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
  1. Rinse each well to be used with 2 mL of DMEM/F-12 and aspirate to remove
  2. Add 0.5 ml per well of EB formation medium or AggreWell Medium (supplemented with Y-27632) to each well of the AggreWell plate
  3. 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
  4. Dissociate cells in the desired well using TrypLE and resuspend in 1-1.5ml of AggreWell Medium (supplemented with Y-27632)
  5. Without removing the EB formation medium added previously, add the appropriate volume of undifferentiated cells from above step
  6. After adding cells, immediately pipette gently several times to distribute the cells evenly throughout the well
  7. Centrifuge the AggreWell plate at 100 x g for 3 minutes to capture the cells in the microwells
  8. Examine the AggreWell plate under a microscope to verify that cells are evenly distributed among the microwells
  9. 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
  10. After 24 hours, EBs should be visible inside the wells and ready to harvest
  11. Gently pipette and transfer everything in one well of aggrewell plate onto one well of 6-well low attachment plate
  12. change media every 1-2 days and transfer onto new well when too many single cells/debris floating in the solution
  13. 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)
  14. use KSR media to mature EBs for total of 6-8 days, then passage/transfer the EBs onto matrigel plate
  15. 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
  16. culture and differentiate the cells with Fibroblast media until confluent, change media every other day
  17. 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]

  1. EB generation and maturation (P0)
  2. passage EB onto matrigel plate to start fibroblast differentiation with media 106+LSGS, usually done at 1:1 ratio (P1)
  3. passage confluent cells onto matrigel or gelatin plates depends on confluency/condition, can be passage at 1:2 or 1:3 ratio (P2)
  4. cells can be passaged onto gelatin plates only and some ES-like colonies might still present, continue the differentiation/maturation until confluent (P3)
  5. At P4 and onward, higher % of differentiated cells express both CD44 and CD13 markers, which CD13 is detected in mature fibroblast only
  6. 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]

  1. passage differentiated fibroblast to desired cell #: 10^4 for 24-well plates and 10^5 for 6-well plates
  2. 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)
  3. 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)
  4. 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
  5. passage reprogrammed (or partially reprogrammed) cells with Relesr (essentially EDTA/PBS solution that digest ESC/iPSC into small aggregate but not differentiated cells)