Nearly every cell nucleus in an adult human contains the complete instructions (i.e., all the genes) to make an entire human. Thus, we each carry about 100 trillion sets of the genetic instructions that could make a genetically identical human. Cells in early embryos, and human embryonic stem cells (hESCs) are able to read all of our genes and therefore are able to produce all the cells of a human. However, during our development, cells become progressively more specialized and lose their ability to read some of these instructions. Each specialized adult cell (a “differentiated” cell) is locked down in such a way that only a subset of the genes can be read; each type of cell uses a different set of these instructions. The process is normally inexorably driven in one direction: the process is not reversed, and one type of differentiated cells does not change into another type. However, experiments in other animals demonstrate that the locks in mature cells can be completely reset in the laboratory, in particular by transferring a nucleus containing the genetic instructions into a fertilized egg. Thus, it is possible to reverse the normal process that creates the locks on the genetic instructions, allowing the instructions from a specialized adult cell to be unlocked and used to generate all other cell types in an individual. Our goal is to develop technologies for directly changing differentiated adult human cells into cells that behave similarly to human embryonic stem cells (hESCs), without the need for transferring nuclei into human embryos. To do so, we propose to identify the genes that code for the locks that prevent genetic instructions from being read in specialized cells and to investigate methods for resetting these locks. We have devised a system that allows us to look through nearly the entire genome of a model animal for such genes. Many of the genes we have found are equivalent to genes in humans that perform the same functions. We will test whether these human genes turn hESCs into differentiated cells whose genetic instructions are locked down. With the goal of resetting the locks, we will also inactivate these genes in mature, differentiated cells and will ask whether this causes these cells to become capable of reading instructions that they normally cannot read and makes them behave similarly to hESCs. Critical for the success of this project are hESC lines that are prohibited under current federal guidelines. This research may make it possible to create better stem cells and could lead to technologies that make it possible to create new hESC-like cells from virtually any cell in a patient without the need for harvesting human embryos or relying on the difficult task of transferring cell nuclei. Because these hESC-like cells would be genetically identical to the other cells in the patient, they would not be rejected when transplanted.
Statement of Benefit to California:
The driving long-range goal of this research is to develop new ways to create hESC-like cells. Successful application of these methods would make it possible to produce hESC-like cells from mature tissue of any patient that could be used to treat a large variety of diseases and injuries. As this research is designed to reveal how cells in embryos are able to produce many different types of adult cells (for example, cells of the brain, spinal cord, heart, muscle, liver, pancreas, etc.) the results of this research will be immediately informative to scientists studying the biology of stem cells and other areas of human development. Thus, this research could spur further discoveries by California scientists in many areas of human health. Our findings will first be communicated to scientists at conferences within the state and will therefore have a direct immediate impact on California science. Developing methods for creating better stem cells (capable of producing a greater variety of replacement cells), or generating new stem cells from mature tissue, will have a dramatic impact on medicine and biotechnology in this state. California would be the state in which centers that use these technologies are first established and clinical trials are first carried out. The citizenry of California would have early access to new clinical treatments for a wide variety of diseases ameliorated by stem cell therapies. Discoveries from the proposed studies might well lead to intellectual property claims, and royalties derived from licensing of the new methods could generate substantial revenue for UC through the UCSB Office of Technology & Industry Alliances. These discoveries could spawn new ventures in the biotechnology industry that would contribute to the California economy. The successful application of this research would make California a focus for a new direction in stem cell research and therapies, which would draw scholars and clinicians, as well as investors, from outside the state. Finally, there are potential ramifications of this research that could benefit the political climate in California. Methods for creating new hESC lines that circumvent the need for human embryos would quell the enduring political conflict between advocates of hESC research and the significant fraction of the electorate opposing such research on moral and religious premises. While hESC-based therapies promise to change the course of human medicine and health, there can be little question that legal challenges, such as those currently obstructing use of the funds earmarked by the passage of Proposition 71, will continue as long as human embryos are needed for such research. If the ultimate goal of this research is achieved, the pace of stem cell-based research and medicine would become limited not by resolution of political and legal conflicts, but instead by the availability of resources and human ingenuity.
