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Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging
Cytotherapy (2004) Vol. 6, No. 6, 621 /625
Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging JA Frank1, SA Anderson1,2, H Kalsih1, EK Jordan1, BK Lewis1, GT Yocum1 and AS Arbab1 1
Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, Clinical Center, and 2Neuroimmunology Branch, National Institutes of Neurologic Disorders and Stroke, NIH, Bethesda, Maryland, USA
Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This
review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.
Introduction
modified by either conjugating Ag-specific internalizing MAb to the surface dextran coating, or cross-linkage of the dextran (CLIO) and conjugation with short HIV-transactivator transcription (Tat) proteins, have been used to facilitate incorporation of USPIO into cells [2,8]. In addition, iron oxide nanoparticles coated with dendrimers, which are used to transfect DNA into cells, have also been used to label cells [3]. When dextran-coated SPIO nanoparticles are added to polycationic transfection agents, a complex is formed that can be used to label cells magnetically [4 /6,9,10]. In this report, we will briefly describe the techniques and experimental studies that have been performed demonstrating the versatility of this labeling approach, and the potential for translation from bench to bedside for ultimate use in clinical trials.
Magnetic resonance (MR) contrast agents are valuable tools in the diagnostic evaluation and follow-up of managed treatment, as well as in the assessment of organ function. Recently, there has been increasing interest in developing and testing novel tools, reagents and methods to image specific molecular pathways in vivo, particularly those that are key targets in disease processes. Molecular and cellular imaging enables monitoring of the trafficking of labeled cells using magnetic, genetic or fluorescent probes, in order to determine further the underlying disease processes or to explore specifically the temporal /spatial migration of cells, and allows the development of novel clinical trials. Various approaches have been developed that use magnetic resonance imaging (MRI) contrast agents to label cells magnetically [1/9]. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles are Food and Drug Administration (FDA)-approved MR contrast agents for hepatic imaging (SPIO), and ultrasmall (U)SPIO has been used in clinical trials as a blood pool agent or for lymphangiography. Following an intravenous injection of cells magnetically labeled with SPIO nanoparticles, tissues appear hypointense (dark) on MRI due to significant T2 and T2* relaxation time shortening. SPIO nanoparticles
Modified SPIO coupled to MAb or Tat protein In 1999, we covalently attached a MAb to the rat transferrin receptor (OX-26) to the USPIO MION-46L, and were able to label rat CG-4 progenitor oligodendroglial cells magnetically in culture through receptormediated endocytosis [1]. These magnetically labeled CG-4 cells were then implanted into myelin-deficient rat spinal cords and, 10 /14 days later at death, the cords were extracted and MR microscopy and staining for myelin
Correspondence to: Joseph A Frank, MD, Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, National Institutes of Health, Building 10, Room B1N256, 10 Center Drive, MSC 1074, Bethesda, MD 20892 /1074, USA. – 2004 ISCT
DOI: 10.1080/14653240410005267
622
JA Frank et al .
performed. Areas of remyelination were well correlated with hypo-intense areas on 3-dimensional T2* images, demonstrating for the first time the ability to monitor the migration of magnetically labeled cells. By covalently attaching the HIV-Tat protein to CLIO, naive lymphocytes and CD34 hematopoietic stem cells (HSC) were magnetically labeled with USPIO nanoparticles, and no alteration in cell function, surface marker expression, viability or colony unit formation was observed in labeled cells compared with unlabeled cells [2,8]. Recently, Kircher et al . [8] were able to track the homing of sensitized CLIO-Tat labeled CD8 T cells to B16 OVA tumor, after intravenous infusion into the tumor, within 12 /36 h by in vivo MRI. These authors demonstrated differences on MRI and immunohistochemical analysis in the B16 OVA tumor compared with contralateral control flank tumors. These authors indicated that by calculating changes in T2 relaxation times of the flank tumors, they were able to detect CD8 T-cell migration into the B16 OVA tumor within 12 h following infusion [8].
