- The here described induction and differentiation conditions are based on the feeder-free 2D culture of human induced pluripotent stem cell (iPSC) lines 201B78 and 409B29 on laminin-511 E8 fragment coated cell culture plates with AK02N being the iPSC-culture and maintenance medium of choice.
- Media are always used after being equilibrated 30 minutes to 1 hour at RT except if stated otherwise.
- Stable and high-quality culture of established human iPSC lines is critical for subsequent successful differentiation of mesodermal cell types or induction of axioloids described in this protocol (Figure 1).
A. Human iPSC culture in feeder-free condition
a) Coating of cell culture dishes & plates for feeder-free iPSC culture
Conditions described hereafter have been adjusted for a single well of a 6 well culture plate and should be adjusted accordingly (see Table 1).
1. Add 1.5 ml of PBS containing iMatrix-511 (0.5 µg/cm2) into each well of a 6 well culture plate.
2. Incubate the plate for at least 1 hour at 37°C, 3 hours at room temperature or overnight at 4°C. Usually coating is performed overnight at 4°C.
3. Aspirate* iMatrix-511 containing PBS and add 1.5 ml of StemFit® AK02N containing Y-27632 (10 µM).
4. Place the plate into a humidified 5% CO2 incubator at 37°C until further use.
*Do not let the plate dry out when changing solutions.
b) Passaging of human iPSCs in feeder-free conditions
Passage should be done when iPSCs cultures are between 50% and 80% cell confluency.
1. Aspirate the culture medium.
2. Wash the cells with 2 ml of PBS.
3. Aspirate the PBS and add 300 µl of 0.5X TrypLETM Select solution (1:1 dilution with 0.5 mmol/l-EDTA/PBS).
4. Gently shake the plate to evenly distribute the solution in the well, and place into a humidified CO2 incubator set to 37°C.
5. After 2 minutes, take out the plate and check the cells under the microscope; if cells have already started to detach move to the next step, otherwise gently shake the plate again to re-distribute the 0.5X TrypLETM Select solution and place into the incubator for another 2 minutes.
6. Aspirate the TrypLETM Select solution and gently wash with 2 ml of PBS.
7. Aspirate the PBS and add 1 ml of StemFit® AK02N with Y-27632 (10 µM).
8. Harvest cells with cell scraper or pipetting into a 1.5 ml microtube.
9. Dissociate the cells by gently pipetting the cell suspension using P1000 pipette (about 15 times) and measure cell density.
10. Seed 1.3x104 cells/well* onto the previously prepared iMatrix-511 coated 6 well culture plate containing 1.5 ml StemFit® AK02N with Y-27632 (10 µM) (for other plates or dishes see Table 1).
11. Gently shake the plate to distribute cells evenly and place in a humidified 5% CO2 incubator at 37°C.
12. On the next day, change the medium to AK02N without Y-27632.
13. Change medium every other day until day 4, then every day until axioloid induction at day 5 or passage**.
* Initial cell seeding density should be adjusted in order for hiPSCs to be at ~60% confluency at day 5 of culture.
** Cells should not be cultured for more than 7 days before being passaged.
B. Induction of axioloids from human iPSCs
a) Initiation of differentiation of hiPSCs under 2D culture conditions
At day 5 of culture, check the quality of cultured iPSCs* and confirm that they reached ~60% confluency (good initial cell density is a critical factor!).
1. Prepare 1.5 ml per well (when using a 6 well plate) induction medium consisting of AK02N-C supplemented with CHIR 5 µM** and bFGF 20 ng/ml.
2. Expose the cells to the induction medium and culture for 24 hours.
* If hiPSC conditions are not good (e.g. confluency too high or too low, many spontaneously differentiated cells are present or colony sizes/shapes are unusual) do not use cells for induction.
** We found that CHIR concentrations need to be slightly adjusted depending on the cell line which is used. In our hands, for most human iPSC lines the optimal CHIR concentration is usually in the range of 4-6 µM.
b) Aggregation step and 3D differentiation of cells
24 hours after initial primitive streak/mesoderm induction with WNT agonist and FGF recombinant protein, treated cells are detached and aggregated under 3D conditions in non-attachment plates.
1. Prepare aggregation medium by supplementing AK02N-C with CHIR (use a concentration identical to the one used for the initial 2D induction phase), bFGF 20 ng/mL, SB431542 10 µM, and Y-27632 10 µM.
2. Wash the cells with 2 ml of PBS.
3. Add 0.3 ml of AccutaseTM and distribute it evenly in the well by gently shaking the plate.
4. Incubate for an initial 2 minutes in a humidified CO2 incubator set to 37°C .
5. Observe the cells under the microscope whether they are starting to detach or not; if not, re-distribute the AccutaseTM and incubate it for another 2 minutes.
6. Neutralize by adding 0.7 ml of aggregation medium.
7. Harvest the cells using a cell scraper or pipetting into a 1.5 ml tube and centrifuge (800 rpm, 22°C, 4 minutes).
8. Aspirate the supernatant and add 1 ml of aggregation medium.
9. Mechanically dissociate the cells by gently pipetting (about 15 times) with P1000 and measure the cell concentration.
10. Prepare a cell suspension of 1.0x104 cells/ml (in aggregation medium).
11. Add 50 µl of this cell suspension per well into a 96 well U-bottom low attachment plate (500 cells/well)* using an electronic pipette or mechanical 12-channel pipette.
