In these studies we have differentiated human embryonic stem cells towards hematopoietic lineages using our ‘spin EB’ protocol in a serum-free medium 
. Compared with previously published protocols 
, we find that the forced aggregation of hESCs generated EBs more uniform in size that differentiated with greater efficiency and synchrony, resulting in the formation of blood cells in over 90% of EBs 
. The culture medium was supplemented with growth factors that induced and patterned mesoderm to a hematopoietic fate for the first 10 days, followed by a cytokine cocktail designed to bias further differentiation towards the Mk lineage. After 13 and 20 days of differentiation, characterisation of cell populations isolated on the basis of their expression of CD34, CD41 and CD45 on the cell surface enabled us to correlate immunophenotype, Mk clonogenic activity and hematopoietic gene expression. At both time points, we found that Mk colonies were confined to cells that expressed either or both CD41 and CD34, with the highest frequency observed in cells that co expressed both surface markers. There was evidence of ongoing maturation of Mk in the collagen based clonal assays, with polyploidization and shedding of platelet-like particles. The platelet-like particles generated in these cultures displayed characteristics similar to platelets derived from cord blood platelets, including responsiveness to ADP.
CD41 (GpIIb) is expressed on a range of hematopoietic precursors during early hematopoietic development in the embryo, from ES cells and in the adult 
. However, in the cytokine milieu present in our studies, after 13 days of differentiation, most of the clonogenic cells in the CD41+
fractions formed Mks. These observations are reminiscent of the findings in adult mouse and human bone marrow and cytokine-mobilized peripheral blood stem cells, in which Mk precursors were most enriched in CD41+
. However, others have suggested that it was CD41−
cells from both cytokine-mobilized peripheral blood and umbilical cord blood that contained the most immature Mk progenitors with the greatest proliferative potential 
. Follow up studies by the same authors examined the engraftment potential of TPO-expanded cord blood cells and demonstrated that the most rapid emergence of human platelets in the peripheral blood of immunocompromised mice followed the transplantation of CD34−
cells, rather than the transplantation of cells expressing identifiable Mk surface markers 
. It is interesting to note that the phenotypic development of Mk from cytokine-mobilized peripheral blood, in which Mk transiently co-expressed CD34 and CD41, differed from Mk development from umbilical cord blood, in which Mk progenitors transiently lost CD34 expression before acquiring CD41 
. In our experiments we also observed a tendency for larger Mk colonies to be formed by d13 CD41−
cells and d20 CD41lo/−
cells (). The fact that we observed Mk progenitors in CD41+
populations and no clonogenic activity in CD41−
cells suggests that our differentiating hESC cultures were more similar in their behavior to cytokine-mobilized peripheral blood.
Generation of Mks and their precursors from hESCs has been demonstrated in several studies in which co-culture of differentiating hESC with OP9 mouse stromal cells was a common feature. Erythroid-megakaryocyte progenitors were first identified in a CD43+
subset of cells after 6 days of differentiation 
. In a later study from another laboratory, a similar erythroid-megakaryocytic precursor present from day 8 of hESC differentiation that co-expressed CD235, CD41a, CD43 and CD34, could be cultured to generate CD235−
Mk progenitors and CD235+
erythroid progenitors 
. Megakaryocytes co-expressing CD41a and CD42 were observed after a 2 week differentiation on OP9 stromal cells in serum containing medium supplemented with TPO, although the phenotype of the progenitor population was not determined in this study 
. In these cultures, even though a high proportion of Mk were polyploid, proplatelet formation was infrequently observed 
Culturing hESC on C3H10T1/2 or OP9 feeder layers in serum containing medium supplemented with exogenous VEGF, Takayama and colleagues observed sac-like structures after two weeks that contained hematopoietic cells, approximately 13% of which co-expressed CD34 and CD41a 
. Further co-culture of the isolated hematopoietic cells on fresh feeder cells for 9 days led to ~50% cells co-expressing CD41a and CD42b and eventually the generation of platelet like particles in the culture supernatant. Double positive CD41a+
cells could be generated from all combinations of CD41a and CD34 expressing sac cells, although fewer arose from the CD34+
. Our observations that Mk colonies were confined to cells that expressed either or both CD41a and CD34 are consistent with these data.
It has been recognized that platelets derived from hiPSCs could be a useful source of patient matched platelets, circumventing the loss of responsiveness to platelet transfusions associated with immunorejection of non-autologous platelets. To explore this possibility, Takayama and colleagues generated Mk from hiPSCs via hematopoietic sacs, using the co-culture protocol they reported previously 
. They serendipitously observed that reactivation of cMYC
, one of the reprogramming genes introduced during iPSC generation, was associated with enhanced generation of Mk progenitors but that persistent cMYC
expression reduced Mk differentiation efficiency 
. They complemented these observations by demonstrating that enhanced platelet production was achieved by transient elevation, followed by reduction, of cMYC
expression. Functional studies of iPSC-generated platelets showed that they responded to ADP activation by binding to PAC-1, spreading on fibrinogen and that transfused platelets adhered to thrombus in vivo. However, this form of platelet production might not be available to most patients needing platelets.
Lu and colleagues cultured differentiated hESCs in methylcellulose to form hematopoietic blast colonies and directed their further differentiation towards the Mk lineage by culturing in medium supplemented with TPO, SCF and IL-11 
. However, the progenitor population that developed into CD41a+
Mks after a few days was not identified. Interestingly, only ~5% of the hESC-derived platelets generated under these feeder- and serum-free conditions co-expressed CD41a and CD42b. Co-culture of Mk with OP9 stromal cells in medium supplemented with TPO, SCF and sodium heparin increased the proportion of CD41a+
cells to ~40%. These hESC-derived Mks produced platelets that were activated by thrombin, spread on fibrinogen- and vWF-coated surfaces and formed fibrin clots.
In conclusion, our study has demonstrated that CD34+ and CD41+ cells differentiated from hESCs in serum-free, feeder cell-free culture, generated clonogenic Mk progenitors in response to thrombopoietic combinations of cytokines. This work complements previous studies that demonstrated the feasibility of generating functional platelets from human pluripotent cells, and represents a further step towards the generation of human platelets under defined conditions for therapeutic use in the future.