Magnetodendrimers and BANG particles Magnetodendrimers (MD-100) were synthesized using a dendrimer macromolecule instead of dextran for the coating of the SPIO, and shown to label stem cells and other mammalian cells efficiently in culture [3]. MD-100labeled oligodendroglial progenitors derived from neural stem cells were transplanted in the ventricles of neonatal dysmyelinated Long Evans Shaker (LES) rats [3]. Migration of labeled cells into the brain parenchyma could be observed up to 42 days following implantation of MD-100labeled cells. In areas of new myelin formation, there was corresponding demonstration of anti-myelin basic protein immunostaining, proving that the MD-100-labeled cells functioned and could also be readily identified at a clinically relevant 1.5 Tesla MR field strength [3]. Hinds et al. [7] reported on an intracellular contrast agent (BANG) that is micron sized, containing an aggregate of iron oxide magnetite mixed with a fluorophore encased in an inert di-vinyl benzene polymer microsphere, and showed that these particles were found in endosomes of mesenchymal stem cells (MSC) and CD34 cells following incubation. These authors demonstrated that BANG particles did not alter the viability, proliferation or differential capacity of BANG-labeled HSC and MSC compared with unlabeled cells, and the labeled cells were detected by MRI and correlated to confocal micro-
scopy. Recently, Hill et al. [11] were able to detect BANGlabeled swine MSC implanted into an infracted area of myocardium by MRI.
Ferumoxides complexed to transfection agents In 2002, our group had developed a technique for magnetically labeling stem cells that did not involve proprietary compounds, unique or complex synthesis or biochemical modification of the dextran-coated SPIO [4,5]. By combining the SPIO ferumoxides (Feridex† , Berlex, Wayne, NJ, USA) with polycationic transfection agents (TA), endosomal incorporation of the FDA-approved SPIO nanoparticles occurs with high labeling efficiency. Ferumoxides (FE) complexed to poly-l-lysine (PLL) were used to magnetically label adherent and suspension cells grown in culture, including multipotent human HSC and MSC, lymphocytes and other mammalian cells [5,6,10]. FE /PLL did not affect long-term cell viability, growth rate or apoptotic indices compared with unlabeled cells [4 /6,10]. In non-dividing MSC, endosomal iron nanoparticles could be detected for more than 6 weeks. However, in rapidly dividing cells, intracellular iron disappeared by five to eight cell divisions [10]. There was no significant change in metabolic and physiologic properties of FE /PLL-labeled cells compared with unlabeled cells. FE /PLL-labeled MSC was shown to differentiate along an apidogenic lineage when stimulated in appropriate media [10]. Improvement in the FE/PLL-labeling approach was accomplished by substituting protamine sulfate (Pro) for PLL, thereby making it possible to label stem cells and other mammalian cells with two FDA-approved agents [9]. Pro has a molecular weight of approximately 4.2 kDa and is commonly used to treat heparin-induced anticoagulation. It has a bulk electrostatic charge (zeta potential) of 7.07 millivolts and complexes with FE through electrostatic interaction. We have shown that adherent cells (e.g. MSC, monocytes, macrophages) and cells grown in suspension (e.g. HSC, T cells) are efficiently labeled with FE/Pro complexes and labeled cells demonstrate no short- or long-term toxicity, changes in function, differentiation capacity or phenotype compared with unlabeled cells [9]. Figure 1 is a graphic display of the technique used to label cells with FE/Pro. In general, FE/Pro is mixed at a ratio of FE 100 mg/mL to Pro 4 /6 mg/mL in serum-free media for approximately 10 min, and then added at a 50:50 volume-to-volume ratio with media of cells in culture and
Magnetic labeling for detection by magnetic resonance imaging
623
Figure 1. Schema for magnetically labeling cells using ferumoxides mixed with protamine sulfate.
incubated from 2 h to overnight depending on cell type. Cells are then washed with PBS containing 3 /10 U heparin sulfate/mL. Using this approach we have found that the iron content is approximately 1 /3 picograms/cell for T cells or CD34 cells, and about 20 picograms/cell for monocytes and MSC [9]. Unlabeled cells contain approximately 0.05 /0.5 picograms of iron. When comparing FE/Pro with FE /PLL complexes for labeling cells, we have found the FE /Pro complex results in a cleaner preparation, especially if heparin sulfate is included in the initial washes. Heparin sulfate appears to compete with the dextran coating of the SPIO nanoparticles for the protamine sulfate, dissolving the FE /Pro complex from the media and also from any remaining FE / Pro attached to the cell surface at the end of the incubation period. Labeling cells with FE /PLL may result in clumps of cells around the iron oxide nanoparticles because of incomplete internalization of the complexes.
