Using Bioengineered Bioluminescence to Track Stem Cell Transplantation In Vivo

Abstract

Bioluminescence imaging enables the real-time detection and tracking of engrafted cells in vivo noninvasively and dynamically. By detecting and quantifying the photons released from the oxidation of luciferin catalyzed by luciferase enzymes, this approach has proven effective in tracking engrafted stem cell survival and retention, making it a powerful tool to monitor cell fate after transplantation without animal sacrifice. Here we describe a protocol that allows luciferase-labeled stem cells to be imaged and tracked in vivo by bioluminescent imaging via an IVIS Spectrum imaging system.

1. Introduction

Bioluminescence imaging (BLI), a technology invented over the past two decades to enable the noninvasive visualization of ongoing biological processes in small laboratory animals, has tremendously facilitated the development of regenerative medicine [1–5]. BLI is generally achieved by utilizing the native light emission from organisms that can generate bioluminescence, including the firefly (firefly luciferase), sea pansy (renilla luciferase), and bacteria such as vibrio fischeri (bacterial luciferase). After the DNA encoding the luminescent protein is incorporated into the stem cell genome, these cells are transplanted into target organs so that their bioluminescence can be detected by BLI to obtain information on the survival and retention of engrafted stem cells [6]. With the development of commercially available bioluminescence tomography (BLT), BLI is now widely used in stem cell research to investigate cell fate and in cancer research to monitor tumor progression and metastasis [7–9]. By providing valuable insights on transplanted stem cell fate and function in vivo, BLI can improve our understanding of the therapeutic mechanisms of stem cells, thereby optimizing cell therapy protocols and improving the therapeutic efficacy of stem cells.

The protocol described here is relevant to BLI in stem cell research. Because the peak emission wavelength of firefly luciferase is about 560 nm and the red-shift of this emission makes detection of firefly luciferase much more sensitive in vivo [10], we chose firefly luciferase (Fluc) to exemplify the application of BLI in stem cell research.

2. Materials

1. Falcon^™ Standard Tissue Culture Dishes

2. Sterile Syringe Filters: PVFD Membrane

3. RW4, mouse embryonic stem cell

4. 129X1/SvJ mice

5. Lenti-X 293T cells

6. pLenti CMV Puro LUC (w168-1) (50 ng/μL in LB Broth medium)

7. LB Broth medium

8. Helper psPAX2 and pMD2.G

9. Mix & Go Competent Cells

10. LB Agar Ampicillin-100 Plates

11. ZR Plasmid Miniprep – Classic

12. Gibco^™ OptiPRO^™ SFM Medium

13. Gibco^™ Essential 8^™ Medium

14. DMEM–Dulbecco’s Modified Eagle Medium

15. Ampicillin

16. Chloroquine diphosphate salt powder

17. Dulbecco’s phosphate-buffered saline (DPBS)

18. jetPEI ^® DNA Transfection, dilute 4 μl of jetPEI ^® in 150 mM NaCl to a final volume of 50 μl

19. Polybrene Infection / Transfection Reagent, 10 mg polybrene per mL sterile Ultra Pure water

20. Puromycin, dissolved in ddH₂O to make a 10 mg/ml stock solution; 0.1 ml of 10 mg/ml puromycin stock solution was added into 499.9 ml Essential 8^™ medium to make Essential 8^™ medium with 2 μg/ml puromycin.

21. UltraPure^™ 0.5M EDTA, pH 8.0, 1:1000 dilution with DPBS to make a 0.5 mM EDTA working solution.

22. Isoflurane Liquid Inhalation 99.9% Glass Bottle 250 mL/Bt

23. D-luciferin stock solution: 15 mg/mL D-luciferin in DPBS sterile filtered. Dissolve D-Luciferin powder (either Potassium or Sodium Salt) in DPBS to a final concentration of 15 mg/mL. Then pre-wet a 0.22 μm filter by drawing through 5-10 mL of sterile H₂O and discard water. Finally, sterilize the D-Luciferin solution by going through the prepared 0.22 μm syringe filter.

24. Excella^® E24 Economical Benchtop Incubator Shaker

25. IVIS Spectrum in vivo imaging system

26. Promega GloMax^® 20/20 Luminometer

27. Caliper LifeScience Living Image^® in vivo imaging software

3. Methods

3.1. Bacterial transformation

We first transformed Mix & Go Competent Cells with the commercially available plasmid LentiCMV Puro LUC (see Figure 1).

Figure 1.

