In 2006, the research group directed by Shinya Yamanaka showed for the first time the possibility to reprogram mouse fibroblasts into induced pluripotent stem cells (iPSC) with properties similar to those of embryonic stem cells (ESC) by forced expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc, since then termed Yamanaka factors (1). After this first reprogramming report, these researchers and other groups have successfully used the same factors, or other combinations, to reprogram a wide range of mouse or human somatic cells into iPSC (2-4). Subsequently, a direct conversion of somatic cells with defined characteristics into cell lineages with different functions (also termed direct lineage reprogramming) was achieved using predominantly pioneer/cell-type specific transcription factors playing key roles in the embryonic specification of the cell fate of interest during development (5). During direct conversions, the transition from one functional cell type to another is thought to be executed without passing through an intermediate pluripotent state. Although tumorigenic risks are associated with cells obtained either by reprogramming somatic cells into iPSC or by direct cell conversion, the bypass of the pluripotent state limits the risks of teratoma formation. Since the original iPSC generation report, great advancements and refinements of the experimental procedures have been made to circumvent some issues raised by the reprogramming processes (6). Currently, the reprogramming of virtually any cell type is possible using different combinations of transcription factors, epigenetic regulators, microRNAs and/or small molecules (7).
Reprogrammed cells are now an invaluable tool for in vitro disease modelling, high-throughput screens for toxicity tests and drug discovery, and reprogramming raises the possibility for the derivation of patient-specific autologous cells for therapeutic purposes (8). However, the translational potential of these cells is still hampered by the inefficiency of the reprogramming processes, the frequent incomplete cell-fate specification of the reprogrammed cells which may have remnants of the original gene expression pattern and cell function, and de novo mutations occurring during the process (6).
Amongst the signalling pathways with essential roles in embryonic development, ESC self-renewal and differentiation, the TGF-β family members ACTIVIN and NODAL are particularly important, acting mostly through the activation of downstream specific transcriptional effectors SMAD2 and SMAD3 (9). ACTIVIN/NODAL instructive roles for embryonic development and complex cell fate decisions are achieved through a precise dosage of their expression levels, an intricate network of interactions with other signalling pathways resulting in the balanced regulation of target genes dependent on SMAD2/3 (9-20). The selective and robust binding of SMAD2/3 to specific loci across the genome and/or their transcriptional activity relies on interactions with other transcriptional factors and epigenetic modulators.
A recent report has established an unprecedented role for SMAD2/3 which may act as general potentiators for human and mouse iPSC reprogramming, and for direct cell lineage conversion, when expressed as constitutively active forms with the adequate reprogramming transcription factors (21). Indeed, constitutively active SMAD2/3 cooperated with Yamanaka factors to accelerate and increase the emerging number of colonies expressing NANOG. Surprisingly, the authors showed that the endogenous SMAD2/SMAD3 are dispensable for mouse embryonic fibroblasts (MEF) conversion into iPSC by Yamanaka factors (21). Constitutively active SMAD3 only amplified the expression of genes pre-activated by Yamanaka factors, and interacted with OCT4 and SOX2, central pioneer transcription factors of the Yamanaka factors (21). Constitutively active SMAD3 was present at OCT4-targeted genes during reprogramming and promoted the recruitment of histone modifiers and chromatin remodelers to facilitate the switch of gene expression pattern during reprogramming. The ACTIVIN/NODAL signalling pathway, through the activation of SMAD2/3, was previously reported to be critical for self-renewal of human ESC and mouse epiblast-derived stem cells (EpiSC) by a direct regulation of NANOG expression (14,15,18,22,23). Human ESC and mouse EpiSC are primed pluripotent stem cells with distinct properties from those of mouse naïve ESC which are derived from the inner cell mass of the blastocyst. Primed human ESC and mouse EpiSC express the core pluripotency-related transcription factors (such as NANOG, OCT4 and SOX2) concomitantly with genes important to drive differentiation (such as T/BRACHYURY, SOX17, GATA6 and FGF5, amongst other genes), and display no expression of genes specifically associated with the metastable naïve pluripotency state (such as ZFP42/REX1, DPPA5, amongst other genes). Apart from its role in the maintenance of pluripotency, ACTIVIN/NODAL-SMAD2/3 pathway in cooperation with WNT and BMP signalling pathways also actively drives the specification of pluripotent stem cells to mesendoderm cells and prevents the neuroectodermal default differentiation process of these cells (13,14,24). These observations imply that SMAD2/3 activation and function are precisely controlled in pluripotent stem cells, and minor changes to the established balance of active SMAD2/3 may have an impact on cell-fate or cell properties. In the report by Ruetz et al. (21), the changes in gene expression promoted by the constitutively active SMAD3 co-expression with Yamanaka factors have indicated a faster reprogramming process, but they do not allow to predict the final cell fate outcome upon completion of the reprogramming process. It would be of interest to evaluate the full potential of constitutively active SMAD2/3 to achieve the complete reprogramming of somatic cells both to naïve and primed iPSC, and test their full differentiation potential to assess unpredictable bias towards specific cell fates.
