June 1, 2011 (Vol. 31, No. 11)
Richard A. A. Stein M.D., Ph.D.
Both Natural and Experimental Processes Stand to Benefit from Greater Understanding of Nongenetic Factors
The mechanisms that ensure that cells from multicellular organisms are genetically homogenous, but structurally and functionally heterogeneous, represent one of the most intriguing areas in biology. Increasing numbers of studies reveal that genetics alone cannot explain all aspects related to development and differentiation. At the same time, the importance of nongenetic or epigenetic factors is supported by several lines of evidence, including health problems that develop in certain cloned animals, possibly as a result of improperly expressed imprinted genes, or the causal link between adversity during pregnancy and adult-onset diseases later in life.
Many embryonic stem cells that are used in regenerative medicine are generated by in vitro approaches, and previous studies have linked in vitro manipulation to certain diseases, such as Angelman and Prader-Willi syndromes, possibly as a result of abnormal genomic imprinting.
“When performing in vitro culturing of cells or embryos, it is very important to check their epigenetic status,” says Izuho Hatada, Ph.D., professor at the laboratory of genome science, Institute for Molecular and Cellular Regulation, Gunma University.
After comparing the methylation pattern and gene-expression status of embryonic stem cells derived from in vivo and in vitro generated blastocysts, Dr. Hatada and colleagues reported that embryonic stem cells established in vitro exhibited, at the very early passages, increased abnormal genomic imprinting as compared to cells that were established in vivo.
“We are interested in learning what causes these abnormal epigenetic states,” explains Dr. Hatada. Dr. Hatada’s group recently found that Gadd45b, a putative demethylation factor that is implicated in the stress signaling pathways, was upregulated during in vitro culturing conditions when compared to in vivo embryonic stem cells, a finding that explains, at least in part, the increased demethylation and decreased methylation that were observed in several genes from early-passage embryonic stem cells.
“One of the most important things in regenerative medicine is to check the epigenetic status of the genes and learn more about how to normalize it before clinical medicine applications.”
One of the most widely used techniques to create a clonal embryo, with applications in regenerative medicine, reproductive cloning, and biomedical research, is somatic cell nuclear transfer. During this in vitro approach, the donor nucleus from a somatic cell is inserted into an ovum from which the nucleus has been removed in advance. Subsequent to the transfer, the donor nucleus is reprogrammed by the host cell and initiates divisions to form a blastocyst. Increasing numbers of studies have revealed that this nuclear reprogramming process is the result of epigenetic changes.
Theodore P. Rasmussen, Ph.D., associate professor in pharmaceutical sciences at the University of Connecticut, and colleagues, recently reported that during somatic cell nuclear transfer, nuclear reprogramming likely occurs by the same chromatin remodeling mechanisms that reshape the genome immediately after fertilization.
This work also revealed that reprogramming of the somatic cell heterochromatin is linked to epigenetic remodeling activities present in the recipient oocyte. An activity that occurs in the ooplasm strips MacroH2A, a unique histone variant originating from the maternal protein pool, which is eliminated shortly after fertilization and starts being synthesized in the embryo three cell divisions later, at the 16-cell stage, when it is assembled into facultative heterochromatin.
Research in Dr. Rasmussen’s lab showed that soon after the somatic cell nuclear transfer, MacroH2A is first stripped from the chromosomes and subsequently degraded in a process that requires intact microtubules and nuclear envelope breakdown. “It is becoming quite clear that a lot of things happen that change the dynamics of chromatin as the zygote transitions to the blastocyst and beyond that stage as well,” explains Dr. Rasmussen. “But exactly how a cell regulates its epigenetic information is still a mystery.”
“The epigenome is becoming a very exciting field of study,” says Jeanne F. Loring, Ph.D., professor in the department of chemical physiology and founding director of the Center for Regenerative Medicine at the Scripps Research Institute. “And it makes perfect sense, because the interest should be in the activity of the genome, not just the genome that is sitting there with its sequence.”
Recently, Dr. Loring and colleagues described a new approach to characterize the global miRNA profile of human embryonic stem cells and several types of differentiated cells. This work, which reportedly provided the most comprehensive set of differentially regulated miRNAs in human embryonic stem cells to date, also revealed that miRNAs associated with human embryonic stem cells occur in genomic clusters. Two clusters that previously had not been associated with this cell type were unveiled in this research.
Oncogenic miRNAs were overrepresented among miRNAs that were upregulated, and tumor suppressor miRNAs were overrepresented among miRNAs that were downregulated in human embryonic stem cells, suggesting certain self-renewal mechanisms that are shared with cancer cells. “We want to understand the remarkable state of pluripotency; no other cells are like that, and we are trying to find out how epigenetics plays a role in their reprogramming and differentiation.”
An important research effort in Dr. Loring’s group focuses on DNA methylation, including the methylation of cytosine residues from CpA and CpG sites. Dr. Loring and colleagues recently conducted a whole-genome comparative analysis of DNA-methylation dynamics in three cell types at progressive differentiation stages: pluripotent human embryonic stem cells, fibroblast-like cells differentiated from them, and primary neonatal foreskin fibroblasts.
