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Recently, molecular imaging has become a conditio sine qua non for cell-based regenerative medicine. Developments in molecular imaging techniques, such as reporter gene technology, have increasingly enabled the noninvasive assessment of the fate and biology of cells after cardiovascular applications. In this context, bioluminescence imaging is the most commonly used imaging modality in small animal models of preclinical studies. Here, we present a detailed protocol of a reporter gene imaging approach for monitoring the viability and biology of Mesenchymal Stem Cells transplanted in a mouse model of myocardial ischemia reperfusion injury.
In the first decade of the 21st century we have been pelted with all sort of cell-based therapies in clinical cardiovascular practice[1–4] stimulated to a good degree by the promising results of some preclinical studies[5–8]. A wide range of adult stem cell types have been used, including Mesenchymal Stromal Cells (MSCs)[9,10], Bone Marrow-derived Mononuclear Cells (BMMNCs) and Hematopoietic Stem Cells (HSCs)[11,12]. However, the bottom line has been a deflated enthusiasm due to insufficient retention and engraftment of the cells, which may limit their regenerative capacity[13–15]. Thus, the need to further investigate in vivo the kinetics of cell integration into the host tissue (both in preclinical and clinical settings) has become critical for the advancement of regenerative medicine. Until recently, assessment of the fate of transplanted stem cells has relied on traditional ex vivo assays and molecular techniques (i.e., histology, western blotting). Although these methods are easy to carry out for the molecular biologists and do not require special instruments, they involve invasive procedures and are limited in their capacity to monitor temporal changes in the living subject. This has inspired the development of novel research strategies devoted to improve the delivery as well as the biology of transplanted stem cells. Within this context, the advent of molecular imaging represents a far-reaching milestone for the noninvasive monitoring of cell fate in vivo[17–20].
Direct (fluorophores, superparamagnetic iron oxide particles, radioisotopes[23,24]) and indirect (reporter genes) labelling[16,25] of stem cells may be used to assess their short and long-term distribution, along with their viability, proliferation and functional interaction with the host microenvironment.
When choosing the appropriate technique for any experimental design, it is critical to keep in mind the biological and biochemical properties as well as the sensitivity of each strategy. While direct labelling is used to monitor cell fate only shortly after transplantation, mainly due to the progressive dilution of the signal - as a consequence of cell division - and cell toxicity issues - which may vary depending on the agent and doses used, reporter gene imaging allows long-term assessment of cell fate with longitudinal and repetitive imaging[26,27]. Using this strategy, cells are engineered to over-express or produce de novo an enzyme, receptor or protein: when this protein interacts with an exogenously administered substrate, it results in a signal that can be used to distinguish not only implanted cells from endogenous cells with high specificity, but also modulations in intracellular functions. However, it is critical to keep in mind that the manipulation of DNA sequences may alter the biology of transplanted stem cells. Furthermore, transgene expression is a very complex process, that involves many molecular steps: this may limit the amount of protein produced as well as the strength of the signal.
During the last ten years, using a reporter gene strategies designed to express the firefly luciferase (Fluc) or renilla luciferase (Rluc) gene, Bioluminescence Imaging (BLI) has been successfully adopted to monitor in vivo stem cell viability and engraftment[29,18,30] as well as to study transplanted stem cell biology and its interaction with the microenvironment (Figure 1). Due to the lack of spatial resolution and tissue depth penetration, the use of BLI is restricted to small animal studies (rats and mice). However, this strategy can be adapted to clinically used imaging modalities, such as Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT), using - for instance - thymidine kinase (TK) or human sodium-iodide symporter (hNIS) as reporter genes, respectively[19,27]. Therefore, BLI may be considered as a starting step in the development of novel imaging strategies in high-throughput preclinical studies.
Here, we describe in detail the materials and methods necessary to perform in vivo and ex vivo BLI of MSCs transplanted to the myocardium of a mouse model of ischemia/reperfusion injury.
Prepare and store all reagents at 4°C or on ice, unless stated otherwise. Prepare at the same time all the necessary controls. Carefully follow all waste disposal regulations for waste materials.
For plasmid tranfection:
For retroviral infection:
1Young cells are required for better efficiency of transfection.
2Recently, novel strategies have been developed for stable genomic integration to facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. These technologies use DNA-binding proteins such as Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALEN) or Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9 systems.
3The efficiency of retroviral infection is enhanced significantly in MSCs, by including polybrene during the infection.
4This step allows the positive selection of those cells that were efficiently infected by the retroviral vector. An alternative could be Puromycin (1–5 μg/ml).
5Autoclave all instruments before surgical session. Sterilize instruments in between animals using a glass beads sterilizer to avoid infections.
6Do not overheat the pad as it may cause burns to the animal.
7The reagent is light sensitive. Minimize exposure to light.
8The enhancer condenses the DNA molecules and the buffer provides optimal salt conditions for efficient DNA condensation.
9The mouse should be deeply anesthetized to avoid pharyngeal reflex (gag reflex), which makes the intubation procedure more difficult. Insert the endotracheal tube only if the trachea and the vocal cords are clearly visible. Do not try to force entry, but change the position of the tube tip. The tube tip is angled and should face upward. Do not perform more than 3 attempts as the trachea may be damaged irreversibly.
10Choose the intercostal space for thoracotomy based on curvature, after first rib that is less curved than the rib above. Be careful avoiding contact with the lungs.
11This requires some expertise. If necessary, carefully lift the atrium. The left anterior descending coronary artery is bright red to orange/pink, as opposed to the veins, which are dark red, is pulsatile and runs from below the left auricle to the apex.
12If the heart rate is too high, temporarily increase the anesthesia to 2.5% isoflurane.
13Avoid entering the LV cavity, but go deep enough to see the LAD pulsate over the needle.
14For a murine model of myocardial infarction perform a permanent ligation of the LAD: close the suture using a double surgeon’s knot, fixed with two extra half hitches. The heart region below the knot should become pale in few seconds. The knot is not released.
15The surgical procedure should last no more than 75 min.
16The presence of fur may decrease the amount of detectable signal.
17High doses of isoflurane may affect cellular metabolism in the living subject. The interaction between substrate (D-luciferin) and reporter protein (Firefly Luciferase) requires oxygen, magnesium and ATP, all co-factors that are available only in viable and metabolically active cells. Therefore, it is critical to avoid deep sedation.
18Due to its absorption/emission properties, the Renilla Luciferase signal cannot be reliably detected by in vivo BLI. Thus, the use of ex vivo luminometry is recommended.
19The imaging procedure should not cause any kind of discomfort to the animal.