Hi

I am using these mediums since one year!! I found them good for generating NPCs. What problem you are facing with these medium?

Thanks

Thank you so much for answering! Are you using the embryoid body formation branch of the protocol or the monolayer one? Is your starting material hiPSCs? In short, the problem is that after I switch to the Neural Progenitor Medium the morphological heterogeneity of the cell population increases dramatically, and after a few passages they start to look...unhealthy. Their proliferation rate is also fluctuating. I am not sure what the root of my problem is. Do you think it could be that I am not selective enough when I pick the neural rosettes? Are you doing this step manually or with the neural rosettes selection reagent? On the other hand, have you noticed a lot of variation with the process from cell line to cell line? If you think we could discuss this in a little more detail, this is my email address [email protected].

Thank you very much for your time and advice, I truly appreciate it!

Hi

I worked on Monolayer protocol and starting material was healthy iPSCs.Initally I have a problem in reference to NPCs proliferation and later it solved. monolayer-based protocol is fast and simple (generating PAX6+ NPCs in 6-9 days), assessment of marker expression is necessary to confirm neural induction (cell morphology is not always a reliable indicator). 

Please review the technical bulletin Monolayer Culture Method for Neural Induction of hPSCs to see if your setup deviates from protocol. I have few questions:

At what time point are the cultures now? 

What Passage number was the maintenance iPSCs?

How was this iPSC generated?

Have they been Karyotyped? 

In NPCs generation, cell number plays a crucial role. The newly generated NPCs survive best when kept in close proximity to each other (they like to be crowded). it is important to use the recommended high seeding density and maintaining cultures to 80% confluent (or until OCT4 the expression is lost and PAX6 expression goes up). 

Hope it works!!

Best

Tanisha

Thank you very much! I have only used the embryoid body formation-based protocol so far. My starting material are hiPSC from either healthy donors or patients. One of the lines was P11 and the other one is around P60. I know this is quite a high passage, but we do not have another option in this particular case and other labs have managed to produce NPCs (with different protocols) from this line even with this high passage. Yes, they have been karyotyped and I have no reason to think that they are not of good quality. I keep them as colonies on Matrigel in mTeSR 1, with manual cleaning of differentiating areas.

I had my cells now as Neuronal Progenitor Cells but I have stopped culturing them because they were looking really bad, so I will have to start the whole process again. What kind of proliferation problems did you have and how were they solved?

I was very careful with the seeding density, maybe even too careful - I never did a split higher than 1:2 or 1:2.5 and I didn't split them before being highly confluent (maybe they were even too confluent, could this be the problem?). Do you keep them on Matrigel or on Laminin+Ornithine?

Did you ever generate neurons from these NPCs? If so, which protocol/media did you use? Was it successful?

Thank you very very much for your time and your interest, I really appreciate it!

- Bianca

Hi

I cultured my NPCs on Matrigel. I never generated neurons from NPCs as I have protocol for neuron generation directly from iPSCs. I just generated NPCs from iPSCs using only monolayer protocol for my experiments. If you never tried Monolayer protocol, Try it once!! May be it works for you!!

passage number and splitting, is fine!!

Good luck!!

Hi Flavia-Bianca Cristian and Tanisha Singh,

I just saw this thread and hence, wanted to join in on the discussion.

Flavia-Bianca, has your issue with the NPCs been resolved?

I am having the same issues especially when passaging the cells from the neural induction medium to the neural progenitor medium. I have tried the protocol twice already and the cells seem to start becoming unhealthy at this transition of the medium. Would you have any recommendations as to go about resolving this issue? For example, what is the seeding density for the cells for one well of a 6-well dish?

For your information, I have been using PLO/Laminin-coated dishes for my cells.

Thanks in advance.

Also, Tanisha Singh, could you please let me know what protocol do you use to derive neurons directly from iPSCs?

We can correspond via mail. My mail id is: [email protected]

Best,

Orthis

Hi, Orthis Saha

Attached is the paper

Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: A novel therapeutic lead to treat frontotemporal dementia

Thanks!

Hi Tanisha Singh , Thank you for the reference to the article.

I am still struggling with the monolayer protocol for now. If you have any suggestions/pointers to this end, I would be happy to discuss them with you. In the meantime, I have begun the embryoid body protocol too so as to be able to derive NPCS at the earliest.

Thanks in advance.

Hi, I just start using this kit, but get into trouble at the beginning of the monolayer protocol. I seeded about 2*10^6 cells in a single well of 6-well plate, but the well was full when I checked 12hours later. What density do you use with the monolayer protocol? And do cell die a lot during differentiation?

Dear Flavia-Bianca Cristian , Tanisha Singh and Orthis Saha

I also wanted to join in this discussion. I'm planning to use the Stemdiff neural induction media to produce NPC from hiPSCs and further differentiate neurons and astrocytes from these NPCs. I would appreciate it if you could tell me about your experiences with this medium to produce NPC. Also, I would like to know if you already differentiated neurons from NPCs and if you had any trouble performing NPC (using monolayer protocol) or even in the neural differentiation step. It would be lovely if you give some bits of advice or protocol that could help me in this quest. This is my email address [email protected].

Thank you in advance.

Hi Sheila Garcia-Rosa , In my experience, I found induction to be successful with the Embryoid Body protocol rather than the Monolayer protocol as suggested by Stem Cell Technologies. For both the induction and the propagation, you need to ensure that cells are as close together as possible and you seed them always at densities higher than the recommended ones. Also, we have found that once you reach the NPC stage, you'll need to keep the cells very confluent between passages. For example, for 1 well of a 6 well-dish, we seed 1 million cells.

Thank you very much for your fast answer and such detailed advice! I was considering using the monolayer protocol because I thought that could be easier since I don't have experience with EBs and neural rosette selection. I found the protocol below that I'm going to use as a reference to standardize neuronal and astrocyte differentiation from NPCs. Did differentiated neurons directly from hiPSC? Do you have a protocol that you could provide?

Article An efficient neuron-astrocyte differentiation protocol from ...

For the differentiation, we stick to 6 weeks of differentiation, using a biweekly change of BrainPhys (+ supplements)..ie..replacing half of the existing BrainPhys with fresh BrainPhys

Hi Flavia-Bianca Cristian :

I am joining late here but here are some thoughts... P60 might be too high of a passage number, there are several ways besides karyotype to check your cell quality. If you are concerned about high-passage number you may expand and cryopreserve NPCs using STEMdiff Neural Progenitor Medium and STEMdiff Neural Progenitor Freezing Medium.

A quick note that monolayer is easier to use, embryoid body method is more robust for difficult cell lines - both can be suitable and result in successful experiments.

We have several new STEMdiff products and methods to culture and co-culture various cell types in the neural system.

Faris Samkari

Hi,

If PAX6 doesn't work, Try for Nestin, SOX1, for estimating Neural population. I am sure you'll find good expression for Nestin if NPCs generated so far are pure!

Hi Faris Samkari

Note that for most PSC cell lines, EB protocol is a little more robust than the monolayer protocol. If you really need to use monolayer for a particular cell line, there are several options to improve the performance. Plating density, multiple passages (3X passages over weeks) in STEMdiff SMADi differentiation media can help. For detailed methods, check out STEMCELL Methods Library here:

https://www.stemcell.com/technical-resources/methods-library/cell-culture-new/ectodermal-cells/neural-cells.html

I think you will find it updated with everything that you are looking for to optimize neural differentiation in the monolayer protocol.

Thanks

Jason