Tina:Cardiomyocyte differentiation in H9 ESCs (CDH9)
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- Purpose: To determine how Metallo Lab's modified cardiomyocyte differentiation protocol works on H9 ESCs.
- Cells: Brainbow H9 constructs
Experiment #1 (CDH9_E1_T1)[edit]
- Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
- Date: 4/10/2013 ~ 5/15/2013
- Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded on 4/10. (1:6 dilution from the second passage of H9 ESCs under a feeder free culture system) OP9 cells were used as a negative control.
- Protocol used: Christian Metallo Lab protocol.
- Two induction medium used: Gln+ induction medium,DMK induction medium
- Two cell density used: 100%, 80% confluency.
12-well plate #1
100% confluency | 1 | 2 | 3 | 4 |
A | Gln, repeat 1 | Gln, repeat 2 | DMK, repeat 1 | DMK, repeat 2 |
B | Gln, repeat 3 | Gln, repeat 4 | DMK, repeat 3 | DMK, repeat 4 |
C | Negative ctrl, repeat 1 | Negative ctrl, repeat 2 | Negative ctrl, repeat 3 | Negative ctrl, repeat 4 |
12-well plate #2
80% confluency | 1 | 2 | 3 | 4 |
A | Gln, repeat 1 | Gln, repeat 2 | DMK, repeat 1 | DMK, repeat 2 |
- Induction: 4/16 (Day 0). Cells reached 100% and 80% confluency, respectively. Aspirate mTeSR1 medium, wash once with PBS, add Gln+ and DMK induction medium accordingly. Pre-mix CHIR99021 (and DMK, for DMK group only) with culture media, then added to each well.
- 4/17 (Day 1). Change medium (pre-mix DMK). Observed lots of cell death. Might due to the extremely high cell density and the sudden withdrawal of serum in differentiation medium.
- 4/18 (Day 2). Still, lots of dead cells. Add 5 ul DMK to DMK group.
- 4/19 (Day 3). Shake wells before change media to remove dead cells. Most of them has been removed after that. Change to combined medium.
- 4/20~4/22. Change medium accordingly. Cells were still dying. But many survived also.
- 4/23 (Day 7). Change to Insulin+ medium. Start to change medium every 3 days from now on.
- 4/26 (Day 10). Observed one beating colony in 80% confluency, Gln+ group (Plate#2, A1). No signs of any other beating cells in other wells yet.
- Pictures:
Experiment #2 (CDH9_E1_T2)[edit]
- Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
- Date: 4/20/2013 ~ 5/20/2013
- Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded on 4/20. (1:3 dilution from the second passage of H9 ESCs under a feeder free culture system).
- Protocol used: Christian Metallo Lab protocol.
- Two induction medium used: Gln+ induction medium,DMK induction medium
- Two cell density used: 100%, 70% confluency.
24-well plate #1
1 | 2 | 3 | 4 | |
A | 100%, Gln, repeat 1 | 100%, Gln, repeat 2 | 100%, DMK, repeat 1 | 100%, DMK, repeat 2 |
B | 100%, Gln, repeat 3 | 100%, Gln, repeat 4 | 100%, DMK, repeat 3 | 100%, DMK, repeat 4 |
A | 70%, Gln, repeat 1 | 70%, Gln, repeat 2 | 70%, DMK, repeat 1 | 70%, DMK, repeat 2 |
B | 70%, Gln, repeat 3 | 70%, Gln, repeat 4 | 70%, DMK, repeat 3 | 70%, DMK, repeat 4 |
- Induction: 4/23 (Day 0). Cells reached 100% and 70% confluency, respectively. Start experiment. Mix CHIR99021 and DMK with medium before adding to each well.
- 4/24 (Day 1). Change medium (pre-mix DMK). Still, observed lots of cell round-up. May be died or detached. can't tell.
- 4/25~5/20. Change medium accordingly. Take pictures.
- Pictures:
- Perform flow analysis on 5/20. Results: Cell numbers were too low. The test was not successful. Flow analysis will be repeat next time.
Experiment #3 (CDH9_E1_T3)[edit]
- Study Purpose: Increase cell number to see if thing goes better.
- Date: 5/1/2013 ~ 5/28/2013
- Cells: H9 ESC line, parental cell (without brainbow construct in it). Cells seeded on 5/1. (1:6 dilution from feeder).
- Protocol used: Christian Metallo Lab protocol.
- Two induction medium used: Gln+ induction medium,DMK induction medium
- Cell density used: 100% confluency.
6-well plate
Well #1: 100% confluency, DMK induction medium, repeat 1
Well #2: 100% confluency, Gln+ induction medium, repeat 1
Well #3: 100% confluency, Gln+ induction medium, repeat 2
Well #4: 100% confluency, DMK induction medium, repeat 2
- Induction: 5/6 (Day 0). Cells reached 100% confluency. Start experiment. Add medium first and then add CHIR99021 and DMK directly into each well.
- 5/7 (Day 1). Change medium (pre-mix DMK). Still, observed lots of cell round-up. But much lesser than before. I can still see many cells attached to the dish this time.
- 5/8 ~ 5/28. Change medium accordingly. Take pictures.
- Cells differentiate extremely well in 6-well plate.
- Pictures:
- Movies on 5/27/2013 (Day 21):
- Flow analysis:
Experiment #4 (CDH9_E1_T4)[edit]
- Study Purpose: Collect spent media every 3 days to determine the Gln consumption level during differentiation.
- Date: 5/16/2013 ~ 6/11/2013
- Cells: H9 ESC line, parental cell (without brainbow construct in it). Cells seeded on 5/16. (1:6 dilution from feeder free).
- Protocol used: Christian Metallo Lab protocol.
- Two induction medium used: Gln+ induction medium,DMK induction medium
- Cell density used: 100% confluency.
12-well plate
100% confluency | 1 | 2 | 3 | 4 |
A | DMK, repeat 1 | DMK, repeat 2 | Gln, repeat 1 | Gln, repeat 2 |
B | DMK, repeat 3 | DMK, repeat 4 | Gln, repeat 3 | Gln, repeat 4 |
C | DMK, repeat 5 | DMK, repeat 6 | Gln, repeat 5 | Gln, repeat 6 |
- Induction: 5/20 (Day 0). Cells reached 100% confluency. Start experiment. Add medium first and then add CHIR99021 and DMK directly into each well.
- 5/21 ~ 6.11. Change medium accordingly. Take pictures. Collect spent media every 3 days after Day 7 (Day 7, 10, 13, 16, 19, 22).
- Pictures: