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 JoVE General

ES Cell-derived Neuroepithelial Cell Cultures

1, 1, 1, 1

1McLean Hospital, Harvard Medical School

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    Summary

    Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

    Date Published: 11/30/2006, Issue 1; doi: 10.3791/118

    Cite this Article

    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).

    Abstract

    ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

    Protocol

    Step 1

    1. Plate mitomycin-C ggrowth-inhibited (10 µg/ml for 2.5 hours) MS5 cells at a density of 70,000/cm2 on gelatin-coated (0.01 % for 30 minutes) 6 well plates in α-MEM media.
    2. When cells are attached and have formed a monolayer (over night growth), switch to SRM.
    3. Manually isolate ES cell colonies from the ES cell cultures using a syringe with a 27 ½ G needle.
    4. Tritrurate the colonies carefully with a 1 ml blue tip and plate at low density on the MS5 cells (usually 2-3 colonies per 6 well plate).

    Note: The ES cells can also be taken from enzymatic propagation using collagenase or trypsin.

    Step 2

    1. Grow the ES cells on the MS5 feeders for 14-21 days until rosette-containing colonies form.


    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

    1. Isolate and pick the rosettes with a 27 ½ G needle under a microscope.

    Note: Avoid cutting and picking the MS5 stromal cells. If colonies are packed with rosettes, one can also isolate the entire colony.


    Step 4

    1. Aspirate the clusters of rosettes, tritrurate them carefully with a 1 ml blue tip, and plate them on poly-L-ornithine (0.001%) and fibronectin (1 µg/ml) or laminin (1 µg/ml) -coated 6 wells in N2-A media usually supplemented with bFGF (20 ng/ml). The rosettes will reform and expand.

    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).

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    Discussion

    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.

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    Disclosures

    The authors have nothing to disclose.

    Materials

    Name Type Company Catalog Number Comments
    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 nonessential amino acid solution GIBCO, by Life Technologies 12383
    DMEM/F12 GIBCO, by Life Technologies 11330
    N2-A supplement Stem Cell Technologies 07152
    Mitomycin-C Sigma-Aldrich M0503
    Gelatine type-A Sigma-Aldrich G1890
    poly-L-ornithine Sigma-Aldrich P4957 0.01% solution
    Laminin Sigma-Aldrich L-2020
    Fibronectin Sigma-Aldrich F2006
    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 nonessential 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

    References

    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).

    Comments

    2 Comments

    Where can we get MS5 stromal cell line?

    Sergey Akimov, JHU SOM
    Reply

    Posted by: Sergey A.June 24, 2010, 12:00 PM

    Just now noticed your comment. I believe the easiest way to obtain MS5 cells is via DSMZ - (German Collection of Microorganisms and Cell Cultures; http://www.dsmz.de). The MS5 line is internal DSMZ no."ACC 441".

    Reply

    Posted by: AnonymousOctober 27, 2011, 8:32 AM

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