وسم الخلايا الجذعية مع Ferumoxytol ، وهو أوكسيد الحديد وافقت عليها الهيئة الجسيمات النانوية
Summary
وصفنا تقنية لوضع العلامات وتعقب الخلايا الجذعية مع ادارة الاغذية والعقاقير التي وافق عليها ، superparamagnetic أكسيد الحديد (سبيو) ، ferumoxytol (Feraheme). هذه التقنية التي تستخدم الخلوية التصوير بالرنين المغناطيسي (MR) التصوير عن التصور ، ويمكن الوصول بسهولة للرصد طويل الأجل وتشخيص ناجحة أو فاشلة engraftments الخلايا الجذعية في مرضى.
Abstract
Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an “off label” use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.
Protocol
Discussion
تحسين فعالية engraftments الخلايا الجذعية هو أمر حاسم لتطوير الطب التجديدي. وهناك تقنية التصور غير الغازية للخلايا الجذعية في الجسم الحي يعزز بشكل كبير من قدرتنا على فهم الآليات التي تؤدي إلى نتائج engraftment ناجحة. وسم المغناطيسي لرؤية MR ، مثل إجراء أننا أظهرنا ، ويسمح <em…
Disclosures
The authors have nothing to disclose.
Acknowledgements
وأيد هذا العمل من خلال منحة من المعهد الوطني لالتهاب المفاصل والعضلات والعظام والأمراض الجلدية : 3R01AR054458 – 02S2.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| D-MEM High Glucose | Sigma | D5648 | Or other base medium for desired stem cell line to be used |
| D-PBS (Ca++, Mg++ free) | GIBCO | 14190-144 | |
| Trypsin-EDTA 0.05% | Invitrogen | 25300-120 | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30071.03 | |
Ferumoxytol (Feraheme) |
AMAG | 59338-0775-01 | |
| Protamine Sulfate | APP Pharm. | 22930 |
References
- Narsinh, K. H., Plews, J., Wu, J. C. Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?. Mol Ther. 19, 635-638 (2011).
- Bulte, J. W. In vivo MRI cell tracking: clinical studies. AJR. Am. J. Roentgenol. 193, 314-325 (2009).
- Henning, T. D., Boddington, S., Daldrup-Link, H. E. Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging. J. Vis. Exp. (13), e685-e685 (2008).
- Tallheden, T., Nannmark, U., Lorentzon, M. In vivo MR imaging of magnetically labeled human embryonic stem cells. Life. Sci. 79, 999-1006 (2006).
- Jung, C. W., Jacobs, P. Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil. Magn. Reson. Imaging. 13, 661-674 (1995).
- Coyne, D. W. Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease. Expert. Opin. Pharmacother. 10, 2563-2568 (2009).
- Li, Z., Suzuki, Y., Huang, M. Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects. Stem Cells. 26, 864-873 (2008).
- Metz, S., Bonaterra, G., Rudelius, M. Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro. Eur. Radiol. 14, 1851-1858 (2004).
- Nedopil, A., Klenk, C., Kim, C. MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis. Invest. Radiol. 45, 634-640 (2010).
- Kraitchman, D. L., Heldman, A. W., Atalar, E. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation. 107, 2290-2293 (2003).
- Stuckey, D. J., Carr, C. A., Martin-Rendon, E. Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart. Stem Cells. 24, 1968-1975 (2006).
- Henning, T. D., Sutton, E. J., Kim, A. The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells. Contrast. Media. Mol. Imaging. 4, 165-173 (2009).
- Arbab, A. S., Yocum, G. T., Kalish, H. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood. 104, 1217-1223 (2004).
- Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects. J. Vis. Exp. (38), e1793-e1793 (2010).
- Arbab, A. S., Yocum, G. T., Wilson, L. B. Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability. Mol Imaging. 3, 24-32 (2004).
- Babic, M., Horak, D., Trchova, M. Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling. Bioconjug Chem. 19, 740-750 (2008).
- Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. Accelerated stem cell labeling with ferucarbotran and protamine. Eur. Radiol. 20, 640-648 (2010).
- Lu, M., Cohen, M. H., Rieves, D. FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease. Am. J. Hematol. 85, 315-319 (2010).
