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.
SYNOPSIS: Thirty six miRNAs have been identified that are expressed in human embryonic stem cells, some of which are down-regulated upon differentiation into embryoid bodies. The functions of these in stem cell self-renewal and differentiation are not known. This application proposes to study the functions of the micro RNA’s both by constitutive overexpression in human ES cells (Aim 1) or by knockdown using antisense oligonucleotides (Aim 2). The applicant will determine the effects of these manipulations on cell renewal, differentiation, and apoptosis. SIGNIFICANCE AND INNOVATION: Cell regulation by microRNAs is now appreciated to be of widespread significance. About thirty six micro RNA’s have been identified that are expressed in human ES cells, but their functional importance has not been determined. It seems highly likely that these regulatory molecules will play important roles in stem cell biology. STRENGTHS: This proposal addresses a very relevant and potentially field-altering scientific question; the role miRNAs may play in either keeping ES cells in an undifferentiated state or informing ES cells to differentiate into discrete types of tissues. The proposed experiments are well within the capabilities of the laboratories that this proposal would fund. The laboratory of the PI has developed a useful way to misexpress miRNAs. In their system, miRNAs are placed in the intronic region of transcript. Upon transfection, the miRNA is spliced allowing for processing of the miRNA. The unique aspect of this system is that the transfection and processing of the miRNA can be monitored by following GFP expression. WEAKNESSES: Reviewer one: The assays that will be used to study stem cell renewal and differentiation are very poorly described (for example, they simply say that they will look to see whether embryoid bodies are formed). The assays to study apoptosis are not described at all. The superficial description of these assays leaves one very uncertain as to whether useful information about the specific biological functions of these microRNA’s will emerge from the proposed studies. A notorious problem with these kinds of experiments is off-target effects. The applicants merely state that they will use RNA profiling to determine if off-target effects are occurring. Not only is this inadequate, but its very hard to understand how microarrays will reveal the presence of off-target effects. The applicants provide no data that knockdown of microRNAs in human ES cells can be achieved by transfection with antisense oligonucleotides. It is well known that the efficacy of this technique is highly variable among different kinds of cells, and therefore evidence of its feasibility in these cells is essential. Reviewer two: A drawback of this proposal is that it is not clear why human ES cells are required to perform the described experiments. The authors note that the miRNAs found in human cells have murine homologues with very similar genomic organization and expression patterns. It would be easier and cheaper to work with mouse or rat ES cells to perform the proposed screen to identify functions for ES cell-specific miRNAs. Once the function of a given miRNA has been determined in mouse or rat, it could then be investigated in detail in hES cells. Since many of the techniques required to perform these experiments are already present in the mouse system, determining the function of ES cell miRNAs could be performed much faster in the mouse system. The author proposes to use microarray analysis to determine target specificity and any “off-target” problems. The idea appears to be that hES cells in which miRNAs have been either misexpressed or removed will be subjected to an array analysis. The array will be used to determine if the introduction (or removal) of miRNAs up-regulates or presumably also down-regulates, genes that are not direct targets. The problem is that no one knows what the complete target list for a given miRNA is. There is also no discussion on how long after transfection cells will be harvested for array analysis. It will be very difficult to identify if the alteration of miRNA levels is caused by off-target effects using arrays. All 36 miRNAs that will be overexpressed in hES cells are already present in this cell population. Since miRNAs have only been reported to downregulate, or in most cases completely abolish gene expression, it is not clear that the overexpression of miRNAs will produce a phenotype (i.e. the targets of these miRNAs are already off in wild type hES cells). The inclusion of a picture of hES cells expressing GFP under the control of the human Oct3/4 promoter demonstrates that these cells are capable of being stably transformed but does not add to the proposal. The investigators cannot use these lines for their proposed experiments since they will use GFP to score for miRNA processing driven from their intronic miRNA expression vector. DISCUSSION: There was no further discussion following the reviewers' comments.