12. Centrifuge the plate (800 rpm, 22°C, 2 minutes).
13. Place the plate in the cell incubator at 37°C for 24 hours.
14. The next day (day 1), add 150 µl per well of AK02N-C medium.
15. The following day (day 2) aspirate delicately 150 µl of the culture medium per well (using a 12-channel pipettes) and replace with 150 µl of fresh AK02N-C medium.
* The initial number of aggregated cells is critical to obtain proper axial elongation and subsequent segmentation and somite formation; carefully homogenize your cell suspension and check for high survival rate of dissociated cells prior to aggregation. Initial cell number to be used might vary and needs to be optimized depending on the used cell line. We usually use 500 cells/well but slightly higher or lower cell numbers will work depending on utilized cell line.
c) Matrigel embedding of hiPSC-derived 3D mesodermal aggregates
72 hours after the initial WNT and FGF pulse, axioloids which have undergone initial symmetry breaking and have started to slightly elongate are embedded into AK02N-C supplemented with 10% Matrigel (MG). To stabilize forming segments and induce epithelialization of somites, retinoid signaling molecules have to be added, i.e. add retinol (10 µM), all trans-retinal (1 µM) or retinoic acid (100 nM) to the MG containing embedding medium*.
1. On ice, prepare embedding medium by mixing cold (4°C) AK02N-C with 10% cold (4°C) Matrigel**.
2. Add 80 µl of 1% BSA in PBS into each well of a 96 well flat-bottom suspension culture plate in order to quench possible adhesion of axioloids to the walls/bottom of the well.
3. Aspirate the BSA solution, place the plate on ice and add 80 µl of embedding medium into each well with an electronic pipette.
4. Carefully transfer*** each axioloid into a well of a 96 well flat-bottom suspension culture plate containing embedding medium. Using a P2 pipette aspirate each axioloid one by one without damaging them and in the smallest volume possible, e.g. 1-1.5 µl****.
5. After 24 or 48 hours, embedded axioloids can be fixed and processed for further staining or differentiation.
* Other molecules, e.g. cytokines, small molecule agonists or inhibitors can also be added directly into the embedding medium.
** Matrigel concentration can be lowered to 5% without any apparent effect on axioloid induction efficiency or morphogenesis of axioloids. Other Matrigel-equivalent ECM compounds or mixtures can be also used but need to be tested and titrated accordingly.
*** Mesodermal aggregates/axioloids are transferred manually; to be able to see small aggregates place your 96 well in front of a white background and tilt it slightly or place an LED light (avoid heat) behind the plate and tilt it to see the aggregates.
****Axioloids to be embedded into Matrigel should be individually selected based on their general morphology. Axioloids with cylindrical elongation features are ideal, nevertheless the more asymmetrical ones can be also chosen if their NMP containing tailbud region (BRA+/SOX2+/MEOX1-) is still large. In general, the tailbud end should always be the same size or bigger than the anterior (MEOX1+) “head part” (see Figures 2 and 3). If you embed inappropriate or suboptimal “starting points” into Matrigel +/- retinoids, axioloids will not elongate or epithelialize properly (Figure 4). Optimal “starting shapes” might differ between utilized PSC lines and need to be determined for optimal outcomes.
C. Analysis of axioloids
a) Live imaging and luminometric analysis of axioloids
Using HES7-Luciferase reporter iPSC line (201B7 Luc)-derived axioloids we can visualize the oscillatory expression of the segmentation clock associated gene HES7. To this end axioloids are embedded into MG-containing medium (+/- retinoids) supplemented with 100 µM of luciferin in specific culture vessels. For Kronos HT luminometer measurements 24 well cell imaging plates are used and for IX-83-based bioluminescent imaging 96 well EZVIEW glass bottom plates coated with 1.5% of PVA are used. Other live imaging experiments are performed in standard cell suspension culture plates used for standard axioloid embedding. Further details on various molecular and functional assessment methods and analytical tools applied for characterization of axioloids can be found in our accompanying manuscript.
b) Collection and fixation of axioloids
- Prepare a wash buffer (BSA 0.1%, EDTA 0.25 mM in PBS).
- Coat any tips or tubes with the wash buffer prior to use.
- Collect axioloids in a pretreated tube.
- Wash 3 times with wash buffer; between each washing step let the aggregates sink to the bottom of the tube.
- Aspirate carefully the supernatant and proceed with fixation process.
Fixation for immunofluorescence (IF):
- Fix with 4% PFA for 30 minutes at RT.
- Remove PFA and wash 3 times with the wash buffer for 5 minutes each.
- Store in PBS at 4°C until further processing.
Fixation for in situ hybridization chain reaction (HCR):
- Fix in 4% PFA 1 hour at RT.
- Wash 3 times with PBST for 5 minutes each.
- Add 1 mL of 100% MeOH per tube.
- Store at -30°C until further processing.