TITLE: Human iPSCs culture and cardiomyocyte subtype differentiation in fully chemically defined conditions 
AUTHORS: Joanna Delimata-Raczek, Natalia Koralewska, Marek Figlerowicz, Ireneusz Stolarek

[DESCRIPTION]
Cardiomyocyte in vitro differentiation represents a pivotal avenue in stem cell research, offering unprecedented insights into cardiac development and disease understanding. Over the years, protocols for inducing cardiomyogenesis have evolved substantially, particularly with the advent of induced pluripotent stem (iPS) cell technology. The transition from intricate and uncontrolled systems to more refined and robust protocols has been paramount, driven by a cumulative understanding of the molecular and cellular cues governing the process.
This protocol addresses the intricacies of cultivating iPS cells and orchestrating their differentiation into distinct cardiomyocyte subtypes. The protocol encompasses not only the fundamental aspects of human induced pluripotent stem cell (hiPSC) culture but also delves into the nuanced stages of cardiomyocyte differentiation, sub-type specification, and the critical maturation phase. The refined methodologies outlined in this protocol contribute to advancing capabilities in disease modeling, drug discovery, developmental biology, and regenerative medicine.

[BEFORE_START]
Keep in mind that while working with cells everything should be kept sterile, every medium or any reagent should be prepared under the sterile hood, autoclaved, or filtered through 0,2um filter.

[STEPS]
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3. iPSC culture
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10. Standard iPSC culture before differentiation. 
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6. iPSC passage
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7. iPSC cryopresevation
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6.1. The optimal time for harvest or passage for iPSCs is typically when cells reach approximately 70-80% confluency.
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7.1. Prepare cryopreservation medium: culture medium without antibiotics with 5%-10%  . The cryopreservation medium should be stored in the fridge and keep cold during the procedure.

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4. Pluripotency characterization

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5. Mycoplasma testing
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3.2. Medium should be changed every day. When using    it is possible to skip weekend medium change by adding 2x more medium on Friday afternoon, but it is only recommended when cells are at low confluency.
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6.2. To start with prepare mild cell detachment solution, for example PBS with 0.5 millimolar (mM) EDTA. Warm it to Room temperature  before adding it to the cells.

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4.1. To guarantee the reliability of the research iPSC lines should be verified for pluripotency markers either by qPCR or immunocytochemistry. The most common pluripotency markers are OCT4, SOX2, SSEA4, TRA-1-60, and alkaline phosphatase.


 



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6.3. Prepare a new Geltrex-coated plate as in steps  

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6.4. Wash cells with warm PBS first, then add 1 mL of cell detachment solution to each well in a 6-well plate format. Transfer the plate to the incubator 37 °C. After 3 min take out the plate and observe the cells under the microscope. iPSCs colonies should look loose and be ready to detach, if not please transfer the plate back to the incubator for one minute or two more, but do not exceed the incubation time and do not let the iPSC colonies detach completely and float in the solution.

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6.6. You can split the cells using ratio for example 1:6 to 1:30 or take a small amount for counting first to have an exact number of cells in a new well.
 

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6.5. Remove the cell detachment solution and rinse the cells with 1 mL of warm culture medium. Pipette the cells a couple of times, but be careful not to over-pipette the cells. The cells should stay in clumps and easily detach from the well. Transfer the appropriate amount to new wells. 

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6.7. Agitate the plate gently from side to side and forwards and backwards to ensure that the cell aggregates are evenly distributed across the surface of the well containing culture medium with theaddition of rock inhibitor.
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6.8. The next day in the morning, exchange the medium to fresh culture medium without rock inhibitor. Continue to change the medium daily until iPSCs reach confluency again.
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7.2. Firstly perform cell detachment as described in steps   but instead of adding the cell suspension to the new well transfer it to 15 mL centrifuge tube.

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7.3. Centrifuge the tube 1400 rpm, 5 min 

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7.4. Remove the supernatant with care not to move the cell debris. Add an appropriate amount of cold cryopreservation medium to the tube, and resuspend the cells. Transfer cryotubes to a freezing container and put them in -80 °C . The next day, you can transfer cryotubes to the liquid nitrogen for longer storage.

 

 





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11. Differentiation to cardiomyocyte subtypes

 

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11.1. In the beginning, prepare two types of medium:
1. Cardiomyocytes induction medium
2. Cardiomyocytes culture medium

 
 

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17. Passaging cardiomyocytes 
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5.1. It is very important to maintain contamination-free cultures. A commonly known good laboratory practice is to regularly check cell cultures for mycoplasma. Mycoplasma contamination is very hard to notice without special tests. This contamination usually does not cause cell death, but it negatively affects the cell metabolism and growth making the research not reliable. The best way to assess mycoplasma contamination in culture is to use available qPCR or immunocytochemistry ready-to-use kits, for example:  

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11.2. Prepare small molecules working solutions of CHIR99021, IWP2 and retinoic acid (RA)

 


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17.1. Prepare a new Geltrex-coated plate   

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12. Atrial subtype differentiation
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12.1. On day 0 change the iPSC culture medium to cardiomyocytes induction medium with an addition of 5 micromolar (µM) CHIR99021 (0.5 µL from 10 millimolar (mM)).

