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

Manipulating Methodologies for Reprogramming Cells

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    Neurons reprogrammed from fibroblasts: The green color labels beta-3 tubulin, which is a specific neuronal marker, and the red color labels synapsin, which is another neuronal marker for synaptic interactions. [Sheng Ding, Ph.D., UC-San Francisco]

    A stride that marked the turn of the century—realizing the possibility of reprogramming embryonic and adult fibroblasts into pluripotent stem cells by the co-expression of only four gene products—opened a vibrant chapter in the biomedical sciences.

    In the short time that has elapsed since that seminal discovery, new and increasingly versatile strategies have been proposed to develop reprogramming protocols that are not only cost- and time-effective, but also safer.

    “Currently, a major interest in the field of stem cell research is the possibility of reprogramming fibroblasts into other tissue-specific cell types without reverting to the pluripotent state,” says Sheng Ding, Ph.D., professor and investigator at the University of California, San Francisco, and the Gladstone Institute of Cardiovascular Disease.

    Because pluripotent stem cells that fail to undergo differentiation are tumorigenic, such a direct reprogramming approach presents a superior safety profile. “We can use this type of reprogramming in tissue culture, ex vivo, to manufacture large numbers of functional cell types,” Dr. Ding says.

    Another strategy is to deliver certain drugs into the region of tissue damage and induce in situ reprogramming. Dr. Ding and his colleagues recently developed two different paradigms for cell reprogramming. One of them relies on cell type-specific factors that can be delivered into fibroblasts to generate the desired cell type. “Another approach that we developed, which provides a more universal paradigm for reprogramming, uses generic reprogramming factors only for a short period of time, to epigenetically activate fibroblasts, and those plastic fibroblasts are subsequently exposed to a different type of signal to fully differentiate,” he explains.

    Using this strategy, Dr. Ding and his colleagues generated cardiac, neural progenitor, and blood vessel endothelial cells, without reverting to pluripotency as an intermediate step. This process very much resembles the one encountered in other species in vivo, wherein regeneration can occur through the transient, low-level expression of stem cell factors. “In ongoing studies, we have additionally shown the possibility of generating pancreatic cells and hepatocytes by direct reprogramming,” he says.

  • Heterogeneity Persists

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    Red blood cells carrying pathologic mitochondria, which are shown by the dark staining iron granules around the circumference of the nucleus. These abnormal red blood cells (sideroblasts) are an indication of bone marrow failure in Pearson’s syndrome, which a team at Children’s Hospital Boston modeled in vitro using iPS cells. [Suneet Agarwal, M.D., Ph.D.]

    “There is long-term value in developing strategies to deliver cells as medicines,” says George Q. Daley, M.D., Ph.D, professor of hematology and oncology at Children’s Hospital Boston.

    Significant research efforts in Dr. Daley’s group are focusing on developing blood stem cells for clinical applications. He and his colleagues recently revealed that the highly conserved Notch signaling pathway, previously implicated in several hematopoietic conditions, plays a critical role in the early stages of hematopoiesis in human embryonic and induced pluripotent stem cells (iPSCs), a finding that is opening new avenues toward understanding hematopoietic fate specification.

    Particularly for blood cell diseases with a known genetic basis, cell-based therapies are emerging as a promising option, but safety considerations—due in part to possible immune responses—are a limiting factor. The use of iPSCs, in which the genetic defect is corrected, may circumvent this concern. However, an insufficiently understood phenomenon in cellular reprogramming revolves around the notion that not all iPSCs are identical. Significant functional variability, defined as differences between embryonic stem cells and iPSCs, as well as clone-to-clone heterogeneity were found, and appear to be shaped by a combination of genetic and epigenetic contributions.

    “It is perplexing how unpredictable some stem cells are, and we do not yet have a good understanding of this phenomenon,” Dr. Daley says.

    Stem cell variability and heterogeneity appear to be more accentuated for certain cell types and pose major roadblocks in implementing clinical applications. Dr. Daley and his colleagues are focusing on translating a technology to generate platelets of sufficient robustness for stem cell-based therapies.

    “We are trying to determine how many cell lines we need to generate, to reasonably expect to find one with the desirable performance properties, and this has to be balanced against the extremely high costs,” he says.

  • Complex Perturbations

    “About a decade ago, Rafael Irizarry [Ph.D.] and I developed a genomic array-based method to look in an unbiased way at methylation over a region encompassing a large fraction of the methylome,” says Andrew P. Feinberg, M.D., professor of molecular medicine at Johns Hopkins University School of Medicine.

    Taking this approach, known as comprehensive high-throughput array-based relative methylation, the researchers revealed that most tissue-specific DNA methylation differences, which historically were thought to be confined at CpG islands, are in fact situated at regions within approximately two kilobases from their boundaries, in the so-called CpG island shores. Whole-genome bisulfide sequencing showed that the sharp demarcation between the high methylation found at CpG islands and the lower methylation found at CpG island shores is lost in cancer, and the shift of the boundaries toward the CpG islands or away from them results in aberrant methylation patterns.

    An additional stride toward understanding genome-wide DNA methylation in disease came about when, in a comparison between colorectal cancer and normal colorectal mucosa from the same patients, the researchers identified large chromosomal blocks of DNA, extending over approximately half the genome, that become hypomethylated in cancer. Largely corresponding to structures known as LOCKs (large organized chromatin lysine modification regions), these regions also show an approximately 80% overlap with lamin-associated domains, which were found microscopically and are thought to be associated with the nuclear membrane. These genomic regions, which correspond to heterochromatin and are highly methylated in normal cells, increase in size during differentiation and are lost in cancer.

    Collectively, these findings unveil a much more complex set of epigenetic perturbations characterizing the malignant state than previously thought. “A general dysregulation of DNA methylation occurs in cancer,” Dr. Feinberg explains.

    These structural insights into genome-wide DNA methylation led to a key observation. “We found an enormous overlap in the regions that are altered in several types of cancer and in those that are changed during stem cell reprogramming, and both of them are very much related to regions where DNA methylation normally changes in a tissue-specific manner,” he says.

    This pointed the researchers toward the possibility that, epigenetically, cancer cells may acquire methylation patterns similar to the ones that are found in other tissue types. By using experimental and modeling approaches to integrate these findings, Dr. Feinberg and his colleagues revealed that the defining epigenetic trait in cancer is not so much the nature of the DNA methylation changes, as their stochasticity.

    “There is some type of randomization of the methylation pattern, and as a result a tumor might be much better defined by its departure from the normal epigenetic pattern than by a switch to a different pattern,” Dr. Feinberg says. This is reminiscent of the stochasticity that can be observed during normal development, where highly variable DNA methylation patterns are reported even in genetically identical animals. “We think that the same is true in cancer, and the opening of this stochasticity allows cancer cells to be selected and to gain a growth advantage at the expense of the host,” he adds.

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