The sequences map of pLenti CMV Puro LUC (w168-1) used in this protocol. This is a lentiviral expression vector of firefly luciferase with CMV promoter, ampicillin resistance gene, and puromycin resistance gene. pLenti CMV Puro LUC (w168-1) was a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17477; http://n2t.net/addgene:17477; RRID:Addgene_17477).

1. To a tube of competent cells thawed on ice, add 1-5 μl plasmid pLenti CMV Puro LUC (50 ng/μL in LB Broth medium), and mix gently for a few seconds.

2. Immediately put the tube on ice and incubate for 2-5 min (up to 60 min).

3. Spread 50-100 μl mixture onto a pre-warmed LB agar ampicillin-100 plate. Incubate the plate at 37 °C overnight or until single colonies appear.

4. Select a single colony and inoculate the picked colony in a flask containing 50 ml of LB Broth medium with 100 μg/ml of ampicillin added.

5. Incubate the flask overnight at 37 °C in a shaker incubator with a speed set at 200 rpm.

3.2. DNA extraction

There are several options and commercially available kits that can be used to extract plasmid DNA. Here, we describe plasmid DNA minipreparation (miniprep) utilizing the ZR Plasmid Miniprep Kit, which enables the isolation of a maximum 25 μg plasmid DNA per preparation. This step involves the use of columns and several centrifugation steps to shear, extract, and precipitate plasmid DNA. For the detailed protocol, please refer to the manufacturer’s instructions.

3.3. Lentivirus production and packaging

This process involves the production of lentivirus from a lentiviral vector incorporated into Lenti-X 293T cells following a polyethylenimine (PEI) transfection protocol. The production of lentivirus (2nd generation protocol) requires the simultaneous transfection of the transfer plasmid (LentiCMV Puro LUC), lentiviral packaging plasmid (psPAX2), and envelope-expressing plasmid (pMD2.G) into packing cells (Lenti-X 293T). The produced LentiCMV Puro LUC lentivirus can be further used for Luc-tagged stable cell line generation.

1. Seed 293T packaging cells at a density of 3.5×10⁶ cells/plate in DMEM complete medium in 10 cm tissue culture plates (see Note 1).

2. Place the cells at 37 °C in a 5% CO₂ incubator for 20 hours.

3. Gently aspirate the medium, add 10 mL fresh DMEM complete medium supplemented with 25 μM chloroquine diphosphate, and incubate for 5 hours.

4. Prepare a mixture of the 3 transfection plasmids:

   Reagent	Amount per 10 cm dish
   psPAX2	3 μg
   pMD2.G	3 μg
   LentiCMV Puro LUC Plasmid	3 μg
   OptiPro SFM to a total volume	490 μl

5. Add the diluted jetPEI (0.108 mM, 10μl) in a dropwise manner into 490 μL plasmid mixture at a ratio of 1 μg DNA : 3 μg PEI in a total volume of 500 μL per 10 cm dish. While adding PEI, gently flick the diluted DNA tube. Incubate the mixture for 15-20 min at room temperature (20-25 °C).

6. Carefully add the transfection mix drop by drop to the Lenti-X 293T packaging cells, taking care not to disturb the cells.

7. Let the cells recover at 4 °C overnight (maximum 18 hours).

8. The next morning, replace the medium with 15 mL of fresh DMEM complete medium.

9. Incubate the cells for another 48 hours (at 37 °C), and then filter and collect the viral supernatant with a 0.45 μm strainer and a 50 ml polypropylene storage tube.

10. While the viral supernatant can be stored at 4 °C temporarily (max one day), to reduce the loss of titer, it is best to immediately aliquot it (4 vials, each 1ml), snap freeze it in liquid nitrogen, and store it at −80 °C immediately (see Note 2).

3.4. Lentiviral vector infection

The lentiviral vectors generated in the preceding step are used to infect RW4 mouse embryonic stem cells. In this protocol, we infected 70% confluent RW4 mouse embryonic stem cells that were cultured in a 10 cm² dish with 10 ml Essential 8^™ medium.

1. Add 10 μl Polybrene (10 μg/μl) to cultured cells so that the final Polybrene concentration is 10 μg/ml.

2. Add 4 ml of filtered viral particles produced in the previous step to cultured cells (see Note 3).

3. Centrifuge the plate at 250 x g for 5 min to increase the physical interaction of viruses with the surface of cells.

4. Place the plate in a 37 °C, 5% CO₂ incubator for 24 hours. After that, change the medium with fresh Essential 8^™ medium and leave the plate at 37 °C, 5% CO₂ for another 24 hours (see Note 3 and 4).

5. To select for transfected cells, antibiotic selection is started by supplementing 2 μg/ml puromycin in the medium.

6. Renew the medium containing 2 μg/ml puromycin every other day. Cell populations that have successfully integrated with the pLenti CMV Puro LUC will expand under puromycin selection conditions.

