Proteome-wide Profiling of Ubiquitination and Sumoylation in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem (hES) cells can serve as a biological repair system, with the potential to develop into many types of specialized cells present in the body. They can theoretically divide without limit to replenish other cells. When a stem cell divides, each daughter can remain a stem cell or adopt a more specialized role such as a muscle, blood or brain cell, depending on the presence or absence of biochemical signals. Thus, any therapeutic application will require the driving of the hES cells’ differentiation into particular specialized cells for transplantation into patients—for instance, beta cells to produce insulin for diabetics or dopamine-producing neurons to treat Parkinson’s disease. Controlling this differentiation process is one of the biggest challenges in stem cell research. Understanding of the mechanisms that regulate different differentiation processes helps to control the fate of hES cells in petri dishes to generate various cell types for regenerative therapy. Inside cells, proteins can be attached covalently to small proteins such as ubiquitin and SUMO under different biological conditions. It has been shown that ubiquitination and sumoylation play critical roles in regulating protein functions after they are linked to ubiquitin or SUMO. Many of the known targets of ubiquitination are the regulators for controlling cell growth, differentiation, proliferation and death. Sumoylation has been shown to be critical for gene transcription. It is reasonable to believe that these pathways will be involved in manipulating the fate of hES cells, affecting their behaviors under different chemical stimulus for differentiation. However, the molecular details of these processes are currently unknown. We propose to identify the cellular factors that are uniquely modified at different stages of hES cell development. The results obtained in this study will provide direct information about the regulatory process in hES cells at a proteome-wide level. This will lead to identification of important proteins that are specifically regulated in hES cell development, and help to design conditions and design therapeutic agents to manipulate ubiquitination or sumoylation pathway to control the growth and differentiation of hES cells. Clearly, this would be an enormous step forward towards clinical application of hES cells. In addition, some of the identified modified proteins can be used as markers for proteome-based screening of different hES cell types, which is critical when regenerative treatment is carried out. In summary, the proposed research will unravel the roles of the key cellular regulatory mechanism in hES cell biology. The outcome of the research can help to develop specific diagnostic tools and will also be important to the understanding and manipulation of hES cell differentiation.
Statement of Benefit to California: 
This proposal will provide unique information of one of the key regulatory pathways controlling the fate of hES cells. The results will help to understand how hES cells is regulated regarding their growth, differentiation and survival. This is critical for generating specialized cell types for regenerative treatment for various human diseases such as diabetes, neurodegenerative disorders, heart diseases, etc. About half of California's families are estimated to have a member who could potentially benefit from stem-cell therapies. In addition, some of the identified modified proteins can be used as markers for proteome-based screening of different hES cell types, which is critical when regenerative treatment is carried out. Furthermore, the results will help us to determine conditions and design therapeutic agents to manipulate ubiquitination or sumoylation pathway to control the growth and differentiation of hES cells. We believe this would be an enormous step forward towards clinical application of hES cells. In summary, the scientific outcome of this proposal will provide a strong basis for inspiring biotech industry in California, providing job opportunities for Californian residents and stimulating economy growth. Exciting research in stem cells will also draw the best young scientists to the area to pursue career and advance science and medicine to keep California competitive in the field of regenerative medicine.
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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