This result was not totally unexpected and is likely unique to hiPSC-initiated hematopoiesis in culture that only lasted for 20 days. two actions: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and growth of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo. genes in hiPSCs produces expandable immature MKs in a procedure that includes coculture with the mouse 10T1/2 stromal cells. Turning off the overexpression of these genes in the immature MKs results in the production of platelets [20]. However, it would be highly desirable if we could generate large numbers of MKs from hiPSCs in the absence of mouse stromal cells and without the need for manipulation of oncogene expression. A recent study demonstrated that this generation of MKs from HPCs is usually achievable under feeder-free conditions [18]. However, that study only examined hESCs and used animal-derived products such as bovine serum albumin (BSA) [18, 21]. These xenogeneic and undefined reagents often cause low reproducibility and conflict with the rigid requirements of clinical or preclinical applications [22, 23]. To search for a strong and efficient culture condition to generate expandable MK progenitors from hiPSCs, we developed a serum-free and feeder-free system of hiPSC differentiation to MKs with a high level of reproducibility. In this two-step differentiation system [21, 24C26] which first generates CD45+CD34+ definitive HPCs followed by MK differentiation, we were able to effectively generate a cell populace enriched for CD41+CD42a+ megakaryoblasts. Moreover, we also used Food and Drug Administration (FDA)-approved pharmacological agents to replace TPO and BSA in the culture medium, an important factor for future clinical applications. Forty-five hiPSC lines Olcegepant hydrochloride from 23 individuals were evaluated in our system, resulting in highly reproducible outcomes. Materials and Methods Maintenance and Growth of hiPSC Lines Human iPSC lines, BC1 and E2, derived from human adult BM hematopoietic cells and mesenchymal stem cells (MSCs), respectively [27C29], were adapted to feeder-free conditions using the E8 medium (Essential 8 medium commercialized by Life Technologies, Carlsbad, CA, http://www.lifetechnologies.com) [30]. The cells Olcegepant hydrochloride were maintained in an undifferentiated state and routinely passaged as small clumps using the EDTA method or as single cells after enzymatic digestion by Accutase (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com). To enhance single cell survival, 10 M ROCK inhibitor Y27632 (Stemgent, Cambridge, MA, http://www.stemgent.com) was added in the medium for the first 24 hours after seeding. Other hiPSC lines were derived from peripheral blood mononuclear cells (MNCs) using nonintegrating episomal vectors, as previously described [26C28, 31C33]. After establishment, they were all expanded in the Essential 8 medium on either Matrigel (1:30; BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) or vitronectin (5 g/cm2, Life Technologies). Generation of MKs and Platelets From hiPSCs Human iPSCs were differentiated into definitive CD34+CD45+ HPCs, using the spin-embryoid body (spin-EB) method in feeder- and serum-free conditions altered from previously described protocols [24, 26, 34]. Single hiPSCs were suspended in serum-free medium (SFM) (supplemental online Table 1). In brief, the SFM contained components that included insulin-transferrin-selenium or BSA. We added 10 M Y27632, 10 ng/ml recombinant human FGF-2 (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) and 10 ng/ml bone morphogenetic protein 4 (BMP4; R&D Systems). Cells (3,000) were seeded into nontreated round-bottom 96-well plates (Corning Costar, Acton, MA, http://www.corning.com/lifesciences) in 50 l per well and centrifuged at 300for 5 minutes. From day 2 to day 14, the cells were cultured in SFM containing Rabbit Polyclonal to Collagen III FGF-2 (10 ng/ml), BMP4 (10 ng/ml), stem cell factor (SCF) (50 ng/ml; R&D Systems), and vascular endothelial growth factor A (VEGF-A) (10 ng/ml; PeproTech, Rocky Hill, NJ, http://www.peprotech.com). TPO (20 ng/ml; PeproTech) or romiplostim (Nplate, Olcegepant hydrochloride 20 ng/ml; Amgen, Thousand Oaks, CA, http://www.amgen.com) was added to the medium at day 11. Nplate is usually a U.S. FDA-approved TPO analog [35]. On day 14, the suspended cells were harvested and then filtered through 100-m cell strainers (BD Biosciences) to remove the EBs. The harvested single cells were seeded into 6-well plates (1 106 cells per well) for MK culture in SFM made up of SCF (20 ng/ml), oprelvekin (IL-11, 10 ng/ml; Pfizer, New York, NY, http://www.pfizer.com), and TPO or Nplate (50 ng/ml) for 5 days. We also substituted BSA in the.