The recent breakthrough in stem cell biology on somatic cell reprogramming offers invaluable tool for modeling human genetic disorders. Using fibroblasts from affected ICF syndrome patients, we have the means to generate induced pluripotent stem cell (iPSC) models. By generating DNMT3B-deficient iPSC lines, we will differentiate them to somatic stem cells and terminally differentiated cell types that are associated with ICF syndrome. It is known that DNMT3B mutations can affect genome instability and cell cycle regulation. Therefore, ICF iPSCs and its somatic derivatives will provide a powerful tool to understand the molecular function of DNMT3B in human cell physiology. Our study will shed light on the pathogenesis of ICF syndrome and to broaden potential therapeutic applicability. This proposal research will be an important step to translate stem cell research into future clinical applications.
Statement of Benefit to California:
Stem cells have emerged as a model system to understand molecular and pathogenic mechanisms underlying many human diseases such as neurodegenerative disorders (Parkinson's disease and Alzheimer's disease) and cancer and immunodeficiency. Many of these disorders are due the dysfunction of DNA methylation, a DNA modificaiton mechanism involved in regulating gene activity. Recent technology demonstrated that patient skin cells can be converted into induced pluripotent stem cells, thus generating patient specific stem cells. We propose to generate human stem cells containing genetic mutations in DNA methylation machinery. This allows to study the basic biology of a human disease due to the defects in DNA methylation. Because perturbation of DNA methylation is associated with many human diseases, we expect to learn many fundamental biology from this human stem cell study. Finally, this study will shed light on the utility of combining gene and cell therapy in curing human diseases. Our proposed research will accelerate the pace in the application of human embryonic stem cells and induced pluripotent stem cells for curing immnuodeficiency and neurological disorders, thus directly benefiting the health care of the State of California and its citizens.
In this proposal, the applicant seeks to create a human cell-based model of ICF Syndrome (Immunodeficiency, Centromeric instability and Facial anomalies), a recessive autosomal disorder caused by mutations in a de novo DNA methyltransferase, DNMT3B. This model will be used explore the molecular basis of this complex syndrome in three specific aims. First, the applicant will generate induced pluripotent stem cells (iPSCs) from ICF patients and characterize potential defects in various cellular processes and epigenetic patterning. The goal of Aim 2 is to rescue observed phenotypes by introducing wild-type DNMT3B into ICF-iPSCs. In Aim 3, the applicant will compare differentiation of control, ICF-iPSCs and rescued ICF- iPSCs into a variety of hematopoietic and neural cell types.
Significance and Innovation:
- Reviewers acknowledged that understanding the basic biology of DNA methylation is important to understanding reprogramming and differentiation.
- The proposed studies are mainly descriptive, and there is minimal innovation in the approaches to be employed.
- There is very little exploration of novel mechanisms or pathways with the potential to significantly advance the field. However, reviewers judged the rescue experiments to be innovative, as they may provide insights about when, and if, cellular phenotypes associated with DNMT3B deficiencies could be reversed.
Feasibility and Experimental Design:
- A major concern is the feasibility of recuing ICF phenotypes using the proposed viral system for DNMT3B expression. While the applicant acknowledged some potential risks, there were no viable alternative plans presented to overcome these challenges. As Aims 2 and 3 hinge on the success of this approach, reviewers considered this lack to be a tremendous oversight.
- A second major concern is the potential difficulty in drawing meaningful conclusions from the proposed experiments. ICF Syndrome is disorder that involves potentially causative changes in epigenetic patterning. However, the reprogramming process used to generate iPSC lines involves epigenetic remodeling in its own right, thereby introducing significant complexity to these studies and their interpretation.
- There is a lack of compelling preliminary data to support the applicant’s ability to derive iPSCs into various mature cell types, particularly those of the hematopoietic lineage.
- The experimental plan is logical and technically achievable, although alternative strategies are inadequately developed.
Principal Investigator (PI) and Research Team:
- The principal investigator (PI), is an acknowledged expert in DNA methylation and the role of DNA methyltransferases in stem cells. He/she has numerous publications in top tier journals.
- The research team has appropriate expertise to conduct the proposed studies, with the notable exception of differentiating iPSCs to hematopoietic cell types.
Responsiveness to RFA:
- The proposal appropriately addresses the goals of the RFA.