Researchers have discovered an added layer of complexity in the network that determines human embryonic stem cell fate. A report publishing online April 30th in the journal Cell, a Cell Press publication, shows that a microRNA known as miR-145 lowers the activity of three key ingredients in the "recipe" for making embryonic stem cells. The discovery may have implications for improving the efficiency of methods designed to reprogram differentiated cells into embryonic stem cell-like cells and for the use of those transformed cells in replacing cells lost to disease or injury, the researchers said.
"The heart of the matter is that before this paper, we knew if you want to maintain a pluripotent state and allow self-renewal of embryonic stem cells, you have to sustain levels of transcription factors, including Oct4, Sox2 and Klf4," said Kenneth Kosik of The University of California, Santa Barbara. "We also knew that stem cells transition to a differentiated state when you downregulate those factors. Now we know how that happens a little better."
Transcription factors are genes that control other genes. On the other hand, microRNAs are single-stranded RNA molecules that control the activity of other genes. When microRNAs in the genome are transcribed from DNA, they target complementary messenger RNAs (mRNAs), which serve as the templates for proteins, to either encourage their degradation or prevent their translation into functional proteins. In general, one gene can be repressed by multiple microRNAs and one microRNA can repress multiple genes, the researchers explained. In a wide variety of developmental processes, microRNAs fine tune or restrict cellular identities by targeting important transcription factors or key pathways.
The new study adds embryonic stem cell identity to that list. Kosik's team found that levels of miR-145 change dramatically when human embryonic stem cells differentiate into other cell types. miR-145 was of particular interest because it had been predicted to target Oct4, Klf4 and Sox2. (Those three factors are perhaps best known as three of four ingredients originally shown to transform adult human skin cells into "induced pluripotent stem cells" (iPS cells), which behave in nearly every respect like true embryonic stem cells (http://www.nurse101.com/news/archives/000038.html). That four-ingredient recipe has since been pared down to one, Oct4, in the case of neural stem cells (http://www.nurse101.com/news/archives/000037.html).)
A rise in miR-145 prevents human embryonic stem cells' self-renewal and lowers the activity of genes that lend stem cells the capacity to produce other cell types, the researchers report. It also sends the cells on a path toward differentiation. In contrast, when miR-145 is lost, the embryonic stem cells are prevented from differentiating as OCT4, SOX2 and KLF4 concentrations rise.
They also show that the control between miR-145 and the "reprogramming factors" goes both ways. The promoter for miR-145 is bound and repressed by OCT4, they found.
"It's a beautiful double negative feedback loop," Kosik said. "They control each other."
Because there is typically less "wiggle room" in the levels of microRNA compared to mRNA, further studies are needed to more precisely quantify the copy numbers of miR-145 and its targets to figure out exactly how this layer of control really works, Kosik said.
The findings in embryonic stem cells might also have importance for cancer.
"There are sets of microRNA that are widely up- or downregulated in cancers," he said, noting that several studies have specifically linked low miR-145 levels to various forms of cancer. "Tumor stem cells are the engines of tumors. If miR-145 is sustaining or maintaining a differentiated state, loss of that may have something to do with malignant transformation."
The researchers include Na Xu, The University of California, Santa Barbara, CA; Thales Papagiannakopoulos, The University of California, Santa Barbara, CA; Guangjin Pan, University of Wisconsin-Madison, Madison, WI; James A. Thomson2, Kenneth S. Kosik, The University of California, Santa Barbara, CA.
A simple recipe—including just four ingredients—can transform adult human skin cells into cells that resemble embryonic stem cells, researchers report in an immediate early publication of the journal Cell, a publication of Cell Press. The converted cells have many of the physical, growth and genetic features typically found in embryonic stem cells and can differentiate to produce other tissue types, including neurons and heart tissue, according to the researchers.
They added, however, that a comprehensive screen of the activity of more than 30,000 genes showed that the so-called “induced pluripotent stem (iPS) cells” are similar, not identical, to embryonic stem cells. "Pluripotent" refers to the ability to differentiate into most other cell types.
