Amy B. Rosen

4th Year Medical Student

Department: SOM

Graduate Program: Biomedical Engineering

Advisor: Dr. Ira Cohen

Abstract:

Title:  Finding Fluroescent Needles in the Cardiac Haystack: Tracking the location and fate of hMSCs with quantum dots for electrical and mechanical repair of damaged myocardium

Amy B. Rosen, Damon J. Kelly, Adam J. T. Schuldt, Irina A. Potapova, Sergey V. Doronin, Richard B. Robinson, Michael R. Rosen, Peter R. Brink, Glenn R. Gaudette and Ira S. Cohen

With the advent of clinical trials for cardiac regeneration employing adult stem cells, it becomes essential for both safety and efficacy to know the spatial distribution of cells delivered in vivo in animal models and in humans. Quantum dots (QDs) are extremely bright semiconductor nanoparticles with unique spectral properties that permit sensitive detection excluding autofluorescence. We have developed an approach to uniformly load QDs into adult human mesenchymal stem cells (hMSCs) via endocytosis. This optimized loading procedure avoids perinuclear aggregation, which can compromise hMSC viability. Both unloaded hMSCs and those with intracellular QDs proliferate and differentiate to terminal lineages similarly. Further, by tracking loaded hMSCs through multiple divisions in vitro, we confirmed that dilution of QDs per cell did not hinder signal detection over 44 days (at least 6 divisions) in vitro. QDs did not cross gap junctions between hMSCs and were not taken up by cardiac myocytes in culture, suggesting that false positives should not pose a serious concern. We have imaged and enumerated QD-loaded hMSCs through the full thickness (100µm) of an extracellular matrix patch, an impossibility with traditional cell tracking agents such as EGFP. To track stem cell location in vivo, we injected QD-loaded hMSCs into the left ventricular wall of the rat heart. One hour later, cardiectomies were performed and 10-µm-thick sections were obtained and imaged for QD fluorescence. Computer algorithms were developed to “stack” the fluorescent images and recreate the three-dimensional distribution of the injected stem cells and analyze their distribution. We have also delivered QD-loaded hMSCs and cardiogenic cells on an extracellular matrix patch to the canine ventricle to replace a full thickness cardiac defect. After 8 weeks, QD-positive cells were identified within the myocardium. Some of these cells expressed markers of endothelial and cardiac myocyte lineages, suggesting that they had differentiated and retained the label in the process. Given their ease of loading, absence of effects on proliferation and differentiation and the low likelihood of false positives, these results suggest it is feasible to use QDs to label stem cells and track their location and fate following in vivo delivery to the mammalian heart.

Publications:
(MSTP-supported publications indicated with an *)

*Cohen, I.S., Rosen, A.B., and Gaudette G.R. (2007). A Caveat Emptor for myocardial regeneration: Mechanical without electrical recovery will not suffice. Journal of Molecular and Cellular Cardiology. 42(2): 285-288.

*Rosen, A.B., Kelly, D.J., Schuldt, A.J.T., Lu, J., Potapova, I.A., Doronin, S.V., Robinson, R.B., Rosen, M.R., Brink, P.B., Gaudette, G.R., and Cohen, I.S. Long term tracking of human mesenchymal stem cells loaded with quantum dots for quantitative in vivo 3-D fluorescence analysis. (Submitted)

Brian P. Helmke, Amy B. Rosen, and Peter F. Davies (2003). Mapping Mechanical Strain of an Endogenous Cytoskeletal Network in Living Endothelial Cells. Biophys. J. 84:2691-2699.

Abstracts from conference proceedings

*A.B. Rosen, Kelly, D.J., Schuldt, A.J.T., Lu, J., Potapova, I.A., Doronin, S.V., Robinson, R.B., Rosen, M.R., Brink, P.R., Gaudette, G.R., and Cohen, I.S. Quantitative 3-D reconstructions of quantum dot-loaded human mesenchymal stem cells injected into the rat ventricle in vivo. Biomedical Engineering Society Meeting. Chicago, IL, 2006.

*A.B. Rosen, Kelly, D.J., Schuldt, A.J.T., Lu, J., Potapova, I.A., Doronin, S.V., Robinson, R.B., Rosen, M.R., Brink, P.R., Gaudette, G.R., and Cohen, I.S. Finding fluorescent needles in the cardiac haystack: reconstructing the three-dimensional distribution of human mesenchymal stem cells injected into the rat ventricle in vivo using quantum dot nanoparticles. Circulation Research. 2006 99(5):E34.

*Potapova, I.A., Doronin, S.V., Kelly, D.J., Rosen, A.B., Schuldt, A.J.T., Lu, Z., Guo, Y., Raptis, N.D., Towner, A.A., Robinson, R.B., Rosen, M.R., Brink, P.R., Gaudette, G.R., and Cohen, I.S. Functional regeneration of the canine ventricle using adult human mesenchymal stem cells committed in vitro to a cardiac lineage. Circulation Research. 2006 99(5):E19.

B. P. Helmke, A. B. Rosen, K. Choi, and Peter F. Davies. Shear Stress–Induced Strain Focusing in the Endothelial Cytoskeleton. Biomedical Engineering Society Meeting. Houston, TX, 2002.

B. P. Helmke, A. B. Rosen, and P. F. Davies. Measurement of Cytoskeletal Strain Field Induced by Shear Stress in Living Endothelial Cells. Arteriosclerosis, Thrombosis and Vascular Biology Conference, American Heart Association. Arlington, VA, 2001.

Brian P. Helmke, Amy B. Rosen, David B. Thakker, Robert D. Goldman, and Peter F. Davies. Spatiotemporal Organization of Cytoskeletal Strain Induced by External Fluid Mechanical Forces. American Society of Mechanical Engineers Bioengineering Conference. Snowbird, UT, 2001.

News:

Congratulations to MSTP's own Amy Rosen!
Amy has been named a finalist in the Collegiate Inventors Competition for her entry entitled, "Tracking the 3-D distribution of delivered stem cells in vivo with quantum dot nanoparticles." Amy could win up to $25,000 for her work! She's traveling tomorrow on all-expenses paid trip to Washington D.C. for a final round of judging and the awards dinner and presentation. More details can be found at the Collegiate Inventors Competition website.

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