Experimental studies using FE /PLL- or FE /Prolabeled sensitized T cells produce similar clinical and pathologic disease compared with unlabeled cells. T cells sensitized to myelin Ag and labeled with FE /PLL have been used in an adoptive transfer model of experimental allergic encephalomyelitis (EAE) to induce disease and EAE lesions that could then be detected on high-resolution MR microscopy in the spinal cords in SJL mice [12]. By labeling mouse BM-derived Sca1 HSC with PLL, we have been able to demonstrate with MRI the incorporation of these cells into the tumor angiogenesis of an implanted RT2 glioma in the mouse model, demonstrating for the first time direct visualization of the developing neovascularization in a growing tumor [13]. We have also shown it is possible to monitor the migration of CD34 AC133 FE/Pro-labeled stem cells into flank tumors implanted into nude mice by MRI and correlate the findings with immunohistopathology [9]. Other studies have shown that
624
JA Frank et al .
MRI can detect the temporal /spatial migration of magnetically labeled human MSC into tumors, in the liver of nude rats, implanted directly into infracted myocardium or implanted neural stem cells migrating into areas of experimental stroke [6,14 /16].
the future, this approach for magnetically labeling cells may be used in conjunction with MRI guidance, to develop innovative treatment trials to guide the initiation of therapy or, if repeated cell infusions or transplantations are needed, to obtain the desired density of cells targeted to the tissue.
Translation from bench to bedside Although FE and Pro are FDA-approved agents, the FE/Pro complex is considered a combination drug subject to regulation and therefore requires toxicity testing and pre-clinical evaluation prior to submission for an investigative new drug application to the FDA. Protocols are under development to scale up the labeling of stem cell and PBMC with the FE/Pro complex using standard equipment, disposables and procedures found in blood banks (E. Read, C. Carter Jr and V. Fellows, personal communication). Research groups wanting to label cells with the FE /Pro complex should consult with the FDA and will probably need to demonstrate the following: j j
j
j
j
the labeling technique is reproducible; labeled cells have similar viability as unlabeled cells without significant loss during the labeling process; the phenotype of the stem cells or other mammalian cells is unaltered as a result of the labeling with FE / Pro; the magnetically labeled cells are able to differentiate and the cells’ functional capacity remains the same as unlabeled cells; there are no toxins or infectious agents present in the resulting product.
Pre-clinical studies in experimental disease models may be required to demonstrate that FE/Prolabeled cells can be detected in the target tissue by MRI and on histopathology. Labeling procedures for pre-clinical studies should be similar to what will be used in clinical trials. Finally, disease-specific indications will probably need to be developed along with appropriate documentation and submitted to the FDA. In conclusion, the approach to labeling cells using a FE/Pro complex has the potential to translate from bench to bedside. MRI can then be used to non-invasively monitor the temporal and spatial migration of stem cells, other cells or genetically engineered cells as part of repair, replacement or therapeutic strategies in clinical trials. In
References 1 Bulte JWM, Zhang SC, van Gelderen P et al . Neurotransplantation of magnetically labeled oligodendrocytes progenitors: MR tracking of cell migration and myelination. Proc Natl Acad Sci 1999;96:15256 /61. 2 Lewin M, Carlesso N, Tung CH et al . Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000;18:410 /4. 3 Bulte JWM, Douglas T, Witer B et al . Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol 2001;19:1141 /7. 4 Frank JA, Zywicke H, Jordan EK et al . Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents. Acad Radiol 2002;9:S484 /8. 5 Frank JA, Miller BR, Arbab AS et al . Clinically applicable labeling of mammalian cells and stem cells by combining (FDA)approved superparamagnetic iron oxides and commonly available transfection agents. Radiology 2003;228:480 /7. 6 Arbab AS, Bashaw LA, Miller BR et al . Intracytoplasmic magnetic labeling of mammalian cells with ferumoxides and transfection agent for cellular magnetic resonance imaging: methods and techniques. Transplant 2003;76:1123 /30. 7 Hinds KA, Hill JM, Shapiro EM et al . Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 2003;102:867 /72. 8 Kircher MF, Allport JR, Graves EE et al . In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic Tlymphocyte trafficking to tumors. Cancer Res 2003;63:6838 /46. 9 Arbab AS, Yocum GT, Kalish H et al . Magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood accessed 20 April 2004;104:1217 /23. 10 Arbab AS, Bashaw LA, Miller BR et al . Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles (ferumoxides) and transfection agent (poly-l-lysine) for cellular MR imaging. Radiology 2003;229:838 /46. 11 Hill JM, Dick AJ, Raman VK et al . Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009 /14. 12 Anderson SA, Shukaliak-Quandt J, Jordan EK et al . Trafficking of magnetically labeled encephalitogenic T-cells in the EAE mouse model by cellular magnetic resonance imaging. Ann Neurol 2004;55:654 /9.
