Подход к анализу миграции эксплантированных клеток (ствол клетки нервного гребня) описывается. Этот метод является недорогим, нежный, и способны различать хемотаксис с обеих хемокинез и других воздействий на миграционный полярности, таких как полученные из межклеточных взаимодействий в рамках первичного ствола нейронной клеточной культуре гребня.
Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9.
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions.
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.
Проведение исследования хемотаксиса на магистральных НКС оказалось сложным для ряда причин. Магистральные НКС представляют собой гетерогенную популяцию стволовых клеток, что будет отличать, если культурный долгосрочные, поэтому ствол НКС должно быть получено от первичного эксплант…
The authors have nothing to disclose.
Мы даем особую благодарность Lino Ким, Стив Гусман и Ujit Satyarthi для оказания технической помощи в ходе разработки этого метода. Мирон Hawthorne, Ричард Spengel, и Роберто Рохас обрабатывается камеры, используемые здесь, и при условии, столь необходимую техническую помощь. Примечательно, что Роберто Рохас производится Рисунок 4. Мы также благодарны за ценные советы Скотт Фрейзер до развития выше анализ хемотаксиса. Эта работа была частично поддержана NIH-МУРЗ SCORE-5S06GM048680-13, Медб и награды от ХСС, Northridge Graduate Program Support Диссертация на CW.
Name of the reagent | Company | Catalogue number | Comments (optional) |
DMEM | Omega Scientific | DM-22 | |
Penicillin Streptomycin Solution | Omega Scientific | PS-20 | 100X Stock Concentration |
L-Glutamine | Omega Scientific | GS-60 | 100X Stock Concentration |
Fetal Bovine Serum | Omega Scientific | FB-11 | Lot# 105247 (or another that is comparable) |
Modified Zigmond chamber | Home made | N/A | Reservoir volume: ~ 160 μl ea; for additional specifications, see Fig. 4 and the supplemental fabrication protocol |
Cell culture dish | Denville | T6040 | 40 x 10 mm |
Fibronectin | BD | 354008 | 10X Stock prepped by diluting 1 mg FN in 1 ml H2O and 9 ml DMEM |
Coverslips | Fisher | 12-548-B | Precleaned; 22 x 22 mm |
L15 medium | Thermo Scientific | SH30525.02 | |
Petroleum Jelly | Comforts | 011110794642 | 100% |
Centrifuge tube | Biologix | 10-9152 | 15 ml |
Dispase | Cell Systems | 4Z0-850 | 10X Stock Concentration |
Syringe | BD | 309602 | 1 ml |
Needle | BD | 305127 | 25 G x 1.5 in. |
Alexa Fluor 488-IgM | Invitrogen | A21042 | Stock is 2 mg/ml; 7 moles dye/mole IgM |
Dissecting Forceps | FST | Misc. | Dumont #5 or 55; straight tipped; stainless steel or titanium |
Tungsten Needle | N/A | N/A | Home made; placed in a pin holder |
Blunt Forceps | Tiemann | 160-18 | Used for transferring embryos to Ringer’s from egg yolk |
Supplemental Protocol: Fabrication of a Modified Zigmond Chamber
Please refer to Figure 4 as a reference for the protocol below: