Epigenetic pre-programming of human ES cells that leads to differentiation bias

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01385
ICOC Funds Committed: 
$0
Public Abstract: 
Human embryonic stem cells (hESCs) carry great potential for cell replacement therapies, since they can differentiate into number of different cell types. Therefore, it is crucial to understand the differentiation and proliferation of hESCs. We previously demonstrated that not all hESCs were created equal by documenting that two NIH hESC lines (i.e., HSF1 and HSF6) generated human neurons with different characteristics when they were differentiated into neural lineages under the same differentiation procedure. Specifically, HSF1 generates cells of forebrain origin while HSF6 gives rise to more posterior and non-forebrain neurons. These findings clearly indicate a differentiation bias between hESCs. Our knowledge on the control of cell-fate specification has been based on studies using animal models. However, not only the human genome is different from those of animals, but also the epigenetic blueprints differ significantly between human and animal stem cells. The epigenetic blueprints determine the specificity, timing, or quantity of expression of particular genes or cohorts of genes. In this proposal, we will reveal the unique epigenetic blueprints of several hESC lines that define their differentiation bias when they are allowed to differentiate along the neural lineage. Our results will reveal the link between the epigenetic blueprint and the differentiation bias of hESCs or induced pluripotent stem (iPS) cells. The approach of this proposal will enable us to predict the differentiation bias of a particular hESC or iPS cell line in the future based on its epigenetic blueprint without laboriously going through differentiation procedures for each lineage to empirically determine whether a particular line can be differentiated into a particular cell lineage. This approach will save tremendous amount of time and resources when the cells are planned to be used for therapeutic applications. Furthermore, our proposed study will reveal whether we can rewrite the epigenetic blueprint in hESCs or iPS cells using number of different factors. Taken together, understanding the genetic and epigenetic mechanisms underlying hESC differentiation bias may shed light on the nature of the developmental program and suggest strategies for controlling hESC and iPS cell differentiation.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Our preliminary findings demonstrated that certain hESC lines show differentiation bias when they are allowed to differentiate along the neural lineage in culture. In our application, we propose to reveal the unique epigenetic blueprints of several hESC lines that define the differentiation decision of hESCs. The unique epigenetic blueprints may arise from embryo-specific genetic variations or stochastic events accumulated during in vitro culturing. Such a problem, though initially surfaced when dealing with different hESC lines, might also apply to pluripotent stem cells that are derived using the induced pluripotent stem (iPS) cell technology. Our proposed studies will help us understand the genetic and epigenetic mechanisms underlying hESC differentiation bias and it may shed light on the nature of the developmental program controlling hESC and iPS cell differentiation. Numerous residents of California suffer from diseases that could potentially be cured by using stem cell based therapies. In order to be able to use hESCs and/or iPS cells for therapy, better understanding of their differentiation is absolutely needed. There is no question that the information obtained from the experiments of this proposal will benefit the residents of California with respect to stem cell therapy.
Progress Report: 
  • Induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine: these cells are similar to embryonic stem cells (ESCs) but can be derived upon “reprogramming” of any mature cell type isolated from a patient. Thus, tissue-specific stem cells derived from iPSCs and re-injected into the same patient may not trigger immune rejection. However, before the full potential of iPSCs is achieved, we need to learn how to better generate these cells, control their maturation into tissue-specific stem cells and progenitors, and harness their tumorigenic potential. Interestingly, ESCs and iPSCs share many characteristics of cancer cells, including their unlimited proliferation potential, and emerging evidence suggests that the mechanisms underlying the infinite proliferation of cancer cells and ESCs are intimately intertwined. Similarly, the progressions stages of tumorigenesis and cellular reprogramming to iPSCs share several characteristics, including changes in the packaging of the chromosomes.
  • Based on these observations, we proposed to directly study the function of a major cancer pathway, the RB pathway, in cellular reprogramming and iPSCs. RB is a key tumor suppressor in humans. RB acts as a cellular brake that restricts cell division but has several other cellular functions, including in the control of cellular maturation. When RB is mutated, cells divide faster and become more immature, two features of cancer cells, but also of cells undergoing reprogramming. We hypothesized that RB is an important regulator of cellular reprogramming and will test this idea using mouse and human cell types in culture. In the last year, we have performed experiments that largely support this hypothesis. We have found that, similar to its role in normal cell cycle, RB acts as a brake to normally restrict the reprogramming of cells into iPSCs. We have also found that RB is regulated in cells by enzymes that normally control the coating structure of chromosomes; these enzymes are thought to play a role in reprogramming, suggesting that RB may be a critical regulator of reprogramming by controlling the ability of reprogramming factors to modify the structure of the DNA. These experiments now provide a powerful system to analyze the molecular mechanisms underlying cellular reprogramming.
  • Our general goal is to better understand the differences and similarities between cancer cells and embryonic stem cells, to prevent tumor formation following stem cell transplantation but also to gain novel insights into the mechanisms of tumorigenesis and into the biology of embryonic stem cells. To this end, we have been studying how a tumor suppressor named Rb controls the dedifferentiation (or "reprogramming") of cells into induced pluripotent stem cells (iPS cells), which are similar to embryonic stem cells. (ES cells).
  • We have found that, similar to other tumor suppressors such as p53, Rb normally restricts the reprogramming process, both in human and mouse cells. We have also found that loss of RB does not change the proliferation rate of cells during reprogramming, suggesting that the enhanced efficiency of reprogramming observed in the absence of Rb is not due to a simple increase cell number. We are currently investigating the mechanisms by which Rb normally restricts the reprogramming process.
  • Our overarching goal is to understand the mechanisms controlling the balance between stem cell pluripotency, self-renewal, and tumorigenesis, to harness the full therapeutic promise of human embryonic stem cells (hESCs). To this end, we study the function of the RB gene family in stem cells. Our initial hypothesis was that RB family genes may control the reprogramming of somatic cells into iPSCs by interacting with chromatin remodeling factors to induce specific changes in the chromatin structure and control the expression of a specific program of genes. We found that loss of RB, but not of its family members p107 and p130, results in enhanced reprogramming of fibroblasts to iPS cells. In the past year, we have investigated this unique function of RB. In particular, we have performed high throughput RNA-seq and ChIP-seq experiments for RB early in the differentiation process to explore the mechanisms by which loss of RB may enhance reprogramming. We have also performed ChIP-Seq experiments with various chromatin marks to explore the relationship between RB loss and change sof the chromatin structure of cells early in reprogramming.
  • During the reporting period, we have pursued our work on the role of the retinoblastoma tumor suppressor during the reprogramming of mouse and human cells into induced pluriptoent stem cells (iPS cells). We have performed and analyzed genome-wide RNA-seq and ChIP-seq experiments to investigate how loss of RB promotes reprogramming. We have also tested candidate downstream mediators of RB in reprogramming using mouse genetics in vivo.

© 2013 California Institute for Regenerative Medicine