Magnetic labeling for detection by magnetic resonance imaging
13 Anderson SA, Glod J, Arbab AS et al . Non-invasive MR imaging of magnetically labeled stem cells to directly identify neovasculature in a glioma model. Blood ; First edition paper; prepublished online August 26, 2004; DOI 10.1182/blood 200406-2222. 14 Arbab AS, Jordan EK, Bashaw LA et al . In vivo targeting iron oxide labeled human mesenchymal stem cell by magnetic field gradients: detection by MRI in rat model. Human Gene Ther 2004;15:351 /60.
625
15 Kraitchman DL, Heldman AW, Atalar E et al . In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003;107:2290 /3. 16 Hoehn M, Kustermann E, Blunk K et al . Monitoring of implanted stem cell migration in vivo ; a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci USA 2002;99:16267 / 72.
Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging JA Frank1, SA Anderson1,2, H Kalsih1, EK Jordan1, BK Lewis1, GT Yocum1 and AS Arbab1 1
Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, Clinical Center, and 2Neuroimmunology Branch, National Institutes of Neurologic Disorders and Stroke, NIH, Bethesda, Maryland, USA
Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This
review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.
Introduction
modified by either conjugating Ag-specific internalizing MAb to the surface dextran coating, or cross-linkage of the dextran (CLIO) and conjugation with short HIV-transactivator transcription (Tat) proteins, have been used to facilitate incorporation of USPIO into cells [2,8]. In addition, iron oxide nanoparticles coated with dendrimers, which are used to transfect DNA into cells, have also been used to label cells [3]. When dextran-coated SPIO nanoparticles are added to polycationic transfection agents, a complex is formed that can be used to label cells magnetically [4 /6,9,10]. In this report, we will briefly describe the techniques and experimental studies that have been performed demonstrating the versatility of this labeling approach, and the potential for translation from bench to bedside for ultimate use in clinical trials.
Magnetic resonance (MR) contrast agents are valuable tools in the diagnostic evaluation and follow-up of managed treatment, as well as in the assessment of organ function. Recently, there has been increasing interest in developing and testing novel tools, reagents and methods to image specific molecular pathways in vivo, particularly those that are key targets in disease processes. Molecular and cellular imaging enables monitoring of the trafficking of labeled cells using magnetic, genetic or fluorescent probes, in order to determine further the underlying disease processes or to explore specifically the temporal /spatial migration of cells, and allows the development of novel clinical trials. Various approaches have been developed that use magnetic resonance imaging (MRI) contrast agents to label cells magnetically [1/9]. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles are Food and Drug Administration (FDA)-approved MR contrast agents for hepatic imaging (SPIO), and ultrasmall (U)SPIO has been used in clinical trials as a blood pool agent or for lymphangiography. Following an intravenous injection of cells magnetically labeled with SPIO nanoparticles, tissues appear hypointense (dark) on MRI due to significant T2 and T2* relaxation time shortening. SPIO nanoparticles
Modified SPIO coupled to MAb or Tat protein In 1999, we covalently attached a MAb to the rat transferrin receptor (OX-26) to the USPIO MION-46L, and were able to label rat CG-4 progenitor oligodendroglial cells magnetically in culture through receptormediated endocytosis [1]. These magnetically labeled CG-4 cells were then implanted into myelin-deficient rat spinal cords and, 10 /14 days later at death, the cords were extracted and MR microscopy and staining for myelin
Correspondence to: Joseph A Frank, MD, Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, National Institutes of Health, Building 10, Room B1N256, 10 Center Drive, MSC 1074, Bethesda, MD 20892 /1074, USA. – 2004 ISCT
DOI: 10.1080/14653240410005267
622
JA Frank et al .
