Talk:Stem Cells 2

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Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis

Am J Physiol Cell Physiol. 2009 Jul;297(1):C43-54. Epub 2009 Apr 29.

Lang KC, Lin IH, Teng HF, Huang YC, Li CL, Tang KT, Chen SL. Source Dept. of Life Sciences, National Central University, 300 Jhongda Rd., Jhongli 32054, Taiwan, ROC.


Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

PMID: 19403798

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells

Cell Res. 2008 Dec;18(12):1177-89.

Liu X, Huang J, Chen T, Wang Y, Xin S, Li J, Pei G, Kang J. Source Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.


Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cell-cycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

PMID: 19030024

Developmental signaling network regulated by endogenous Yamanaka factors.

Human embryonic stem cells: preclinical perspectives

J Transl Med. 2008 Jan 29;6:7.

Deb KD, Sarda K. Source Embryonic Stem Cell Program, Manipal Institute of Regenerative Medicine, #10 Service Road, Domlur, Bangalore 560071, India. Abstract Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

PMID: 18230169

Human embryonic stem cells (hESCs) without the use of additional human embryos

  1. Reprogramming of adult cell nucleus - use existing hESCs to fuse with an adult somatic cell, generating a cell line that retains ESC specific properties and yet has the genotype of the somatic cell donor. However, there is no technology available to selectively remove all the ESC chromosomes while retaining the somatic cell chromosomes. In addition this removal of chromosome needs to be timed to occur only after the hybrid cell has been reprogrammed to take the properties of the stem cells. Development of such technologies is potentially expensive and will presumably take years.
  2. ESCs from embryo like entities - use of somatic cell nuclear transfer (SCNT) to produce developmentally compromised embryo-like structures, with the help of genetically premodified deficient nuclei which cannot support development. The zygote produced by such nuclear transfer undergoes cleavage in-vitro and produce ICM cells, which would be used for deriving ESCs, but would not proceed further in development. A proof of principle to this was accomplished by generating mouse ESCs, using a donor nucleus which was silenced for Cdx2 gene. This is ethically correct for those who believe that fetal life begins only after the embryo implants. However, one need not go for creating a mutation to achieve this target, as a blastocyst cannot develop into a complete human life in vitro, irrespective of the presence or absence of any kind of genetic alterations.
  3. ESC lines from single blastomeres - a single cell can be isolated from the cleavage stage embryo, a technique well established for preimplantation genetic diagnosis (PGDs), and used to create a cell line from it; the rest of the embryo can be transferred back to the uterus to give rise to a fetus. Robert Lanza's group has shown that ESC lines could be established from single cell biopsies of the mouse and human embryos. However, this technique is very difficult to translate to human being. Also, the fate of the residual embryos if they are transferred is largely unknown, as there is a lack of long term studies supporting the health of babies born following PGD.
  4. ESC lines from induced somatic cell dedifferentiation - adult somatic cells are genetically modified and reprogrammed to undergo a process of dedifferentiation, by inducing the expression of pluripotency related genes. Recently, induced pluripotent stem cell lines have been derived by allowing trans-acting factors present in the mammalian oocytes to reprogram somatic cell nuclei to an undifferentiated state. They have demonstrated that four factors OCT-4, SOX-2, Nanog and LIN28 are sufficient to reprogram human somatic stem cells to pluripotent stem cells. Whereas, Takahashi and Yamanaka (2006) induced somatic cells into pluripotent stem cells by introducing four factors OCT-4, SOX-2, c-Myc and KLF-4. These cells designated as induced pluripotent stem cells (iPS) exhibit morphology of embryonic stem cells and express ES cell markers. Although, Takahashi and Yamanaka (2006) and Yu et al., (2007) carried out astonishing experiments by reprogramming somatic cells into pluripotent stem cells, several technical limitations such as use of retrovirus or lentiviruses for transfecting OCT-4, Nanog, SOX-2, C-MYC, LIN28 or KLF4 restrict the use of such cell lines for clinical applications (Hanna et al., 2007).
  5. Embryonic like stem cells from alternative sources - adult stem cells similar to blastomeres of the preimplantation stage embryos have been identified and isolated by Henry Young and coworkers. These cells called the blastomere-like stem cells (BLSCs) are found to be totipotent due to their potential to give rise to all tissue types including the gametes. These BLSCs can be induced to differentiate in a unidirectional manner to form pluripotent embryonic-like stem cells (ELSCs). It is also claimed that these cells do not express the MHC class-I or HLA DR-II cell surface markers. More recently Meng et al., (2007) have discovered a population of stem cells in the menstrual blood. These cells named as the "Emdometrial Regenerative Cells" are shown to be capable of differentiating into 9 tissue lineages namely: cadiomyocytic, respiratory epithelial, neurocytic, myocytic, endothelial, pancreatic, hepatic, adipocytic, and osteogenic.

(Text modified from <pubmed>18230169</pubmed>| J Transl Med.)