Dear Malay!

Please You look at following article and protocol:

Article Chemically Defined and Small Molecule-Based Generation of Hu...

Cardiac differentiation of hiPSCs

Both hiPSCs and hESCs (>p20) were split at 1:10 or 1:12 ratios, using EDTA as above and grown for 4 days, at which time they reached ~85% confluence. Medium was changed to CDM3, consisting of RPMI 1640 (11875, Life Technologies), 500 µg/mL Oryza sativa-derived recombinant human albumin (A0237, Sigma-Aldrich, 75 mg/mL stock solution in WFI H2O, stored at −20 °C), and 213 µg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, 64 mg/mL stock solution in WFI H2O, stored at −20 °C). Medium was changed every other day (48 h). For d0-d2, medium was supplemented with 6 µM CHIR99021 (LC Laboratories). On d2, medium was changed to CDM3 supplemented with 2 µM Wnt-C59 (Selleck Chemicals). Medium was changed on d4 and every other day for CDM3. Contracting cells were noted from d7.

Other additions to cardiac differentiation media tested were 10.7 µg/mL recombinant human transferrin, 14 µg/mL sodium selenite, 1 µg/mL linoleic acid, 1 µg/mL linolenic acid, 2 ng/mL triiodo-l-thyronine, 2 µg/mL L-carnitine, 1 µg/mL D,L-alpha-tocopherol acetate, 100 ng/mL retinol acetate, 1 µg/mL ethanolamine, 20 ng/mL corticosterone, 9 ng/mL progesterone, 47 ng/mL lipoic acid, 100 ng/mL retinol, 1 µg/mL D,L-alpha-tocopherol, 100 ng/mL biotin, 2.5 ug/mL catalase, 2.5 µg/mL glutathione, 2.5 µg/mL superoxide dismutase, 2 µg/mL L-carnitine, 15 µg/mL D(+)-galactose, 16.1 µg/mL putrescine, 450 µM 1-thioglycerol, 55 µM 2-mercaptoethanol, and 64 µg/mL L-ascorbic acid 2-phosphate (all from Sigma-Aldrich).

Pay attention to the highlighted components.

Dear Malay,

Did you manage to get good iPSC-CM differentiation earlier for these isogenic iPSC clones or you are trying to set it up for the first time ? If you are establishing it for the first time for these iPSC lines then you might need to optimise several parameters (cell density at d0, concentration of Wnt activator/inhibitor etc.) .

Also, I am not sure which RPMI composition you are using.

My first suggestion would be to pls make sure that the RPMI medium contains GlutaMAX and HEPES. In my experience, it makes a huge difference for the efficiency and survival during iPSC -CM differentiation.

In case you are already using RPMI with GlutaMAX and HEPES, can you pls specify on which day you observe cell death or what is the pattern of cell death?

I noticed that you mentioned that cells without CHIR are also dying, but that might occur if iPSCs are extremely confluent without any iPSC maintenance medium (mTeSR).

So my suggestion would be to first check RPMI composition and if that doesn't help then check the matrix (geltrex or matrigel), cell density at d0 of differentiation and concentration of CHIR.

Good luck !

Hi @ Sambhabi, first of all, I would like to thank you for the suggestions.

Yes, I am trying to establish it for the first time in my lab. I am also working from scratch. However, I did not encounter any problem during iPSC maintenance time in mTeSR Plus media.

A few of my colleagues from neighbor labs gave me a recommendation that I should use >90% confluence before starting the differentiation with CHIR99021-HCl. I have tried to titrate the exact concentration of CHIR but ended up cell death. So I did not get any conclusive result regarding CHIR concentration.

I got complete cell death after 48hrs of CHIR treatment. I noticed moderate cell death at 24hrs of CHIR treatment and especially low (0uM or 1uM) and hi (8uM) CHIR concentration killed more cells after 24hrs of treatment.

