Alice:Single cell tracing and RNA-seq Lab Notes/hiPSC secondary reprogramming
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hiPSC differentiation and secondary reprogramming[edit]
- the goal of this project is to understand single cell transcriptomic profile during secondary reprogramming
- EB generation is using Aggrewell 400 (8 well total with each well contains approximately 1,200 microwells) from Stem Cell Technologies
Fibroblast differentiation protocol[edit]
- Culture each 1 well of 6-well plate to 80-90% confluency, then generate EBs following manufacturer's protocol, briefly outlined below:
- Aliquot and pre-warm human EB formation medium or AggreWell Medium to room temperature (15 - 25°C)
- Supplement EB formation medium with Y-27632 (Dihydrochloride) to a final concentration of 10 μM
- Pre-warm DMEM/F-12 to room temperature (15 - 25°C)
- the amount of cells needed per well and total EB that will be generate is outlined below:
DESIRED NUMBER OF CELLS PER EB |
REQUIRED NUMBER OF CELLS PER WELL
|
50 |
6 x 10^4 cells
|
|
|
100 |
1.2 x 10^5 cells
|
|
|
200 |
2.4 x 10^5 cells
|
|
|
500 |
6 x 10^5 cells
|
|
|
1,000 |
1.2 x 10^6 cells
|
|
|
2,000 |
2.4 x 10^6 cells
|
|
|
3,000 |
3.6 x 10^6 cells
|
- Rinse each well to be used with 2 mL of DMEM/F-12 and aspirate to remove
- Add 0.5 ml per well of EB formation medium or AggreWell Medium (supplemented with Y-27632) to each well of the AggreWell plate
- Centrifuge the AggreWell plate at 2000 x g for 5 minutes in a swinging bucket rotor that is fitted with a plate holder to remove any air bubbles from the microwells
- Dissociate cells in the desired well using TrypLE and resuspend in 1-1.5ml of AggreWell Medium (supplemented with Y-27632)
- Without removing the EB formation medium added previously, add the appropriate volume of undifferentiated cells from above step
- After adding cells, immediately pipette gently several times to distribute the cells evenly throughout the well
- Centrifuge the AggreWell plate at 100 x g for 3 minutes to capture the cells in the microwells
- Examine the AggreWell plate under a microscope to verify that cells are evenly distributed among the microwells
- Incubate the cells at 37°C with 5% CO2 for 24 hours, 48 hours is also fine but nutrients are more likely to be depleted if seeded with large amount of cells/well
- After 24 hours, EBs should be visible inside the wells and ready to harvest
- Gently pipette and transfer everything in one well of aggrewell plate onto one well of 6-well low attachment plate
- change media every 1-2 days and transfer onto new well when too many single cells/debris floating in the solution
- use Aggrewell media for day 1, then on day 2, change to 1/2 aggrewell and 1/2 KSR media (DMEM/F12, 20% KOSR, 1% glutamax, 1% NEAA, 0.1% B-me)
- use KSR media to mature EBs for total of 6-8 days, then passage/transfer the EBs onto matrigel plate
- culture the cells with KSR media on day 1, then switch to 1/2 KSR and 1/2 (media 106+ LSGS ) fibroblast media (Fisher Scientific) on day 2
- culture and differentiate the cells with Fibroblast media until confluent, change media every other day
- passage the confluent cells onto gelatin coated plates and continue to differentiate/enrich fibroblast cells until P5 or P6 (>90% differentiated, CD13+)
Fibroblast expansion[edit]
- EB generation and maturation (P0)
- passage EB onto matrigel plate to start fibroblast differentiation with media 106+LSGS, usually done at 1:1 ratio (P1)
- passage confluent cells onto matrigel or gelatin plates depends on confluency/condition, can be passage at 1:2 or 1:3 ratio (P2)
- cells can be passaged onto gelatin plates only and some ES-like colonies might still present, continue the differentiation/maturation until confluent (P3)
- At P4 and onward, higher % of differentiated cells express both CD44 and CD13 markers, which CD13 is detected in mature fibroblast only
- Also, cells can be passaged at much lower density such as 1:6 starting from P4, and should take less than 7 days to become confluent
Secondary reprogramming[edit]
- passage differentiated fibroblast to desired cell #: 10^4 for 24-well plates and 10^5 for 6-well plates
- roughly 2 days after passage, the cells should be at 40% confluency, change to reprogramming media (DMEM/F12, 10% KOSR, 10%FBS, 1% glutamax, 1% NEAA, 0.1% B-me)
- add DOX (final concentration: 2 ng/ml) and bFGF (final concentration: 10 ng/ml) to the media for 14-20 days (or however long depends on experimental setup)
- The cells will become very confluent (80-100%) by the end of reprogramming period, so it is better to passage the cells at this point
- passage reprogrammed (or partially reprogrammed) cells with Relesr (essentially EDTA/PBS solution that digest ESC/iPSC into small aggregate but not differentiated cells)