Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).
Step 1
Note: The ES cells can also be taken from enzymatic propagation using collagenase or trypsin.
Step 2
Note: Change media often. The rosettes are usually at the outer edges of the colonies and their formation can be greatly enhanced if 300-500 ng/ml Noggin is added to the SRM (3). In some differentiation protocols, SRM can be exchanged with N2-A media and supplemented with growth or other factors to promote cell specification (2-5).
Step 3
Note: Avoid cutting and picking the MS5 stromal cells. If colonies are packed with rosettes, one can also isolate the entire colony.
Step 4
Note: To enhance cell survival, one can plate the small clusters in droplets to increase cell density. This step can also be modified by supplementing the N2-A media with additional growth or other factors to promote cell specification (2-5).
This protocol demonstrates the different steps in generating and isolating neuroepithelial cells from human ES cells using SDIA. The application of this method is manifold and has been used in many protocols to produce specified neurons (e.g. 1, 2, 5-9). The rosettes are thought to resemble neural tube cells with an anterior phenotype (2, 5, 10) and also contain neural crest progenitors (11, 12). In addition, they retain a certain level of plasticity, since they can be patterned by specific factors in defined culture conditions. Thus, the SDIA-derived neural progenitors can give rise to many cell types from the central and peripheral nerve system making them a useful tool for the derivation of different neural cell populations in ES cell differentiation paradigms.
The authors have nothing to disclose.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
L-glutamine | Gibco | 25030 | ||
alpha-MEM | Gibco | 12571 | ||
penicillin/streptomycin | Gibco | 15140 | ||
Knockout-DMEM | Gibco | 10829 | ||
Knockout serum replacement | Gibco | 10828 | ||
MEM non-essential amino acid solution | Gibco | 12383 | ||
DMEM/F12 | Gibco | 11330 | ||
N2-A supplement | Stem Cell Technologies | 07152 | ||
mitomycin-C | Sigma | M0503 | ||
gelatine type-A | Sigma | G1890 | ||
poly-L-ornithine | Sigma | P4957 | 0.01 % solution | |
laminin | Sigma | L-2020 | ||
fibronectin | Sigma | F2006 | ||
basic fibroblast growth factor (bFGF) | Invitrogen | 13256 | ||
1 ml syringe with 27 1/2 G needle | Becton Dickinson | 309623 | ||
N2-A media | medium | DMEM/F12 + 1% N2-A supplement | ||
Serum replacement media (SRM) | medium | Knockout-DMEM + 20 % Knockout serum replacement +1% MEM non-essential amino acid solution + 2 mM L-glutamine | ||
α-MEM media | medium | α-MEM + 10 % FBS + 2 mM L-glutamine + 1%penicillin/streptomycin | ||
MS5 | cell line | stromal cells | ||
Microscope | ||||
6 well plates | for tissue culture |