ENGINEERED MANIPULATIONS OF HUMAN EMBRYONIC STEM CELLS USING NANO-NEEDLES AND NANO-PARTICLES

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Stem cell therapy can be useful for treating various diseases, such as spinal cord injury, Parkinson’s disease, Alzheimer’s disease and diabetes. However, the use of human embryos raises ethical as well as supply issues. There is also a major possibility of undesirable rejection of the transplanted stem cells administered to patients who have these diseases. While some recent advances offer promising results, such techniques currently have some critical flaws, hence new innovative approaches are needed to resolve the encountered or anticipated problems. The proposed research contains an effort to lay the ground work for resolving some of the major hurdles using nanotechnology and physical science approaches. To expedite therapeutic applications of stem cells, better understanding of the many factors which influence stem cell behavior, and new methods to cause differentiation of stem cells need to be developed. While there have been significant research investigations of stem cell behavior using cell biology approaches, there have been few studies to explore new ways to direct the fates of stem cells using physical science methods. Therefore, in this proposal of interdisciplinary research, we will introduce forefront nanotechnology to engineer desirable and beneficial changes in the behavior of human embryonic stem cells (hESCs). With recent nanotech advances, nanoscale manipulations (i.e., 1/80,000 the width of a human hair) of very small particles, materials, molecules are possible, inside the cells as well as inside the site where genes are located, the nucleus. Nanotechnology has not yet been seriously applied to stem cell science to advance clinical therapies. The significance of the proposed research lies in the possible control of differentiation pathways (e.g. stem cell to nerve cell), specifically at the level of the nucleus, in order to create therapeutically useful and ethically acceptable stem cells. The specific aims are thus directed toward the demonstration that bioengineered manipulations of stem cells using nanotechnology will accomplish what has not been previously possible with traditional laboratory methods. To validate the nanotech approach, experiments for controlled intracellular and intranuclear stem cell manipulations will be applied, and their effects on stem cell differentiation into a variety of mature cell types (pluripotency), will be investigated. Advances in stem cell manipulations using nanotechnology will provide significant and powerful ways to accelerate realistic therapeutic applications of stem cells.
Statement of Benefit to California: 
For some patients with diseases, such as spinal cord injury, Parkinson’s disease, Alzheimer’s disease and diabetes, stem cell therapy offers a great hope for efficient therapeutic treatment of the patients. To expedite therapeutic applications of stem cells, an understanding of the key parameters which influence the stem cell behavior, and efficient technical means to induce differentiation into well-defined lineages are needed. While there have been significant worldwide research efforts for studies of stem cell behavior via biological approaches, with some recent advances offering promising approaches for future stem cell therapeutics, there has been little effort to explore new avenues of possible stem cell controls using physical science technology approaches. The proposed research contains an effort, using nanotechnology and associated innovative approaches, to lay the ground work for resolving some of the major hurdles or anticipated problems in the practical medical application of the current stem cell research advances. The successful outcome of the proposed research will benefit many California residents suffering from Alzheimer’s disease, diabetes, spinal cord injury, Parkinson’s disease. The estimated cost of healthcare in California related to the Alzheimer’s type disease alone is several hundred million dollars every year. There are nearly 1.5 million California residents diagnosed with diabetes with additional a few million people at risk. There is tremendous financial impact on California with the introduction of stem cell therapy for cure of these diseases. The proposed research on nanoscale stem cell manipulations is also substantially dependent on utilization of nanotechnology and materials, devices, processes being employed for semiconductor industries. The State of California is very strong in semiconductor industry including many silicon valley based high-tech companies. The State is also one of the significant leaders in the forefront biotechnology. The success of the proposed stem cell manipulation and control techniques, and their eventual commercialization for stem cell therapeutics, can foster expansion of the business scopes and revenues for some of the biotech as well as nanotech companies in California, thus creating jobs and enabling the State of California to become a world leader in stem cell science, technology, and medical applications. Such blossoming technical and medical advances will have profound positive effects on the vision of young scientists in California. This will also help many of the State’s large medical institutes to become world leaders in their respective therapeutic treatments of various diseases.
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