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 is to apply and optimize these labeling techniques for a sensitive depiction of human embryonic stem cells (hESC) with OI and MRI. Experimental Design: hESC will be 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 will be proven by mass spectrometry (quantifies the iron oxides) and fluorescence microscopy (detects fluorescent dyes). The labeled hESC will undergo imaging studies and extensive studies of their viability and ability to differentiate into specialized cell types.Imaging studies: Decreasing numbers of 1×105 – 1×102 contrast agent-labeled hESC and non-labeled controls will be 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 will be 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 will be performed before and after the labeling procedure in order to prove an unimpaired viability and function of the labeled cells.Image analysis and histopathology: For quantitative analyses, MR signal intensities and mean fluorescence signal intensities of the cell samples and the image background will be measured and compared for significant differences between different groups (labeled cells and non-labeled controls, different contrast agents, labeling techniques, different cell numbers, different time points after labeling) using dedicated statistical tests. These quantitative data will be compared with results from mass spectrometry and histopathology.Significance: The derived data should establish and optimize hESC labeling with contrast agents for a non-invasive depiction of the labeled cells with MR and OI imaging techniques. Our method would be in principle readily applicable for monitoring of hESC -based therapies and direct correlations between the presence and distribution of hESC in the target organ and functional improvements. The results of this study will be the basis for subsequent NIH grant applications.
The ability to depict transplanted stem cells non-invasively with imaging techniques is crucial for monitoring of virtually any stem cell based therapy. A better understanding of the signal behavior of contrast agent labeled hESC on MR images will lead the way to a rational and more effective use of hESC-based therapies in preclinical and clinical applications. Since a large state-supported research program is initiated with the proposition 71, we anticipate a variety of evolving applications for our imaging technique in particular in California. Of note, our cell tracking techniques would not only be applicable to hESC, but also to adult stem cells (after tailoring our labeling protocols to certain stem cell populations), thereby providing a key technique for a non-invasive and repetitive monitoring of stem cell based therapies. It is possible, that we may develop new labeling techniques for hESC, which could be patented and, subsequently, be of financial benefit for the state of California via related royalties and licenses.Potential applications of our imaging technique comprise comparative investigations of the in vivo differentiation properties of embryonic and adult stem cells, investigations of the engraftment potential of various stem cell subtypes or genetically engineered hESCs and assessments of therapy effects on hESC differentiation outcomes. Results should be immediately helpful in preclinical assessments of new hESC-based therapies, in the design of related clinical trials, and later, in the assessment of those hESC-based therapies in clinical practice. We expect, that these numerous potential applications of our imaging technique will attract additional federal and private research funding. This could result in increased research activities and associated investments in California.Since we use clinical applicable contrast agents and MR scanners for the proposed study, our findings should be in principle readily translatable to clinical applications. Following transplantation of iron oxides labeled hESC into target organs, the presence and grade of potential clinical improvement could be correlated with the presence and quantity of hESC at the site of disease. Thus, our imaging technique may help to establish and monitor new hESC-based therapies to cure otherwise chronic, long standing or devastating diseases. This could ultimately result in large scale reductions in health care costs in California.
SYNOPSIS: This proposal deals with the imaging of hESC’s or their progeny, using iron oxide nanoparticles and fluorescent dyes. The goal is to follow the labeled progeny of hESC’s in patients non-invasively. The applicant raises the possibility that such labeling might impair the viability and differentiation capacity of the target cells. To address this, they will explore techniques that will allow labeling of cells with the least impact on the survival and differentiation of hESC’s. The applicant details three specific aims. In aim 1, she will determine the most effective method for labeling hESC’s that is without effect on cell viability, using iron oxide nanoparticles and fluorescent tags to label the cells. Specifically, she will use three different iron oxides nanoparticles to magnetically label two different human embryonic stem cell (hESC) lines, one approved and one non-approved. The migration and homing of these magnetically labeled hESC can be tracked by magnetic resonance imaging (MRI). One of the three nanoparticles will also be tagged with fluorescent dye and can be tracked by optical imaging technique (OI). Aim 2 will explore the differentiation into neurons, chondrocytes and cardiomyocytes of cells labeled as in aim 1. Aim 3 will investigate the long term ability to measure signal from cells and whether the intracellular labelled nanoparticle has any long-term effect on cell differentiation and survival. The aims of the investigations will be accomplished by in vitro studies.
