Marie Csete MD, PhD
More than 1.5 million allogeneic platelet products in the United States and about twice as many in Europe are administered each year . About 3 percent of platelet infusions are complicated by allergic reactions ranging from mild to severe . Alloimmunization to platelets as well as nonimmune reactions can result in refractoriness to further platelet transfusions . In practice, human leukocyte antigen, or HLA, matching is not routinely performed before platelet transfusions, and HLA matching for platelet refractory patients may not be the complete answer to refractoriness . With AB (universal platelet) donors representing only about 4 percent of the population, a source of platelets that do not elicit an immune response is certainly an unmet medical need.
An obvious source of manufactured platelets is the hematopoietic stem cell, or HSC, but HSCs are difficult to expand in vitro, limiting the necessary first step in generating large numbers of platelets for transfusion. As an early proof of concept model, successful generation of megakaryocyte progenitors, or MPs, from human cord blood cultures was recently reported as part of a phase I clinical trial in China . Cord blood mononuclear cells were expanded in suspension cultures in medium supplemented with thrombopoietin, interleukins-3 and -6, and stem cell factor, resulting in a median expansion of 73.5-fold. CD41 was used to identify MPs. Twenty-four patients received a median dose of 5.45 million CD41+ cells (and 14.8 million mononuclear cells) without apparent serious adverse events. The study was not designed or reported in a way that efficacy could be easily evaluated, but demonstrates the possibility that clinical-grade expansion of cord blood hematopoietic progenitors to relatively large numbers of MP is possible. Cost of manufacture also was not reported.
Pluripotent stem cells, both embryonic stem cells, or hESC, and induced pluripotent stem cells, or iPSC, are objects of study as sources for clinical-grade platelets. Beau Mitchell’s lab at the New York Blood Center has taken a systematic approach based on the step-wise normal differentiation of platelets, addressing the technical challenges distinct to each step (summarized in ). In vivo HSCs first generate a megakaryocyte-erythroid bipotential precursor before commitment to the megakaryocyte lineage. Mature megakaryocytes are polyploid, and the overall level of ploidy is important to platelet production. Mitchell’s group showed that rho kinase inhibition was useful in increasing polyploidy at this stage of differentiation .
A more complicated approach and one with a more difficult regulatory pathway is generation of megakaryocyte cell lines from pluripotent stem cells. Koji Eto’s laboratory, which has been very active in this space, developed megakaryocyte lines using expression of c-MYC fused to a destabilization domain that could be blocked by a small molecule and overexpression of BMI1, using a human embryonic stem cell source . They generated an immortalized megakaryocyte line which could undergo sustained expansion. An important feature of this work is verification of platelet function in animal models after differentiation from the megakaryocyte line .
Another approach to producing platelets from hESC involves co-culture of hESC on either OP-9 or C3H10T1/2 (mesenchymal cell line) in the presence of vascular endothelial growth factor. Under these conditions, the hESC undergo morphologic changes into sac-like structures that provide a conducive environment for hematopoiesis and, with addition of thrombopoeitin, platelet differentiation . These results were reported in 2008 and used as part of optimized methods published more recently .
Since the introduction of reprogramming technologies to generate pluripotent stem cells, several groups have taken developmental shortcuts to find critical factors that allow direct reprogramming to a differentiated cell type without the need to go back in developmental time to the fully pluripotent state. Not surprisingly, direct reprogramming to megakaryocytes also has been reported using three factors — p45NF-E2, Maf G, and Maf K — identified in a differentiation screen . Direct reprogramming into megakaryocytes is faster than first reprogramming to an iPSC state followed by megakaryocyte differentiation, but platelet production from the directly reprogrammed cells is quite inefficient .
In later stages of platelet differentiation, extensions of the megakaryocyte cytoplasmic processes undergo morphologic transformation into proplatelets and branching of these structures is important for generation of non-nucleated platelets. Throughout this differentiation process, the interactions of platelet lineage cells with specific extracellular matrix proteins in precise niches, and shear stress in sinusoidal vessels are critical signals for proper platelet differentiation. Platelet release requires regulated shear stress, so as a consequence engineering adjuncts to traditional tissue culture methods have been critical in increasing efficiency of platelet production from pluripotent stem cells. Bioreactors designed to help control adhesion of differentiating cells and extension of cytoplasmic processes through pores of defined diameter in an engineered scaffold under controlled shear stress have been critical to advancing ex vivo platelet production . Other labs have designed different bioreactor platforms to support this later stage of differentiation.
Overall, though cost estimates are not well-reported, platelet generation from stem cell sources appears to be more cost-efficient than red blood cell manufacturing from pluripotent stem cells. It seems likely that combinations of the various strategies used to push platelet differentiation through the various developmental steps will be needed for a cost-effective platelet product to reach clinical reality. Despite the relatively few numbers of labs working on this problem, the field has advanced to the stage when more clinical trials are likely in the next few years.
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