The TGF-β superfamily is consisted with more than 100 proteins including a multifunctional cytokine TGF- β, activin, nodal, inhibin, lefty, bone morphogenetic proteins (BMPs), one class of growth and differentiation factors (GDFs). Receptors of TGF-β family are classified as three different classes such as type I (TGFRI, also termed activin-like kinases (ALKs)), type II (TGFRII) and type III (TGFRIII) (1). Receptor type I and type II contains intracellular domain with serine threonine kinase activity (2-4). Receptors are activated by the binding of TGF-β family ligands and interact with the family of Smad protein to activate them by phosphorylation within the cytoplasm, and then translocate the phosphorylated Smad proteins into nucleus (Fig. 1). In the nucleus, Smad proteins function as transcriptional cofactors to activate target genes to determine the cell fate upon external stimulation. Depending upon their structure and on their function, Smad proteins are divided into three groups; receptorregulated “R-Smads” (Smad1, Smad2, Smad3, Smad5, Smad8), “Co-Smads”(Smad4), and inhibitory “I-Smads” (Smad6, Smad7) (5).

  The BMP ligands bind to type I receptor ALK2/3/6 and leads to phosphorylation of the transcription factors Smad1/5/8, which are subsequently translocated into nucleus. In the other branch, Activin/Nodal/TGF-β ligands activates type I receptors, ALK4/5/7, and phosphorylates Smad2/3 (9)

Among the TGF-β superfamily members, Lefty is the only inhibitor that is highly enriched in stem cells. Lefty locus contains two genes with the same transcriptional orientation in human, mice, and zebrafish. Human Lefty1 is identical to mice LeftyB and Lefty2 is identical to human LeftyA (10-13). LeftyB has 96% sequence identity with LeftyA and these two proteins differ only in 16 amino acids. However, LeftyB has only 82% sequence identity with Lefty1, which suggests that Lefty proteins has been evolved independently in mouse and humans after the duplication of a single Lefty gene (10).

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  Nodal signals are also contributes to maintain the ES cell identity by the finding that Nodal-deficient mouse embryos exhibit an epiblast with very low levels of Oct-3/4 expression (17, 18). Moreover, nuclear localization of phosphorylated Smad2, which is induced by TGF-β, activin, or nodal signaling were observed in hESCs and it is decreased upon differentiation of the cells (19). Microarray analysis shows that activin supports the maintenance of pluripotency of hESCs possibly by inducing the expression of Oct4 and Nanog (20). Recently, it was reported that Actin/Nodal signaling maintains pluripotency of hESCs and mouse EpiSCs by controlling Nanog expression, which leads to block neuroectoderm differentiation of pluripotency cells (21). Furthermore, TGF-β/ activinmediated SMADs directly binds to the Nanog promoter in hESCs (Fig. 2) (22).

Consistently, chemical inhibition of Smad2 phosphorylation with SB-431542 results in decreases of the expression of markers of undifferentiated ESCs (19, 23). These results suggest that Activin or Nodal produced by ESCs itself functions to promote the self-renewal of mESCs.

 Lefty protein is another TGF-β family member that is highly expressed in human and mouse ESCs. Transcriptional expression of Lefty gene is regulated by Oct3/4, Sox2 and Klf4, which are core transcription factors in maintaining stemness of ESCs (24). Klf4 acts as a mediating factor that coorporates with Oct3/4 and Sox2 and occupy the proximal element of the Lefty1 promoter to activate the gene. Recently, it was shown that activation of canonical WNT signaling by inactivating GSK-3β leads to the high expression of Nodal, LeftyA and LeftyB in hESCs (25). Induction of Lefty expression by inhibiting WNT signaling requires ALKs4/5/7 (25). Inhibition of WNT pathway leads to the activation of Smad signaling (26). There are five potential binding sites for the Smad2/ 3 heterodimer in the promoter region of LeftyA and Lefty2 (25). Activation of Smad2/3 by the treatment of differentiating hESCs with Activin leads to the expression of Nodal, LeftyA, and LeftyB in hESCs (27). Consistently, inhibition of ALK4/5/7 by the kinase inhibitor, SB431542, which blocks activation of Smad2/3, downregulates the expression of LeftyA and B in the undifferentiated stem cells (25). These findings suggest that expression of Lefty in undifferentiated ESCs is mediated by ALK4/5/7- and Smad2/3- signaling pathway.

