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.