इन विट्रो में और vivo में मानव भ्रूण स्टेम कोशिकाओं के bioluminescence रिपोर्टर जीन इमेजिंग में
Summary
स्टेम सेल उपचार में बढ़ती रुचि के साथ, आणविक इमेजिंग तकनीक निगरानी स्टेम सेल प्रत्यारोपण के बाद व्यवहार के लिए आदर्श होते हैं. Luciferase रिपोर्टर जीन गैर इनवेसिव, सेल अस्तित्व, स्थान, और vivo में प्रसार के दोहराव आकलन सक्षम है. यह वीडियो कैसे एक जीवित चूहे में hESC प्रसार को ट्रैक करने के लिए प्रदर्शन करेंगे.
Abstract
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.
Protocol
Discussion
पीईटी और एमआरआई जैसे अन्य रूपरेखा की तुलना में, bioluminescence सीमित स्थानिक संकल्प और कम ऊतक पैठ उत्सर्जित फोटॉनों की अपेक्षाकृत कमजोर ऊर्जा (2-3 eV) के कारण है, इन कारणों के लिए यह इस प्रकार दूर किया गया है बड़े जानवरों मे?…
Acknowledgements
टिम डोयल, पीएचडी और bioluminescence इमेजिंग के साथ सहायता के लिए Vivo इमेजिंग में लिए स्टैनफोर्ड सेंटर के लिए धन्यवाद. इसके अलावा मैट्रिक्स समाधान के साथ स्टेम सेल सह इंजेक्शन पर उसकी तकनीक को साझा करने के लिए Ngan हुआंग, पीएचडी के लिए धन्यवाद. अंत में, स्टीव के लिए धन्यवाद पीएच.डी., लगा पशु चिकित्सा जानवरों की देखभाल के साथ सहायता के लिए.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Dulbecco’s Modified Eagle’s Medium (DMEM) | HyClone | |||
| BD Matrigel™ Basement Membrane Matrix | Growth factor reduced (optional: phenol-red free) | BD Biosciences | ||
| mTeSR1 Maintenance Medium for Human Embryonic Stem Cells | StemCell Technologies | |||
| Phosphate Buffered Saline (PBS) | ||||
| D-Luciferin Firefly, potassium salt | Biosynth AG | |||
| Collagenase IV solution | Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius). | |||
| Baked Pasteur pipets | ||||
| 6-well tissue culture-treated plates | TPP | 92006 |
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