A bioinformatic approach to study context-specific signaling by the stem cell receptor Kit

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01502
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
A major challenge facing all stem cell therapies is regulating the behavior of stem cells – their survival, proliferation, self-renewal and differentiation – to regenerate the desired cell types and tissues. The success of these therapies depends on understanding how extracellular signals, such as growth factors, present in the stem cell niche regulate stem cell behavior through the activation of cell surface receptors. The receptor Kit is present on the surface of multiple human stem cell types, both pluripotent (embryonic and induced pluripotent) and adult (hematopoietic, cardiac and neural) as well as certain mature cell types. In addition, Kit is required for the maintenance of these cells. Our work will address the following question: how does a receptor that is expressed in stem and non-stem cells, function specifically in the context of stem cells to regulate self-renewal and survival? We hypothesize Kit achieves context-specific functioning by interacting with and activating other cell surface receptors, each of which is specific to a different stem cell type. Our previous work, combining bioinformatics predictions with experimental validation, has already identified IL-4R as a novel Kit-activated receptor in hematopoietic stem cells. Our proposed research will first extend this work to find similar Kit-activated receptors in the other stem cell types. To understand the significance of the above Kit-activated receptors, we will compare normal stem cells to those lacking one of these receptors and evaluate the contribution of Kit as well as each Kit-activated receptor to stem cell function. Our final objective is to study the role of microRNAs, a class of small RNA molecules, in the mechanisms by which Kit activation elicits changes in stem cell behavior. A rapidly growing body of evidence suggests that microRNAs play key roles in regulating cellular differentiation but their role in stem cell biology remains unclear. The research outlined in this proposal is aimed at uncovering a novel paradigm for context-specific activity of the pan-stem cell receptor Kit in multiple human stem cell types. In addition to investigating the mechanisms underlying Kit activity, our work may suggest new stem cell-targeted therapies using combinations of growth factors, that exploit the potential synergy between Kit and the Kit-activated receptors.
Statement of Benefit to California: 
The goal of this proposal is to investigate novel mechanisms of action of the stem cell receptor Kit, which is present on embryonic and multiple tissue-specific adult stem cells and known to play a central role in regulating survival, proliferation and self-renewal. Our research is likely to both advance our knowledge of mechanisms underlying stem cell biology and motivate the development of novel stem cell-targeted therapies. The natural environment of stem cells within different tissues in the body provide a variety of cues in the form of growth factors to promote the survival and self-renewal of stem cells and, when needed, their proliferation and differentiation into various types of mature cells. The successful clinical use of human stem cells requires the ability to maintain and grow these cells, and to stimulate them to develop into the desired tissues. This requires a thorough understanding of the mechanisms by which stem cells respond to extracellular cues, which is the focus of our current and proposed work. We seek to uncover novel interactions between Kit and other stem cell receptors and determine the role of these interactions in regulating stem cell behavior. Our scientific method based on combining bioinformatics with experimental validation, has proven to be much faster than conventional approaches, having accelerated by several years our finding of a novel interaction between Kit and the IL-4 receptor in bone marrow stem cells. Our results are likely to inform novel stem cell-targeted therapies. Currently, administration of Kit ligand, also known as Stem Cell Factor, at doses necessary to stimulate bone marrow stem cells results in severe adverse effects due to the action of Kit on certain non-stem cells. Our findings on interactions between Kit and other stem cell receptors may provide a basis for combination therapies that utilize much lower doses of Kit ligand together with other growth factors to specifically target the desired stem cells while diminishing the adverse effects.
Progress Report: 
  • The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of work has described the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we are examining how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We are characterizing the programs of regulation controlled by these proteins. In the past funding period we adapted and applied two new genomewide methods of RNA analysis. The first uses new technologies for high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we can extract information on all the splicing changes occurring during a developmental transition. The second method called CLIP examines the sites of RNA binding by PTB and nPTB in the RNA of each cell type. These methods are now ready to apply to ESC, NPC and motor neurons that we have derived in culture. From this work, we will advance our understanding of how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.
  • The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of work has described the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we are examining how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We are characterizing the programs of regulation controlled by these proteins. In the past two funding periods, we adapted and applied two new genomewide methods of RNA analysis. The first uses new technologies for high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we have extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of RNA binding by PTB and nPTB in the RNA of each cell type. These methods have been applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we learning new events determining how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.
  • The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where a particular cell type or cellular function has been lost. For example, the devastating neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by a progressive loss of motor neurons and consequent paralysis. Cell replacement for diseases of the nervous system poses special challenges because many neurons lose their ability to proliferate and thus regenerate after embryonic differentiation. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. A second dramatic change in the program of gene expression occurs when these progenitor cells cease cell division and differentiate into neurons. A great deal of work has described the DNA level changes affecting gene expression in ES cells and during their transition to NPC and neurons. However, the gene expression pathway is also highly regulated at the RNA level, as the DNA gene is transcribed into RNA and the RNA then translated into protein. An important regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. This processing includes pre-mRNA splicing, and alternative splicing patterns in the pre-mRNA determine the final protein output of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we have examined how two important splicing regulators, the polypyrimidine tract binding protein (PTBP1) and its neuronal homolog PTBP2, affect splicing in normal ES and NP cells, and have characterized their regulatory networks. We adapted and applied two new technologies for RNA analysis. The first used high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of binding by PTBP1 and PTBP2 in the RNA of each cell type. These methods were applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we identified many new genetic events that determine how ES cells differentiate into neurons. These alternative splicing events controlled by the PTB proteins affect numerous functions essential to the development and survival of neurons. We are now examining several of new genetic regulatory events in more detail to understand how pre-mRNA splicing controls cell function in normal development and in disease.
  • The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where a particular cell type or cellular function has been lost. For example, the devastating neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by a progressive loss of motor neurons and consequent paralysis. Cell replacement for diseases of the nervous system poses special challenges because many neurons lose their ability to proliferate and thus regenerate after embryonic differentiation. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. A second dramatic change in the program of gene expression occurs when these progenitor cells cease cell division and differentiate into neurons. A great deal of work has described the DNA level changes affecting gene expression in ES cells and during their transition to NPC and neurons. However, the gene expression pathway is also highly regulated at the RNA level, as the DNA gene is transcribed into RNA and the RNA then translated into protein. An important regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. This processing includes pre-mRNA splicing, and alternative splicing patterns in the pre-mRNA determine the final protein output of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we have examined how two important splicing regulators, the polypyrimidine tract binding protein (PTBP1) and its neuronal homolog PTBP2, affect splicing in normal ES and NP cells, and have characterized their regulatory networks. We adapted and applied two new technologies for RNA analysis. The first used high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of binding by PTBP1 and PTBP2 in the RNA of each cell type. These methods were applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we identified many new genetic events that determine how ES cells differentiate into neurons. These alternative splicing events controlled by the PTB proteins affect numerous functions essential to the development and survival of neurons. We are now examining several of new genetic regulatory events in more detail to understand how pre-mRNA splicing controls cell function in normal development and in disease.

© 2013 California Institute for Regenerative Medicine