Hello,

We work with dopaminergic neurons (mDAN) differentiation (Diff).

I understand how frustrating is start with the diff because there are to much ways do make it and protocols aren’t full described.

I will try help you with our experience.

QUESTION 1: I am going to induce neural/astrocyte differentiation of my iPSC cells, but I have some doubts about the protocol (based on the article from Chailangkarn et al., 2016). Do I have necessarily to use a poly-ornithine/laminin coated plate to expand the neural progenitor cells?

To mDAN diff, majority researches uses poly-ornithine/laminin/fibronectin (PLF) coated plates. How there are to much protocols variations, some researchers uses only one or combine 2 of these matrices.

We tested PLF, only laminin (with different concentrations) and Geltrex (has laminin in it composition):

- PFL coat with mDAN, to much cells pelled off from the well

- Laminin let cells migrate, after 24 hours of iPSC or neural progenitor cell (NPC) passage we saw clusters, about DIV 10 all cells migrated to the center of the well. This impossible few experiments.

- Geltrex showed be better do mDAN diff: iPSC and NPC had low migration, low cell dettachment and immunohistochemical analysis with mDAN markers (TH, Tuj1, Girk2, PitX3, ….) showed more strong staining.

QUESTION 2: I read that it is possible to buy the poly-ornithine solution.

Yes I know ThermoFisher and Sigma sell it ready to use.

I believe there are other companies that sell too….

QUESTION 3: What is the best to do? How many plates, on average, are necessary to expand NPCs and differentiate them into neurons and/or astrocytes?

Depend of 3 factors:

1- your neural diff rendiment (cell number by well diff)

2- wells size and cell concentration (cells/cm2), because probably will need seed in right concentration.

3- number of wells to make your experiments

Keep in your mind that how you start iPSC culture could influence in your differentiation. In our mDAN Diff, we tested start iPS culture with E8, mTeSR1 and 1/2 E8 and ½ mTeSR1. We tested 2 matrices: Geltrex and Matrigel.

Our best results was start iPSC culture with E8 and Geltrex (again we saw less cell detachment and the immunohistochemical analysis with mDAN markers (TH, Tuj1, Girk2, PitX3, ….) showing more strong staining.

But you should test what is best to your proposes

Wasn’t published yet, but our Springer Nature book chapter was accepted. Is VERY DETAILED STEP BY STEP mDAN Diff protocol with what worked and not, I strong recommend it when be accessible:

"Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells" Marcel Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, © Springer Science+Business Media, LLC, part of Springer Nature 2019

I hope helped you, if you have more doubts let me know.

Good Luck !!!!

All of bellow protocols has catalog numbers and medium, reagents tips.

- The main protocol followed (not step by step):

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Kriks S1, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L. Nature. 2011 Nov 6;480(7378):547-51. doi: 10.1038/nature10648.

- Articles with step by step and tips (about mDAN Diff but could help you):

Directed dopaminergic neuron differentiation from human pluripotent stem cells. Zhang P1, Xia N2, Reijo Pera RA3. J Vis Exp. 2014 Sep 15;(91):51737. doi: 10.3791/51737.

Midbrain dopamine neurons from hESCs. Tomishima M.; 2012.StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute. https://www.ncbi.nlm.nih.gov/books/NBK133274/

Neural induction – Dual SMAD inhibition.Tomishima M. StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute; 2008-.2012 Jun 10. https://www.ncbi.nlm.nih.gov/pubmed/23658995

Neural induction – Dual SMAD inhibition. Tomishima M. 2014. SKI Stem Cell Research Facility (http://stemcells.mskcc.org): link: http://coredinates.org/resources/DSi.pdf,

- Tips about medium, cytokines, etc….

Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nolbrant S1, Heuer A1, Parmar M1, Kirkeby A1,2. Nat Protoc. 2017 Sep;12(9):1962-1979. doi: 10.1038/nprot.2017.078. Epub 2017 Aug 31.

