Human development begins at fertilization when the human embryo begins to divide and differentiate. During the process of
embryo development, the totipotent embryo generates the diverse cell types that make up the organs and tissues of the adult.
As cells specialize their functions their patterns of gene expression change to support their specific roles in different tissues and
organs; for example, pancreatic cells begin to produce insulin while cardiac cells express the genes required for heart muscle
contraction. Fundamental to the specialization of different cell types are the changes that occur to control the switch in gene
expression patterns. A major component of this process is the reorganization of the cell nucleus to activate or repress different
genes. The goal of our work is to understand how stem cells change the organization of their nuclei in order to differentiate into
specific cell types.
A major research focus in stem cell biology is the generation of stem cells from adult tissues that can be used for therapeutic
purposes. These induced pluripotent cells may be ideal for stem cell based therapies because they are capable of generating the
tissues from which they were derived and they can be generated from adult tissues. The nuclear organization of stem cells is
globally open and permissive for gene expression but as stem cells differentiate they compact different regions of the genome.
During the generation of these induced pluripotent cells the genome changes its organization from the compact state of the
tissue specific cell to the open state of the stem cell. An important aspect of our proposal is to identify the molecular factors that
control genome reorganization so that we can improve the efficiency of induced pluripotent cell generation from adult tissues.
Our research has the potential to significantly improve the development of patient specific therapies based upon tissue specific
stem cell generation.
Statement of Benefit to California:
This research plan will benefit the State of California by improving the methods to generate induced pluripotent stem cells.
Improved methods for tissue and donor specific pluripotent cell generation will circumvent the problems of immune rejection of
cells and provide a patient specific therapeutic approach to disease treatment. Our experimental approach should be applicable
to any tissue type and thus should facilitate stem cell generation for a broad spectrum of diseases. Our research will further the
basic biological understanding of the processes of stem cell maintenance and cell differentiation. Insight into the biology of
stem cells will lay the groundwork for future studies and the development of novel, stem cell based, therapeutic approaches.
The goal of this proposal is to identify proteins that affect heterochromatin formation by screening for those that alter the cellular distribution of a known heterochromatin-binding protein in human cells. The applicant will then analyze the roles of the identified proteins in human stem cell maintenance, differentiation and reprogramming. A High Content Screen (HCS) utilizing RNAi and imaging technologies that were developed in Drosophila will be applied to an immortalized human cell line to identify these novel proteins. Once identified, the expression, localization, and function of the proteins in human embryonic stem cells (hESCs), normal human dermal fibroblasts, and induced pluripotent cells (iPSCs) derived from those fibroblasts will be studied and the contribution of these proteins to iPSC generation will be analyzed.
The reviewers agreed that this proposal targets a highly significant yet understudied problem in the stem cell field that will need to be addressed to fully understand the mechanisms involved in stem cell differentiation and in reprogramming. If the experimental plan is successful, it is likely that the investigator will uncover cellular factors important in these processes. The reviewers appreciated the highly innovative HCS approach utilized in this well written proposal.
By contrast to the reviewers’ appreciation for the potential impact of this proposal, they raised several serious concerns regarding its feasibility. They did appreciate the preliminary data validating the use of the HCS in Drosophila, but felt that the preliminary data was not sufficient to support the use of this technology in human cells, and reviewers worried that it would be more difficult to use the imaging technologies and interpret the results in human cells. Specifically, reviewers pointed out that the heterochromatin-binding protein exists in three different isoforms in human cells., which are biochemically similar but functionally very different, and this could undermine the premise of the screen, especially since some of the isoforms can be associated with euchromatin. Furthermore, reviewers felt that changes in intercalary heterochromatin, a small yet important form of heterochromatin, would not be detectable in the HCS. The reviewers were concerned that the investigator did not acknowledge any of these potential pitfalls in utilizing the HCS in human cells nor did the investigator offer any alternative strategies in adapting this assay for human cells. Additionally, the reviewers indicated that the proposal does not contain sufficient detail regarding the criteria that will be used to select candidate genes for further characterization. Finally, reviewers found the use of an immortalized cell line for the HCS problematic, especially given the differences between heterochromatin states in differentiated cells when compared to stem cells.
The reviewers praised the investigator’s strong track record with considerable expertise in chromatin biology and an evident desire to enter the human stem cell biology field. The reviewers appreciated that the investigator acknowledged a lack of experience working with human cells and thought the assembled team contained the necessary expertise to carry out the aims. The reviewers appreciated the plan to provide stem cell culture training to the individuals involved in this project.
Overall, reviewers felt this was a high risk proposal that could have a significant impact on understanding reprogramming of somatic cells if successful. However, reviewers were not convinced of the feasibility of the research plan, and hoped a future resubmission would benefit from improved preliminary studies and from addressing some of the reviewers’ concerns.