Functional characterization of mutational load in nuclear reprogramming and differentiation
One of the most potentially powerful aspects of regenerative medicine is stem cell therapy. In this therapy, healthy tissues derived from stem cells will be implanted into patients with damaged tissue in order to restore function. However, there is currently a risk of immune rejection. Human induced pluripotent stem (hiPS) cells have the potential to revolutionize regenerative medicine. By reprogramming a patient's own cells into pluripotent stem cells, stem cell therapies can be performed with little to no risk of rejection. However, this nuclear reprogramming process is not well understood at a mechanistic level. Also, all procedures developed to date use cancer-related genes. This has raised fears that current hiPS cells could potentially have a high cancer risk if used therapeutically. In this proposed research, we intend to study the mutation load associated with reprogramming and the functional consequences of the mutations. We will use the obtained knowledge about transformation to develop safer methods of generating hiPS cells.
We will perform large-scale screening of somatic mutations that have potential deleterious effects during the reprogramming of human primary cells into hiPS cells, and the differentiation of hiPS cells into somatic cell types. We will also characterize whether mutations occurred have any function, including the increase of cancer risk. These information will help us to understand how do the mutations occur and propagate.
This proposed project will not only help us to gain additional mechanistic insights on nuclear reprogramming, but also allow great progress towards functional stem cell therapy, as safe hiPS cells will be available for therapeutic use.
Over the past decade, California has made great progress in stem cell research thanks to Proposition 71. Research into stem cell properties and applications has made the promise of regenerative cell therapies almost a reality. Human induced pluripotent stem (hiPS) cells offer the promise of treatments or cures for diseases that affect millions of people without a risk of immune rejection, including Alzheimer's disease, heart disease, organ failure, and spinal cord injury. However, before these cells can be used therapeutically in the clinic, a better understanding of the mechanisms of generating hiPS and the safety of the resultant cells must be gained. Our work will greatly improve understanding of the reprogramming process with respect to genomic mutations and integrity by determining the relative safety of a variety of available hiPS reprogramming techniques. We will work towards creating hiPS reprogramming methods that have been proven to be non-tumorigenic. Through this research, we hope to clear one of the biggest hurdles stopping hiPS cells: the risk of cancer. Due to the huge potential of stem cell therapies in regenerative medicine, this work has applications to a large number of diseases and genetic disorders that affect Californians, from infants to senior citizens.
In the first year of the funded period, we have performed genetic screening on a large panel of human induced pluripotent stem cell lines, and identified mutations in all lines tested. Functional characterization of these mutations showed that the majority of them did not facilitate the reprogramming of somatic cells towards the pluripotent state.
In the second year of this project we continue our efforts on characterizing genetic abnormalities occurred in the conversion of adult cell types into pluripotent stem cells. We have greatly expanded the scale of the efforts, in terms of number of samples and the regions of genome analyzed.
In this 3-year project, we used DNA sequencing to characterize the accumulation of genetic abnormalities during the process of reporgramming adult cells back to induced pluripotent stem cells. We found that genetic mutations can occur at multiple stages of this process, and are present in different regions of the genome. The majority of such mutations do not seem to have a detectible function.
- Stem Cell Reports (2014) Mouse SCNT ESCs Have Lower Somatic Mutation Load Than Syngeneic iPSCs. (PubMed: 24749065)
- Cell Res (2013) Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during germ cell reprogramming. (PubMed: 23399596)
- Nat Commun (2013) Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming. (PubMed: 23340422)
- Cell Rep (2013) The Presenilin-1 DeltaE9 Mutation Results in Reduced gamma-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells. (PubMed: 24239350)
- Nature (2012) Tet1 controls meiosis by regulating meiotic gene expression. (PubMed: 23151479)
- Proc Natl Acad Sci U S A (2012) Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. (PubMed: 22991473)