In vitro comparison of the biological effects of three transfection methods for magnetically labeling mouse embryonic stem cells with ferumoxides

Abstract

In vivo MRI of stem cells (SCs) is an emerging application to evaluate the role of cell therapy in restoring the injured myocardium. The high spatial and temporal resolution combined with iron-oxide-based intracellular labeling techniques will provide a sensitive, noninvasive, dual imaging modality for both cells and myocardium. In order to facilitate this novel imaging approach, much effort has been directed towards developing efficient transfection methods. While techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the fundamental biological effects of these methods on mouse embryonic SCs (mESC) have not been investigated systematically. In this study a longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted. No significant difference was seen in these biological parameters among the three transfection methods. However, cardiac differentiation was most attenuated by ELP, and iron Growing evidence from preclinical and clinical studies suggests that cell therapy can improve cardiac function (1-4). Although this therapeutic option is a promising approach to repair the injured myocardium, the exact mechanism of functional restoration of the injured myocardium is unknown. While differentiation of embryonic stem cells (ESC) into functional cardiomyocytes has been attributed as one of the possible underlying mechanisms in restoring the injured myocardium, a more fundamental issue regarding cellular viability following delivery into the myocardium also needs to be addressed (5,6). At present, most cell therapy protocols require histological analysis to determine viable engraftment of the transplanted cells. The development of sensitive, noninvasive technologies to monitor this fundamental engraftment parameter will aid clinical implementation of cell therapy. Imaging modalities such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and optical bioluminescence imaging (BLI) have been reported to be capable of evaluating cellular engraftment parameters (4,7,8).In vivo MRI of SCs is an emerging application to monitor SC engraftment. The high spatial and temporal resolution combined with robust iron-oxide-based intracellular labeling enables dual assessment of myocardial function and cellular location. Much effort has been directed towards developing efficient transfection methods to facilitate intracellular magnetic labeling with superparamagnetic iron oxide (SPIO). Although techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the effects of these three transfection methods on engraftment parameters of ESC have not been investigated systematically (9 -11). In this study a comparison of the engraftment parameters of magnetically labeled mouse embryonic SCs (mESC) using the three transfection methods was conducted. Fundamental biological ...

In Vivo Stem Cell Imaging

“…MRI can provide high spatial resolution (ranging from 50um in animal and up to 300um in whole body clinical scanners), high temporal resolution and extract physiologic and anatomic information simultaneously [12,16]. Labeling stem cells with magnetic agents for MRI is a suitable approach to monitor the distribution, migration, survival and differentiation when transplanted into animals over weeks in vivo [1,12,13,[23][24][25].…”

“…Labeling stem cells with magnetic agents for MRI is a suitable approach to monitor the distribution, migration, survival and differentiation when transplanted into animals over weeks in vivo [1,12,13,[23][24][25]. By tracking labeled stem cell with MRI, it is helpful to establishing the optimal number of transplanted cells, define therapeutic windows and monitor cell growth and possible side effects for regenerative therapies [1].…”

“…Usually, investigators manipulate cells ex vivo either by incorporating different exogenous imaging contrast agents or by transfecting different reporter genes. Stem cells carrying contrast agents (direct labeling) can be then detected by optical, magnetic resonance imaging (MRI) or radionuclide imaging methods, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) [10][11][12][13][14][15]. In contrast to direct labeling techniques, reporter gene techniques for stem cell labeling offer an attractive alternative because imaging signals are generated only from viable cells of interest.…”

Multimodality Evaluation of the Viability of Stem Cells Delivered Into Different Zones of Myocardial Infarction

“…The mESCs were maintained in an undifferentiated state on a feeder layer of irradiated mouse fibroblasts with maintenance medium containing Dulbecco modified Eagle medium (Invitrogen, Carlsbad, Calif) supplemented with 15% fetal bovine serum (Hyclone, Logan, Utah), 0.1 mmol/L 2-mercaptoethanol (Sigma, St Louis, Mo), 0.1 mN nonessential amino acids (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 2 mmol/L L-glutamine (Invitrogen), and 1000 U/mL mouse leukemia inhibitory factor (ESGRO, Chemicon, Temecula, Calif) at 37°C, as described previously. 16 …”

Magnetosonoporation: Instant magnetic labeling of stem cells

“…This approach incubates cells with an MR contrast agent while adding an additional transfection reagent (4), such as lipofectamine (5), poly-L-lysine (3,6), and protamine sulfate (7) or with no transfection agents (8). The transfection agents coat MRI contrast agents and convert the negatively charged contrast agents to be positive, which thus facilitates the binding of contrast agents to the anionic cell membrane, followed by internalization of contrast agents into the cells.…”