performed. Areas of remyelination were well correlated with hypo-intense areas on 3-dimensional T2* images, demonstrating for the first time the ability to monitor the migration of magnetically labeled cells. By covalently attaching the HIV-Tat protein to CLIO, naive lymphocytes and CD34 hematopoietic stem cells (HSC) were magnetically labeled with USPIO nanoparticles, and no alteration in cell function, surface marker expression, viability or colony unit formation was observed in labeled cells compared with unlabeled cells [2,8]. Recently, Kircher et al . [8] were able to track the homing of sensitized CLIO-Tat labeled CD8 T cells to B16 OVA tumor, after intravenous infusion into the tumor, within 12 /36 h by in vivo MRI. These authors demonstrated differences on MRI and immunohistochemical analysis in the B16 OVA tumor compared with contralateral control flank tumors. These authors indicated that by calculating changes in T2 relaxation times of the flank tumors, they were able to detect CD8 T-cell migration into the B16 OVA tumor within 12 h following infusion [8].
Magnetodendrimers and BANG particles Magnetodendrimers (MD-100) were synthesized using a dendrimer macromolecule instead of dextran for the coating of the SPIO, and shown to label stem cells and other mammalian cells efficiently in culture [3]. MD-100labeled oligodendroglial progenitors derived from neural stem cells were transplanted in the ventricles of neonatal dysmyelinated Long Evans Shaker (LES) rats [3]. Migration of labeled cells into the brain parenchyma could be observed up to 42 days following implantation of MD-100labeled cells. In areas of new myelin formation, there was corresponding demonstration of anti-myelin basic protein immunostaining, proving that the MD-100-labeled cells functioned and could also be readily identified at a clinically relevant 1.5 Tesla MR field strength [3]. Hinds et al. [7] reported on an intracellular contrast agent (BANG) that is micron sized, containing an aggregate of iron oxide magnetite mixed with a fluorophore encased in an inert di-vinyl benzene polymer microsphere, and showed that these particles were found in endosomes of mesenchymal stem cells (MSC) and CD34 cells following incubation. These authors demonstrated that BANG particles did not alter the viability, proliferation or differential capacity of BANG-labeled HSC and MSC compared with unlabeled cells, and the labeled cells were detected by MRI and correlated to confocal micro-
scopy. Recently, Hill et al. [11] were able to detect BANGlabeled swine MSC implanted into an infracted area of myocardium by MRI.
