in iPSC generation, indicating the important role of Sox2/Oct4-miR-200 cluster interactions in plruripotent stem cells [69]. In line with this, the knockdown of miR-200c suppresses the expression of SOX2 as well as

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20 other pluripotent stem cell factors in hESCs [107]. The crucial role of SOX2-miR200 clusters interaction in ESCs pluripotency requires further investigation. Beside their role in sustaining pluripotency, the microRNA-pluripotent stem cell factors interactions could also drive the cell to differentiate into different fates by targeting the mRNA of SOX2 and other pluripotent stem cell factors and negatively regulate their expression [104, 108]. For example miR-134, miR-296, and miR-470 binds to different coding sequences in Nanog, Oct4, and Sox2 and inhibit their expression [108]. Moreover, miR-145 targets the 3’UTRs of Sox2, Oct4, and Klf4 mRNAs and thus represses the ESC pluripotent state [109]. All the above mentioned examples show that through diverse mechanisms, the regulatory network of miRNA-pluripotency factors interactions are likely to be critical for the fidelity of pluripotent stem cell status.

Conclusions and perspectives The importance of SOX TFs in stem cell biology has recently received great interest and a substantial effort has been dedicated to understand the role of these factors in regulating the cell fate decision in PSCs. In particular, we have focused in this review on the existence and functions of Sox TFs in preimplantation embryos and PSCs, their important role as reprogramming factors to induce pluripotency, and their interaction with other trans-acting factors to perform specific functions in PSCs. It has become apparent that the expression patterns and the dose of Sox factors in PSCs play an important role in determining the destiny of these cells. Some SOX factors, such as SOX2, SOX3, and SOX15 are expressed in undifferentiated PSCs, where they play a role in maintaining their self-renewal and pluripotency (Table 2). Other SOX factors such as SOX7, SOX17, and SOX21 are upregulated upon differentiation of PSCs,

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21 where they are responsible to drive the differentiation into specific lineages. Maintaining appropriate levels of Sox proteins in PSCs are crucial to sustain their abilities in either selfrenewal or differentiation. In this regard, it is interesting to address the mechanisms underlying the different roles of Sox TFs within the same pluripotent cell and determine whether other Sox TFs perform similar functions. Also, further studies are needed to clarify the mechanism by which SOX TFs define specificity for their target genes, particularly the identification of binding partners. Understanding these mechanisms will provide insights into how to control PSCs in vitro as well as their differentiation into certain lineages, which will eventually help in clinical applications. In PSCs, SOX TFs are part of an integrated network that is involved in controlling stem cell fate by activating genes that maintain self-renewal and pluripotency or repressing other genes that trigger differentiation (Table 2). It has been deeply discussed in this review how these SOX proteins gene regulatory functions are based on this circuit of interactions that include SOX factors association with partner transcription factors such as OCT4 and NANOG as well as their specific binding to the pluripotent stem cell-specific miRNA clusters. The details of this binding mechanism(s) and how SOX TFs define the specificity for their target genes, particularly the identification of binding partners, is a great question to be resolved. From the time iPSCs technology was first used, the role of SOX TFs in the generation of iPSCs has been highlighted. Importantly, the recent discovery of replacing the SOX2 with the reengineered Sox factors (SOX7EK and SOX17EK) has substantially improved the efficiency of the reprogramming process [71, 72] (Table 2) and open up new possibilities for the involvement of other SOX TFs in somatic cell reprogramming. This might help to reduce the number of the TFs required for the iPSC generation and exclude the oncogenic factors, such as C-MYC, from

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22 the reprogramming cocktail. Therefore, other studies on iPSC generation are required to examine the combination of different TFs and their effect in the reprogramming process. Doing this kind of analysis will broaden our understanding of the reprogramming process and the transcriptional regulatory networks in PSCs.

Acknowledgment We would like to thank Dr. Balasubramanian Moovarkumudalvan from Qatar Biomedical Research Institute, Qatar Foundation, for helping us in preparing Figure 1 in this review.

Author Disclosure Statement No competing financial interests exist.

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Berzal-Herranz and P Menendez. (2008). Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter. Mol Cell Biol 28:6609-19. 103.

Card DA, PB Hebbar, L Li, KW Trotter, Y Komatsu, Y Mishina and TK Archer. (2008).

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Gill JG, EM Langer, RC Lindsley, M Cai, TL Murphy, M Kyba and KM Murphy. (2011). Snail

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Figure Legends

Figure 1: The superposition of DNA-bound SOX HMG domain structures including SOX2 (red) [11], SOX4 (blue) [12], SOX9 (yellow) [13], and SOX17 (green) [14].

