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Dual Transfer of GFP Gene and MGd into Stem-Progenitor Cells

Posted Apr 30, 2010 Updated Apr 7, 2023
By Bensheng Qiu PhD , Piper Treuting DVM , Xiangcan Zhan PhD , Daohai Xie MD PhD , Charles W. Frevert DVM ScD , Xiaoming Yang MD PhD
6 min read

Rationale and Objectives

The aim of this study was to develop a new technique, the use of magnetic resonance (MR) imaging (MRI) to monitor gene/MR–cotransferred stem-progenitor cells (SPCs) recruited to atherosclerosis.

Materials and Methods

First, a green fluorescent protein (GFP) gene and a T1 MR contrast agent (motexafin gadolinium [MGd]) were cotransferred into neural or bone marrow (BM)–derived SPCs. GFP expression and MGd signal were confirmed by fluorescent microscopy and quantified by flow cytometry. Cell viability and proliferation were then evaluated by trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and GFP/MGd–transferred cells were imaged using 1.5-T and 9.4-T MR scanners. For in vivo validation, GFP/MGd–cotransferred β-galactosidase–BM SPCs were transplanted to apolipoprotein E–knockout mice, and cell migration to atherosclerotic aortas was monitored using 9.4-T micro-MRI with subsequent histologic correlations.

Results

Fluorescent microscopy demonstrated simultaneous GFP expression and MGd signals in cotransferred-cells. Quantitative flow cytometry showed GFP-positive cells at 47 ± 25% and 56 ± 12% and MGd-positive cells at 96 ± 6% and 57 ± 11% for neural stem cells and BM cells, respectively. Cell viability and metabolic rates of cotransferred cells were 86 ± 4% and 84 ± 12%, respectively. In vivo MRI revealed high MR signals of the aortic walls in GFP/MGd–transferred mice, which were confirmed by histologic correlations.

Conclusion

This study has initially proven the new concept of MRI for plaque-specific, cell-mediated gene expression of atherosclerosis.

A characteristic feature of atherosclerotic cardiovascular disease is its diffuse involvement of arteries across the entire human body and the presence of multiple and simultaneous atherosclerotic lesions. Endovascular interventional procedures, such as balloon angioplasty and stenting, are currently used as routine “local” treatments of atherosclerotic arteries. However, these local interventional approaches do not treat multiple diffuse atherosclerosis. Thus, it is essential to seek alternatives to treat all atherosclerotic arteries at once.

Hematopoietic stem-progenitor cells (SPC) can give rise to vascular progenitor cells that migrate or home to atherosclerotic arteries and differentiate into either smooth-muscle cells or endothelial cells . Intravenously transfused hematopoietic SPCs circulate in the blood system, flow through the entire body, and thus home to all atherosclerotic lesions. Gene therapy is an exciting frontier in cardiovascular medicine . The transfer of therapeutic genes into hematopoietic SPCs prior to their transplantation to the body may enable the SPC-mediated plaque-specific delivery of therapeutic genes to diffuse multiple atherosclerotic lesions.

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Materials and methods

Study Design

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In Vitro Evaluation

Agents

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Dual Transfer of GFP and MGd Into the Cells

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Laboratory Conformations

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Evaluation of Cell Viability and Proliferation

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In Vitro MRI

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In Vivo Validation

Extraction of Donor β-galactosidase SPCs

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Figure 1, Schematic diagrams of the experimental steps for in vivo validation study, including (1) creating atherosclerotic plaques in the ascending aorta of the recipient apolipoprotein E–knockout (ApoE) mouse by atherogenic diet; (2) extracting bone marrow (BM) cells (BMCs) from the donor β-galactosidase (LacZ) mouse; (3) cotransferring LacZ/BMCs by green fluorescent protein (GFP) gene and motexafin gadolinium (MGd) in vitro; (4) transplanting GFP/MGd–cotransferred BMCs to the recipient atherosclerotic mouse, which is lethally irradiated; (5) GFP/MGd–cotransferred BMCs migrating to and homing in aortic atherosclerotic plaques; (6) molecular magnetic resonance (MR) detection of MGd-created MR signals at atherosclerotic plaques migrated with GFP/MGd–transferred cells; and (7) confirming the successful migration of cotransferred cells to the plaques by histologic examinations and correlations.

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Cell Transplantation

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In Vivo MRI

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Histologic Correlation

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Image Analysis

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Results

In Vitro Evaluation

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Figure 2, Fluorescent microscopic imaging of nontransferred cells (top row) and the cells cotransferred with green fluorescent protein (GFP) gene and motexafin gadolinium (MGd) (bottom row). Images are obtained at the blue channel for nuclei with 4′,6-diamidino-2-phenylindole stain (a,e) , the green channel for GFP (b,f) , the red channel for MGd (c,g) , and the combination of three colored images (d,h) . Green fluorescence (f) (due to GFP expression) and red-colored spots (g) (due to MGd labeling) surrounding the nuclei are detected in GFP/MGd–transferred cells, while only autofluorescence from the cells themselves is visualized in nontransferred cells (b,c) .

Figure 3, (a) Photo taken immediately after centrifuging cells at the bottoms of tubes, showing the success of motexafin gadolinium (MGd)–transferred cells as dark brown color, which is not seen with nontransferred control cells. (b) Representative 1.5-T magnetic resonance (MR) image of cell pellets at bottoms of tubes, demonstrating a brighter signal in transferred cells compared to nontransferred cells. (c) T1-weighted MR imaging of the nontransferred and MGd-transferred cells suspended in 4% gelatin, showing a brighter signal in transferred cells. (d) Comparison of average MR signal intensities (arbitrary units [au]) between nontransferred and transferred bone marrow cells, demonstrating higher average signal intensity in MGd-transferred cells.

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In Vivo Validation

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Figure 4, (a) Representative magnetic resonance imaging (MRI)–histology correlation. From top, a normal mouse and three apolipoprotein E–knockout mice with no cell transplantation (CT), green fluorescent protein (GFP) CT, and GFP/motexafin gadolinium (MGd) CT. In vivo 9.4-T MRI shows MGd-enhanced portions (P, arrow) of the ascending aortic wall with GFP/MGd CT, which are not seen in three control aortas (A,F,K). Histology with hematoxylin and eosin (H&E) staining shows a normal aorta (B) and atherosclerotic aortas with plaques (P) in all apoE −/− mice (G,L,Q). In the normal control aorta and the apoE −/− control aorta (with no CT), histology confirmed no GFP-positive, MGd-positive, or β-galactosidase (LacZ)–positive cells (C–E and H–J). However, in the aorta treated with GFP cells, GFP-positive (M, arrows) and LacZ-positive cells (O, blue spots) are detected. In the aorta with GFP/MGd CT, histology detects GFP-positive and MGd-positive cells (R,S, arrows), as well as LacZ-positive cells (T, blue spots). Magnification 20×. (b) Comparison of average MR signal intensities of aortic walls between four different animal groups with various treatments. The MGd-created MR signal intensity ratio is significantly higher in the animal group with transplantation of GFP/MGd–transferred cells than in other three control animal groups. m, media. ∗ P < .05.

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Discussion

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Acknowledgment

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Academic Radiology, Volume 17, Issue 5
Journals General Radiology
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