Materials and reagents

Biological materials

1. Stealth RNA vector (SRV)^TM iPSC vector; SRV iPS-2 vector (encoding OCT4, KLF4, SOX2, and C-MYC; TOKIWA-Bio Inc., Ibaraki, Japan, catalog number: S1011694A) based on the Sendai virus

2. SRV^TM iPSC vector; SRV iPS-4 vector (carrying OCT4, KLF4, SOX2, C-MYC, NANOG, and LIN28; TOKIWA-Bio Inc., catalog number: S1011696A)

3. Adipose-derived mesenchymal stem cells (Lonza Bioscience, Walkersville, MD, USA, catalog number: PT5006)

4. Human peripheral blood mononuclear cells (PBMCs) (e.g., derived from healthy donors or patients)

1. MesenPRO RS^TM medium (Thermo Fisher Scientific, catalog number: 12746012)

2. KBM 501 medium (Kohjin Bio, catalog number: 16025015)

3. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10270-106)

4. TrypLE Select (Thermo Fisher Scientific, catalog number: 12563-011)

5. Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190144)

6. UltraPure^TM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, catalog number: 15575020)

7. StemScale^TM PSC suspension medium (StemScale) (Thermo Fisher Scientific, catalog number: A4965001)

8. DAPT, gamma-secretase inhibitor (Abcam, catalog number: ab120633)

9. EPZ004777 (iDOT1L) (Tocris, catalog number: 5567)

10. Y27632 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 036-24023)

11. STEM-CELLBANKER^® GMP grade (ZENOAQ, catalog number: 11924)

12. RNeasy Mini kit (Qiagen, catalog number: 74904)

13. SuperScript IV VILO Master Mix (Thermo Fisher Scientific, catalog number: 11756050)

14. TaKaRa Ex Taq DNA Polymerase (Takara Bio Inc., catalog number: RR001B)

15. Primer pair for β-ACTIN and SRV (e.g., Sigma custom oligo; oligo sequence is below)

    1. β-ACTIN (131 bp)

       Forward: 5-TCCCTGGAGAAGAGCTACG-3

       Reverse: 5-GTAGTTTCGTGGATGCCACA-3

    2. SRV (500 bp)

       Forward: 5-ATATGGAGTACGAGAGGACC-3

       Reverse: 5-CCTCAGGTTGGAGAGAGTCA-3

16. Agarose gel (e.g., Nacalai Tesque Inc., catalog number: 01158-85)

17. Ethidium bromide (e.g., FUJIFILM Wako Pure Chemical Corporation, catalog number: 315-90051)

1. Cell dissociation solution (see Recipes)

2. PBMC medium (see Recipes)

3. Reprogramming medium (see Recipes)

1. Cell dissociation solution

   Reagent	Final concentration	Amount
   TrypLE Select	0.5×	25 mL
   DPBS	0.5×	25 mL
   EDTA	0.25 mM	25 µL
   Total		50 mL




2. PBMC medium

   Reagent	Final concentration	Amount
   KBM 501 medium	90%	45 mL
   FBS	10%	5 mL
   Total		50 mL




3. Reprogramming medium

   Reagent	Final concentration	Amount
   StemScale	n/a	100 mL
   DAPT	5 µM
   EPZ004777	3 µM
   Y27632	10 µM
   Total		100 mL

   n/a, not applicable

Laboratory supplies

1. Microtube 1.5 mL (e.g., Sumitomo Bakelite Co., Ltd., catalog number: MS-4265M)

2. 15 mL centrifuge tube (e.g., IWAKI, catalog number: 2324-015)

3. 30 mL disposable bioreactor (ABLE Biott, catalog number: ABBWVS03A-6)

4. 6-well dish (e.g., IWAKI, catalog number: 3810-006N)

5. 96-well U-bottom dish (e.g., Corning, catalog number: 7007)

6. PCR tube (e.g., Eppendorf, catalog number: 30124359)

7. Cryotube (e.g., Thermo Fisher Scientific, catalog number: 377267)

Equipment

1. CO₂ incubator (e.g., Thermo Fisher Scientific, catalog number: 3110, Forma^TM series II)

2. -80 °C freezer (e.g., Panasonic, catalog number: MDF-U33V-PJ)

3. Centrifuge (e.g., TOMY, catalog number: NIX521)

4. Bioreactor magnetic stir system base (Able Corp. & Biott Co., catalog number: ABBWBP03N0S-6)

5. Laser microscope (e.g., Keyence, catalog number: BZ-X810)

6. Thermal cycler (e.g., Thermo Fisher Scientific, catalog number: 4484073)

7. Cell counter (e.g., Beckman Coulter, Vi-CELL XR Cell Viability Analyzer System)

Procedure