Differentiation of human induced-pluripotent stem cells into smooth muscle cells

Here we describe a protocol for the generation of SMCs from Hutchinson-Gilford Progeria Syndrome (HGPS)- induced pluripotent stem cells (iPSCs) and wild type iPSCs to study their vulnerability. The protocol below has been developed to differentiate iPSCs into SMCs. It comprises 3 steps. First, iPSCs are cultured for 10 days as embryoid bodies (EBs) in suspension, followed by the isolation of CD34 + cells. Cells are then cultured for 4 passages using endothelial growth medium-2 (EGM-2) containing PDGF BB (induction medium) followed by their culture for additional 4 passages in Smooth Muscle Growth Medium-2 (SmGM-2) (maturation medium), for additional 4 passages.


Introduction
The protocol below has been developed to differentiate iPSCs into SMCs. It comprises 3 steps. First, iPSCs are cultured for 10 days as embryoid bodies (EBs) in suspension, followed by the isolation of CD34 + cells. Cells are then cultured for 4 passages using endothelial growth medium-2 (EGM-2) containing PDGF BB    806552; Sigma-Aldrich). The solution must be freshly prepared before medium preparation. βmercaptoethanol is toxic. Avoid inhalation, ingestion, and skin contact.
Maintenance of iPSCs 1. Defrost cryopreserved vials containing mitomycin-inactivated CF-1 MEFs in a 37°C water bath and transfer the cells to a 15 mL conical tube with 4 mL of DMEM medium (for MEFs). Centrifuge the cells 3 min at 300 × g, room temperature, and carefully aspirate the supernatant. Resuspend the pellet in DMEM medium (for MEFs) and plate the cells at a density between 7 × 10 4 /cm 2 and 8 × 10 4 /cm 2 into a 100 mm diameter Petri dish coated with gelatin (0.1%). Incubate the cells for at least 24 h before plating the iPSCs.
2. Defrost cryopreserved vials containing iPSC in a 37°C water bath and immediately transfer the cells to a 15 mL conical tube containing 4 mL of iPSC medium. Centrifuge the cells 3 min at 200 × g, room temperature, and carefully aspirate the supernatant. Resuspend the pellet in iPSC medium supplemented with 10 ng/mL β-FGF and plate the cells into the previously prepared mitomycin-inactivated CF-1 MEFs.
Approximately 10 ml of medium should be used per plate.
4. Upon reaching 80% con uency, aspirate the medium, add 4 mL of 1 mg/mL collagenase type IV solution to the petri dish, and incubate for 15 min at 37°C or until the boundaries of the colonies start to detach.
5. Aspirate the collagenase type IV solution and add 4 mL of iPSC medium supplemented with 10 ng/mL β-FGF per petri dish. Scrape the cells with a 5 mL sterile plastic pipette, transfer the contents of the petri dishes to 50 mL conical tube, and dilute 4 times in iPSC medium. Wash each petri dish with 4 mL of medium and transfer to the same 50 mL conical tube.
6. Centrifuge the iPSCs, 3 min at 200 × g, room temperature, and resuspend the pelleted colonies in iPSC medium at a ratio of 1:3 or 1:4.
7. Carefully aspirate the medium from Petri dishes containing recently prepared mitomycin-inactivated MEFs and add 5 mL of iPSC medium per petri dish. Next, add 5 mL per plate of the cell suspension containing the iPSC colonies, according to the passage ratio.
SMC differentiation of iPSCs 1. Culture iPSCs on 100 mm petri dishes and wait until they reach 80% con uence. Aspirate the medium and add 6 mL of 1 mg/ml type IV collagenase for 2 h at 37°C.
2. After 2 h, add 6 mL of iPSC medium using a 10 mL sterile pipette and pipette up and down a few times to detach cell aggregates. Pool the aggregates (approximately 2 petri dishes per 50 mL conical tube), centrifuge 3 min at 200 × g, room temperature, carefully aspirate and discard the supernatant, then add 6 ml of iPSC medium supplemented with 10 ng/mL β-FGF.
3. Transfer the undifferentiated iPSC aggregates, at a ratio of 2:1, to 100 mm low attachment plates (Corning) containing 10 mL of EB medium.
4. During 10 days, transfer the cell culture medium containing the EBs every 2 days to a 15-mL conical tube. Let EBs settle by gravity, discard the supernatant, resuspend the EBs in fresh differentiation medium (10 mL), and re-plate in the same low-attachment 100 mm petri dish.
Magnetic labeling of cells