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13. Ventricular subtype differentiation
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13.1. On day 0 change the iPSC culture medium to cardiomyocytes induction medium with an addition of 5 micromolar (µM) CHIR99021 (0.5 µL from 10 millimolar (mM)).
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14. Nodal subtype differentiation
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14.1. On day 0 change the iPSC culture medium to cardiomyocytes induction medium with an addition of 12 micromolar (µM) CHIR99021 (1.2 µL from 10 millimolar (mM)).
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12.2. After 48h on day 2 change the medium to fresh cardiomyocytes induction medium with an addition of 5 micromolar (µM) IWP2 (1 µL from 5 millimolar (mM)).

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12.3. On day 3, without changing the medium add 1 µL RA  1 micromolar (µM) from 1 millimolar (mM).

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12.4. On day 4 change the medium to fresh cardiomyocytes induction medium with an addition of1 micromolar (µM) RA.
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12.5. On day 6 change the medium to fresh cardiomyocytes induction medium.
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12.6. From day 8 change the medium to cardiomyocytes culture medium and continue media change every 2-3 days.


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13.2. After 48h on day 2 change the medium to fresh cardiomyocytes induction medium with an addition of 5 micromolar (µM) IWP2 (1 µL from 5 millimolar (mM)).
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13.3. On day 4 change the medium to fresh cardiomyocytes induction medium.
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13.4. On day 6 change the medium to fresh cardiomyocytes induction medium.
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13.5. From day 8 change the medium to cardiomyocytes culture medium and continue media change every 2-3 days.


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14.2. After 24h on day 1 change the medium to fresh cardiomyocytes induction medium.
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14.3. On day 3 change the medium to fresh cardiomyocytes induction medium with an addition of of 5 micromolar (µM) IWP2 (1 µL from 5 millimolar (mM) .)
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14.4. On day 5 change the medium to fresh cardiomyocytes induction medium with an addition of 3 micromolar (µM) CHIR99021 (0.3 µL from 10 millimolar (mM)).
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14.5. From day 7 change the medium to cardiomyocytes culture medium and continue media change every 2-3 days.
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17.3. Aspirate the medium and wash cells with warm PBS. Add warm   to the well (1 mL ) and incubate 37 °C 5 min-10 min

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17.4. After 5 minutes observe the cells under the microscope to see if they are ready to detach, if not continue to incubate up to 10 minutes maximum. When cells look ready to be detached pipette the solution a couple of times to detach the cells from the well, be careful not to overpipette the cells, because it may disrupt them. 
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17.5. Transfer them to 15 mL centrifuge tube, add an appropriate amount of culture medium (1:1), and centrifuge the tube 1400 rpm, 5 min

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17.6. After centrifugation aspirate the supernatant and resuspend the cells in cardiomyocytes culture medium. You can take asmall portion for cell counting and place desired amount of cells on a new geltrex-coated plate with addition of 10 micromolar (µM) rock inhibitor for better cell recovery. 



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17.7. Agitate the plate gently from side to side and forwards and backwards to ensure that the cell aggregates are evenly distributed across the surface of the well. Put the plate in the incubator. The next day in the morning change the medium to fresh culture medium and continue media change every 2-3 days.
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15. Longer culture
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15.1. Cardiomyocytes and their specific subtypes usually start contracting after 8-10 days from the beginning of the differentiation. After 12-15 days they become defined enough to be safely passaged or cryopreserved. Cells can be kept in a culture for at least 1 month or even longer if such experiments are desired. 
 

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16. Characterization of cardiomyocytes

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18. Cryopreservation of cardiomyocytes
 

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18.1. After centrifugation from step   you can aspirate the supernatant and resuspend cells in cold cryopreservation medium. 

