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

Richard A. A. Stein M.D., Ph.D.

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.

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]

Heterogeneity Persists

“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.

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.]

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.

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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