Embryonic stem cells are undifferentiated and unspecialized cells that have the ability to become any type of adult cell in the body. Because these cells have the unique ability to multiply indefinitely when they are cultured in a laboratory setting, they have the potential to generate an unlimited supply of specialized adult cells, such as bone, cartilage, heart, muscle, liver or blood cells.
Human embryonic stem cells are derived from a fertilized zygote (egg) less than 1 week old. Using blastocysts (5 - 7 day old fertilized egg) obtained from donated, surplus embryos produced by in vitro fertilization, stem cell lines were established. The cell lines are capable of prolonged, undifferentiated proliferation in culture and yet maintained the ability to develop into a variety of specific cell types.
Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. The internal signals are controlled by some genes, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. Amongst these genes are the Id genes that act as master switches within the cells and that regulate the expression of important intracellular proteins called transcription factors. Previous work in our laboratory determined that Id genes regulate growth and differentiation in many different tissues. In this proposal, we will determine how early are the Id genes expressed during human embryonic development. We will also investigate their potential important function during the gain of function of undifferentiated human embryonic stem cells into specialized adult cells.
Addressing these questions is critical because the answers may lead to new ways of controlling stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes including cell-based therapies.
Statement of Benefit to California:
The potential to generate any type of human cells in the body will give scientists and physicians the opportunity to understand and treat a number of cell-based diseases. Illnesses occur because of defects in specific cell types. In the future, it may be possible to use cells derived from human embryonic stem cells to repair the damage caused by disease and injury. Repairing and healing fractured bones by injecting precursors of bone cells into the fracture site is a good example of how embryonic stem cells might be used in the future. However, in order to regenerate organs rather than simply treating the symptoms of disease, it will be necessary to understand the molecular events that direct tissue specificity.
Transcription factors are key actors regulating cell differentiation. Amongst these factors is the important family of basic helix-loop-helix (bHLH) proteins. Our goal is to determine the precise role of their negative regulators, the HLH Id proteins, during human embryonic development. Through their interaction with bHLH factors, Id proteins may have the potential to govern tissue differentiation. Specifically, we propose that Id proteins are dominant factors at the top of the hierarchy of transcriptional regulation in embryos.
We expect that the benefits of this research to the field of regenerative medicine in California, i. e., repairing or replacing diseased or defective tissues or organs in the human body, will be significant. The human body is capable of limited self-repair and does so to heal or repair injuries and age-related wear and tear of tissues and organs. While major advances have been made in treatments using biomaterials, antibodies, growth factors, or hormones, there are several diseases and debilitating conditions that cannot be treated using these approaches. For example, in cases of severe injury or damage to organs such as the heart or the spinal cord, the human body is only capable of limited healing, thus leaving the patient in a debilitated or paralyzed state. The promise of stem cells in regenerative medicine is to find new therapeutic avenues for diseases and conditions that currently have limited or no treatment options.
Understanding the role of HLH transcriptional regulators in human embryonic stem cells will be extremely valuable for study of the phenotype that is controlled by this family of genes and may provide an opportunity to screen for small molecules that may modulate the expression of these HLH proteins during the progression of many diseases, e.g., cancer, neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, motor neuron disease, and multiple sclerosis. This strategy may result in the identification of new candidate drugs that could eventually result in treatments that reduce, or control, disease conditions.
SYNOPSIS: The long-range goal of the proposed research is to identify key transcriptional regulators of early embryonic development, with a focus on the basic helix-loop-helix family of DNA-binding transcription factors. Two research Aims are designed to understand the potential roles of the four members of the Id protein family, HLH-proteins that bind the bHLH transcription factors and inhibit their activity by preventing binding to DNA. The first Aim will determine which Id family members are present in human embryos from the pronuclear stage to day-6 blastocysts obtained from collaborators at the Pacific Fertility Center. Aim 2 will analyze the expression of the Id genes in cultured human H1 embryonic stem cells and determine whether the expression patterns change during in vitro differentiation into several different cell lineages. To determine the relevance of expression patterns, Id gene expression will be inhibited by antisense or RNA interference technologies.
SIGNIFICANCE AND INNOVATION: The Id proteins certainly control aspects of cellular differentiation in many settings. Discovering a regulatory role for these proteins in the decision of stem cells to renew or differentiate would have important implications for the effective production of differentiated human cells for transplant therapies
Analyzing the role of Id and bHLH genes in hESC self renewal and differentiation. Lacks innovation.
STRENGTHS: Access to early staged human embryos from the Pacific Fertility Center to verify patterns of gene expression obtained during the differentiation of human ES cell lines in vitro is a valuable asset.
Prior experience of Investigator in analyzing Id gene expression and function.
WEAKNESSES: Although the experiments to examine expression patterns and to perturb Id expression in cultured human ES cells should tell whether Id proteins could play a regulatory role, the results would not reveal what role they may play, nor lead to the identity of the target bHLH factor(s). A summary of published reports of Id expression in embryonic stem cells and the role proposed for Ids in perpetuating the self-renewal state would have helped define the goals of the project better.
Human embryonic stem cells are difficult to grow, to differentiate, and to transfect or treat effectively with inhibitory RNAs. Without prior expertise in these areas or the assistance of experienced collaborators, the experiments of the second aim will be difficult to complete successfully. Much of the success depends on the ability to direct the differentiation of human ES cells using many of the ten listed protocols, although none have been tried in the applicant’s laboratory. Moreover, methods of analysis for Id expression by Northern hybridization or immunoblotting, which sample populations rather than individual cells, likely will not answer whether Id gene expression is confined to specific lineages, because the in vitro differentiation programs do not produce homogeneous cell populations. Rather the use of double hybridization in situ or double immunodetection for individual cells to co-localize Id mRNAs or proteins with differentiation markers specific for each of the protocols would be necessary.
1. No compelling hypothesis.
2. Aim 1 is descriptive and Aim 2 proposes a superficial analysis of developmental functions of Id genes in differentiation of hESC.
DISCUSSION: There was no further discussion following the reviewers' comments.