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Chan S.M.,Stanford University | Majeti R.,Stanford University | Majeti R.,Stanford Institute for Stem Cell Biology and Regenerative Medicine
International Journal of Hematology | Year: 2013

Aberrant changes in the epigenome are now recognized to be important in driving the development of multiple human cancers including acute myeloid leukemia. Recent advances in sequencing technologies have led to the identification of recurrent mutations in genes that regulate DNA methylation including DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 2 (TET2), and isocitrate dehydrogenase 1 (IDH1) and IDH2. These mutations have been shown to promote self-renewal and block differentiation of hematopoietic stem/progenitor cells. Acquisition of these mutations in hematopoietic stem cells can lead to their clonal expansion resulting in a pre-leukemic stem cell (pre-LSC) population. Pre-LSCs retain the ability to differentiate into the full spectrum of mature daughter cells but can become fully transformed with the acquisition of additional driver mutations. Here, we review the effects of mutations in DNMT3A, TET2, and IDH1/2 on mouse and human hematopoiesis, the current understanding of their role in pre-LSCs, and therapeutic strategies to eliminate this population which may serve as a cellular reservoir for relapse. © 2013 The Japanese Society of Hematology.

Sage J.,Stanford Institute for Stem Cell Biology and Regenerative Medicine
Genes and Development | Year: 2012

Stem cells play a critical role during embryonic development and in the maintenance of homeostasis in adult individuals. A better understanding of stem cell biology, including embryonic and adult stem cells, will allow the scientific community to better comprehend a number of pathologies and possibly design novel approaches to treat patients with a variety of diseases. The retinoblastoma tumor suppressor RB controls the proliferation, differentiation, and survival of cells, and accumulating evidence points to a central role for RB activity in the biology of stem and progenitor cells. In some contexts, loss of RB function in stem or progenitor cells is a key event in the initiation of cancer and determines the subtype of cancer arising from these pluripotent cells by altering their fate. In other cases, RB inactivation is often not sufficient to initiate cancer but may still lead to some stem cell expansion, raising the possibility that strategies aimed at transiently inactivating RB might provide a novel way to expand functional stem cell populations. Future experiments dedicated to better understanding how RB and the RB pathway control a stem cell's decisions to divide, self-renew, or give rise to differentiated progeny may eventually increase our capacity to control these decisions to enhance regeneration or help prevent cancer development. © 2012 by Cold Spring Harbor Laboratory Press.

Reimer A.,Max Planck Institute for Infection Biology | Seiler K.,Max Planck Institute for Infection Biology | Seiler K.,Stanford Institute for Stem Cell Biology and Regenerative Medicine | Tornack J.,Max Planck Institute for Infection Biology | And 2 more authors.
Immunology Letters | Year: 2012

Efficiencies of the generation of induced pluripotent stem (iPS) cells from either mouse embryonic fibroblasts (MEF) or from mouse fetal liver (FL) derived preB cells and their hematogenic potencies were compared. In 10 days approximately 2% of the MEFs transduced with Sox-2, Oct-4 and Klf-4 developed to iPS cells, while only 0.01% of transduced FL-preB cells yielded iPS cells, and only after around 3 weeks. Subsequently, the generated iPS cells were induced to differentiate into hematopoietic cells in vitro. On day 5 of differentiation MEF-iPS yielded numbers and percentages of Flk-1 + mesodermal-like cells comparable to those developed from embryonic stem (ES) cells. Compared to ES cells further differentiation to hematopoietic and lymphopoietic cells was reduced, possibly because of persistent expression of the reprogramming factors. By contrast, FL-iPS cells developed lower numbers and percentages of Flk-1 + cells, and no significant further development to hematopoietic or lymphopoietic cells could be induced. These results indicate that the efficiencies of iPS generation and subsequent hematopoietic development depends on the type of differentiated cell from which iPS cells are generated. © 2012 Elsevier B.V.

Corces-Zimmerman M.R.,Stanford Cancer Institute | Majeti R.,Stanford Cancer Institute | Majeti R.,Stanford University | Majeti R.,Stanford Institute for Stem Cell Biology and Regenerative Medicine
Leukemia | Year: 2014

Cancer has been shown to result from the sequential acquisition of genetic alterations in a single lineage of cells. In leukemia, increasing evidence has supported the idea that this accumulation of mutations occurs in self-renewing hematopoietic stem cells (HSCs). These HSCs containing some, but not all, leukemia-specific mutations have been termed as pre-leukemic. Multiple recent studies have sought to understand these pre-leukemic HSCs and determine to what extent they contribute to leukemogenesis. These studies have elucidated patterns in mutation acquisition in leukemia, demonstrated resistance of pre-leukemic cells to standard induction chemotherapy and identified these pre-leukemic cells as a putative reservoir for the generation of relapsed disease. When combined with decades of research on clonal evolution in leukemia, mouse models of leukemogenesis, and recent massively parallel sequencing-based studies of primary patient leukemia, studies of pre-leukemic HSCs begin to piece together the evolutionary puzzle of leukemogenesis. These results have broad implications for leukemia treatment, targeted therapies, minimal residual disease monitoring and early detection screening. © 2014 Macmillan Publishers Limited. All rights reserved.