SYNOPSIS: In this proposal the investigators wish to determine whether signal pathways identified in C. elegans that govern mesendoderm differentiation of multipotent stem cells play a role in maintaining human embryonic stem cells (hESCs) undifferentiated vs allowing differentiation to mesendoderm, and could be used to dedifferentiate mature cells to an ES-like phenotype. An miRNA screen initiated in C.elegans will be completed and a number of candidate genes identified that govern these steps. In Aim 2, the expression of such putative differentiation vs self-renewal genes will be evaluated in hESCs, and genes thought to induce differentiation in C. elegans overexpressed in hESCs, to determine whether they play a role in lineage commitment of human cells. In Aim 3, the investigators will inhibit expression of key genes in differentiated cells (osteoblasts) and determine if this reprograms cells to a more pluripotent state. SIGNIFICANCE AND INNOVATION: Insights gained in genes and signaling pathways that govern differentiation vs self-renewal of stem cells may yield important tools to maintain ESCs undifferentiated and may provide insights on factors that could govern de-differentiatioon. As such, gene pathways are likely conserved throughout different species; insights gained from model organisms such as C. elegans may well yield information that could aid in maintaining undifferentiated hSCs and human cell reprogramming. The use of results in model organisms to inform studies in hESCs is of merit, and clearly has numerous precedents in other areas of research. The PI's laboratory has much expertise in characterizing developmental regulatory processes in C.elegans. These investigators have identified endoderm-regulating GATA factors END1/3 that can convert non-endodermal progenitors to an endodermal fate during a transient developmental time window. A time window during which a pluripotency-to-commitment switch occurs has been identifed and RNAi screens have been performed to identify gene-products that are required for this switch. The application of RNAi technology, and results obtained in the C.elegans studies is innovative and highly significant. STRENGTHS: The major strengths of this proposal are the high level of expertise in C.elegans biology in the PI's laboratory, and the partnering and integration of this expertise with hESC expertise in Dr. Thompson's satellite laboratory in California. This collaboration has the potential of applying insights from C. elegans to hESC. It is highly likely that important results will be obtained. Finally, the importance of the question to be addressed. WEAKNESSES: The major weakness of this proposal is the broad scope of the proposed research. It is much more feasible to perform screens in C.elegans than in mammalian systems, particularly ones as complex as hESCs. There is concern that the proposed studies are somewhat overly ambitious. It would have been more opportune if studies in Aim 1 which are solely a C.elegans screen would have been finished, and a final list of candidate genes identified. It is unclear how many genes identified in aim 1 will be tested in Aims 2 and 3, and how the investigators will prioritize such studies. As there will likely be multiple genes to be evaluated, the proposal lacks a high throughput screening approach. Overexpression studies in Aim 2 may be time-consuming - for each gene transferred the possibility of gene silencing will need to be addressed. The number and class of genes to be tested in Aim 3 is ill-defined. As the invetsigators would like to reprogram differentiated cells, they will likely at least need some or all of the key transcription factors oct4, sox2 and nanog, known to be indispensable for maintaining hESCs undifferentiated and required in different studies (Yamanaka and Smith) to reprogram neural cells and fibroblasts. DISCUSSION: This proposal is from an outstanding C. elegans investigator who has identified genes that regulate pluripotency in the worm and who suggests that the information learned can translate into hESCs. This proposal aims to apply signalling information from C. elegans to hESC studies. Can decisions made by C. elegans inform the fates of hESCs? The GATA factor screen was previously done in worms. While these pathways may be conserved, the screen and other research are too broad, and it is not clear that orthologs can be clearly defined. The group found 50-100 targets in C. elegans, and the screen is still incomplete. Aim 3 is a longshot from a feasibility perspective; it is unclear what criteria will be used to determine which genes to choose. This proposal is thought to be overly ambitious; a huge amount of work is required just to carry 50 genes forward. It would have been better to finish the C. elegans screen and prioritize a list for the human work. In addition, the hypothesis that a single gene will have the ability to de-differentiate osteocytes is unlikely to be true.