Bright Ferritin—a Reporter Gene Platform for On-Demand, Longitudinal Cell Tracking on MRI

Background. Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo . Aim. To develop a reporter gene that permits in vivo stem cell labeling. We examined the sodium-iodide symporter (NIS), a protein that is not expressed in the heart, but promotes cellular uptake of 99m Tc or 124 I, thus permitting cell tracking by SPECT or PET imaging, respectively. Methods. The human NIS gene ( h NIS) was expressed in rat cardiac derived stem cells (rCDCs) using lentivirus driven by the CAG or CMV promoter. NIS function in transduced cells was confirmed by in vitro 99m Tc uptake. Eleven rats were injected with 1 or 2 million rCDCs intramyocardially immediately after LAD ligation; 6 with CMV-NIS and 5 with CAG-NIS cells. Dual isotope SPECT imaging was performed on a small animal SPECT/CT system, using 99m Tc for cell detection and 201 Tl for myocardial delineation, 24 hrs after cell injection. PET was performed on a small animal PET scanner using 124 I for cell tracking and 13 NH 3 for myocardial delineation, 48hrs after cell injection. Contrast Ratio (CR) was defined as [(signal in the cells)-(signal in blood pool)]/signal in blood pool. High resolution ex vivo SPECT scans of explanted hearts (n=3) were obtained to confirm that in vivo signal was derived from the cell injection site. The presence of h NIS mRNA was confirmed in injected hearts after animal sacrifice (n=2), by real-time RT-PCR. Results. NIS expression in rCDCs did not affect cell viability/proliferation (p=0.718, ctr vs NIS). In vitro 99m Tc uptake was 6.0±0.9% vs 0.07±0.05, without and with perchlorate (specific NIS blocker), respectively. NIS-transduced rCDCs were easily visualized as spots of 99m Tc or 124 I uptake within a perfusion deficit in the SPECT and PET images. CR was considerably higher when cells were transduced by the CMV-NIS virus in comparison to the CAG-NIS virus (70±40% vs 28±29%, p=0.085). Ex vivo small animal SPECT imaging confirmed that in vivo 99m Tc signals were localized to the injection sites. PCR confirmed the presence of h NIS mRNA in injected hearts. Conclusion. NIS expression allows non invasive in vivo stem cell tracking in the myocardium, using both SPECT and PET. This reporter gene has great potential for translation in future clinical applications.

Download Full-text

Ferritin as a reporter gene of in vivo stem cell tracking by 9.4-T cardiac MR in a rat model of myocardial infarction

Journal of Cardiovascular Magnetic Resonance ◽

10.1186/1532-429x-15-s1-p31 ◽

2013 ◽

Vol 15 (S1) ◽

Author(s):

Eun-Ah Park ◽

Whal Lee ◽

Panki Kim ◽

Hoe Suk Kim

Keyword(s):

Myocardial Infarction ◽

Stem Cell ◽

Reporter Gene ◽

Rat Model ◽

Cell Tracking ◽

Cardiac Mr ◽

Stem Cell Tracking

Download Full-text

System for the Validation of Cell-Tracking Algorithms Using On-Demand Simulated Optical Microscope Images

2006 International Conference of the IEEE Engineering in Medicine and Biology Society ◽

10.1109/iembs.2006.259528 ◽

2006 ◽

Author(s):

Bechara Moufarrej ◽

Sylvain Martel

Keyword(s):

Cell Tracking ◽

Optical Microscope ◽

On Demand ◽

Tracking Algorithms ◽

Microscope Images

Download Full-text

Camelid reporter gene imaging: a generic method for in vivo cell tracking

EJNMMI Research ◽

10.1186/s13550-014-0032-8 ◽

2014 ◽

Vol 4 (1) ◽

Cited By ~ 3

Author(s):

Lode RY Goethals ◽

Tomas J Bos ◽

Luc Baeyens ◽

Frank De Geeter ◽

Nick Devoogdt ◽

...

Keyword(s):

Reporter Gene ◽

Cell Tracking ◽

Reporter Gene Imaging

Download Full-text

A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

10.1101/2020.02.10.942672 ◽

2020 ◽

Cited By ~ 3

Author(s):

John J Kelly ◽

Moe Saee-Marand ◽

Nivin N Nyström ◽

Yuanxin Chen ◽

Melissa M Evans ◽

...

Keyword(s):

Reporter Gene ◽

Cell Tracking ◽

Integration Site ◽

Reporter Genes ◽

Safe Harbor ◽

Enhanced Mri ◽

Contrast Enhanced ◽

Targeted Integration ◽

Contrast Enhanced Mri

AbstractImaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 µl subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.

Download Full-text

Development of endogenous beta-galactosidase and autofluorescence in rat brain microvessels: implications for cell tracking and gene transfer studies.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.7.8014479 ◽

1994 ◽

Vol 42 (7) ◽

pp. 953-956 ◽

Cited By ~ 25

Author(s):

B Lal ◽

M A Cahan ◽

P O Couraud ◽

G W Goldstein ◽

J Laterra

Keyword(s):

Rat Brain ◽

Reporter Gene ◽

Cell Tracking ◽

Fluorescent Dyes ◽

Tumor Biology ◽

Lacz Reporter ◽

Lacz Reporter Gene ◽

Specific Staining ◽

Adult Rat ◽

Beta Galactosidase

Cell transplantation is commonly used in studies of CNS development, tumor biology, and gene therapy. Fluorescent dyes and the E. coli lacZ reporter gene are used to identify transplanted cells in host tissues. The usefulness of these methods depends on host autofluorescence and beta-galactosidase (beta-Gal) activity. Our interest in the CNS vasculature led us to examine vascular autofluorescence and beta-Gal activity in postnatal and adult rat brains. Brains were perfusion-fixed (3.7% paraformaldehyde), cryoprotected, and cryostat-sectioned (12 microns). Autofluorescent vessel profiles were quantitated in sections using rhodamine filter sets and beta-Gal-positive vessels were quantitated under bright-field after incubation of sections with X-Gal chromogenic substrate for 1-18 hr at 37 degrees C. Multifocal vessel autofluorescence appeared in postnatal Day (PND) 18 Lewis rats (0.6 +/- 0.4 vessels/field) and increased tenfold in adults (6.8 +/- 0.3/field). The numbers of beta-Gal-positive vessels in PND 18 and adult sections incubated with X-Gal for 18 hr were 21.1 +/- 1.7 and 119 +/- 17, respectively. Host beta-Gal staining was similar to that produced by implanted endothelial cells expressing the bacterial lacZ reporter gene. Reducing incubation times in X-Gal to less than 4 hr eliminated endogenous staining and retained lacZ-specific staining. The presence of vascular autofluorescence and endogenous beta-Gal activity must be considered when either fluorescence- or lacZ-dependent cell markers are used in rat brain.

Download Full-text