I have been trying neuronal differentiation from human embryonic stem cells (hESCs; H9) via the hESC aggregate method from Zhang's lab (Methods Mol Biol. 2010;584:355-66. doi: 10.1007/978-1-60761-369-5_19; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948208/)
Instead of laminin, I seeded my aggregates on Matrigel on day 7 (as Matrigel also contains laminin). However, I was not able to observe neural rosettes at day 10, and now it is at day 16 but I still could not see any neural rosettes although I see some cells on the periphery of the attached aggregates are neural stem cells and some have differentiated into neurons.
I would like to ask if there any important points to note for neural differentiation. Also, would Matrigel be ok for neural differentiation?
Thank you.
Please follow http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0024027 for neural rosette formation.
In my suggestion matrigel works great for hESC colonies. For grwoing EBs to Rosette
please use Laminin/Poly-Orithine
see the protocol above. I have used this for my project and works amazing.
Let me share my opinion after 25 years working with progenitors and stem cells. ANY UNDIFFERENTIATED STEM CELL WILL MAKE IT. Only tissue specific stem cells, since they have been instructed during their natural development can genrate a true neuron of a given type, or any other specialized cell types. All the rest is pseudo-science, faked neurons
I appreciate the concerns regarding validity of stem-cell derived neurons.
For me, a neuron is defined by its function
Please see:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0085932
http://www.jneurosci.org/content/34/11/4070.short?sid=910462bc-a4cd-4ac6-88f7-34d9b6e16405
Kindest regards
Hi Soumya,
I would like to ask for the reference you gave me, at day 10, the paper says transfer to laminin coated (10 ug/mL), do you need to coat poly-ornithine?
For seeding of R-NSCs, they seed on PLO(10 ug/mL) and Laminin (1 ug/mL), do you follow 1 ug/mL laminin or do you use 10 ug/mL laminin?
And I would also like to ask what kind of laminin do you use? Do you use the human placental type or mouse laminin?
Thanks a lot!
I'm just adding this answer in 2020 just for those who are still wondering if Matrigel works for getting neural rosettes: it works! about 60% of Matrigel is Laminin and that's sufficient for plating differentiating EBs. I'm currenctly using N2B27 + bFGF/EGF to differentiate H9 hESC towards neurons and I pass through EBs and plate them at day 10 on matrigel coated plates for Neural rosette enrichment. based on the quality of your culture you should see the rosettes 2-5 days after plating (sometimes you can see them on EBs before plating). I also keep Neural Stem cells on Matrigel and they are fine.
you dont need matrigel (you dont have to use matrigel). In fact if you plate EBs on plastic or plastic coated with gelatin (for better attachment of aggregates), you will get nice rosettes from EBs, which differentiated neural. I suggest you move to differentiating in monolayers to achieve uniform differentiation and have more cells for downstream applications, e.g. PMID: 19609935, PMID: 26283078
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