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Sep 1, 2013 (Vol. 33, No. 15)

Manipulating Methodologies for Reprogramming Cells

  • Early-Stage Cells

    “We found a variety of proteins that are selectively secreted by early-stage pancreatic lesions,” says Kenneth S. Zaret, Ph.D., professor of cell and developmental biology at the University of Pennsylvania Perelman School of Medicine.

    The absence of good disease progression markers, frequent delays in detection, and the five-year survival rate that approximates 6%, are major challenges for the management of pancreatic adenocarcinoma.

    To establish an experimental system that facilitates the identification of blood markers associated with disease progression, Dr. Zaret and his colleagues allowed pancreatic ductal adenocarcinoma cells to undergo reprogramming to the pluripotent state, and hypothesized that some cells would subsequently redifferentiate into adult pancreatic tissue and recapitulate early stages of cancer development.

    While cells derived from most of the initial tumor samples did not exhibit malignant genotypes, one cell line generated pancreatic intraepithelial neoplasia precursor cells that, in a mouse model, progressed to the invasive stage.

    “This approach enabled us to obtain a very rare cell line after scanning many tumors, and additional changes might have allowed these rare events to be identified,” Dr. Zaret says.

    Introducing early-stage human cancer cells into the mouse model could allow the longitudinal identification of markers that could predict progression to invasiveness. “In this case, the animal would serve as a surrogate for humans to determine blood markers,” says Dr. Zaret.

    The in vitro culturing of early-stage cells provided the opportunity to perform proteomic analyses to identify secreted proteins that could serve as potential markers. “We wanted to determine which proteins are stably detected in the bloodstream of mice harboring human lesions that have progressed to the invasive stage,” he says.

    This strategy revealed that the combined detection of protein products of the HNF4α, TGFβ, and integrin networks could represent an early noninvasive marker to predict pancreatic adenocarcinoma progression.

    “After establishing this system in the animal model, we want, together with other investigators, to analyze how well some of these candidate markers can predict invasiveness in humans,” Dr. Zaret says. This system also helps circumvent one of the major challenges hindering the study of cancer progression in humans, the heterogeneity of the lesions that are present upon diagnosis in patients. “But because we can use human cells that progress from early to invasive stages, we are in a position to longitudinally assess dynamic changes, and gain new insights into disease progression,” he adds.

  • Allelic Exclusion

    “Allelic exclusion has historically been a poorly understood topic,” says Howard Cedar, M.D., Ph.D., professor of biochemistry and genetics at the Hebrew University of Jerusalem.

    Described in cells of the immune, olfactory, and other systems, allelic exclusion refers to the process of expressing only one allele of a gene, while the other one remains silent. The human immune system is able to generate an estimated 10 billion different types of antibodies, a recombinatorial diversity that is mostly controlled at the level of DNA rearrangements. “But every antibody-producing cell ends up synthesizing only one type of antibody, even though the existence of two alleles makes it theoretically possible to produce a different type of antibody from each allele, so there is a paradox here,” says Dr. Cedar.

    Allelic exclusion at the immunoglobulin locus, intensely debated over time, was initially thought to occur through a feedback process, in which rearrangements at one allele could signal to switch the other allele off. “It turns out that a different mechanism is in place,” Dr. Cedar says.

    He and his colleagues have revealed, for the first time, the possibility to identify the allele that will undergo rearrangements and become functional based on particular chromatin signatures that involve specific histone acetylation and methylation patterns. “We can already identify the allele that is marked to be used for recombination, in a cell that does not even know yet that it will produce antibodies,” he explains.

    Dr. Cedar and colleagues further observed that in the region encoding the immunoglobulin genes, one allele always replicates early during the cell cycle, while the other one replicates late, a phenomenon termed asynchronous replication.

    “It is always the early-replicating allele that will undergo rearrangement,” Dr. Cedar says. In pre-B and B cells this phenomenon is clonal, meaning that the same replication pattern will be maintained for all the progeny of a cell and after subsequent cell divisions, as well.

    Not all cells exhibit this pattern, though. “Adult hematopoietic stem cells do not maintain the same allelic replication pattern as they divide,” Dr. Cedar says.

    Even though these cells also exhibit asynchronous replication, one of their distinguishing features is that the early- and late-replicating alleles alternate during successive generations. “This makes adult hematopoietic cells pluripotent in terms of their allelic choice,” he explains.

    This phenomenon reflects a new dimension of stem cell plasticity. “Maternal or paternal alleles can be expressed until a certain point during stem cell development, until they make a decision, and after that this pattern becomes fixed and it cannot change any longer,” Dr. Cedar continues.

    As these and many other advances illustrate, cellular reprogramming transcends interdisciplinary boundaries and is set to answer biological questions from areas that, historically, were not thought to be associated with stem cell biology or with development. Along with regenerative medicine, fields such as immunology, oncology, and chromatin biology also stand to benefit from advances in cellular reprogramming. While translating research findings to clinical benefits still has to surpass significant hurdles, the prospect to reshape the therapeutic arena is witnessing a transformative era.

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