The chemical cocktail used in the new study is identical to one the team showed could produce iPS cells from adult mouse cells in another Cell report last year. That came as a surprise, said Shinya Yamanaka of Kyoto University in Japan, because human embryonic stem cells differ from those in mice. Those differences had led them to suspect "that some other factors might be required to generate human iPS cells,” he said.
The findings are an important step forward in the quest for embryonic stem cell-like cells that might sidestep the ethical stumbling blocks of stem cells obtained from human embryos. He emphasized, however, that it would be “premature to conclude that iPS cells can replace embryonic stem cells.”
Embryonic stem cells, derived from the inner cell mass of mammalian blastocysts--balls of cells that develop after fertilization and go on to form a developing embryo--have the ability to grow indefinitely while maintaining pluripotency, the researchers explained. Those properties have led to expectations that human embryonic stem cells might have many scientific and clinical applications, most notably the potential to treat patients with various diseases and injuries, such as juvenile diabetes and spinal cord injury.
The use of human embryos, however, faces ethical controversies that hinder the applications of human embryonic stem cells, they continued. In addition, it is difficult to generate patient or disease-specific embryonic stem cells, which are required for their effective application. One way to circumvent these issues is to induce pluripotent status in other cells of the body by direct reprogramming, Yamanaka said.
Last year, his team found that four factors, known as Oct3/4, Sox2, c-Myc, and Klf4, could lend differentiated fibroblast cells taken from embryonic or adult mice the pluripotency normally reserved for embryonic stem cells. Fibroblasts make up structural fibers found in connective tissue. Those four factors are “transcripton factors,” meaning that they control the activity of other genes. They were also known to play a role in early embryos and embryonic stem cell identity.
The researchers have now shown that the same four factors can generate iPS cells from fibroblasts taken from human skin. “From about 50,000 transfected human cells, we obtained approximately 10 iPS cell clones,” Yamanaka said. “This efficiency may sound very low, but it means that from one experiment, with a single ten centimeter dish, you can get multiple iPS cell lines.”
The iPS cells were indistinguishable from embryonic stem cells in terms of their appearance and behavior in cell culture, they found. They also express genetic markers that are used by scientists to identify embryonic stem cells. Human embryonic stem cells and iPS cells display similar patterns of global gene activity.
They showed that the converted human cells could differentiate to form three “germ layers” in cell culture. Those primary germ layers in embryos eventually give rise to all the body’s tissues and organs. They further showed that the human iPS cells could give rise to neurons using a method earlier demonstrated for human embryonic stem cells. The iPS cells could also be made to produce cardiac muscle cells, they found. Indeed, after 12 days of differentiation, clumps of cells in the laboratory dishes started beating.
The human iPS cells injected under the skin of mice produced tumors after nine weeks. Those tumors contained various tissues including gut-like epithelial tissue, striated muscle, cartilage and neural tissue. They finally showed that iPS cells can also be generated in the same way from other human cells.
“We should now be able to generate patient- and disease-specific iPS cells, and then make various cells, such as cardiac cells, liver cells and neural cells,” Yamanaka said. “These cells should be extremely useful in understanding disease mechanisms and screening effective and safe drugs. If we can overcome safety issues, we may be able to use human iPS cells in cell transplantation therapies.”
The researchers include Kazutoshi Takahashi, Kyoto University, in Kyoto, Japan; Koji Tanabe, of Kyoto University, in Kyoto, Japan; Mari Ohnuki, of Kyoto University, in Kyoto, Japan; Megumi Narita, of Kyoto University, in Kyoto, Japan, and the Japan Science and Technology Agency, in Kawaguchi, Japan; Tomoko Ichisaka, of Kyoto University, in Kyoto, Japan, and the Japan Science and Technology Agency, in Kawaguchi, Japan; Kiichiro Tomoda, of the Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA; and Shinya Yamanaka, of Kyoto University, in Kyoto, Japan, the Japan Science and Technology Agency, in Kawaguchi, Japan; and the Gladstone Institute of Cardiovascular Disease, in San Francisco, CA, USA
The simple recipe scientists earlier discovered for making adult stem cells behave like embryonic-like stem cells just got even simpler. A new report in the February 6th issue of the journal Cell, a Cell Press publication, shows for the first time that neural stem cells taken from adult mice can take on the characteristics of embryonic stem cells with the addition of a single transcription factor. Transcription factors are genes that control the activity of other genes.