Figure 2: Development of preimplantation embryos and generation of embryonic stem cells. During early development (prior to implantation), the zygote undergoes a number of mitotic divisions. In human, at the day 5-8 (3.5 days in mouse), the blastocyst stage embryo is formed, which consists of inner cell mass (ICM), surrounded by an outer layer of the trophectoderm (TE). In vivo, the ICM will form all the three germ layers, while the TE will form the placenta. Embryonic stem cells (ESCs) are derived from the ICM of the blastocyst stage embryos and under appropriate culture conditions in vitro, they can self-renew indefinitely while maintaining their pluripotency. ESCs depend on an intrinsic network of transcription factors, which are also expressed in the ICM, to maintain their pluripotent identity. ESCs are able to differentiate into the three germ layers (endoderm, mesoderm, and ectoderm).

Figure 3: The relationship between SOX2 and SOX3 in hESCs. Diagram represents the relationship that exists in undifferentiated hESCs between SOX2 and SOX3. Normally hESCs express high level of SOX2, and low level of SOX3. Suppressing SOX2 in hESCs induces activation of SOX3 expression leading to maintain the pluripotency, but not self-renewal. Inhibition of both factors (SOX2 and SOX3) in hESCs leads to suppression of pluripotency and self-renewal as well as induce hESC differentiation.

34


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35 Figure 4: Outline of SOX2 regulatory network for mediating pluripotency. (A) A protein association network illustrates the most important interactions between SOX2 and other transcription factors that regulate pluripotency. Thicker lines represent stronger associations. This protein interaction map was done using STRING database version 9.1 based on medium confidence events [110]. (B) SOX2 partners with different transcription factors (TFs) to control the expression of different regulatory factors that maintain the balance between self-renewal and differentiation. This TF complex mediates stem cell fate through activation of genes that maintain self-renewal or suppressing them to upregulate specific genes that direct stem cell differentiation to specific cell lineages.

35


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36

Figure 5: Differential assembly of OCT4 with SOX2 and SOX17. The preferential binding of SOX2 and SOX17 on canonical and compressed cis elements involved in pluripotency and endodermal programs, respectively. The compressed motif has a missing base at position 18, which is highlighted in the orange square.

Figure Legends

36

Stem Cells and Development The SOX transcription factors as key players in pluripotent stem cells (doi: 10.1089/scd.2014.0297) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 37 of 47

37

Figure 1: The superposition of DNA-bound SOX HMG domain structures including SOX2 (red)

[11], SOX4 (blue) [12], SOX9 (yellow) [13], and SOX17 (green) [14].

37


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38

38


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39

Figure 2: Development of preimplantation embryos and generation of embryonic stem cells. During early development (prior to implantation), the zygote undergoes a number of mitotic divisions. In human, at the day 5-8 (3.5 days in mouse), the blastocyst stage embryo is formed, which consists of inner cell mass (ICM), surrounded by an outer layer of the trophectoderm (TE). In vivo, the ICM will form all the three germ layers, while the TE will form the placenta. Embryonic stem cells (ESCs) are derived from the ICM of the blastocyst stage embryos and under appropriate culture conditions in vitro, they can self-renew indefinitely while maintaining their pluripotency. ESCs depend on an intrinsic network of transcription factors, which are also

39


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40

expressed in the ICM, to maintain their pluripotent identity. ESCs are able to differentiate into

the three germ layers (endoderm, mesoderm, and ectoderm).

40


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41

Figure 3: The relationship between SOX2 and SOX3 in hESCs. Diagram represents the relationship that exists in undifferentiated hESCs between SOX2 and SOX3. Normally hESCs express high level of SOX2, and low level of SOX3. Suppressing SOX2 in hESCs induces activation of SOX3 expression leading to maintain the pluripotency, but not self-renewal. Inhibition of both factors (SOX2 and SOX3) in hESCs leads to suppression of pluripotency and self-renewal as well as induce hESC differentiation.

41


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Figure 4: Outline of SOX2 regulatory network for mediating pluripotency. (A) A protein association network illustrates the most important interactions between SOX2 and other transcription factors that regulate pluripotency. Thicker lines represent stronger associations. This protein interaction map was done using STRING database version 9.1 based on medium confidence events [110]. (B) SOX2 partners with different transcription factors (TFs) to control

42


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43 the expression of different regulatory factors that maintain the balance between self-renewal and differentiation. This TF complex mediates stem cell fate through activation of genes that maintain self-renewal or suppressing them to upregulate specific genes that direct stem cell differentiation to specific cell lineages.

Figure 5: Differential assembly of OCT4 with SOX2 and SOX17. The preferential binding of SOX2 and SOX17 on canonical and compressed cis elements involved in pluripotency and endodermal programs, respectively. The compressed motif has a missing base at position 18, which is highlighted in the orange square.