This work revealed certain features that are shared by undifferentiated and differentiated cells, such as the association between promoter hypomethylation and gene hypermethylation on one hand, and increased transcription on the other. An unexpected finding emerging from this analysis is the increased methylation of exons relative to introns and the sharp methylation transitions at exon-intron boundaries, pointing toward the potential involvement of differential methylation in the coupling between transcription and gene splicing. Other epigenetic features, such as global methylation and non-CpG methylation levels, correlated with the developmental stage and were highest in human embryonic stem cells.
“It is important to remember that the epigenome is impossible to interpret if we do not know a lot about the genome. We are still in those gray days, when we are looking at the actual DNA sequence, lots of data is coming out, and we are discovering a lot of sequence variation. But one major question is how to reliably identify changes that are associated with disease,” says Dr. Loring.
One of the challenges associated with generating specific cell types from embryonic stem cells is the need to first differentiate them into homogenous cellular populations that can be maintained long-term. Recently, Sheng Ding, Ph.D., senior investigator and professor at Gladstone Institutes and The University of California, San Francisco, and colleagues, were able to convert human embryonic stem cells grown as a monolayer into homogenous primitive neuroepithelial cells, and most significantly developed a small molecule cocktail that can maintain the primitive nature of the neural cells over unlimited generations.
This method allowed the uniform capture and stable maintenance of these cells and, in addition, emerges as the fastest and most efficient approach to generate primitive neural stem cells from human embryonic stem cells to date.
An exciting development in Dr. Ding’s group is the use of small molecule inhibitors for cellular reprogramming. “We recently developed a new reprogramming paradigm that allowed us to convert fibroblasts into cardiomyocytes by a method that is fundamentally different from what people have done before.”
By briefly reactivating and overexpressing four murine transcription factors, Dr. Ding and colleagues were able to reprogram embryonic and adult fibroblasts into beating heart cells over an 11–12 day period and demonstrated, by several methods, that a pluripotent embryonic-like stem cell intermediate was not involved. This innovative approach allowed differentiated cells to be generated almost three times faster than by alternative methods and has profound therapeutic implications beyond cardiogenesis, for several clinical areas.
Still at an early stage, the possibility of reprogramming cells in this manner promises important therapeutic applications. “We have lots of proof-of-concept demonstrations that cells can be reprogrammed by small molecules under defined conditions,” explains Dr. Ding. “Those conditions are not perfect, they are not yet practical for clinical applications, but we are moving forward and we are getting better conditions to generate high-quality cells.”
“It is important to think about reprogramming from the natural side, rather than only from the experimental side, because embryonic pluripotent stem cells and early embryos appear to have some capacity to reprogram their own epigenomes,” explains Wolf Reik, M.D., head of the epigenetics laboratory at the Babraham Institute.
Dr. Reik’s laboratory focuses on natural epigenetic reprogramming, a phenomenon that was first discovered a little over 10 years ago when several groups described genome-wide DNA-methylation loss that occurs subsequent to fertilization in the pre-implantation mouse embryo, up to the blastocyst stage.
“We have been trying to examine natural reprogramming from a mechanistic point of view and to understand its biological significance.” Investigators in Dr. Reik’s laboratory recently described the active demethylation of 5-methylcytosine to generate 5-hydroxymethylcytosine as a novel modification visualized in the paternal pronucleus of mouse, bovine, and rabbit zygotes. The conversion of 5-methylcytosine to 5-hydroxymethylcytosine in embryonic stem cells is mediated by TET1 and TET2, enzymes that are highly expressed in these cells.
In an analysis of the genome-wide pattern of methylation and hydroxymethylation during differentiation of murine embryonic stem cells, Dr. Reik’s team found decreased hydroxymethylation along with increased methylation and gene silencing at embryonic stem cell promoters. This indicates that 5-hydroxymethylcytosine plays important roles in genome-wide methylation reprogramming, and the balance between global hydroxymethylation and methylation appears to be tightly linked to the balance between pluripotency and lineage commitment.
Understanding epigenetic modifications during natural reprogramming has multiple clinical applications. Regenerative medicine is one area that will benefit, and assessing the epigenetic status of embryonic stem cells promises interventions to therapeutically change reprogramming when needed.
“A broader area is in common adult diseases in humans, where genetic explanations are still not satisfactory,” says Dr. Reik. While genetic variants were unveiled and characterized for many adult-onset complex human diseases, most frequently, a large part of the risk cannot be explained by genetic factors alone. “It is this area where knowledge about epigenetic variants, and how they contribute, and epigenetic modifiers, will be important.”
Epigenetics promises to unveil fundamental mechanistic details about natural and experimental cellular reprogramming during differentiation and development. Some of the most recent developments in this field underscore the central role of epigenetic and genetic factors that, in combination, shape these processes. A better understanding of the players and signaling pathways promises not only to unravel the mysteries that surround cellular reprogramming, but also to open important therapeutic avenues.