Stress response signaling pathways in hESCs

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
A major challenge in the development of regenerative medicine strategies involving human embryonic stem cells (hESCs) remains the routine and reproducible culturing of hESCs in the laboratory without loss of their developmental potential. Outside their natural environment, cell culture conditions in the laboratory represent artificial cellular environments that cause cellular stress and may harm the cell physiology. Most human cells respond to cellular stress conditions by activating mechanisms that are capable of reducing the damage to the cell. However, activating these cellular stress response mechanisms may often also have consequences for the proliferation and developmental capacity of the cells. To response DNA damage, most cells for example inhibit the progression of the cell cycle until the DNA has been repaired. In previous work, our laboratory has characterized the regulatory proteins and mechanism that control stress responses in mammalian immune cells. We are able use computational simulations of these complex regulatory mechanisms to gain insights and direct specific experiments. Here we propose to determine how stress responses are regulated in hESCs. We will make quantitative measurements regarding the presence of the key regulatory proteins and construct a computational model of the regulatory networks as it pertains to hESCs. This allows us to compare different hES cell lines, provide important insights on their physiological regulation, and predict the effects of pharmacological treatments. As the very first molecular and computational description of important regulatory mechanisms, the results will also form the basis for future studies that monitor the molecular changes during tissue development and the role these molecular components play in such tissue development. In addition, we propose to engineer molecular tools that allow us to monitor the molecular stress level in the cell. Engineered proteins inserted into hESCs will be used as sensors of cellular stress. By monitoring their activity we will optimize laboratory methods and techniques related to growing stem cells in the laboratory. These studies may have result in improving current technologies and make a critical step in developing regenerative medicine more reliable and routine.
Statement of Benefit to California: 
A major challenge in the development of regenerative medicine strategies involving human embryonic stem cells (hESCs) remains the routine and reproducible culturing of hESCs in the laboratory without loss of their developmental potential. Outside their natural environment, cell culture conditions in the laboratory represent artificial cellular environments that cause cellular stress and may harm the cell physiology. The proposed research is focused on characterizing the molecular stress responses in hESCs. The research will significantly contribute to an understanding of stem cell biology and the molecular mechanism that regulate the capacity for self-renewal and for tissue development. The molecular characterization will contribute to an understanding how different hESC lines differ and what their respective advantages may be. And finally the proposed research will result in improved laboratory methodologies for the handling of hESCs. The research will benefit the State of California and its citizens by (1) enhancing the scientific knowledge base of the molecular mechanisms that control hESC physiology and developmental capacity (2) improving the methodologies for culturing and manipulating hESCs thereby increasing the likelihood of successful of regenerative medicine strategies (3) making California a leader in stem cell systems biology by constructing a computational model that allows for virtual cell studies by simulations (4) training a junior PI and two talented postdoctoral fellows with proven scientific track records in stem cell biology, enabling them to contribute to Regenerative Medicine in their own future laboratories.
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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