Generating Blood Platelets from iPSCs without Using Serum or a Mouse Feeder Cell Line

Step 1

1. Reprogrammed human iPSC cell lines are cultured on a Matrigel –coated surface with mTeSR1 medium
2. Confluent iPSCs are dissociated using cell dissociation buffer (CDB) and resuspended in fresh mTeSR1 medium + 10 µM Y27632
3. Cells are seeded on human collagen IV-coated plates (5 µg/cm2)
4. Incubate at 37°C for 24hrs at 5% CO₂ / 20% O₂
5. After 24hrs change media to an animal –component free media for human hematopoietic expansion + 50 ng/ml of human VEGF, BMP4 and FGF Basic
6. Culture 4 days under hypoxic conditions - 5% CO₂ / 5% O₂, then 2 days at 5% CO₂ / 20% O₂
   50 – 60% of attached cells should be CD31+ indicative of differentiated hemogenic endothelial-like cells

Step 2

1. Change media to an animal-component free media for differentiating human ESCs and iPSCs + 25 ng/ml of  human TPO, human SCF, human Flt-3 ligand, 10 ng/ml of IL-3 and IL-6, and 5 U/ml of heparin
2. Culture 1- 5 days
3. The majority of floating cell should be Megakaryotype Progenitor Cells  (MKPs) with the following surface marker expression: CD31+, CD34+, CD43+, CD41a+, CD13+, CD14-, CD42+/-
4. Collect and seed in new plates for megakaryocyte maturation and platelet generation

Step 3

1. MKPs should be cultured in ultra-low attachment plates with animal component free media for hematopoietic expansion + TPO, SCF, IL-6, IL-9 and heparin
2. Cells should be cultured for 4 – 9 days, at 7% CO₂ and 39°C, using fresh media each day for at least the first 4 days. For the first 3 days the media should include 5µM Y27632
3. Once proplatelet morphology has occurred (day 6-9) platelets should be collected and analyzed for CD41a+/CD42b+ purity

Step 4: Blood platelet collection

1. Large megakaryocytes should be removed first by centrifuging at low speed 50 x g for 10 minutes
2. Next remove small megakaryocytes and proplatelets by centrifuging 300 x g for 10 minutes
3. iPSC-generated platelets can then be collected by centrifuging at 1,000 – 2,500 x g for 10 minutes in the presence of 1µM PGE1
4. iPSC pellets should then be purified using a BSA gradient centrifugation

Protocol adapted from: