Stem cells are multipotent, meaning that they can develop into any cell type of the human body. Biomedical applications propose that, after introduction into humans, stem cells could replenish damaged or lost cells in human bodies and thereby cure human diseases such as Parkinson, Alzheimer’s, and diabetes. One prerequisite for the success of the biomedical application of stem cells are tools that actively control the development of a stem cell into any given cell type, such as neurons, muscle cells, or insulin-producing pancreatic cells. However, the tools directing stem cell differentiation are only now being discovered. The proposed research project intends to fill this gap and uses a novel research approach to develop tools, which can control to development of cells (cell differentiation) into any desired cell type.
The research approach is based on studies, indicating that a novel group of non-coding RNAs plays an important role in cell differentiation in the fruit fly and mice. The non-coding RNA originate from and control the expression of genes, whose activities control cell differentiation. We have shown that the introduction of non-coding RNA into cells changes the developmental fate of cells, suggesting that the non-coding RNA represent tools that control the differentiation of cells including stem cells. We have identified 32 non-coding RNAs in human cells, which originate from different regulators of cell differentiation and are transcribed in differentiated but not human stem cells. Thus, the specific working hypothesis of the research project is that non-coding RNA control the differentiation of human stem cells.
To test that hypothesis, we shall assess whether non-coding RNA can induce stem cell differentiation. First (Aim 1), we shall test whether the introduction of non-coding RNA into human stem cells activates the expression of genes, whose activities control cell differentiation. To confirm the results of Aim 1, we shall assess whether non-coding RNA-mediated expression of key regulatory genes of cell differentiation coincides with the recruitment of regulatory proteins, which establish and maintain the expression of the key regulatory genes throughout the entire life. This is important, as an actively controlled progression of stem cell to differentiated cell is only then successful, when the differentiated cell maintains its identity throughout the entire life. Third, we shall elucidate whether activation of key regulatory genes by non-coding RNA induces cell differentiation. In summary, the proposed project will provide novel insights into the molecular mechanisms underlying stem cell differentiation and novel molecular tools to control stem cell differentiation. Our efforts will significantly contribute towards the development of biomedical applications that allow the utilization of stem cells in the treatment of human diseases.
The long-term goal of the proposed project is the development of molecular tools, which are capable to actively control the differentiation of stem cells. California will benefit from the proposed research project in several ways.
ResearchThe proposed research project represents a novel avenue of stem cell research. Tools, which can actively control stem cell differentiation, remain elusive. Thus, the proposed development of such tools represents a major advancement in stem cell research and provides California with cutting-edge technology. In addition, the research project provides training opportunities for the next generation of stem cell researchers.
EconomyThe proposed research project will benefit the economy of the State of California. The proposed research provides employment opportunities for researchers. In addition, the developed technology can be transferred into a company setting (e.g., in form of a start-up company) that provides additional employment opportunities and tax revenue for the State of California. The generated revenue will provide compensation for the State of California that is likely to exceed the amount of the initial investment by the Stare of California into the proposed research proposal by many magnitudes.
Public HealthThe proposed project has the potential to make a significant contribution to stem cell research in particular the biomedical application of stem cells. In the long-term, technology derived from this project will contribute to the development of assays to treat human diseases. Thus, the proposed project will provide the citizens of California with novel biomedical assays capable of treating diseases such as diabetes, Parkinson and Alzheimer’s.
SYNOPSIS: This proposal addresses the functions and activities of non-coding RNAs emanating from homeotic genes (hom-ncRNA). The Principal Investigator (PI) has identified such RNAs in Drosophila where they appear to facilitate recruitment of epigenetic activators to host genes and oppose repression of homeotic genes by polycomb components. The PI has identified 32 such hom-ncRNAs in humans. These are not expressed in human embryonic stem cells (hESCs). In the first aim the effects of expression of hom-ncRNAs on homeotic gene expression in hESCs will be examined. Subsequently the recruitment of epigenetic regulators (eg MLL) to homeotic genes will be assessed by chromatin immunoprecipitation (IP). In the third aim, markers of differentiation will be assayed in hESCs expressing the hom-ncRNAs. The hope is that these studies will provide novel insights into control of differentiation in hES cells.
INNOVATION & SIGNIFICANCE: The proposal is novel in its focus on hom-ncRNAs. The significance of studying control of hESC differentiation is high. How study of hom-ncRNAs will impact this area is less clear. This proposal seeks to determine whether the conserved pathway of homologous non-coding RNA-regulated HOX genes can be harnessed to control differentiation of hESCs. This is a highly innovative approach that could be employed to drive differentiation into defined lineages using small RNA transgenes to induce a more differentiated chromatin state at selected HOX genes. The model system chosen to prove the approach is hematopoietic differentiation, which is already well described, but is important for determining if the approach extends beyond existing approaches for protein overexpression.
STRENGTHS: This proposal has outstanding merit as it is based on a solid hypothesis and the research team is highly qualified to carry out the proposed experiments. In particular, Dr Sauer’s lab has recently shown homologous non-coding RNAs regulate epigenetic control of HOX genes in the Drosophila model system. They have extended these observations in mammals to show that nearly all the murine HOX cluster genes have non-coding RNAs that recruit the activating histone methyltransferase MLL concomitant with gene activation. Aims 1 and 2 extend these observations by asking whether human non-coding RNA can be transiently introduced into hESCs to induce HOX gene transcription dependent upon MLL binding. Aim 3 gets to the heart of the biology of the system, and addresses whether this regulatory pathway can be harnessed to selectively induce lineage specification.
In summary, the strengths of this proposal include (1) the PI's experience with hom-ncRNAs and preliminary identification in humans; (2) a collaborator within the PI's department for hESC research; and (3) the potential of developing new insights into how differentiation genes are repressed in hESCs.
WEAKNESSES: The main weakness in this proposal is that the first two aims are not at all risky, because they only modestly extend the high profile recent observations from the PI's lab. The heart of the interesting science is in Aim 3. Other weaknesses include: (1) lack of a priori reasons to study hom-ncRNAs; (2) since hom-ncRNAs are not expressed in hESCs, it is uncertain what may be learned from their expression; and (3) the proposal may be construed only to meet the requirements of the RFA.
DISCUSSION: This is an interesting topic where the PI is trying to move from work on the fruit fly Drosophila melanogaster into hESC research. One reviewer felt that perhaps there was no reason a priori to study homeotic gene ncRNA since they are not expressed in hESCs. Another reviewer was more enthusiastic, stating that the PI has the expertise to perform this work and understands the important questions. This reviewer felt that even though there is not much preliminary data, the first part of the proposal is almost certain to work. The HOX5A model for hematopoiesis is good, and the expectations are clear.