43


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Table 1: Sox family proteins are divided into groups SoxA-SoxH based on the degree of amino acid identity within the HMG-box.

Group

Subgroup

Members

Reference

SRY

[17]

SOXB1

SOX1, SOX2, AND SOX3

[18]

SOXB2

SOX14 AND SOX21

[19]

SOXC

SOX4, SOX11 AND SOX12

[20]

SOXD


[23]

SOXE


[21]

SOXF


[22]

SOXG

SOX15

[24]

SOXH

SOX30

[25]

SOXA SOXB

44

Stem Cells and Development The SOX transcription factors as key players in pluripotent stem cells (doi: 10.1089/scd.2014.0297) as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro

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45 Table 2: The role of different SOX TFs in preimplanatation embryos, pluripotent stem cells, and iPSC generation SOX TFs SOX1

SOX2

Preimplantation embryos

PSC pluripotency

PSC self-renewal

 Is capable to replace SOX2 during the reprogramming process (highly efficient) [62]  Essential for reprogramming (high efficiency) [3, 4]

Not reported

Not reported

Not reported

 Expressed in preimplantation embryos and essential for embryo development [28, 29, 31]

 Essential for pluripotency in both hESCs and mESCs [42, 54, 55]

 Essential for self-renewal in both hESCs and mESCs [42, 54, 55]

Not reported

 Maintains pluripotency in the absence of SOX2 in hESCs [58]  No essential role in pluripotency of mESCs [59]

 Does NOT maintain selfrenewal in the absence of SOX2 in hESCs [58]  No essential role in selfrenewal of mESCs [59]

 Is capable to replace SOX2 during the reprogramming process (high efficiency) [62]

Not reported

Not reported

Not reported

Not reported

 No essential role in pluripotency of both hESCs and mESCs [58, 59]

 No essential role in selfrenewal of both hESCs and mESCs [58, 59]

 Not effective during the reprogramming process [62]  Reengineered SOX7 (SOX7EK) is capable to replace SOX2 during the reprogramming process (very high efficiency) [73]  Is capable to replace SOX2 during the reprogramming process (Moderate efficiency) [62]

 Expressed in preimplantation embryos and regulates differentiation during development [33]

 No essential role in pluripotency

 No essential role in selfrenewal

Not reported

Not reported

Not reported

SOX3

SOX7

SOX15

SOX17

iPSC generation

SOX18 45

 Not effective during the reprogramming process [62]  Reengineered SOX17 (SOX17EK) is capable to replace SOX2 during the reprogramming process (very high efficiency) [71, 73]  Is capable to replace SOX2 during the reprogramming process (Low efficiency) [62]  Reengineered SOX18 (SOX18EKC17) is

Stem Cells and Development The SOX transcription factors as key players in pluripotent stem cells (doi: 10.1089/scd.2014.0297) as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro

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capable to replace SOX2 during the reprogramming process (Moderate efficiency) [73]

46

Stem Cells and Development The SOX transcription factors as key players in pluripotent stem cells (doi: 10.1089/scd.2014.0297) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 47 of 47

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47

Tumor microenvironment: bone marrow-mesenchymal stem cells as key players.

Reprogramming of mouse cochlear cells by transcription factors to generate induced pluripotent stem cells.

Bioinformatic analysis of the four transcription factors used to induce pluripotent stem cells.

RNA polymerase III transcriptomes in human embryonic stem cells and induced pluripotent stem cells, and relationships with pluripotency transcription factors.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

Transcription factor heterogeneity in pluripotent stem cells: a stochastic advantage.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.

Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

MicroRNA and Transcription Factor: Key Players in Plant Regulatory Network.

Epigenetic Silencing of the Key Antioxidant Enzyme Catalase in Karyotypically Abnormal Human Pluripotent Stem Cells.

Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors.

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells.

STAT transcription factors in normal and cancer stem cells.

Induced pluripotent stem cells as a model for diabetes investigation.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

A Chemical Biology Study of Human Pluripotent Stem Cells Unveils HSPA8 as a Key Regulator of Pluripotency.

Network analysis of transcription factors for nuclear reprogramming into induced pluripotent stem cell using bioinformatics.

Exploring the utility of organo-polyoxometalate hybrids to inhibit SOX transcription factors.

Mesenchymal stem cells and induced pluripotent stem cells as therapies for multiple sclerosis.

Germline and Pluripotent Stem Cells.

Role of SOX family of transcription factors in central nervous system tumors.

Neural transcription factors: from embryos to neural stem cells.

Reprogramming sertoli cells into pluripotent stem cells.