A significant finding reported by the authors is that constitutively active SMAD2/3 cooperates with a variety of cell-type transcription factors to promote distinct direct cell conversions, suggesting that constitutively active SMAD2/3 may act as general potentiators of transcription factor-triggered cell reprogramming (21). Indeed, constitutively active SMAD2/3 cooperated with CEBPα to convert from mouse B lymphocytes into macrophages, with CEBPα and PRDM16 to convert mouse myoblast cells into adipocytes, and with ASCL1, BRN2A, MYTII and NEUROD1 to generate mature neurons from human fibroblasts (21).
Together, the positive results obtained in cell reprogramming mediated by the forced co-expression of adequate transcription factors and constitutively active SMAD2/3 paves the way for further exploration of the potential of SMAD2/3 for direct conversion of somatic cells into other cell types yet not tested. It would be of interest to assess the potential of proteins interacting with SMAD2/3 and/or modulating their function to improve of the efficiency and specificity of the conversion processes, in combination with activated SMAD2/3 and the appropriate reprogramming factors. In particular, factors known to be critical for the maintenance of pluripotency or the function of the desired cell type to be reprogrammed. For instance, the SMAD2/3 co-repressors SNON (SKIL) and SIP1 which contribute to the maintenance of human ESC pluripotency and mouse EpiSC by inhibiting the transcriptional activity of SMAD2/3 at mesendodermal gene promoters (13,17). Interestingly, the expression of SNON itself in human ESC is under the conjoint control of NANOG, OCT4, SOX2 and SMAD2/3 (13,17). CITED2 is another interesting SMAD2/3 co-activator with potential to synergize with these proteins in reprogramming processes (25). Indeed, CITED2 is important for mouse ESC self-renewal through the regulation of NANOG expression, and for cardiac differentiation (26,27). Moreover, CITED2 co-expression with Yamanaka factors was reported to accelerate and increase the efficiency of iPSC generation (27,28). Additionally, the co-expression of CITED2 with Yamanaka factors reduced the variability of the expression of some core pluripotency-related genes, particularly if reprogramming pre-senescent MEF which are refractory to the process (27,28). Thus, the combined co-expression of constitutively active SMAD2/3 with the Yamanaka factors, and CITED2, SNON or/and SIP1 may even hold a greater potential for a rapid and efficient cell reprogramming than just SMAD2/3 in combination with Yamanaka factors.
The original idea by Shinya Yamanaka to reprogram somatic cells into pluripotent stem cells (1), by forced expression of factors playing critical roles in the maintenance of ESC identity and functions, has established solid fundaments for cell line reconversion. However, this powerful method still presents associated concerns that need to be addressed to efficiently and reliably originate cell-types of interest. As described recently for constitutively active SMAD2/3 (21), other transcription factors, epigenetic regulators, microRNAs and/or small molecules have been shown to cooperate, complement or substitute Yamanaka factors for reprogramming. This recurrent revisitation of Shinya Yamanaka’s pioneer work by researchers worldwide is constantly improving cell engineering processes and bringing reprogrammed cells closer to safe clinical applications.
Funding: The generous funding by the national Portuguese Fundação para a Ciência e a Tecnologia (FCT) for the project PEst-OE/EQB/LA0023/2013, and by the FCT and the Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) for the project ALG-01-0145-FEDER-28044 are acknowledged.
Conflicts of Interest: The author has no conflicts of interest to declare.
- Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 2006;126:663-76. [Crossref] [PubMed]
- Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008;22:1987-97. [Crossref] [PubMed]
- Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008;132:567-82. [Crossref] [PubMed]
- Lengerke C, Daley GQ. Disease Models from Pluripotent Stem Cells. Ann N Y Acad Sci 2009;1176:191-6. [Crossref] [PubMed]
- Morris SA. Direct lineage reprogramming via pioneer factors; a detour through developmental gene regulatory networks. Development 2016;143:2696-705. [Crossref] [PubMed]
- Takahashi K, Yamanaka S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 2016;17:183. [Crossref] [PubMed]
- Xu J, Du Y, Deng H. Direct Lineage Reprogramming: Strategies, Mechanisms, and Applications. Cell Stem Cell 2015;16:119-34. [Crossref] [PubMed]
- Kimbrel EA, Lanza R. Current status of pluripotent stem cells: moving the first therapies to the clinic. Nat Rev Drug Discov 2015;14:681-92. [Crossref] [PubMed]
- Gaarenstroom T, Hill CS. TGF-Î2 signaling to chromatin: How Smads regulate transcription during self-renewal and differentiation. Semin Cell Dev Biol 2014;32:107-18. [Crossref] [PubMed]
- Brown S, Teo A, Pauklin S, et al. Activin/Nodal Signaling Controls Divergent Transcriptional Networks in Human Embryonic Stem Cells and in Endoderm Progenitors. Stem Cells 2011;29:1176-85. [Crossref] [PubMed]
- Bertero A, Brown S, Madrigal P, et al. The SMAD2/3 interactome reveals that TGFÎ2 controls m6A mRNA methylation in pluripotency. Nature 2018;555:256. [Crossref] [PubMed]
- Bertero A, Madrigal P, Galli A, et al. Activin/Nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark. Genes Dev 2015;29:702-17. [Crossref] [PubMed]
- Chng Z, Teo A, Pedersen RA, et al. SIP1 Mediates Cell-Fate Decisions between Neuroectoderm and Mesendoderm in Human Pluripotent Stem Cells. Cell Stem Cell 2010;6:59-70. [Crossref] [PubMed]
- Vallier L, Mendjan S, Brown S, et al. Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development 2009;136:1339-49. [Crossref] [PubMed]
- Brons IGM, Smithers LE, Trotter MWB, et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 2007;448:191-5. [Crossref] [PubMed]
- Guzman-Ayala M, Lee KL, Mavrakis KJ, et al. Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells. PLoS One 2009;4. [Crossref] [PubMed]
- Tsuneyoshi N, Tan EK, Sadasivam A, et al. The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells. Genes Dev 2012;26:2471-6. [Crossref] [PubMed]
- Tesar PJ, Chenoweth JG, Brook FA, et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007;448:196. [Crossref] [PubMed]
- Robertson EJ. Dose-dependent Nodal/Smad signals pattern the early mouse embryo. Semin Cell Dev Biol 2014;32:73-9. [Crossref] [PubMed]
- Sun LT, Yamaguchi S, Hirano K, et al. Nanog co-regulated by Nodal/Smad2 and Oct4 is required for pluripotency in developing mouse epiblast. Dev Biol 2014;392:182-92. [Crossref] [PubMed]
- Ruetz T, Pfisterer U, Di Stefano B, et al. Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming. Cell Stem Cell 2017;21:791-805.e9. [Crossref] [PubMed]
- Pauklin S, Vallier L. Activin/Nodal signalling in stem cells. Development 2015;142:607-19. [Crossref] [PubMed]
- Tanaka A, Yuasa S, Mearini G, et al. Endothelin-1 Induces Myofibrillar Disarray and Contractile Vector Variability in Hypertrophic Cardiomyopathy-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. J Am Heart Assoc 2014;3. [Crossref] [PubMed]
- Vallier L, Reynolds D, Pedersen RA. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol 2004;275:403. [Crossref] [PubMed]
- Chou YT, Wang H, Chen Y, et al. Cited2 modulates TGF-beta-mediated upregulation of MMP9. Oncogene 2006;25:5547-60. [Crossref] [PubMed]
- Pacheco-Leyva I, Matias AC, Oliveira DV, et al. CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells. Stem Cell Reports 2016;7:1037-49. [Crossref] [PubMed]
- Kranc KR, Oliveira DV, Armesilla-Diaz A, et al. Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells. Stem Cells 2015;33:699-712. [Crossref] [PubMed]
- Charneca J, Matias AC, Escapa AL, et al. Ectopic expression of CITED2 prior to reprogramming, promotes and homogenises the conversion of somatic cells into induced pluripotent stem cells. Exp Cell Res 2017;358:290-300. [Crossref] [PubMed]
Cite this article as: Bragança J. SMAD2/3, versatile molecular tools for cellular engineering. Stem Cell Investig 2018;5:24.