7. Verify the successful incorporation of luciferase plasmid by measuring luciferase activity or checking the in vitro BLI to ensure the bioluminescence signal is detectable before attempting in vivo (see Figure 3 and Note 5).

   1. For luciferase activity measurement, the cell lysate is prepared in 1X lysis reagent provided by Promega Firefly Luciferase Activity Kit following the manufacturer’s instructions.
   2. 20 μl of cell lysate is mixed with 100 μl of luciferase assay reagent in a 1.5 ml Eppendorf tube (both the lysis reagent and luciferase assay reagent are included in the Promega Firefly Luciferase Activity Kit).
   3. The emitted light signal is detected by Promega GloMax^® 20/20 Luminometer.
   4. For in vitro BLI, 2×10⁵ transfected cells are seeded into 24-well black plates overnight.
   5. The next day, cells are detached by 0.25 mM EDTA and suspended in 500 μl DPBS.
   6. After incubation with 150 ng/μl D-luciferin, cells are imaged using a charge-coupled device (CCD) camera within IVIS Spectrum in vivo imaging system.

Figure 3.

Luciferase verification in cells. (A) Luciferase activity in cell lysates measured by Promega GloMax^® 20/20 Luminometer. (B) In vitro bioluminescence imaging.

3.5. Implantation of luciferase-labeled cells

In this protocol, we implant luciferase-labeled RW4 mouse embryonic stem cells into the left adductor muscle of a 129X1/SvJ mouse by suspending the cells in 30 μl medium and carefully injecting cells with a 29-gauge insulin syringe. 129X1/SvJ mice are chosen as the recipient mice because they share the same genetic background with RW4 mouse embryonic stem cells, which minimizes the potential for immune rejection and allows the long-term tracking of cell populations in vivo.

1. After removing the culture medium, wash cells with 10 ml 1x DBPS twice and detach attached cells with 2 ml 0.5 mM EDTA in DPBS. Place the EDTA-treated cells into an incubator (37 °C, 5% CO₂) and wait for 2-5 min.

2. Transfer 2 ml cell mixture to a 15 ml Falcon tube containing 4 ml Essential 8^™ medium. Calculate the cell density using a hemocytometer.

3. Centrifuge cells at 200×g, remove the supernatant, and then resuspend 10⁶ cells in 30 μl of Essential 8^™ medium. Draw off 30 μl cell suspension with a 100 μl syringe and put the syringe on ice to wait for mice anesthetization.

4. Anesthetize mice in a chamber that provides 5% (v/v) inhaled isoflurane mixed with 1 L/min of oxygen (see Note 6).

5. Inject the 30 μl cell suspension into the left adductor muscle of 129X1/SvJ mice with a 27-gauge needle and a 100 μl syringe.

3.6. Bioluminescence imaging

In vivo BLI is performed to track the survival and retention of engrafted RW4 mouse embryonic stem cells.

1. Mice are anesthetized with 5% (v/v) inhalant isoflurane in 1 L/min of oxygen (see Note 6).

2. After anesthetization, mice are intraperitoneally injected with 10 μL of Luciferin stock solution per gram of body weight (normally ~200 μL for a 20 g mouse for a standard 150 mg/kg dose; 25-27-gauge needle is recommended here).

3. Five min later, mice are placed inside the CCD camera box. Use the control panel to either manually set or use the software’s Imaging Wizard (under sequence setup) to automatically set imaging parameters. Note that luminescent images often require longer exposure time and a lower F-stop (a wider aperture allows more light to reach the CCD, which is usually set at 1 in BLI) than fluorescent images. Ideally, images should take no longer than 5 min of exposure time. Here, the image-acquiring sequence step is programmed at 2-min intervals pending the recognition of the peak signal (see Figure 4 and 5, see Note 7 and 8).

4. Fixed-area region of interests (ROIs) are carefully drawn and photons released from the ROIs are assessed by photons/second/cm²/steradian (P·s⁻¹·cm⁻²·sr⁻¹) using the Living Image software.

Figure 4.

The standard setting for IVIS software’s imaging parameters.

Figure 5.

In vivo bioluminescence imaging. (A) Representative BLI time course (every 2 min) image of mice injected with Fluc-positive RW4 mouse embryonic stem cells. (B) The graph illustrates the bioluminescence saturation of the region of interest (ROI) shown in (A).

Figure 2.

Schematic diagrams depicting 5 steps required to successfully achieve bioengineered bioluminescence for tracking cell populations in vivo.