The roles of non-coding RNAs in the self-renewal and differentiation of pluripotent stem cells

The roles of non-coding RNAs in the self-renewal and differentiation of pluripotent stem cells

Funding Type: 
New Faculty II
Grant Number: 
RN2-00923
Approved funds: 
$1,406,823
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
There are thousands of cell types in the animal body, many of which can be derived from embryonic stem cells (ES cells), a pluripotent cell type that thrive in cell culture condition. ES cells differentiate into various cell type in a tissue culture dish in response to different growth factor/cytokine treatment, which can be transplanted back into animals for regenerative medicine application. In recent years, scientists have generated another type of pluripotent stem cell population, designated as induced pluripotent stem cells (iPS cells). These iPS cells are derived from adult cell types, and capable of differentiating into a variety of cell types in a tissue culture dish. Both ES cells and iPS cells not only provide a unique paradigm to study early mammalian development, but also hold great promise for regenerative medicine. Therefore, understanding the molecular network that regulate stem cell maintenance and stem cell differentiation of these cell types are very important for their application in regenerative medicine. In the studies we proposed, we will examine a novel class of gene regulators for their functions to maintain the stem cell population, as well as to trigger their differentiation into specific lineages. The focus of our studies is non-coding RNAs, which are RNA molecules that do not have capacity to encode proteins. Among the best studied non-coding RNAs are the microRNAs, which are small RNA molecules that are potent regulator for gene expression. These small RNAs often have the capacity to each regulate hundred of genes, therefore, regulating diverse developmental processes and physiological processes. If the biogenesis of these small RNAs are removed, stem cells exhibit multiple defects both in the tissue culture dish and in animal development. In this proposal, we aim to investigate the roles of non-coding RNAs in the regulation of stem cell maintenance and differentiation, and to identify novel non-coding RNA regulators that may impact stem cell biology. It is worth noting that RNA therapies, particularly those using synthetic small RNAs or their inhibitors, bypass the need for conventional gene therapy, and provide great promise for clinical application. The methods to delivery small RNAs into the cells and animals have been improved significantly over the past decade, and it is very likely that small RNAs and their inhibitors will be soon utilized for therapeutic applications. Therefore, our proposed research will generate exciting findings to stem cell biology and may lead to the development of novel diagnostic markers and therapeutic approaches for regenerative medicine.
Statement of Benefit to California: 
The studies proposed here explore the functions of novel non-coding RNAs in the self-renewal and differentiation in pluripotent stem cells. This is a new area of stem cell research, which, in funded, can benefit the state of the California to develop new diagnostic markers and therapeutic approaches in regenerative medicine. to gain knowledge on the molecular basis for pluripotency, and to train young scientists in stem cell biology. In what follows, the benefit of proposed research to the State of California and its citizens are summarized in details. Pluripotent stem cells not only provide a unique paradigm to study early mammalian development, but also hold great promise for regenerative medicine. RNA therapies are an area of intense investigation, and the in vitro and in vivo delivery methods for RNA molecules have been greatly improved over the past few years. Therefore, it is possible in the near future to use RNA therapy, particularly those involving small RNAs or their inhibitors, for regenerative medicine. RNA therapy is fundamentally different from the “conventional” growth factor or cytokine delivery to modulate stem/progenitor cells. RNA therapy by itself, or in combination with conventional therapy, may provide a novel approach for regenerative medicine. In addition, studies of non-coding RNAs in stem cells may give rise to new markers for stem cells, as well as their differentiated lineages. The proposed research will carefully examine the roles of non-coding RNAs in regulating the pluripotency of stem cells, which is a field in its infancy. Not only the knowledge acquired from this study will enhance our understanding on the molecular basis for stem cell maintenance and differentiation, but the novel research tools developed from this study will also benefit the stem cell research in the state of California and its people. In addition, the proposed research will allow young scientists to be well trained in the stem cell field, and to combine two exciting fields in biology, i.e., non-coding RNA biology and stem cell biology. And such training will be critical for the state of California to have its own research force on stem cell biology in the future.
Progress Report: 

Year 1

The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a novel miRNA, Esdmir-1, whose loss-of-function significantly promotes the reprogramming of iPS cells. This is an important finding, not only does it set up a paradigm for our future studies, it also provides an attractive methodology to improve iPS reprogramming in human. Our future effort in the next funding period (year 2) will be focused on completing the studies on Esdmir-1, evaluating the functions of additional candidate miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.