Ferumoxides complexed to transfection agents In 2002, our group had developed a technique for magnetically labeling stem cells that did not involve proprietary compounds, unique or complex synthesis or biochemical modification of the dextran-coated SPIO [4,5]. By combining the SPIO ferumoxides (Feridex† , Berlex, Wayne, NJ, USA) with polycationic transfection agents (TA), endosomal incorporation of the FDA-approved SPIO nanoparticles occurs with high labeling efficiency. Ferumoxides (FE) complexed to poly-l-lysine (PLL) were used to magnetically label adherent and suspension cells grown in culture, including multipotent human HSC and MSC, lymphocytes and other mammalian cells [5,6,10]. FE /PLL did not affect long-term cell viability, growth rate or apoptotic indices compared with unlabeled cells [4 /6,10]. In non-dividing MSC, endosomal iron nanoparticles could be detected for more than 6 weeks. However, in rapidly dividing cells, intracellular iron disappeared by five to eight cell divisions [10]. There was no significant change in metabolic and physiologic properties of FE /PLL-labeled cells compared with unlabeled cells. FE /PLL-labeled MSC was shown to differentiate along an apidogenic lineage when stimulated in appropriate media [10]. Improvement in the FE/PLL-labeling approach was accomplished by substituting protamine sulfate (Pro) for PLL, thereby making it possible to label stem cells and other mammalian cells with two FDA-approved agents [9]. Pro has a molecular weight of approximately 4.2 kDa and is commonly used to treat heparin-induced anticoagulation. It has a bulk electrostatic charge (zeta potential) of 7.07 millivolts and complexes with FE through electrostatic interaction. We have shown that adherent cells (e.g. MSC, monocytes, macrophages) and cells grown in suspension (e.g. HSC, T cells) are efficiently labeled with FE/Pro complexes and labeled cells demonstrate no short- or long-term toxicity, changes in function, differentiation capacity or phenotype compared with unlabeled cells [9]. Figure 1 is a graphic display of the technique used to label cells with FE/Pro. In general, FE/Pro is mixed at a ratio of FE 100 mg/mL to Pro 4 /6 mg/mL in serum-free media for approximately 10 min, and then added at a 50:50 volume-to-volume ratio with media of cells in culture and
Magnetic labeling for detection by magnetic resonance imaging
623
Figure 1. Schema for magnetically labeling cells using ferumoxides mixed with protamine sulfate.
incubated from 2 h to overnight depending on cell type. Cells are then washed with PBS containing 3 /10 U heparin sulfate/mL. Using this approach we have found that the iron content is approximately 1 /3 picograms/cell for T cells or CD34 cells, and about 20 picograms/cell for monocytes and MSC [9]. Unlabeled cells contain approximately 0.05 /0.5 picograms of iron. When comparing FE/Pro with FE /PLL complexes for labeling cells, we have found the FE /Pro complex results in a cleaner preparation, especially if heparin sulfate is included in the initial washes. Heparin sulfate appears to compete with the dextran coating of the SPIO nanoparticles for the protamine sulfate, dissolving the FE /Pro complex from the media and also from any remaining FE / Pro attached to the cell surface at the end of the incubation period. Labeling cells with FE /PLL may result in clumps of cells around the iron oxide nanoparticles because of incomplete internalization of the complexes.
Experimental studies using FE /PLL- or FE /Prolabeled sensitized T cells produce similar clinical and pathologic disease compared with unlabeled cells. T cells sensitized to myelin Ag and labeled with FE /PLL have been used in an adoptive transfer model of experimental allergic encephalomyelitis (EAE) to induce disease and EAE lesions that could then be detected on high-resolution MR microscopy in the spinal cords in SJL mice [12]. By labeling mouse BM-derived Sca1 HSC with PLL, we have been able to demonstrate with MRI the incorporation of these cells into the tumor angiogenesis of an implanted RT2 glioma in the mouse model, demonstrating for the first time direct visualization of the developing neovascularization in a growing tumor [13]. We have also shown it is possible to monitor the migration of CD34 AC133 FE/Pro-labeled stem cells into flank tumors implanted into nude mice by MRI and correlate the findings with immunohistopathology [9]. Other studies have shown that
624
JA Frank et al .
MRI can detect the temporal /spatial migration of magnetically labeled human MSC into tumors, in the liver of nude rats, implanted directly into infracted myocardium or implanted neural stem cells migrating into areas of experimental stroke [6,14 /16].
the future, this approach for magnetically labeling cells may be used in conjunction with MRI guidance, to develop innovative treatment trials to guide the initiation of therapy or, if repeated cell infusions or transplantations are needed, to obtain the desired density of cells targeted to the tissue.