- The main ways to make mDAN Diff:

To mDAN Diff, there are more than 100 articles. For one main method kind another researchers did variations making very confused for who will start with neural diff.

How to make a midbrain dopaminergic neuron. Arenas E1, Denham M2, Villaescusa JC3. Development. 2015 Jun 1;142(11):1918-36. doi: 10.1242/dev.097394.

Hello Isabela Ichihara de Barros

I think Fabiano Tófoli gave a very good and detailed answer.

But I just want to add a short comment regarding to your first question.

I think you need to combine that poly-ornithine solution with laminin. Because I don't think it would be enough alone.

Laminins are key proteins of the basement membrane and you need to imitate the natural, cell-specific cell-matrix interaction for the attachment of stem cells at first and then for their accurate morphology, proliferation, migration and differentiation. I think you can use Poly-L-lysine or Matrigel alone and also different combinations but I've never read anything about using poly-ornithine alone. (I was using laminin alone and my cells were dying after a while. Then I realized I was using a very low concentration for a coating. So, once again, I am convinced that proper coating is really essential if you want your cells to stay alive.)

Good luck!

Elmas

Thakns for the tip Elmasnur Yilmaz !

I agree with Elmasnur Yilmaz's reply.

For the first tests, I recommend start use same matrix and concentration as the original article.

About mentioned article, Chailangkarn et al., 2016 (A model of human neurodevelopment for Williams syndrome), they did two differentiations: neural progenitor cells (NPCs) and cortical neurons (the last, following Marchetto et al., 2010):

- NPCs were used for RNA Seq

- Co-cultures of neurons and astrocytes for functional analysis

What cells you want to study? NPCs or cortical neurons?

Only one answer your questions? Need work with both?

If you will work with astrocytes, co-culture can be a contamination for your analysis. Already were published articles only atrocites differentiation (sure is hard only have one kind of neurons in differenciated culture, but you will have less cell contamination).

I think you must be sure if can work with imature neurons (NPCs) and a pure cell culture.

The last author (Alysson R. Moutri) of the two articles is Brazilian (works with Rett Syndrome), we know him and is accessible. Maybe it's a good idea you to talk with him.

If you have time, can change and test new matrix (eg: geltrex) to see if it is more efficient for differentiation. I like to use Geltrex because has laminin in the composition and lower variation among samples.

Matrigel I have not seen any protocol using it for the mDAN culture, but I don´t know for other neurons. It is used to made coat on the EBs or organoids and cannot change by another matrix.

Regards,

Hello Isabella,

I found these articles, I guess could help you:

Article Comparison of 2D and 3D neural induction methods for the gen...

Article Highly efficient methods to obtain homogeneous dorsal neural...

Our work with dopaminergic neurons diff:

Chapter Midbrain Dopaminergic Neurons Differentiated from Human-Indu...

Regards,

Thanks for you help, Fabiano Tófoli !

there were some good suggestions indeed. The question mentioned "expand" and "differentiate". Laminin induces neuronal differentiation. You will automatically have astroglia after passaging NPCs for several passages. you will get a lot of neurons from early passage NPCs, and then you will get a lot of astros and some oligos. However, the question was also how to expand. To expand, don't break rosettes into single cells, this will prevent you from expanding your derived NPCs (because they will start differentiating quickly). Culture in clumps, e.g., see PMID: 19609935. change half of the media (not 100% of the media), in this way you will enable your culture maintain progenitor state for a long(er) time. Don't use trypsin, but rather papain or even mechanical dissocation of rosettes into clumps (not single cells). Culturing them on gelatin or peptide hydrogel is likely to be better than laminin to expand the culture. Polyotnithine/Laminin will help with differentiation. Observe ## of neurons vs glia, when you dissociate your rosette culture at passage #2 vs 5-6, you will see many more astros at later passages.

Thank you a lot for your great tips Igor O. Nasonkin