INNOVATION AND SIGNIFICANCE: The proposed work is in an important area of science and medicine. The ability to track labeled cells in the body following their administration or transplantation is of great significance to many future therapies. The application of magnetic resonance imaging (MRI) and optical imaging (OI) to human ES cells has novelty and the approaches, while not totally innovative, are thoroughly and systematically presented. Although the iron oxides labeling techniques have been in use for last few years, systematic investigations concerning the effects of iron oxides label on the proliferation, functional and differential capacities of hESCs is lacking. The proposed investigation is novel but not innovative; however, systematic investigation is warranted to know the effects of magnetic label on hESCs. Optimization of labeling conditions with iron oxides in respect to cellular toxicity and long term imaging prospects is essential for any given type of cells. The rigorous evaluation of the techniques involved in labeling, the contrast agents used and the effects that they have on cell survival and differentiation, as applied to human ES cells, are some of the most original aspects of this work.
With the future prospect of hESC for the treatments of different diseases, it would be necessary to track the implanted cells at regular intervals using any in vivo imaging techniques. Among the current in vivo imaging modalities MRI has the highest resolution and can be used to track the very small number of magnetically labeled hESC. MRI has advantages over nuclear medicine techniques in respect of resolution, and radiation doses to the cells (labeled cells) and patient. In this respect this study is timely and highly significant to translate this technique to human studies.
The proposal is well written and logical with a thorough and rigorous research flow. The PI has established expertise in the field of labeling of cells for imaging and in imaging cells both in in vitro and in vivo and has established a collaborations with a well qualified stem cell investigator, Dr. Pera. The investigator is in an excellent academic setting with all required infrastructure – MRI, Stem Cell Core, etc. The aims of the study are clearly described and can be accomplished without any major impedance. In addition the time line to complete the study is well explained.
Applicant has acknowledged that some of the strategies are scientifically risky. It is an open question whether the cardinal feature of human ES cells, that is their high mitotic rate will lead to dilution of the label leading to an inability to image the cell/s. Other investigators already have indicated that intracellular iron will disappear by 5-8 cell divisions in rapidly growing cells. hESCs are very rapidly growing cells and have tendency to form layers. If labeled hESCs are kept on MEF for growth, the labeled hESCs will loose intracellular iron rather rapidly and there may not be any iron left within the cells by day 14. In that case, the long-term follow up and toxicity study will not be possible. To overcome the rapid loss of intracellular iron, PI may consider making EB soon after labeling hESCs. These EB can be kept in culture and disintegrated at specific interval to investigate the effects of iron label and to study the detectability threshold by MRI. PI should consider making a growth inhibited hESCs (in vitro) model that would mimic the in vivo scenario where incorporated hESCs may not grow.
Major weakness of the proposal is the inconsistency regarding the starting doses of iron for labeling hESCs. In the section “Rationale and Significance” the PI has indicated that the doses of iron oxides that have been in use to label cells (including adult stem cells) might be high and would be more efficient for hESCs, and higher dose may initiate apoptosis. However, in the proposal the PI has mentioned to start labeling with a starting dose of 50 µg Fe/ml, which is rather high for 1x106 cells. PI should start labeling cells with a smaller dose. In addition, it is not clear to this reviewer about the implication of making embryoid body (EB). Will PI convert labeled hESCs to EB or EB be produced before labeling? If PI is planning to label EB, there will be unlabeled cells at the center of the EB. If PI has a plan to label hESCs, when they are in a single layer on MEF, how labeled MEF can be separated from hESCs?
DISCUSSION: This was a thorough and systematic proposal on imaging. No one has done this type of work with hESCs. Both reviewers, however commented on the issue of the high rate of mitotic division in hESCs which makes the labeling risky. Although the PI didn't consider this issue in the application, they should be able to explore this issue and perhaps give a better idea of the number of divisions that can be analyzed relative to the MR imaging technique used. There was some skepticism over the limit of resolution (number of cells) of MRI as an imaging technology for hESC (and derivitives), and whether the imaging would ultimately be useful for replacement therapy and integration. Detecting single cells may require a concentration of iron oxide that would prove to be toxic.