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Multiple roles for Oct4 in induced pluripotency from MEFs and mouse myoblasts

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From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Original reports describing the generation of induced pluripotent stem cells (iPSCs) set the foundations of the reprogramming process as the exogenous expression of four transcription factors Oct4, Sox2, Klf4, c-Myc and/or Lin28 and Nanog. Subsequent work has shown that with the use of small molecules direct reprogramming can also be achieved by overexpression of a subset of these factors, in some cases with exogenous Oct4 only. Oct4 expression seems to be integral to the reprogramming process and two articles in the March 2011 issue of Stem Cells now reveal additional roles for Oct4. The first article, by Yuan et al.1 from Sheng Ding’s laboratory at the Scripps Research Institute in California, reports a new small molecule which can facilitate reprogramming. The authors screened 100 small molecules under TGF-β receptor inhibition in mouse embryonic fibroblasts (MEFs) that had been retrovirally transduced with Oct4 and their results show that AMI-5, an inhibitor of protein arginine methyltransferase (PRMT) activity, could greatly facilitate the reprogramming process. After 30-40 days this treatment generated colonies of iPSC (miPSC-Os) that were morphologically and genetically similar to mESC and which had silenced the expression of exogenous Oct4. Bisulphite sequencing demonstrated high demethylation of the Nanog and Oct4 promoter regions in miPSC-Os, akin to mESCs, indicating that the appropriate epigenetic remodelling associated with pluripotency had occurred. Embryoid body (EB) formation from miPSC-Os resulted in the generation of cell types from all germ layers and following blastocyst injection, miPSC-Os generated chimaeras and were able to contribute to the genital ridge germline. Further, tetraploid complementation assays performed by injection of miPSC-Os into CD-1 tetraploid embryos generated pups, albeit at a low frequency, that were derived entirely from miPSC-Os, although none survived past the early postnatal period. This is the first report of such stringent pluripotency testing in a study using a single pluripotency factor to generate iPSCs facilitated by small molecules, in this case an inhibitor of PRMTs activity. PRMTs catalyse the methylation of arginine side chains which is associated with chromatin remodelling and transcriptional regulation as well as processes such as DNA repair and signal transduction. One family member, PRMT5, has recently been linked to pluripotency by its ability to repress differentiation genes in ESCs.2 The current study now implicates the regulation of protein arginine methylation in Oct4-mediated reprogramming and also that the processes occurring downstream of this small molecule induction of reprogramming is sufficient to derive bona fide iPSCs.

The second article, from Watanabe et al.3 at the University of Minnesota and Oregon Health and Science University, reveals a further role for Oct4 in the reprogramming process, this time in mouse skeletal muscle progenitor cells (myoblasts). As a cell differentiates the transcriptional state and epigenetic markers of the cell shift towards a profile that is typical of the mature phenotype and the further down this maturation process a cell travels, generally the harder it becomes to achieve reprogramming. Myoblasts are committed muscle cell progenitors derived from satellite cells, the myogenic stem cells for muscle regeneration. MyoD is a master regulator of myogenic specification and differentiation and myoblasts derived from mice lacking the MyoD gene (MyoD-/-) show enhanced proliferation and delayed final differentiation. To examine the reprogramming potential of myoblasts and the role of MyoD in iPSC induction, Watanabe et al.3 FACS sorted satellite cells by their expression of integrin-α7 and β1 from wild type, Oct4-GFP mice and MyoD-/- adult mice and cultured them under myogenic differentiation conditions. Myoblasts, confirmed by their Pax7 and Myf5 expression, were transfected with a retrovirus encoding Oct4, Sox2, cMyc and Klf4 or these factors in combination with MyoD. ESC-like colonies (with 0.002% efficiency) were observed after two weeks that expressed pluripotency marker and gene expression similar to ESCs, and which lacked parental MyoD/Pax7 and exogenous retroviral vector expression. Further, by EB formation, teratoma formation and chimaerism with germ-line transmission, myoblast-derived iPSCs demonstrated their pluripotent nature. The authors also observed that the generation of iPSCs was almost three-fold more efficient by transfection of myoblasts from MyoD-/- mice.

It may not be a great surprise that the forced expression of a key regulator of myogenic fate might interfere with the process of reprogramming and that its downregulation would be a necessary step in the path to reprogramming for sufficient induction of pluripotency to occur. Infection of wild type myoblasts with a vector expressing a solitary reprogramming factor revealed that exogenous Oct4 expression caused significant downregulation of myogenic genes MyoD and Pax7, while Sox2 also caused this effect but to a lesser extent. In addition, forced expression of Oct4 initiated the upregulation of endogenous pluripotent genes Sox2, Oct4 and Nanog, but was not sufficient to induce pluripotency. The effects of Oct4 expression could be overcome under forced MyoD expression allowing the recovery of myogenic differentiation potential, even in the presence of Oct4. Using luciferase reporter constructs the authors demonstrated that Oct4 does not directly suppress MyoD protein function or compete for target DNA binding. Chromatin Immunoprecipitation (ChIP) assays indicated the repressional activity of Oct4 on MyoD upstream enhancer regions by direct binding to the MyoD core enhancer and distal regulatory regions, however Oct4 did not prevent MyoD binding overall. This implicates Oct4 as a transcriptional suppressor of MyoD, or MyoD as a negative regulator of reprogramming.

Elucidation of the molecular mechanisms governing the reprogramming process and eradication of the residual DNA methylation signatures associated with parental somatic cell types will no doubt help us to achieve induced pluripotency more completely and will also address some of the issues currently associated with iPSCs, such as limited differentiation capacity. The use of small molecules may not only enhance iPSC generation but facilitate the development of less risky protocols to direct somatic cells towards a pluripotent state. Both articles provide key information to this end. On the other hand, perhaps we can turn the perceived disadvantages of iPSCs derived from differing somatic cell types into an advantage to achieve our aims; as stated in the second article by Watanabe et al.3 the use of myoblast-derived iPSCs may have better differentiation capacity over hESC-derived myogenic cell types for muscular disease, and therefore may present a better resource of these cells for cell based therapies aimed towards muscle regeneration.

 

References

1Yuan X, Wan H, Zhao X, Zhu S, Zhou Q, Ding S. Combined Chemical Treatment Enables Oct4-Induced Reprogramming from Mouse Embryonic Fibroblasts. Stem Cells. 2011.

2Tee WW, Pardo M, Theunissen TW, Yu L, Choudhary JS, Hajkova P, Surani MA. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev. 2010 Dec 15;24(24):2772-7.

3Watanabe S, Hirai H, Asakura Y, Tastad C, Verma M, Keller C, Dutton JR, Asakura A. Myod Gene Suppression By Oct4 is Required for Reprogramming in Myoblasts to Produce Induced Pluripotent Stem Cells. Stem Cells. 2011.