Translation from bench to bedside Although FE and Pro are FDA-approved agents, the FE/Pro complex is considered a combination drug subject to regulation and therefore requires toxicity testing and pre-clinical evaluation prior to submission for an investigative new drug application to the FDA. Protocols are under development to scale up the labeling of stem cell and PBMC with the FE/Pro complex using standard equipment, disposables and procedures found in blood banks (E. Read, C. Carter Jr and V. Fellows, personal communication). Research groups wanting to label cells with the FE /Pro complex should consult with the FDA and will probably need to demonstrate the following: j j
j
j
j
the labeling technique is reproducible; labeled cells have similar viability as unlabeled cells without significant loss during the labeling process; the phenotype of the stem cells or other mammalian cells is unaltered as a result of the labeling with FE / Pro; the magnetically labeled cells are able to differentiate and the cells’ functional capacity remains the same as unlabeled cells; there are no toxins or infectious agents present in the resulting product.
Pre-clinical studies in experimental disease models may be required to demonstrate that FE/Prolabeled cells can be detected in the target tissue by MRI and on histopathology. Labeling procedures for pre-clinical studies should be similar to what will be used in clinical trials. Finally, disease-specific indications will probably need to be developed along with appropriate documentation and submitted to the FDA. In conclusion, the approach to labeling cells using a FE/Pro complex has the potential to translate from bench to bedside. MRI can then be used to non-invasively monitor the temporal and spatial migration of stem cells, other cells or genetically engineered cells as part of repair, replacement or therapeutic strategies in clinical trials. In
References 1 Bulte JWM, Zhang SC, van Gelderen P et al . Neurotransplantation of magnetically labeled oligodendrocytes progenitors: MR tracking of cell migration and myelination. Proc Natl Acad Sci 1999;96:15256 /61. 2 Lewin M, Carlesso N, Tung CH et al . Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000;18:410 /4. 3 Bulte JWM, Douglas T, Witer B et al . Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol 2001;19:1141 /7. 4 Frank JA, Zywicke H, Jordan EK et al . Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents. Acad Radiol 2002;9:S484 /8. 5 Frank JA, Miller BR, Arbab AS et al . Clinically applicable labeling of mammalian cells and stem cells by combining (FDA)approved superparamagnetic iron oxides and commonly available transfection agents. Radiology 2003;228:480 /7. 6 Arbab AS, Bashaw LA, Miller BR et al . Intracytoplasmic magnetic labeling of mammalian cells with ferumoxides and transfection agent for cellular magnetic resonance imaging: methods and techniques. Transplant 2003;76:1123 /30. 7 Hinds KA, Hill JM, Shapiro EM et al . Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 2003;102:867 /72. 8 Kircher MF, Allport JR, Graves EE et al . In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic Tlymphocyte trafficking to tumors. Cancer Res 2003;63:6838 /46. 9 Arbab AS, Yocum GT, Kalish H et al . Magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood accessed 20 April 2004;104:1217 /23. 10 Arbab AS, Bashaw LA, Miller BR et al . Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles (ferumoxides) and transfection agent (poly-l-lysine) for cellular MR imaging. Radiology 2003;229:838 /46. 11 Hill JM, Dick AJ, Raman VK et al . Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009 /14. 12 Anderson SA, Shukaliak-Quandt J, Jordan EK et al . Trafficking of magnetically labeled encephalitogenic T-cells in the EAE mouse model by cellular magnetic resonance imaging. Ann Neurol 2004;55:654 /9.
Magnetic labeling for detection by magnetic resonance imaging
13 Anderson SA, Glod J, Arbab AS et al . Non-invasive MR imaging of magnetically labeled stem cells to directly identify neovasculature in a glioma model. Blood ; First edition paper; prepublished online August 26, 2004; DOI 10.1182/blood 200406-2222. 14 Arbab AS, Jordan EK, Bashaw LA et al . In vivo targeting iron oxide labeled human mesenchymal stem cell by magnetic field gradients: detection by MRI in rat model. Human Gene Ther 2004;15:351 /60.
625
15 Kraitchman DL, Heldman AW, Atalar E et al . In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003;107:2290 /3. 16 Hoehn M, Kustermann E, Blunk K et al . Monitoring of implanted stem cell migration in vivo ; a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci USA 2002;99:16267 / 72.