HLH Factors in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • CIRM Grant – Public Abstract:
  • Non-invasive imaging techniques for an in vivo tracking of transplanted stem cells offer real-time insight into the underlying biological processes of new stem cell based therapies, with the aim to depict stem cell migration, homing and engraftment at organ, tissue and cellular levels. We showed in previous experiments, that stem cells can be labeled effectively with contrast agents and that the labeled cells can be tracked non-invasively and repetitively with magnetic resonance imaging (MRI) and Optical imaging (OI). The purpose of this study was to apply and optimize these labeling techniques for a sensitive depiction of human embryonic stem cells (hESC) with OI and MRI.
  • Experimental Design: hESC were labeled with various contrast agents for MRI and OI, using a variety of labeling techniques, different contrast agent concentrations and different labeling intervals (1h – 24h). The cellular contrast agent uptake was proven by mass spectrometry (quantifies the iron oxides) and fluorescence microscopy (detects fluorescent dyes). The labeled hESC underwent imaging studies and extensive studies of their viability and ability to differentiate into specialized cell types.
  • Imaging studies: Decreasing numbers of 1 x 10^5 - 1 x 10^2 contrast agent-labeled hESC and non-labeled controls were evaluated with OI and MRI in order to determine the best contrast agent and labeling technique as well as the minimal detectable cell number with either imaging technique. In addition, samples of hESC were investigated with OI and MRI at 1 min, 2 min, 5 min, 1h, 2h, 6h, 12h, 24h and 48 h in order to investigate the stability of the label over time. Viability and differentiation assays of the hESC were performed before and after the labeling procedure in order to prove an unimpaired viability and function of the labeled cells.
  • Results: The FDA-approved contrast agents ferumoxides and indocyanine green (ICG) provided best results for MR and optical imaging (OI) applications. The cellular load with these labels was optimized towards the minimal concentration that allowed for detection with MR and OI, but did not alter cell viability or differentiation capacity. The ferumoxides and ICG-labeled hESCs as well as stem cell derived cardiomyocytes and chondrocytes provided significantly increased MR and OI signal effects when compared to unlabeled controls. ICG labeling provided short term labeling with rapid excretion of the label from the body while ferumoxides labeling allowed for cell tracking over several weeks.
  • Significance: The derived data allowed to establish and optimize hESC labeling with FDA approved contrast agents for a non-invasive depiction of the labeled cells with MR and OI imaging techniques. Our method is in principle readily applicable for monitoring of hESC -based therapies in patients and allows for direct correlations between the presence and distribution of hESC-derived cells in the target organ and functional improvements. The results of this study will be the basis for a variety of in vivo applications and associated further grant applications.

© 2013 California Institute for Regenerative Medicine