This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.
Labeling of mesenchymal stem cells with the fluorescent dye DiD
Labeling of human embryonic stem cells with the fluorescent dye ICG
OI is a relatively new imaging technique, based on the detection of fluorescence. OI is as sensitive as radiotracer-based imaging techniques, but not associated with any irradiation exposure. OI provides an effective means of tracking cells non-invasively and repetitively, thereby providing insight into cell migration to the target site. One major limitation of the technique is the limited tissue penetration of fluorescent probes in vivo. Thus, a tracer accumulation in deep tissues, more than about 5-10 cm from the skin surface, may not be detected. Current clinical applications are limited to superficial techniques such as endoscopic, cardiovascular and retinae imaging (Funovics et al. 2003) but this domain of research will continue to expand through the recognition of the vast possibilities offered by in vivo cell tracking.
This project was supported by a Leon J. Thal SEED grant from the California Institute for Regenerative Medicine. Tobias Henning was funded by a Research stipend from the German Research Association (DFG, HE 4578/1-2). We want to gratefully acknowledge Juanito Meneses for his advice on the culture of human embryonic stem cells.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Indocyanine Green (IR-125) | Reagent | Fisher Scientific | AC41254-1000 | |
DMSO | Reagent | Sigma | D-2650 | |
D-MEM High Glucose | Reagent | Sigma | D5648 | |
Gelatin | Reagent | Sigma | G1890 | Dilute to final concentration of 0.1% in PBS |
PBS, Mg and Ca free | Reagent | GIBCO | 14190-144 | |
Trypsin-EDTA 0.05% | Reagent | Invitrogen | 25300-120 | |
Trypsin-EDTA 0.25% | Reagent | Invitrogen | 25200-114 | |
FCS, characterized | Reagent | Hyclone | SH30071.03 | |
Cell Strainer (40 μm Nylon) | Reagent | BD Falcon | 352340 | |
DiD | Reagent | Molecular Probes | V-22887 | |
Knockout DMEM | Reagent | GIBCO | 10829018 | |
Knockout Serum Replacer | Reagent | GIBCO | 10828028 | |
b-Mercaptoethanol | Reagent | Sigma | 7522 | |
Non-essential Amino Acids | Reagent | GIBCO | 11140050 | |
FGF-2 | Reagent | R&D Systems | 233-FB-025 | |
Glutamine 200mM | Reagent | GIBCO | 25030081 | |
Penicillin/Streptomycin | Reagent | GIBCO | 15140-122 | |
Protamine Sulfate | Reagent | American Pharmaceutical Partners Inc. |