The discovery follows a 2006 report also in the journal Cell that showed that the introduction of four ingredients could transform differentiated cells taken from adult mice into "induced pluripotent stem cells" (iPS) with the physical, growth, and genetic characteristics typical of embryonic stem cells. Pluripotent refers to the ability to differentiate into most other cell types. The same recipe was later shown to work with human skin cells as well.
Subsequent studies found that the four-ingredient recipe could in some cases be pared down to just two or three essential ingredients, said Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Germany. "Now we've come down to just one that is sufficient. In terms of the biology, it's really quite amazing."
The discovery sheds light on centuries-old questions about what distinguishes the embryonic stem cells that give rise to egg and sperm from other body cells, Schöler said. It might also have implications for the use of reprogrammed stem cells for replacing cells lost to disease or injury.
Other researchers led by Shinya Yamanaka showed that adult cells could be reprogrammed by adding four factors – specifically Oct4, Sox2, Klf4, and c-Myc. Recently, Schöler and his colleagues demonstrated that Oct4 and Klf4 are sufficient to induce pluripotency in neural stem cells.
By omitting Klf4 in the new study, they have now established that Oct4 is the "driving force" behind the conversion of the neural stem cells into iPS cells. The lone transcription factor is not only essential, but it is also sufficient to make neural stem cells pluripotent.
Those cells, which Schöler's team calls "1F iPS" can differentiate into all three germ layers. Those primary germ layers in embryos eventually give rise to all the body's tissues and organs. Not only can those cells efficiently differentiate into neural stem cells, heart muscle cells, and germ cells, they show, but they are also capable of forming tumors when injected under the skin of nude mice. Those tumors, or teratomas, contain tissue representing all three germ layers. When injected into mouse embryos, the 1F iPS cells also found their way into the animals' developing organs and were able to be transmitted through the germ line to the next generation, they report.
The results show that adult stem cells can be made pluripotent without c-Myc and Klf4, both of which are "bona fide" oncogenes that can help turn normal cells into cancer cells, Schöler said. Limiting the number of factors is also a bonus because it means fewer genes must be inserted into the genome, where they can potentially have detrimental effects.
"Strikingly, Oct4 alone is sufficient to induce pluripotency in neural stem cells, which demonstrates its crucial role in the process of reprogramming…" the researchers concluded. "Future studies will show whether other sources of neural stem or progenitor cell populations such as mouse or human bone marrow-derived mesenchymal stem cells or dental pulp can be reprogrammed to iPS cells and whether expression of Oct4 can be induced by non-retroviral means, a prerequisite for the generation of iPS cells of therapeutic value."
The researchers include Jeong Beom Kim, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Vittorio Sebastiano, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Guangming Wu, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Marcos J. Arauzo-Bravo, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Philipp Sasse, University of Bonn, Bonn, Germany; Luca Gentile, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Kinarm Ko, Max Planck Institute for Molecular Biomedicine, Munster, Germany; David Ruau, RWTH Aachen University Medical School, Aachen, Germany; Mathias Ehrich, SEQUENOM Inc., San Diego, CA; Dirk van den Boom, SEQUENOM Inc., San Diego, CA; Johann Meyer, Hannover Medical School, Hannover, Germany; Karin Hubner, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Christof Bernemann, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Claudia Ortmeier, Max Planck Institute for Molecular Biomedicine, Munster, Germany; Martin Zenke, RWTH Aachen University Medical School, Aachen, Germany; Bernd K. Fleischmann, University of Bonn, Bonn, Germany; Holm Zaehres, Max Planck Institute for Molecular Biomedicine, Munster, Germany; and Hans R. Scholer, Max Planck Institute for Molecular Biomedicine, Munster, Germany.