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Rowena Mittal — 2008-09 Fellow

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Project Description
One of the most promising advances in recent regenerative research has been the isolation of pluripotent stem cells, embryonic and adult, capable of differentiating to multiple tissues. However, technical hurdles must be overcome in the study of stem cell location and fate in vivo before practical stem cell based therapies will be approved by the Food and Drug Administration. One of the most critical hurdles to be overcome is assessing the fate of transplanted stem cells, both in experimental animals and ultimately in the clinic. Current techniques used for studying stem cell biology such as immunohistochemical difference analysis, genetic manipulation, and dye-labeling are extremely limited in their ability to provide qualitative or quantitative assessments noninvasively on stem cell behavior in live animal models, much less during clinical applications. Presently, there are no effective noninvasive toolsets available to monitor stem cells. If an advanced level of understanding of stem cell location and differentiative fate in vivo can be reached, researchers may be able to cure diseases such as Parkinson’s disease, diabetes, heart disease, multiple sclerosis, spinal cord injuries, and other degenerative diseases. We propose to develop a quantitative toolset, using novel multimodal nanoparticles, which will represent the first system capable of noninvasively monitoring transplanted stem cell fate using fluorescence and micro CT with subsequent histological validation upon biopsy or necropsy. This toolset will significantly contribute to the practical application of stem cell methodologies. This research aligns well with the CIT/ICES strategic focus because it is a novel technique requiring a multidisciplinary and collaborative approach with profound value for the scientific and medical research communities and significant potential for commercial development.

Approach and Methodology
Researchers of the Molecular Bioesensor and Imaging Center (MBIC) at CMU have recently developed quantum dots, or nanoparticles of various semiconductor materials, capable of having measurable x-ray contrast in conventional CT scanners as well as infrared fluorescence. Due to their small size, stability under physiological conditions, and our ability to functionalize their surfaces with biointeractive molecules; internalized nanoparticles provide a large signal which can be measured noninvasively without interfering with cell biology. However, these novel particles need to be validated in live models and optimized for fluorescence and x-ray absorbance before they can become a marketable, conventional tool for stem cell research. In this research, novel nanoparticles developed by MBIC will be validated for tracking of stem cell location and differentiative fate in live small-animal studies.

With co-advisor expertise of Marcel Bruchez in quantum dot development for imaging applications and Phil Campbell in the application of quantum dots in various tissue-engineering models, I will investigate this novel research toolset with the following approach. Novel nanoparticles will be calibrated for CT and fluorescence across a range of concentrations, and in a variety of formats or “phantoms.” These phantoms include solutions, gels, cells in vitro, and engineered tissue scaffold implanted subcutaneously in mice. After calibration, properties of the nanoparticles must be optimized for performance. Such properties are interdependent, and include per particle contrast, per particle fluorescence, cellular uptake efficiency, and toxicity in vitro and in vivo. To improve contrast, particle size can be increased; however particle size is limited by the restraints of cellular uptake biology. To improve fluorescence, fluorescent dyes such as cyanine can be conjugated to these novel nanoparticles, also allowing for flexibility in imaging setup. The safety of these nanoparticles for in vivo applications needs to be assessed, and assays will be conducted in vitro to observe the effect of particle uptake on cell biology. The nanoparticles that will be used for studies during this fellowship are of two basic types—the traditional CdSe nanoparticles available commercially, which have some significant safety considerations in clinical application, and recently discovered and prepared novel nanoparticles prepared by Dr. Bruchez’s group, which have substantially lower potential toxicity concerns. We will assess both of these materials in a variety of formats for effects on cell proliferation, apoptosis, and differentiation using a combination of biochemical methods and time-lapse microscopic imaging methods. A collaborative effort with Dr. Bruno Peault of the Stem Cell Research Center at Children’s Hospital of Pittsburgh will be taken to apply this toolset to answer clinically relevant questions regarding stem cells in vitro and in vivo.

Additionally, this research will develop methods to quantify the cellular uptake of nanoparticles, which will add significantly to the state-of-the-art. Currently, there is no method for determining the number of particles taken per cell. Without this value, optimizing a toolset for tracing stem cell fate will be difficult; literature speculates that nanoparticles are divided between daughter cells during mitosis, and potentially lost or transferred in solution after apoptosis. It is essential to understand these processes quantitatively to develop a more broadly applicable toolset for tracking cell fate and history in relevant clinical model systems. We will develop analytical and quantitative methods based on elemental analysis of digested cells, and establish correlations between these labor intensive and destructive methods and more practical methods that can be easily implemented in routine cell-biology experimentation, such as flow cytometry.