Name: in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells
Uploaded: 2008-05-02T17:59:00
Description: Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

Thanks to Tim Doyle, PhD and the Stanford Center for In Vivo Imaging for assistance with bioluminescence imaging. Also thanks to Ngan Huang, PhD for sharing her technique on stem cell co-injection with matrix solution. Lastly, thanks to Steve Felt, Ph.D. for assistance with veterinary animal care.

<ol>
<li>Construction of Double Fusion Reporter Gene<ol>
<li>In order to perform bioluminescence imaging of human embryonic stem cells, you first need to obtain cells that stably express a luciferase reporter gene such as firefly luciferase driven by a constitutive promoter like Ubiquitin or EF1a.</li>
<li>The focus of this protocol is on reporter gene applications, so detailed procedures are not provided here. However, our lab's general strategy is to use a double fusion construct vector that contains firefly luciferase (fluc)...

<ol>
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Compared to other modalities such as PET and MRI, bioluminescence has limited spatial resolution and reduced tissue penetration due to the relatively weak energy of emitted photons (2-3 eV); for these reasons it has thus far not been applicable in large animals. However, bioluminescence has the advantage of being low-cost, high-throughput, and non-invasive, making it highly desirable for in vivo stem cell tracking in small animals. Non-bioluminescence reporter genes such as PET and fluorescence constructs may be used in ...

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<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
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<td>&nbsp;</td>
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<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
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<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
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<td>6-well tissue culture-treated plates</td>
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<td>TPP</td>
<td>92006</td>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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