 

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18.3. Post-thaw recovery
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18.2. Add an appropriate amount of cold cryopreservation medium to the tube, and resuspend the cells. Transfer cryotubes to a freezing container and put them in -80 °C . The next day, you can transfer cryotubes to the liquid nitrogen for longer storage.
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17.2. For better cell survival post-passaging it is recommended to add 10 micromolar (µM)   to the culture medium approximately 1 hour before passage.
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16.1. Successfully differentiated cells display completely different morphology, with characteristic net-like structure. The most important feature is their ability to contract. It is also recommended to perform PCR analysis or immunocytochemistry to confirm theexpression of key cardiac markers such as TNNT2 and NKX2.5.
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16.2. Immunocytochemistry confirmation of key cardiac markers expression in differentiated cardiomyocyte-like cells using commercially available kit   
 


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18.4. Prepare the previously coated 6-well plate by adding 2 mL of warm culture medium to each well and 2 µL  from 10 millimolar (mM)     to minimize the cell death.
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18.5. Take out the desired cryovial from the liquid nitrogen and quickly transfer it to 37 °C water bath. Be careful not to submerge the cap of the cryovial and keep a little ice cube in the cryovial before taking it out. Disinfect the cryovial with 70% ethanol. 
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18.6. Add 9 mL of warm cardiomyocyte culture medium to 15ml centrifuge tube. Centrifuge 1400 rpm, 5 min 

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18.7. After centrifugation aspirate the supernatant and resuspend the cells in culture medium. You can take small portion for cell counting. 
 

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18.8. Transfer the cells to a new plate, agitate the plate gently from side to side and forwards and backwards to ensure that the cell aggregates are evenly distributed across the surface of the well. Put the plate in the incubator. Next day change the medium without rock inhibitor and continue media change every 2-3 days.
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8. Standard iPSC culture before differentiation. 
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8.1. When cells become confluent passage them to prepared geltrex-coated 12-well plate. 


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2.6. Gently rock plate side to side, and back and forth to spread the cells evenly across the well. Transfer the plate to the incubator. The next day in the morning change the medium to fresh culture medium without rock inhibitor.

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2.4. Transfer 1 mL of thawed cell suspension to the 15 mL centrifuge tube and add 9 mL of warm culture medium very gently drop by drop, swirling the tube to minimize osmotic shock to the cells.
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2.5. Centrifuge the cell suspension 1400 rpm, 5 min. Carefully remove and discard the supernatant. Gently add 1 mL of medium to the remaining cell pellet. Gently resuspend cells in medium and seed into 2 wells of a previously prepared 6-well plate slowly drop by drop.

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2.2. Prepare the previously coated 6-well plate by adding 2 mL of warm culture medium to each well and 2 µL  from 10 millimolar (mM) of  to minimize the cell death.
   
 

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3.1. Incubate the cells at 37 °C/ 5% CO2  incubator and observe their growth and confluence under the microscope daily. Perfect undifferentiated iPSC colonies should look compact and present distinct borders. Cells present large nuclei and smaller cytoplasm region. It is essential to check iPSCs for the presence of spontaneous differentiation. If any signs of differentiation occur, colonies should be discarded immediately.

 





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2.3. Take out the desired cryovial containing frozen cells from the liquid nitrogen and quickly transfer it to 37 °C water bath. Be careful not to submerge the cap of the cryovial. You can swirl the tube a little to help it thaw quicker but pay attention to save a little ice cube inside the cryovial before taking it out. Disinfect the cryovial with 70% ethanol and put it under the hood. 

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2.1. 
Prepare culture medium using the following kit:  : add 48.5 mL of Essential 8 flex medium to the 50 mL centrifuge tube and  1 mL of 10x supplement. It is not necessary, but it is advised to also add 500 µL  of   to prevent cell culture from contamination.

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2. iPSC thawing
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1.3. Coat 6-well culture plate by adding 1 mL of coating medium to each well and cover the entire surface of each well. Incubate the plate in the incubator for at least  30 min before proceeding further.


 

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1.2. Resuspend single Geltrex aliquot in cold   medium to obtain desired dilution. Store diluted coating medium in the fridge.
 


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1.1. Thaw  on ice 4 °C . It is prohibited to thaw Geltrex at room temperature or in a water bath. Remember to keep it on ice during the whole aliquoting procedure and use only ice-cold pipette tips and tubes. 

 

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1. Coating culture plates with ECM proteins for enhanced attachment of iPSCs.
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9. Differentiation of hiPSCs into cardiomyocytes using commercially available kit  

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9.1. On day 0 of the differentiation remove iPSC culture medium from well and replace it with Cardiomyocyte Differentiation Medium A from the kit.
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9.2. On day 2 replace the medium with Cardiomyocyte Differentiation Medium B from the kit. 
 

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9.3. On day 4 replace the medium with Cardiomyocyte Maintenance Medium. Change the medium every 2-3 days with Cardiomyocyte Maintenance Medium. Starting from day 8 cells should begin to contract.
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8.2. View cells under microscope every day to estimate their confluence. When cells reach desired confluence between 60%-80% they are ready for differentiation.
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10.1. When cells become confluent passage them to prepared geltrex-coated 12-well plate.