 Taken together, TGF-β family signals play crucial roles in the maintenance of ESC’s self-renewal and pluripotency both in human and mouse.

  When the culture condition that maintain the undifferentiated state of ESCs are changed to induce differentiation of the cells, ESCs start to generate progeny that consists of derivatives of all three germ layers (28). For this reason, ESCs have been used as an in vitro experimental model to understand early stages of embryogenesis, which generates ectoderm, mesoderm and endoderm during gastrulation.

  Differentiation of ESCs into ectoderm is often referred to as the default pathway, since neuroectoderm rapidly develops from ESCs in the culture media without serum. Recently, it was reported that endogenous retinoic acid (RA) production from vitamin A in the medium promotes development of neural progenitors from mESCs by suppressing Wnt-dependent Nodal signaling (29). In mESCs, BMPs inhibit the formation of neuroectoderm and induce its epidermal differentiation (30).

  When BMP is added to mESCs in the presence of VEGF, it is able to induce hematopoietic differentiation of the cells in serum free culture (31). ER71, a number of the Ets transcription factor family, is a downstream target of BMPs and functions to regulate the generation of Flk⁺ blood and vessel progenitors (32). BMP signals initially induce mesoderm and later specify blood lineages via activation of the Cdx-Hox pathway and Wnt signaling (33).

  BMP signals also promote the proliferation of mESCs derived endothelial cells by inducing the expression of Tie2 and Flk1, both of genes are known to promote endothelial- cell proliferation (34). In addition, TGF-β and activin inhibit the proliferation of endothelial cells by inducing the expression of p21 (35). These results suggest that TGF-β family signals play crucial roles during vascular development from mESCs. At the early state of ESC’s differentiation, primitive streak-like populations are formed, which are further differentiated into endoderm by the sustained activation of Nodal/activin signals. Endoderm progenitors are generated by culturing ESCs in the presence of activin in serum-free conditions and subsequent culture of progenitors with BMP-4, activin, and fibroblast growth factor (FGF) produce highly enriched hepatic populations that expressing albumins (35). When activin and BMP-4 are added into ESCs’ differentiation culture media at various steps, Pdx1 (pancreatic and duodenal homeobox gene  1) positive cells are generated at a high efficacy (30%) (37).

  During the formation of mesendoderm lineage, Smad2 mediates Activin/Nodal signaling in mESCs (38). Taken together, these findings suggest that ectoderm is differentiated from human and mouse ESCs in the absence of TGF-β signal, while mesoderm and endoderm is differentiated by a combination of TGF-β family signals.

 Conclusion 

 ESCs are useful sources for the therapeutic application of stem cells. To account for the unique properties of pluripotent ESCs or reprogrammed induced pluripotent stem cells, it will be necessary to understand the endogenous signaling network, which is connected with transcriptional regulatory mechanism responsible for maintaining ESC’s identity. Unlike the conserved roles of core transcription factors like Nanog, Oct3/4, Sox2, and c-Myc etc to maintain pluripotency, mouse and human ESCs operate different internal mechanism to extrinsic signals. Among those systems, TGF-β family members have been recognized as crucial for both human and mouse ESC’s cell fate determination. Therefore, understanding the effects and mechanisms of TGF-β family signals in human and mouse ESCs as well as reprogrammed iPS cells both in pluripotent state and in differentiation state is essential for applying ESCs to therapeutic cell therapy.