I am trying to differentiate neural stem/precursor cells derived from human ES cells (HuES 7 line) via rosette formation, into main brain cell types viz. neurons, oligodendrocytes and astrocytes. I am using DMEM/F12+N2+B27+Glucose(2 mg/ml)+Fibronectin (2.5ug/ml) as the differentiation medium on polyornithine/laminin-coated dishes. But the cells are coming off within 2-3 days and don't look healthy. It would be quite helpful if anybody suggests a better protocol, which is easier and faster?
You could try the 3D cultures as done by the Vaccarino group and others:
Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12770-5. doi: 10.1073/pnas.1202944109. Epub 2012 Jul 3.
Hi Ashaq,
I am using DMEM/F12+B27+Pen/Strep+HEPES+1%FBS as the differentiation media on poly-L-Lysine coated wells and it is working well for me (except that i am working with E13.5 mouse cortical cells derived neurospheres). Initially, i was facing a similar problem like you, when i dont wash of the excess coating material clearly and without drying the plate completely before plating the cells. I dont know whether this could be the problem or something else. I hope this may help you.
Dear Ashaq,
You may consider using the NeuroCult Human NS-A Differentiation Kit: http://www.stemcell.com/en/Products/All-Products/NeuroCult-NSA-Differentiation-Kit-Human.aspx
The procedure is very simple and straight-forward, and it also has been optimized to provide constant quality performance.
In terms of ratio of differentiation, you would usually get more astrocytes (about 80%), oligos and followed by neurons.
Here is the link to the technical manual/protocol: http://www.stemcell.com/~/media/Technical%20Resources/3/28724MAN_3_0_0.pdf, (starting from page 15). The protocol lists out very detailed steps from harvesting your neural stem/progenitor cells to differentiating and immunolabeling them.
I hope this helps.
Thank you,
Dunstan
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