The role of RNA in regulating de novo DNA methylation in hESC differentiation

Funding Type: 
Basic Biology II
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
iPS Cell
Public Abstract: 
Our goal is to understand the processes that control how human embryonic stem cells differentiate to become various distinct cell types. The control of several important genes that determine this process relies on a process known as DNA methylation. DNA methylation involves the reversible "decoration" of our DNA at specific gene locations, resulting in the silencing of that gene. This is a key step in the commitment of these cells to particular differentiated endpoints. Understanding what controls the DNA methylation and silencing of key genes in human embryonic stem cell differentiation is the goal of this proposal. RNA can regulate how proteins function in the cell, and based on many examples involving DNA methylation, we will test if RNA controls when and where DNA methylation occurs during the differentiation of human embryonic stem cells. We propose to use methods relying on the use of human embryonic stem cells and purified enzymes that carry out the DNA methylation itself. The identity of RNA molecules that bind to these enzymes will be determined by two distinct methods, and the biological importance of these RNA molecules to embryonic stem cell differentiation will be determined. The mechanism of how these RNA molecules alter the function of the the DNA methyltransferase enzymes will be determined. The realization of our goals is anticipated to provide the basis for controlling the function of the critical DNA methyltransferases, which has already been demonstrated to be important in influencing the cellular differentiation process.
Statement of Benefit to California: 
The ability to use human embryonic stem cells depends on being able to control their differentiation. Thus, while these cells have the potential to become a different cells within different human tissues, our current ability to direct such changes is limited. This proposal will benefit the State of California and its citizens by providing improved ways of achieving this goal, of being able to direct these cells to particular differentiated cell types.
Progress Report: 
  • Public Summary of Scientific Progress
  • The discovery of induced pluripotent stem (iPS) cells has generated much excitement, because, like embryonic stem (ES) cells, they have the potential to differentiate into every cell type in the body, yet their derivation is straightforward technically and does not require the use of human embryos. There are three related challenges that must be met before iPS cells can be applied to regenerative therapy: 1) Identify methods that improve the efficiency of iPS cell generation and the quality of derived iPS cells, 2) determine whether iPS cells can function identically to ES cells, which remain the gold standard, and 3) identify methods that can control the differentiation of iPS cells into the cell types desired for regenerative therapy. Current methods for iPS cell derivation are highly inefficient: only a small percentage of any human adult cell population can be successfully de-differentiated to an ES-like state. Directing the differentiation of iPS or ES cells is also highly inefficient: in every directed-differentiation method, only a small percentage of cells differentiate into the desired cell type. Therefore, understanding the behavior of individual cells within a mixed population is the level of detail required to understand the processes that will ultimately improve the methodologies utilized for their application to regenerative medicine.
  • A new proteomic technology called mass cytometry, developed by Dr. Scott Tanner at the University of Toronto, was recently applied in the Nolan lab to measure over 40 (eventually 100) parameters simultaneously at the single cell level. Mass cytometry uses rare earth metal-tagged antibodies to label cells both on the surface and inside the cell, then vaporizes those cells at 13,000 degrees Fahrenheit and counts how many of each of the different tags a cell had at the time of its demise. During this year a critical aspect of this study was the development of new software tools to organize and interpret the high-dimensional data sets generated by mass cytometry. An effective and informative means for displaying this multi-dimensional data was the application of Spanning-tree Progression Analysis of Density normalized Event (SPADE analysis), which creates an intuitive 2D representation of multi-dimensional data. SPADE recovers the underlying branching and continual structure of high-dimensional cytometry data by recognizing the subtle differences between cellular phenotypes in n-dimensional space (where n is the number of markers acquired) and tracing them in a straightforward manner. The entire n-dimensional hierarchy is visualized in two dimensions as SPADE trees. Furthermore, in this past year we developed “Time-SPADE” analysis in which trees are built sequentially by time point, allowing for time course analysis of the changing cell populations that occur during the highly dynamic processes of iPS cell derivation and ES/iPS cell differentiation.
  • In our continuing collaboration with Dr. Marius Wernig at Stanford, a recognized leader in the biology and methodologies of iPS cell reprogramming, we applied mass cytometry and SPADE analysis to study the behavior of single cells during reprogramming. Using specific metal-tagged antibody panels that we developed, we were able to trace the molecular path of de-differentiation to the ES-like state, and we showed that the co-expression of the four Yamanaka factors at both the mRNA and protein level is critical for successful reprogramming. To study differentiation in addition to de-differentiation, we applied mass cytometry and Time/SPADE methodologies to trace the behaviors of single cells within an entire sample as they undergo differentiation into defined cell types. For these experiments we were able to trace ES cells as they underwent changes into mesoderm, ectoderm and endoderm, the three cell lineages that give rise to all of our organs. These experiments set the stage for using protocols to induce differentiation into a specific cell type such as neuronal, hematopoietic, cardiac, and pancreatic as just some of the possibilities. Studying these processes at the single cell level with unprecedented detail will facilitate our optimization of iPS cell derivation and differentiation for human regenerative therapy.
  • In the past year, we have leveraged the power of high-dimensional single cell analysis to map the transitions that occur during iPS cell reprogramming, a novel method that allows for the creation of patient-specific pluripotent stem cells with great potential for regenerative medicine. We have also mapped the transitions that occur during differentiation of these iPS cells to cell types such as neural and cardiovascular, a critical step for their eventual application to regenerative medicine therapies. Our step-by-step mapping of these long, multi-step processes at the single cell level reveals multiple new points of control that allow for manipulation and optimization tailored to each patient’s specific therapy needs.
  • Our research is focused on iPS cell reprogramming, a method that allows for the creation of patient-specific pluripotent stem cells with great potential for regenerative medicine. The process of iPS cell reprogramming is long (> 2 weeks) and inefficient (<1%), and therefore time course analysis at the single cell level single cell level is required to identify and characterize the minority population of successfully reprogramming cells. In the past year, we have leveraged the power of high-dimensional single cell analysis to map the transitions that occur during cellular reprogramming. We have identified a key intermediate stage of cellular reprogramming, and we have isolated and characterized this key intermediate population. We have also identified a late-stage non-productive reprogramming trajectory, and characterized the cellular signaling requirements for successful reprogramming to the pluripotent state. We anticipate that this work will lead to improved reprogramming protocols that produce enriched, higher-quality pluripotent stem cells for use in regenerative therapy.

© 2013 California Institute for Regenerative Medicine