The Role of NF-kappaB in Human Embryonic Stem Cell Survival and Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00280
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Because of their ability to develop into most of the specialized cells and tissues of the body, stem cells have the potential to replace diseased or dysfunctional cells with healthy functioning ones. It is the hope of the scientific and medical communities that the use of stem cell based therapies to treat diseases such as Parkinson’s disease, diabetes, heart disease and rheumatoid arthritis, etc. will one day be routine. Because this research field is still in its infancy, a number of scientific challenges must be overcome before the promise of stem cells can be harnessed. To this end, efforts aimed at increasing our understanding of the growth conditions, cellular biology and genetic events involved in stem cell survival and differentiation are key. While over 100 distinct stem cell lines have been derived, less than 20 are available in sufficient quantities for research purposes and of these, only a very limited number have been studied with respect to understanding how stem cells grow and develop into target cells. Clearly there is a great need to significantly expand the number of cell lines to allow comparative analysis of growth conditions, signaling and gene expression processes. These studies will help clarify how these cells can be grown to sufficient quantites to be used clinically and will also help determine at what stage these cells have maximum therapeutic potential. We are interested in understanding how proteins called NF-kappaB factors regulate the expression of genes involved in stem cell survival, growth and differentiation. In adult stem cells, NF-kappaB has been implicated in promoting cell survival. In contrast, almost nothing is known about the action of these factors in embryonic stem cells and whether they play a similar protective role. We propose to generate stem cell lines carrying a potent inhibitor of all NF-kappaB action and use these cells to assess the impact on stem cell survival and progression to cells that make up the central nervous system, namely, neurons and glia. These cells will provide a powerful experimental platform to explore the biology of stem cell survival and neuronal differentiation as it relates to a specific gene regulation program. Using the limited number of stem cell lines currently available, researchers have demonstrated that despite sharing some key characteristics, these lines also differed markedly. This highlights the importance and necessity of studying how certain genes are turned on or off in order to maintain both the survival and differentiation of stem cells. The studies proposed herein will provide important insights into how NF-kappaB regulates stem cell survival and differentiation. This information will ultimately advance our efforts at generating stem cells with therapeutic potential for use in the clinic.
Statement of Benefit to California: 
Experts predict that stem cell research holds the potential to help up to half of all Americans who suffer from diseases, including Parkinson’s and Alzheimer’s diseases, stroke, spinal cord injury, heart disease, arthritis and cancer. Because of their ability to develop into most of the specialized cells and tissues of the body, stem cells have the potential to replace diseased or dysfunctional cells with healthy functioning ones. This regenerative medical technology represents one of the most exciting medical advances to date and may be the only hope for those suffering from what we now refer to as 'incurable diseases'. Despite its infancy, early results from stem cell therapy trials have prompted significant optimism in the scientific community that these therapies will one day be routine. However, there remain several scientific challenges that must be overcome before promise of stem cells can be harnessed. One major challenge involves identifying the desired stem cell type and once identified, determining the optimal culture conditions to form progenitor cells that will ultimately differentiate into the desired therapeutic cell type. A second challenge will be to determine how embryonic stem cell progression through the various differentiation stages is regulated and at what stage these cells posess maximum therapeutic potential. The studies proposed herein are aimed at advancing our understanding of the molecular mechanisms governing viability, pluripotency and differentiation of embryonic stem cells. Using a variety of biochemical, molecular biological and bioinformatics approaches, we will explore the mechanisms by which NF-kappaB regulates specific genes in both undifferentiated human embryonic stem cells and differentiated neuronal progenitor cells. Together, these studies will provide important insights into how NF-kappaB contributes to embryonic stem cell biology and ultimately to neuronal development and repair to treat neurological disorders. Notwithstanding the obvious enormous health and quality-of-life benefits that would accompany the development of effective stem cell therapies, the financial health care savings for the state of California could be sizable. As such, we believe these studies will benefit the citizens of California personally and financially, as well as positively impacting society at large.
Progress Report: 
  • A prominent subset of white blood cells, named CD4 helper T cells, are critical in modulating the immune response against viral and bacterial pathogens. During HIV infection, the CD4 compartment is selectively reduced, suppressing the activity and response of cytolytic CD8 T cells, needed to abolish cells infected with the virus. Pharmaceutical therapies have been developed but they are not consistently effective and multidrug resistant viral strains are increasingly prevalent. Similarly, in vitro manipulated human dendritic cells are now being explored to tolerize against autoimmune disease or to stimulate antitumor responses. However, the number of dendritic cells that can be isolated form patients using current technologies is small. Consequently, different approaches need to be developed to enhance T cell reconstitution. In principle, multipotent hematopoietic progenitors could be derived from hESCs without long-term in vitro culture. A drawback is that the number of human hematopoietic progenitors derived from human ES cell cultures is limited using current culture conditions. Consequently, a subset of studies involving in vitro manipulated human cells would be difficult to perform. The transduction of human progenitor cells can be achieved using a tissue culture system in which human cord blood progenitors are differentiated in the presence of stromal cells that express the Notch ligand DL-1 towards the T cell lineage. However, the efficiency by which human progenitor cells differentiate into the T lineage cells is low. In the original application we proposed to develop a novel strategy that would permit the generation of large numbers of human T cell progenitors (up to 109) from human hematopoietic stem cells. To accomplish this objective we would target a critical regulator of early hematopoieisis, named E2A. Indeed during the two years period funded by CIRM we have demonstrated that murine hematopoietic progenitors that overexpress an inhibitor of E2A, named Id2, can be grown indefinitely in culture without losing their ability to generate many different types of white blood cells in the laboratory. This strategy is unconventional since it would permit the growth and isolation of large numbers of T cell progenitors, which has not been achieved so far by conventional culture conditions. We will continue these studies and use the same strategy to establish a long-term culture of human hematopoietic progenitor cells. If successful the approach would enable clinicians to reconstitute the hematopoietic compartments of patients carrying invading pathogens, including HIV infected patients, with large numbers of T cells that either express either a wild-type TCR repertoire or TCRs with specificities directed against invading pathogens. I expect this to succeed since we have already achieved this objective using murine progenitors as demonstrated during the past two years using funds obtained form the CIRM.

© 2013 California Institute for Regenerative Medicine