News Article
Site: www.biosciencetechnology.com

All living things require proteins, members of a vast family of molecules that nature “makes to order” according to the blueprints in DNA. Through the natural process of evolution, DNA mutations generate new or more effective proteins. Humans have found so many alternative uses for these molecules — as foods, industrial enzymes, anti-cancer drugs — that scientists are eager to better understand how to engineer protein variants designed for specific uses. Now, Stanford researchers have invented a technique to dramatically accelerate protein evolution for this purpose. This technology, described in a paper published online Dec. 7 in Nature Chemical Biology, allows researchers to test millions of variants of a given protein, choose the best for some task and determine the DNA sequence that creates this variant. “Evolution, the survival of the fittest, takes place over a span of thousands of years, but we can now direct proteins to evolve in hours or days,” said Jennifer Cochran, Ph.D., an associate professor of bioengineering, who shares senior authorship of the paper with Thomas Baer, Ph.D., executive director of the Stanford Photonics Research Center. The lead author is Bob Chen, a graduate student in bioengineering. “This is a practical, versatile system with broad applications that researchers will find easy to use,” Baer said. By combining Cochran’s protein engineering know-how with Baer’s expertise in laser-based instrumentation, the team created a tool that can test millions of protein variants in a matter of hours. “The demonstrations are impressive, and I look forward to seeing this technology more widely adopted,” said Frances Arnold, Ph.D., a professor of chemical engineering at Caltech who was not affiliated with the study. The researchers call their tool µSCALE, for Single Cell Analysis and Laser Extraction. The “µ” stands for the microcapillary glass slide that holds the protein samples. The slide is roughly the size and thickness of a penny, yet in that space a million capillary tubes are arrayed like straws, open on the top and bottom. The power of µSCALE is how it enables researchers to build upon current biochemical techniques to run a million protein experiments simultaneously, then extract and further analyze the most promising results. The researchers first employ a process termed “mutagenesis” to create random variations in a specific gene. These mutations are inserted into batches of yeast or bacterial cells, which express the altered gene and produce millions of random protein variants. A µSCALE user mixes millions of tiny opaque glass beads into a sample containing millions of yeast or bacteria and spreads the mixture on a microcapillary slide. Tiny amounts of fluid trickle into each tube, carrying individual cells. Surface tension traps the liquid and the cell in each capillary. The slide bearing these million yeast or bacteria, and the protein variants they produce, is inserted into the µSCALE device. A software-controlled microscope peers into each capillary and takes images of the biochemical reaction occurring therein. Once a µSCALE user identifies a capillary of interest, the researcher can direct the laser to extract the contents of that tube without disrupting its neighbors, using an ingenious method devised by Baer. “The beads are what enable extraction,” Baer said. “The laser supplies energy to move the beads, which breaks the surface tension and releases the sample from the capillary.” Thus, µSCALE empties the contents of a single capillary onto a collector plate, where the DNA of the isolated cell can be sequenced and the gene variant responsible for the protein of interest can be identified. “One of the unique features of µSCALE is that it allows researchers to rapidly isolate a single desired cell from hundreds of thousands of other cells,” said Chen, who wrote the software to examine and detect signs of interesting protein activity within the test tubes. Promising variants can be collected and reprocessed through µSCALE to further evolve and optimize the protein. “This is an exciting new tool to answer important questions about proteins,” Cochran said, likening µSCALE to the way that high-throughput tools for gene analysis have allowed researchers to unlock key features of biology underlying human disease. The project began five years ago when Baer and study co-author Ivan Dimov, Ph.D., a visiting instructor and Siebel Fellow at the Stanford Institute for Stem Cell Biology and Regenerative Medicine, developed the first instrument. They showed how to identify cell types in a microcapillary array and extract a single capillary’s contents using glass beads and a focused laser. About three years ago, Cochran and Baer joined forces to develop µSCALE for protein engineering, and the team devised three experiments to showcase µSCALE’s utility and flexibility. In one experiment, researchers sifted through a protein library produced in yeast cells to select antibodies that bound most tightly to a cancer target. Antibodies with a high target-binding affinity are known to be effective against cancer. In a second example, they engineered a bright orange fluorescent protein biosensor. Using µSCALE, they did this almost 10 times faster than previous methods. Such biosensors are often used as tags in a wide variety of biology experiments. A third experiment, carried out with Daniel Herschlag, Ph.D., professor of biochemistry and a co-author of the study, used µSCALE to improve upon a model enzyme. “This system will allow us to explore the evolutionary and functional relationships between enzymes, guiding the engineering of new enzymes that can carry out novel beneficial reactions,” Herschlag said.

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