Aging and Renewal of iPS Cells During Reprogramming, Propagation and Differentiation

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01502
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The recent discovery of human embryonic stem (ES) cells offers great promise to medicine by allowing for the first time, the potential to produce of any of the cell types of the human body. In the future, this could lead to a host of new therapies in the treatment of diseases of the blood, vascular, heart, nervous, endocrine, muscle, skeletal, and other systems of the human body. However, transplant rejection has long been considered to be one of the great obstacles in successfully implementing these new therapies in the emerging field of regenerative medicine. The recent discovery that cells from the body, such as skin cells, can be returned to an ES-like state simply by the expression of 4 genes (induced pluripotent stem (iPS) cell technology) has revolutionized the field of regenerative medicine by showing a path to make patient-specific cells with the potential of ES cells to differentiate into all the cell types of the body. While iPS technology can restore an ES-like functionality to a patient’s own cell, our preliminary results show that this technology may not reliably restore a youthful proliferative lifespan to the cells. This apparent defect relates to the length of the linear ends of the DNA strands, a region of the DNA called the “telomere”. Telomere length determines how many times body cells can divide, and our initial results indicate that iPS technology may lead to cells that are prematurely old (short telomeres). This problem would be predicted to severely limit the ability of companies to scale up product from iPS cells and could similarly limit the therapeutic usefulness and potentially even the safety of such cells in humans. We propose a detailed study of telomere lengths during iPS and the testing of alterations in the procedure targeted toward correcting this problem. We describe a program of research aimed at measuring telomeres in a variety of iPS conditions using a novel system where the initial cells are genetically identical to ES cells, allowing for a careful measurement of telomere lengths during the iPS process. We then propose to modify specific conditions in the iPS process based on the understanding of the mechanisms that regulate telomere length. The final goal of the proposed research is to design and implement improvements in the iPS process that allows telomeres to lengthen back to an embryonic length. The successful restoration of youthful telomere length in iPS protocols would enable iPS-derived cells to have sufficient replicative lifespan to withstand expansion and purification during commercial manufacture, as well as provide robust cells of many kinds with potentially enormous benefits to patients in need of cell-based therapies, in particular an aging population facing age-related degenerative disease.
Statement of Benefit to California: 
New technologies in regenerative medicine offer the opportunity to develop novel therapies for a broad spectrum of life-threatening conditions. Additionally, these new technologies may offer solutions for chronic degenerative diseases, thereby potentially saving the people of the State of California enormous expenditures annually. The realization of this opportunity is dependent on technological advances, such as the development of technologies to deal with obstacles in the clinical utility of such technologies, such as transplant rejection. A recently-developed technology called “induced pluripotent stem (iPS) cell technology offers a relatively simple solution to transplant rejection by allowing scientists to transform an easily-accessible cell type, such as skin cells, back to cells similar to embryonic stem (ES) cells that have the ability to become any of the cell types of the human body. The use of iPS technology circumvents the transplant rejection issue and offers the exciting prospect of making patient-specific cells of many different types for a myriad of human therapeutic applications. We propose a program of research that aims to solve one critical problem of iPS technology relating to the resetting of cell lifespan to aged cells. We show data to indicate that existing technologies have limitations that could restrict the commercial scalability of product from iPS cells and negatively impact patient safety. Safety is critical to the development of any new drug candidate and is even more essential when considering cellular therapies where cells can persist in the body for years. Thus, the primary benefit of our proposed project of generating well-characterized cell lines with a normal cell lifespan is to provide more effective and safer cell therapies. By providing California researchers a means to improve iPS technology in the manner described, we will help overcome the cell lifespan bottleneck that is currently blocking the successful translation of iPS stem cell research to clinical applications. A key beneficial outcome of our project will be to shorten the time it takes to get stem cell therapies from the research laboratory and into the hands of physicians to treat patients suffering from degenerative diseases and injuries. By accelerating the translation of research to drug approval, more Californians that are currently in need of treatment will have the opportunity to benefit from stem cell therapies and Californians will see a more rapid return on their investment in the form of reduced health care costs.
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