New Strategies to Understand Reprogramming Events in the Donor Nuclei Following Somatic Cell Nuclear Transfer

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The possibility of making embryonic stem cells (NT-ESC) using a process called somatic cell nuclear transfer (SCNT), popularly known as “therapeutic cloning,” has captured the imagination of the scientific community because of the technology’s remarkable potential. Patient–specific embryonic stem cell lines have the potential to revolutionize regenerative medicine. When cells, tissue or even entire organs are damaged by disease, we may soon be able to make repairs using replacement cells created through the therapeutic cloning process. This technology involves removing the genetic material inside a donor egg (oocyte) and replacing it with a mature cell to generate embryos, and subsequently disease- or person-specific embryonic stem cell lines. For NT-ESC research to make any significant progress, a substantial number of eggs and embryos will be required. Because there is such a shortage, the eggs and embryos that are donated must be utilized in the most efficient way possible. Unfortunately past SCNT experience has taught us this technique can create embryos that appear normal, but because of improper cell development, are defective. A critical time in that development comes during the first few seconds after the replacement cell and empty egg come together. This project will focus on developing a new method of monitoring the molecular events during these important moments. This time frame is significant because genetic material in the donor cell must be rejuvenated (be returned to an immature state) for proper development of the embryo and the subsequent successful creation of an ESC line. Finding the source of defects will depend on observing how the replacement cells interact with the egg. Having the ability to observe this interaction without retarding further development would be an important scientific advance, one that could allow researchers to improve the success rate of SCNT, thus maximizing the limited genetic resources of eggs and embryos. Support for this project is intended to train an early career scientist who will collaborate with highly skilled individuals from a number of research disciplines, including human embryology, SCNT, non-toxic fluorescent imaging of cells and the assessment of developmentally important genes in cloned embryos. It presents a tremendous opportunity to bring a fresh mind into this research and train them under some of the foremost experts in the field. Although the proposed research is not primarily intended to lead to a specific therapeutic application (although it might), it is designed to provide a strong scientific basis for any such research in the future because the findings will be made available to all scientists through expected publication in scientific journals.
Statement of Benefit to California: 
The projected annual economic impact of Alzheimer’s, Parkinson’s and other presently incurable degenerative diseases is staggering. Alzheimer’s disease alone is projected to cost California’s economy $10 Billion per year. These degenerative diseases, along with cystic fibrosis, spinal cord injury, macular degeneration, and others, are all potential targets for the development of treatments developed through the research of embryonic stem cells. Perhaps the most promising type of stem cell research is somatic cell nuclear transfer (SCNT), also referred to as “therapeutic cloning.” It involves removing the genetic material (DNA) from a donor egg (oocyte), and replacing it with an adult cell from a person with a degenerative disease who might benefit from stem cell therapy. After this new cell develops and multiplies, it may be used to create embryonic stem cells that would not be rejected by the person requiring treatment. These stem cells could then be used to replace brain cells destroyed by Alzheimer’s, nerve cells damaged by stroke or Parkinson’s, abnormal lung tissue from cystic fibrosis, or spinal cord nerves damaged in a traumatic injury, and, as has been shown recently in a study from the University of Washington, retinal cells damaged by macular degeneration. The efficient use of human eggs donated to the program is essential if the full potential of therapeutic cloning is to be realized. Unfortunately past research on SCNT has taught us that this technique can create embryos that appear normal, but because of improper cell development, are in actuality defective. A crucial time in development comes during the first few seconds after the replacement cell and donor egg come together. Critical to this process is an understanding of the events that transpire during this time. The newly created cell must resume the correct pattern needed for early embryonic development and the subsequent isolation of embryonic stem cell lines with therapeutic potential. This research is targeted at developing a new non-destructive technique that would allow these events to be monitored while permitting the embryo to continue its normal development. In this way, the usefulness of different cell types and procedures can be directly evaluated to improve the efficiency of SCNT and to reduce or eliminate those with abnormal development. The resulting cells will be carefully assessed using recognized procedures for assessing chromosome number and correct expression of developmentally important genes. An important additional benefit of this type of research is that it may assist in the development of more effective and efficient treatments for the many Californians with infertility, by providing a greater understanding of methods used to detect normal and abnormal early embryonic development. By identifying and utilizing only normal embryos for treatment, higher pregnancy rates with fewer embryos utilized may be possible.
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