McLean Hospital, Harvard Medical School
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Karki, S., Pruszak, J., Isacson, O., Sonntag, K. C. ES Cell-derived Neuroepithelial Cell Cultures. J. Vis. Exp. (1), e118, doi:10.3791/118 (2006).
Note: The ES cells can also be taken from enzymatic propagation using collagenase or trypsin.
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).
Note: Avoid cutting and picking the MS5 stromal cells. If colonies are packed with rosettes, one can also isolate the entire colony.
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.
|L-glutamine||GIBCO, by Life Technologies||25030|
|alpha-MEM||GIBCO, by Life Technologies||12571|
|penicillin/streptomycin||GIBCO, by Life Technologies||15140|
|Knockout-DMEM||GIBCO, by Life Technologies||10829|
|Knockout serum replacement||GIBCO, by Life Technologies||10828|
|MEM non-essential amino acid solution||GIBCO, by Life Technologies||12383|
|DMEM/F12||GIBCO, by Life Technologies||11330|
|N2-A supplement||Stem Cell Technologies||07152|
|poly-L-ornithine||Sigma-Aldrich||P4957||0.01 % solution|
|basic fibroblast growth factor (bFGF)||Invitrogen||13256|
|1 ml syringe with 27 1/2 G needle||BD Biosciences||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|
|6 well plates||for tissue culture|
1. Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31-40 (2000).
2. Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101, 12543-12548 (2004).
3. Pruszak, J. & Isacson, O. Directed differentiation of human embryonic stem cells into dopaminergic neurons. Human Embryonic Stem Cells: The Practical Handbook Sullivan, S., Cowan, C., Eggan, K. (eds.); John Wiley & Sons (2007).
4. Pruszak, J., Sonntag, K.C., Aung, M.H., Sanchez-Pernaute, R. & Isacson, O. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 25, 2257-2268 (2007).
5. Sonntag, K.C. et al. Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 25, 411-418 (2007).
6. Kawasaki, H. et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 99, 1580-1585 (2002).
7. Ko, J.Y. et al. Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons. J Neurochem 103, 1417-1429 (2007).
8. Hong, S., Kang, U.J., Isacson, O. & Kim, K.S. Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons. J Neurochem (2007).
9. Zhang, S.C. Neural subtype specification from embryonic stem cells. Brain Pathol 16, 132-142 (2006).
10. Pankratz, M.T. et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells 25, 1511-1520 (2007).
11. Lazzari, G. et al. Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro. Stem Cells 24, 2514-2521 (2006).
12. Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B. & Goldstein, R.S. Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells 23, 923-930 (2005).