Non-coding RNA as tool for the active control of stem cell differentiation
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.
Epigenetic mechanisms play pivotal roles in cell fate determination during development. Long non-coding RNAs (ncRNAs) are in involved in epigenetic gene expression by recruiting epigenetic regulators to target genes. We are dissecting the role of ncRNAs in the stemcellness and differentiation of human embryonic stem cells (hESC). We have used biochemical and molecular approaches to identify ncRNAs, which associate with epigenetic regulators in hESC and are involved in the regulation of the expression of homeotic (HOX) genes. HOX genes encode for key regulators of cell differentiation in Arthropods and Chordates. We have identified 32 ncRNAs, which are transcribed during hESC differentiation. The detailed dissection of the role of two of the identified ncRNAs in HOX gene expression has resulted in novel insights into the role of ncRNAs in hESC differentiation. The ncRNA Mistral plays an important role in ectoderm development, one of the three germ layers that give rise to all cells, tissues and organs. Inactivation of Mistral ncRNA during hESC development prevents ectoderm development, indicating that Mistral is a key regulator of ectoderm development. The second ncRNA Scirocco is involved in epigenetic activation of HOX genes, controlling mesoderm development.
The obtained results support a model in which ncRNAs regulate important steps during hESC differentiation. Our results lay a foundation for the application of ncRNAs in hESC differentiation and the development of diagnostic and therapeutic assays to detect and manipulate the differentiation of hESC. The obtained results open novel areas in the filed of stem cell research by providing tools and assays that actively control the differentiation of hESC. These assays represent valuable additions to the efforts aimed at the active control hESC differentiation in order to obtain desired cell types.
The establishment and maintenance of mitotically and meitotically stable -epigenetic- gene expression patterns is paramount for cell proliferation and differentiation. Long non-coding RNAs (ncRNAs) have emerged as important regulators of epigenetic gene expression, in particular epigenetic gene silencing. NcRNAs have been associated with imprinting, gene dosage compensation, gene silencing and metastasis. NcRNAs silence gene expression by recruiting epigenetic repressors of the Polycomb group family to target genes. However, the functional importance of ncRNAs in stem cell biology remains unknown.
This project is aimed at the dissection of the role and function of ncRNAs in stem cell differentiation. During the funding period, we have focused on the functional characterization of one ncRNA. The ncRNA is transcribed in differentiating embryonic stem cells (ESCs) and facilitates activation of Hox genes. Hox genes are key regulators of cell differentiation and establish the developmental fate of cells. Destruction of Mistral through RNAi attenuates the differentiation of ESCs. Collectively, our results uncover a role for the ncRNAin epigenetic activation of gene expression and stem cell differentiation and identify ncRNAs as important regulators of stem cell differentiation. The obtained results establish a foundation for the development of novel tools and assays that actively control the differentiation of stem cells and direct the differentiation of stem cells into a desired cell type.