Characterization of MicroRNA (miRNA) Functions in Human Embryonic Stem Cells (hESCs)

Funding Type: 
SEED Grant
Grant Number: 
RS1-00327
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Despite advances in human embryonic stem cell (hESC) research, at the present time cell-renewal and differentiation remains an unsolvable problem. Self-renewal is the ability of hESCs to produce an identical copy of themselves when they divide (self-renew) whereas cell differentiation is the process by which a stem cell becomes specialized in order to perform a specific function such as in the case of a liver cell, a blood cell, or a neuron. Although a large number of genes have been identified that are able to affect the status of cultured hESCs, these cells almost always become various cell lineage of differentiated cells. The cell lineage is the developmental history of individual stem cells from the first specific cell division to their ultimate becoming special cells of tissues and organs. At present it is unclear what accounts for this change of status of hESCs. Recent discoveries that microRNAs (miRNAs) used to be found in eggs were found in hESCs may provide both new insights and new approach to solve this problem. MiRNAs are a form of single-stranded RNA which is typically 21-23 nucleotides long, and is thought to regulate the expression of other genes, therefore, miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. Our laboratories have recently reported a technology to silence the miRNA in stem cells and observed change of cell division and apoptosis. We believe that this technique can be adapted for investigating the roles of miRNAs in regulations of self-renewal and differentiation of hESCs. At present 36 miRNAs have been identified in hESCs and there is strong and rapidly growing evidence suggesting that the change in miRNA regulation is associated with hESC self-renewal and differentiation. For instance, most of these newly identified miRNAs are specifically expressed in hESCs but down-regulated during the formation of embryoid bodies (EBs). We will determine the function of each of these 36 miRNAs in hESCs using the technology we developed. Since the regulation of self-renewal and differentiation are critical to hESC research, we postulate that our novel approaches may selectively modulate gene function in hESCs, either maintaining consistent self-renewal or causing cell differentiation. The regulation of hESC status by miRNAs could contribute to developing specific cell lineage for specific diseases, leading to the development of diagnostic tools. The outcome of these studies should be a miRNA expression fingerprint leading to development of new tools for potential therapies in various diseases. Indeed, we will be able to use the hESC-specific miRNAs that control genes and microenvironment in the targeting of hESC self-renewal and differentiation to maintain a long-term self-renewal hESC line and to target differentiation along a desired lineage for stem cell based therapies.
Statement of Benefit to California: 
The control of self-renewal and differentiation is the current focus on hESC research because a pluripotent stem cell line usually lasts about 5-8 days and the very next step is cell differentiation. If we can control the threshold of entering cell differentiation, we would have the tool to direct the cells into a specific type for replacing or repairing diseases or damaged tissues in patients with specific diseases, including Parkinson’s disease, heart disease, diabetes, spinal-cord injury and other conditions. Every year an estimated 15,000 persons suffer spinal cord injuries in the United States. An estimated 500,000 people are currently paralyzed from the waist or neck down, and one million are estimated to have spinal cord-associated deficiencies with less severe implications. Many of these patients are veterans. The results of spinal cord injury can be severe deficits that lead to profound decrease in quality of life for the patient. The population of California is about 12.2% of that of the United States, therefore, about 61,000 Californian citizens are suffering from this disease. The case described above is only one of the diseases that the proposed research will benefit the State of California and its citizens. Although treatments using stem cells are not yet available, the proposed research will lay the foundation leading to potential treatments on other human diseases including Parkinson’s Disease, stroke, diabetes, liver disease, heart disease, hemophilia, muscular dystrophy, sickle cell disease and so on because embryonic stem cells are capable of becoming all of the body’s various cell types. The proposed experimental technique aims to explore the role of microRNA which would shed new light for novel tools to direct the hESC to become various cells in the body. Certainly, hundreds of thousands or even millions of Californians who suffer from chronic and debilitating diseases would stand to benefit from the proposed research.
Progress Report: 
  • Human ES cells are routinely grown on feeders with medium containing serum or serum replacements supplemented with bFGF. Although progress has been made in improving culture conditions, the pathways involved in the maintenance of human ES cell self-renewal remain largely unknown. The main purpose of this project was to decipher the requirements for sustaining human ES cell self-renewal and to understand the molecular basis of these requirements. So far we have made the following findings: 1. Sustained activation of STAT3 supports mouse ES cell self-renewal in the absence of feeders ,whereas activation of STAT3 induces differentiation of human ES cells and epiblast-derived stem cells (EpiSCs); 2. Self-renewal of mouse/rat ES cells, but not human ES cells or EpiSCs, is sustained by inhibition of glycogen synthase kinase-3 (GSK3) and mitogen activated protein kinase (MAPK); 3. Activation of integrin pathway enhances self-renewal of human ES cells, but not mouse cells; 4. bFGF supports human ES cell self-renewal through an Erk1/2-dependent pathway. Our findings suggest that the requirements for sustaining self-renewal of human and rodent ES cells are fundamentally different and that human ES cells are most likely analogous to rodent EpiSCs. We are currently generating and characterizing human cells that resemble mouse/rat ES cells. Understanding the basic mechanisms involved in human ES cell maintenance will eventually lead us to develop better methods for the growth of human ES cells, which is clearly important if these cells are to be used clinically.

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