Generating Large Numbers of Human Megakaryocytes from Pluripotent Stem Cells

Abstract The process of hemostasis and blood coagulation relies heavily on a sufficient supply of platelets (PLTs, also known as thrombocytes) within a person's bloodstream. Platelet transfusion is an effective treatment for thrombocytopenia-related diseases, yet paucity of supply and limited shelf-life (5 - 7 days) remain challenging. PLTs are generated by the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) into megakaryocytes (MKs), a rare subset of large polyploid bone marrow cells. Methods to produce MKs in vitro by inducing mesodermal specification and hematopoietic differentiation of human pluripotent stem cells (hPSCs) could provide a reliable and safe supply of PLTs for transfusion and would also be amenable to gene editing for generation of HLA-null universal PLTs. Culture methods to generate hPSC-derived MKs and PLTs have been described, yet published protocols lack standardization, are PSC line dependent and/or promote differentiation of other lineages, resulting in low MK cell yields and purity. The use of feeder cells and/or viral vectors also limits the clinical and scale-up applicability. Here, we describe an efficient feeder cell-free and serum-free culture system that promotes the selective differentiation of hPSCs from multiple ES and iPS lines into polyploid MKs with high purity and yields and ability to generate platelets. The 17-day protocol includes two stages: a 12-day stage to differentiate hPSCs into megakaryocytic-biased HSPCs through endothelial-to-hematopoietic transition (H-phase), and a 5-day stage to further differentiate HSPCs into mature MKs (MK-phase). at the start of the H-phase, hPSC aggregates were plated in mTeSR TM media on Matrigel ®-coated plates at 16 aggregates (100 - 200 µm in diameter, ~100 cells per aggregate) per cm 2 to allow attachment overnight (Day -1). The cells were then cultured in mesoderm-induction medium for 3 days (Day 0 - 3), and subsequently in hematopoietic specification medium for 9 days (Day 3 - 12). During this phase, PSC-derived HSPCs emerged from an adherent layer of endothelial cells and were released into suspension. On day 12 these nonadherent cells were harvested and seeded at 1 - 3.5 × 10 5 cells/mL in MK maturation medium containing thrombopoietin (TPO) and other hematopoietic growth factors and cultured for 5 additional days (MK-phase, Day 12 - 17). At the end of H-phase (day 12) and MK-phase (day 17) the cells were counted and assessed for HSPC markers (CD34/CD45), the erythroid marker glycophorin A (GlyA), MK markers (CD41a/CD42b), DNA ploidy profile and PLT production by flow cytometry and immunofluorescence microscopy. Two embryonic stem (ES) cell lines (H1 and H9), and two induced pluripotent stem (iPS) cell lines (WLS-1C and STiPS-R038) were used in this study. At the end of H-phase (Day 12), on average 48% (range: 34 - 72%) of cells released into suspension co-expressed CD41a and CD42b markers, with an average yield of 93 CD41a +CD42b + cells per seeded hPSC (range: 30 - 200, n = 4 for H9/1C, n = 3 for H1/R038). The cells also expressed CD34 (average of 78% CD34 + cells) and GlyA (average of 71% GlyA + cells), indicating that the H-phase may support differentiation of PSCs to megakaryocyte-erythroid progenitors. At the end of MK-phase (Day 17), on average 82% of the cells expressed CD41a (range: 70 - 99%), 62% of the cells co-expressed CD41a and CD42b (range: 40 - 85%), and an average of 253 CD41a +CD42b + cells were generated per seeded hPSC (range: 70 - 700 MKs, n = 11 for H1/H9/R038, n = 7 for 1C). Of note, less than 5% of cells expressed GlyA, showing that the culture system is specific for megakaryocytic differentiation. The DNA ploidy profile of the CD41a +CD42b + cells generated on Day 17 showed that on average 26% and 9% of cells had 4N and 8N+ DNA ploidy, respectively (n = 11). Multinucleated MKs could also be readily observed by immunofluorescence microscopy. These PSC-derived MKs produced an average of 3.5 PLTs (range: 1 - 10 PLTs, n = 11) based on viable CD41a +CD45 -GlyA - PLT-like particles with a similar size and CD41 expression as control PLTs prepared from fresh blood. In conclusion, we have developed a simple two-step, yet robust serum- and feeder-free culture system for generating high numbers of hPSC-MKs that are large, polyploid, and capable of shedding PLTs. This culture method provides a platform to study thrombopoiesis and is amenable to scale-up method development. Disclosures No relevant conflicts of interest to declare.

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