TITLE: Reprogramming fibroblasts and mononuclear blood cells into iPSCs using Sendai virus
AUTHORS: Dmitry Ovchinnikov

[DESCRIPTION]
Reprogramming primary blood mononuclear suspension cells into induced pluripotent stem cells using a non-integrating RNA viral (Sendai) reprogramming factor delivery method. This ™ CytoTune-iPS 2.0 is a self-replicating RNA virus-based reprogramming approach which utilizes 3 pre-packaged viral particles  expressing human KLF-4+OCT4/POU5F1+SOX2, hKLF-4 and hc-Myc proteins respectively. Use of Sendai viral particles for delivery allows for an efficient non-viral reprogramming of suspension blood and a wide range of other cells into induced pluripotent stem cells (iPSCs). This protocol describes the preparation of primary cell (blood-derived) cultures and their subsequent reprogramming into iPSCs using Sendai virus transduction.

[STEPS]
SECTION: Preparation of Reagents and Materials
1. 
 
Fibroblast culture medium:

The following example is for preparing 50 mL of Fibroblast Culture Medium. If preparing other volumes, adjust accordingly.
Combine the following:
45 mL of DMEM (+Glutamate) base medium
5 mL of fetal bovine serum (FBS), ES-qualified
NEAA 1x (Gibco or like)
Optional:Pen/Strep

Pre-warm to room temperature (20 °C - 25 °C) before use. Store Fibroblast Culture Medium at 2 °C - 8 °C for up to 4 weeks.


SECTION: Preparation of Reagents and Materials
2. Reprogramming using Cyto Tune Sendai vectors:

Count the cells using the desired method (e.g., Countess™II Automated Cell Counter), and calculate the volume of each virus needed to reach the target MOI using the live cell count and the titer information on the CoA.

  

 

SECTION: Preparation of Reagents and Materials
3. (optional) ReproTeSR™ medium for maintenance of adherent cells undergoing reprogramming:

Preparing 500 mL of mTeSR™. If preparing other volumes, adjust accordingly.
Combine the following:
100 mL  5x mTeSR™ supplement (stored ≥-20°C and thawed overnight in TC fridge).
400 mL mTeSR™ for blood reprogramming base medium.

Pre-warm to room temperature (23 °C – 25 °C, not 37 °C) before use.

SECTION: Reprogramming Process notes
4. Transduce somatic cells with the Sendai virus from the ™ CytoTune™-iPS 2.0   kit at day 0, and culture in FIBROBLAST Medium.
SECTION: Reprogramming Process notes
5. After Day 6, culture the cells in mTeSR™ for the remainder of the reprogramming induction phase until iPS cell colonies emerge.
SECTION: Reprogramming Process notes
6. 
 
Day - 2: Replate fibroblasts on TCP or Matrigel-coated TCP surface.
SECTION: Reprogramming Process notes
7. Day – 1: Change with a fresh fibroblast medium.
SECTION: Reprogramming Process notes
8. Day 0: Transduce the cells using the ™ CytoTune™ 2.0 Sendai reprogramming vectors at the appropriate MOI. Incubate the cells  . 

SECTION: Reprogramming Process notes
9. Day 1: Replace the medium with fresh complete fibroblast medium to remove the ™ CytoTune™ 2.0 Sendai reprogramming vectors.
 

SECTION: Reprogramming Process notes
10. Day 4: Start transitioning into an mTeSR™ family medium by replacing half of the fibroblast medium with mTeSR™ Medium.
SECTION: Reprogramming Process notes
11. Day 5: Replace the entire medium with mTeSR™ Medium to conclude the transitioning, and continue culturing cells on rhLamini-521 coated culture dishes.
SECTION: Reprogramming Process notes
12. Day 6–21: Replace spent medium with fresh mTeSR™ Medium every day and monitor the culture vessels for the emergence of iPSC colonies. When iPSC colonies are ready for transfer, perform live staining, and pick and transfer undifferentiated iPSCs onto fresh vitronectin-coated culture dishes for expansion.
SECTION: Reprogramming Process notes
13. Isolate single cell-derived iPS colonies and maintain by manual passaging into mTeSR1 or mTeSRplus media as per the manual passaging SOP.
SECTION: Points of critical importance/focus
14. 
 

SECTION: Potential hazards
15.