Year 2

The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a family of novel miRNAs that promote differentiation in pluripotent stem cells. This is an important finding because we demonstrated that repression of these miRNAs significantly enhances reprogramming efficiency. miRNAs functions can be modulated by transient transfection of oligonucleotide based antagonist, therefore, our findings are likely to lead to an approach to greatly promote iPSC generation in clinical application. Our future effort in the next funding period (year 3) will be focused on functional characterizations of additional miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.

Year 3

So far, our CIRM funded project have provided strong evidence suggesting miRNAs as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells, thus playing an important role in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for year 3. In short, we have carefully characterized the roles of miR-34 miRNAs in somatic reprogramming and indicated the importance of miR-34 inhibition in promoting stem cell self-renewal and somatic reprogramming. In addition, we completed two functional screens to identify miRNAs whose overexpression or inhibition regulates ES cell self-renewal and ES cell differentiation. Functional validation has been performed for majority of the hits from the screen. In particular, we have identified a miRNA, miR-meso, which specifically promotes mesoderm differentiation and represses ectoderm differentiation. Finally, using recombineering technology, we have constructed a BAC with Flag-tagged Lin28 at its endogenous locus. We have also constructed a piggy bac vector that allows us to generate stably integrated ES cell line that expresses Flag-tagged LIN28 for identification of novel LIN28 bound non-coding RNAs. These progresses significantly improved our understanding on the roles of non-coding RNAs in regulating stem cell self-renewal and pluripotency, and may lead to novel strategy for generating completely pluripotent human stem cells for clinical applications. In 2011, a portion of our results funded by the CIRM project were published in Nature Cell Biology as a cover story, and we also filed a patent application reporting a novel strategy to increase somatic reprogramming efficiency.

Year 4

Our CIRM funded project have provided strong evidence suggesting microRNAs (miRNA), and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Although non-coding RNAs, including miRNAs, do not have protein coding capacity, but they possess strong effects in regulating Using genomic approaches, embryonic stem cell culture and induced pluripotent stem cell culture and molecular biology, we were able to screen for, identify and characterize a number of non-coding RNAs, particularly, microRNAs, as important regulators for stem cell self-renewal and differentiation.Our results can have profound implications on the role of ncRNAs in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for the year 4 of our CIRM funded project. . Last year, we have successfully carried out a functional screen to identify miRNAs with important roles in regulating ES cell self-renewal. Among the miRNA candidates emerged from this screen, we primarily focused on the functional and mechanistic characterization of several important miRNA regulators for ES cell pluripotency. We investigated roles of miR-34 miRNAs, whose deficiency significantly promotes somatic reprogramming. We studied the role of miR-34 in the epigenetic remodeling during somatic reprogramming. We also studied the function of mir-290-295 cluster, which constitute the majority of miRNA species in the ES cells. We generated ES cell deficient for the mir-290-295 miRNA cluster to characterize its effects on ES cell self-renewal, and we also explored the functional redundancy of the mir-290-295 family miRNAs by functionally characterized related miRNA family, including the mir17-92 miRNAs. In the previous funding period, we also carried out a screen to identify miRNAs or non-coding RNA with important roles in regulating ES cell differentiation. Here, we functionally characterized the effects of miR-meso in mesoderm differentiation both in vitro and in vivo, using teratoma assays and KO ES cells. In addition, we identified a long ncRNA that exhibit an interesting pattern of expression during ES cell differentiation. We characterized its unique expression alteration during ES cell differentiation, and we are currently generating the knockout ES cells for this long ncRNA to further investigate its function. Finally, we characterized miRNA expression profiles during ES cell to Epiblast stem cell differentiation, and identified additional candidate miRNAs that regulate the exit of pluripotency of ES cells during their differentiation. Finally, we successfully established the biological system to generate human iPSCs from human dermal fibroblasts, and will use this system to explore the role of specific ncRNAs in human somatic reprogramming. Our previous results using mouse ES cells and iPSCs have prepared us well in exploring the significance of our finding in human. This will be a main focus to achieve in our last funding period.

Year 5

Our CIRM funded project have provided strong evidence suggesting miRNAs, and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Consistently, these ncRNAs play an important role in the generation of induced pluripotent stem cells (iPSCs). Using expression studies, functional screening and candidate approaches, we have identified and characterized the roles of multiple miRNAs and long ncRNAs (lncRNAs) in regulating the self-renewal and differentiation of pluripotent stem cells. These non-coding RNAs are essential component of a regulatory network that regulate stem cell cell fate potential, self-renewal and differentiation potential.

© 2013 California Institute for Regenerative Medicine