I do not understand your response regarding RPMI composition. Which one is good RPMI with Glutamax or RPMI with HEPES? Could you explain it?

I used Matrigel for the matrix.

Alexandr Chernov Thanks we are using the same protocol but our iPS is different.

Dear Malay,

Regarding the RPMI you should use both GlutaMAX and HEPES. I always use RPMI basal medium from Gibco (Catalog number:  72400047) - https://www.thermofisher.com/order/catalog/product/72400047?de&en#/72400047?de&en

GlutaMAX is basically a more stable form of L-Glutamine and hence you can avoid toxic effects that might be caused due to spontaneous degradation of L-Glutamine. You can read more here- https://www.thermofisher.com/de/en/home/life-science/cell-culture/mammalian-cell-culture/media-supplements/glutamax-media.html#:~:text=and%20GlutaMAX%20supplement%3F-,L%2Dglutamine%20is%20an%20essential%20nutrient%20in%20cell%20cultures%20for,protein%20and%20nucleic%20acid%20synthesis.&text=acid%20as%20byproducts.-,GlutaMAX%20supplement%20is%20a%20dipeptide%2C%20L%2Dalanine%2DL%2D,and%20does%20not%20spontaneously%20degrade.

Matrigel should work very well for the differentiation for iPSC into CMs.

As you mentioned that your iPSCs are growing well, I am assuming that you are able to split them at regular intervals in uniform and consistent splitting ratios (eg. every 5-6 days).

Also I hope your cells have been checked for any possible mycoplasma contamination.

Regarding CHIR and cell density- Here is what I could suggest based on what you have mentioned.

First, I would suggest to try 4uM CHIR and 5uM CHIR for 48hours followed by Wnt inhibitor (2uM).

Second, cell density at the time of adding CHIR is extremely crucial. Since you are establishing the protocol for the first time I will suggest to compare various cell densities in parallel. This will help you to find out the best conditions.

I would suggest to try cell density of 70%, 85% and 90% for both CHIR concentrations (4uM and 5uM).

Maybe try 3 wells per condition.

(So 6 wells with 70% density where 3 wells will receive 4uM CHIR and other 3 wells will receive 5uM CHIR.)

Based on the conditions that are able to survive you can follow up further on thise densities and CHIR concentrations.

I know that it is quite a task to set up differentiation protocols for every cell line but once you have it optimised then it should run pretty smooth after that.

Hope this will help.

Shambhabi Chatterjee Thanks a lot.

FGF dicchis? concentration koto? essential amino acid gulo thik ache to?

Prosun Das Hello Dr. Das (Prasun Da)

We are using mTeSR Plus media cGMP grade but I have figure out the problem. It was related to the wrong RPMI media stock (without glucose) ordered by one of the previous postdocs of our lab before my joining and I used it mistakenly. Anyway, I got my differentiated cardiomyocytes and they are beating now and happy with the right medium.

Shambhabi Chatterjee Hi Shambhabi, I am using Glutamax RPMI but without HEPES. Does HEPES makes any difference in terms of pH in the media?

Hi Darshini Desai ,

I would recommend to include HEPES in your media.

I have experienced much better results (less cell death and higher yields) of CM differentiation when the media is supplemented with HEPES.

Based on my experience I will say that you will be able to achieve CM diff even without HEPES but the yields will improve considerably if you include HEPES.

Adding HEPES will help to stabilize the pH of the media.

there are publications which mention the use of HEPES for CM diff and EHT preps- https://www.nature.com/articles/nprot.2017.033?WT.feed_name=subjects_stem-cells

Thank you for your suggestions and insights, I will include HEPES into the media.

RPMI (recommend Gibco as previously stated) + B27 (- insulin) + CHIR for day 0 to 2 of CM diff. Test different amounts of CHIR to asses cell death and the tolerance of your cell lines. Trisomy 21 genotype may be a confounding variable on diff efficiency and cell death due to chir exposure. Have you tried running diff with control cell lines, H7 WT for example?