DOI: 10.1148/radiol.2473071885
(Radiology 2008;247:914-915.)
© RSNA, 2008
Iron Oxide–Transfection Agent Complexes Are Not Expected to Coat the Cell Membrane and Prevent CD71 Expression
Branislava Janic, PhD and
Ali S. Arbab, MD, PhD
Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, 1 Ford Place, Detroit, MI 48202
e-mail: saali{at}rad.hfh.edu
Editor:
We read with great interest the article by Dr Schäfer and colleagues (1), in the August 2007 issue of Radiology, on transferring receptor upregulation in ferucarbotran-labeled rat mesenchymal stem cells (MSCs). However, the presented work raised questions.
The authors labeled rat MSCs by incubating cells for 4 hours with the superparamagnetic iron oxide (SPIO) agent ferucarbotran complexed to transfection agents (TAs) (ie, DOSPER or jetPEI [1]) and concluded that ferucarbotran-TA complexes remained attached to cell membrane and were not taken up by the cells. However, recent work has clearly demonstrated that overnight incubation of human MSCs with ferumoxides complexed to poly-L-lysine resulted in significant intracellular uptake and subsequent endosomal and lysosomal degradation (2). Therefore, it is possible that 15-hour incubation would have resulted in intracellular uptake of the ferucarbotran-TA complex instead of coating the cell surface. The authors' (1) explanation for coating does not agree with the physiochemical properties of SPIO-TA complexes' attraction to the cell surface (3) or the mechanistic of endocytosis. The authors did not provide any evidence that ferucarbotran-TA complexes have blocked endocytosis pathways.
Similar methods should have been used (ie, concentrations of iron and incubation times) for labeling cells with ferucarbotran or ferucarbotran-TA complexes before comparing the expression of CD71. The increased expression of CD71 in ferucarbotran-treated cells could be due to high amounts of extracellular iron (4). In addition, it is well documented that increased intracellular iron decreases the expression of transferrin receptors, possibly by a negative feedback mechanism (5). Since the effect of ferucarbotran alone and ferucarbotran-TA complexes on CD71 expression was the main focus of this work, it would have been appropriate to analyze CD71 expression at the total protein and/or messenger RNA levels.
The authors indicated a positive association between intracellular iron and expression of transferrin receptors during monocyte-macrophage maturation (6). Pawelczyk et al (5) demonstrated the increase in messenger RNA and protein transferrin receptor expression in ferumoxides–protamine sulfate labeled monocytes and macrophages. These results were expected considering the scavenger function of these cells in phagocytosing debris from the microenvironment. Do the authors imply that the rat MSCs have differentiated toward the monocyte-macrophages lineage? In summary, the data presented insufficiently support the authors' conclusions with regard to the effect of labeling agents on the CD71 expression.
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References
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- Schäfer R, Kehlbach R, Wiskirchen J, et al. Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide. Radiology 2007;244:514–523.[Abstract/Free Full Text]
- Arbab AS, Wilson LB, Ashari P, Jordan EK, Lewis BK, Frank JA. A model of lysosomal metabolism of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles: implications for cellular magnetic resonance imaging. NMR Biomed 2005;18:383–389.[CrossRef][Medline]
- Kalish H, Arbab AS, Miller BR, et al. Combination of transfection agents and magnetic resonance contrast agents for cellular imaging: relationship between relaxivities, electrostatic forces, and chemical composition. Magn Reson Med 2003;50:275–282.[CrossRef][Medline]
- Hoepken HH, Korten T, Robinson SR, Dringen R. Iron accumulation, iron-mediated toxicity, and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate. J Neurochem 2004;88:1194–1202.[CrossRef][Medline]
- Pawelczyk E, Arbab AS, Pandit S, Hu E, Frank JA. Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging. NMR Biomed 2006;19:581–592.[CrossRef][Medline]
- Chan RY, Seiser C, Schulman HM, Kuhn LC, Ponka P. Regulation of transferrin receptor mRNA expression: distinct regulatory features in erythroid cells. Eur J Biochem 1994;220:683–692.[Medline]
Response
Richard Schäfer, MD *,
Rainer Kehlbach, PhD 
, and
Jakub Wiskirchen, MD
* Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tübingen, Germany
Department of Diagnostic Radiology, University Hospital Tübingen, Germany
e-mail: rainer.kehlbach{at}med.uni-tuebingen.de
We thank Drs Pawelczyk and Frank and Drs Janic and Arbab for their comments on our recently published article on transferrin receptor upregulation on rat MSCs (1). We would like to address several issues they have raised. Drs Pawelczyk and Frank mentioned the increasing rate of senescence and malignant transformation of long-term cultured human MSCs (although in the current literature this issue is controversially discussed [2]) and concluded that investigations on long-term cultured rat MSCs lack of clinical relevance. We are convinced that investigations on possible effects of SPIO labeling of stem cells should not be restricted to a short period after the labeling procedure. In the clinical setting, the transplanted cells must survive after the transplantation to evoke their beneficial effects in the host. Consequently, long-term effects of SPIO labeling on stem cells are clinically highly relevant.
The transferrin receptor (CD71) is usually expressed on human MSCs; rat MSCs have been shown to be CD71 negative (3). Pawelczyk et al (4) reported on the downregulation of CD71 on human MSCs after labeling with ferumoxides–protamine sulfate. We agree that the downregulation of CD71 after iron uptake into cells is expected, although in the respective article the internalization of the iron particles has not been confirmed by means of electron microscopy. To date we can not elucidate the molecular mechanisms leading to the SPIO-mediated CD71 upregulation in rat MSCs. However, two completely independent teams have demonstrated that labeling with iron particles can have an impact on stem cell biology, which is the essential point. After transplanting labeled MSCs in humans, the integrity of functionality including issues of safety is crucial for the patients, so critical and all-embracing research is mandatory. The previous work, especially from the authors of the two letters, is encouraging and valuable, but further investigations on interactions of stem cell labeling are required, since even for TAs like poly-l-lysine not all safety concerns (5,6) have been adequately addressed.
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References
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- Schäfer R, Kehlbach R, Wiskirchen J, et al. Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide. Radiology 2007;244(2):514–523.[Abstract/Free Full Text]
- Bernardo ME, Zaffaroni N, Novara F, et al. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 2007;67(19):9142–9149.[Abstract/Free Full Text]
- Jiang W, Ma A, Wang T, et al. Homing and differentiation of mesenchymal stem cells delivered intravenously to ischemic myocardium in vivo: a time-series study. Pflugers Arch 2006;453(1):43–52.[CrossRef][Medline]
- Pawelczyk E, Arbab AS, Pandit S, Hu E, Frank JA. Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging. NMR Biomed 2006;19(5):581–592.[CrossRef][Medline]
- Strand BL, Ryan TL, In't Veld P, et al. Poly-l-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines. Cell Transplant 2001;10(3):263–275.[Medline]
- Choi YR, Chae SY, Ahn CH, et al. Development of polymeric gene delivery carriers: PEGylated copolymers of l-lysine and l-phenylalanine. J Drug Target 2007;15(6):391–398.[CrossRef][Medline]
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