Adhesion Molecule Function in Human Hematopoietic Development

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Bone marrow transplantation provides a source for human blood stem cells. These cells have been used for years in the treatment of immunodeficiency, cancer and other genetic disorders. Unfortunately, human blood stem cells have a short life span and cannot be easily expanded in the laboratory setting. These shortcomings can be circumvented by the use of human embryonic stem cells (hESC). Human ESC can produce every tissue of the body (including blood), and can be expanded indefinitely in the laboratory. The goal of our work is to understand the cues that control hESC generation of blood cells. In particular, we want to define the role played by adhesion molecules, molecules that mediate cell-to-cell interactions, in the generation of blood cells from hESC. To achieve our goals, we need to develop new tools that will allow us to follow the hESC as they progress down the pathway of becoming a blood cell. In particular, we propose to endow hESC with a reporter molecule that will identify cells that have decided to follow the blood cell path. Moreover, we will develop novel culture methods in the laboratory to assay the hESC’s capacity to produce blood cells. These innovative tools will permit us to determine which adhesion molecules are present in hESC and how the expression of these molecules fluctuate during the process of blood generation. Understanding the process of blood generation from hESC will allow us to develop new therapeutic strategies for the treatment of immunodeficiency and cancer. This aspect of Regenerative Medicine will have major impacts on the way that bone marrow and blood stem cell transplantation is done, hopefully reducing or completely eliminating the detrimental side effects of this therapy.
Statement of Benefit to California: 
California has been at the forefront of scientific discovery. It is known throughout this country for its cutting edge approach to scientific research. The work described in this proposal will help increase our understanding of the process of blood cell generation from human embryonic stem cells. Ultimately, this knowledge will allow us to develop new therapeutic strategies for the treatment of many diseases, including transplantation of blood stem cells to treat immunodeficiency, genetic deficiencies and cancer. California will benefit directly from these discoveries at multiple levels. First, it will support the state reputation as a beacon of scientific discovery. This will positively influence the recruitment of students, scientists and medical doctors to the state, thereby increasing the intellectual pool that will further feed California’s scientific and intellectual engine. Second, discoveries of this nature will foster more entrepreneurial and biotechnological developments within the state, resulting in the creation of companies that will provide needed jobs and help the state’s economy. Lastly, the creation of these companies will result in the promotion of these scientific discoveries to the clinical setting, ultimately benefiting California’s citizens that desperately need these life saving therapies.
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.

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