STEMdiff Megakaryocyte Kit

STEMCELL Technologies developed the STEMdiff™ Megakaryocyte Kit for the serum- and feeder-free differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into polyploid megakaryocytes that express CD41a and CD41b and are capable of shedding platelets. The 17-day monolayer protocol proceeds in two stages: a 12-day hematopoietic specification phase followed by a 5-day megakaryocyte maturation phase. Three media formulations are used sequentially—Medium A (days 0–3), Medium MK1 (days 3–12), and Medium MK2 (days 12–17)—prepared from the included Hematopoietic Basal Medium, Hematopoietic Supplement A, Megakaryocyte Supplement MK1, Megakaryocyte Supplement MK2, and StemSpan™ SFEM II.

On day -1, hES or hiPS cells maintained in mTeSR™1, mTeSR™ Plus, or TeSR™-AOF are plated as aggregates (100–200 µm diameter) at 10–20 aggregates/cm² on Corning® Matrigel®-coated plates. After overnight attachment, the protocol is initiated once 4–10 colonies/cm² are confirmed adhered. On day 12, harvested hematopoietic progenitor cells are replated in Medium MK2 at 1–3.5 × 10⁵ cells/mL for the final 5 days of culture.

By day 17, the average frequency of viable CD41a+CD42b+ cells ranges between 56% and 77%, and the average yield of CD41a+CD42b+ cells per seeded hPSC ranges between 223 and 425 (n > 12 across four ES/iPS lines; results are cell line-dependent). The resulting megakaryocytes display mature features including cellular enlargement and polyploidization, with average ploidy distributions on day 17 of 66% (2N), 24.5% (4N), and 9.5% (8N+). The kit also supports proplatelet formation, yielding an average of 3.2–5.1 platelet-like particles per megakaryocyte.