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Challenges and promises for the development of donor-independent platelet transfusions

Michele P. Lambert, Spencer K. Sullivan, Rudy Fuentes, Deborah L. French and Mortimer Poncz

Abstract

Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo–generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo–generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo–generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo–derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.

Introduction

Platelets have important and well-characterized roles in hemostasis and thrombosis,1 the maintenance of vascular integrity,2 the development of the lymphatic system,3 and the innate immune response.4 Platelets also contribute to the pathophysiology of inflammation and tumor growth and metastasis.5 In both normal biology and disease, their actions are mediated primarily through their adhesive properties and the release of stored hemostatic factors, cytokines, and growth factors.1,3-5 Patients with bone marrow failure, hematologic malignancies, and inherited platelet disorders, and those undergoing chemotherapy or radiotherapy for cancer or stem cell transplantation, often require platelet transfusions.

The use of transfused platelets has increased significantly in the past decades, as exemplified by a 16.8% increase between 2006 and 2008.6 One of the biggest challenges to maintaining a supply of platelets is the dependence upon donors willing to undergo prolonged apheresis for the collection of single-donor platelet units. Recent data suggest that a near-static donor pool in many parts of the United States has not kept pace with demand.6 Compounding the supply issue are the variation in number and functionality of each unit and the differences in surface markers.7,8 Moreover, platelets must be stored at room temperature to maintain function,9 significantly increasing the risk of product bacterial contamination. Platelets become outdated and are discarded after 5 days of storage, creating significant waste (∼13% of apheresis units and ∼36% of whole blood–derived units).6 Infused platelet half-life is 1.5 to 3 days, and infusion of platelets with improved platelet half-life may decrease transfusion needs, although perhaps not as significantly in settings with high rates of platelet destruction, such as sepsis. Generation of an efficient, nondonor-dependent system to continuously provide platelets would be ideal. In this review, we examine the current status of a number of such approaches and identify key areas for continuing research in this rapidly developing field.

Megakaryopoiesis and thrombopoiesis

Hematopoietic stem cells differentiate into megakaryocytes, a rare cell type within the bone marrow nucleated cell population (0.14% in mice10). Megakaryocyte precursors arise in the periosteal bone niche, and then differentiate as they migrate into the perivascular niche in a process called megakaryopoiesis.11 Mature megakaryocytes are sizable, reaching >50 microns in diameter,12 and are polyploid, with a DNA content up to 128N.10 Mature megakaryocytes release proplatelets when they reach the perivascular space11 by a process called thrombopoiesis, shedding platelets, proplatelets, large cytoplasmic fragments, and whole cells into the circulation.13 Where and how the majority of final mature platelets are generated is not clear, but older observations suggest that megakaryocytes or cytoplasmic fragments may release final-sized platelets when entrapped in the pulmonary microvascular bed14,15 or mature in the circulation.16 In an adult human, megakaryopoiesis and thrombopoiesis are very efficient, resulting in the generation of approximately 1011 platelets per day with estimates of ∼103-4 platelets produced per human megakaryocyte.14 Most platelet transfusions involve from 3× 1011 to 4 × 1011 platelets to achieve a relevant platelet count increase in a thrombocytopenic adult.

The identification and purification of thrombopoietin (TPO), the major positive regulator of megakaryocyte proliferation and differentiation and a key cytokine for stem cell maintenance,17 has allowed the generation of ex vivo systems to study the complex processes leading to platelet formation. Further development of these ex vivo systems in the past several years has raised the possibility of deriving platelets for clinical application by ex vivo generation from cultured megakaryocytes or earlier precursor cells or by in vivo platelet release from infused megakaryocytes.

Candidate starting cells useful in generating ex vivo–derived megakaryocytes

Hematopoietic stem and progenitor cells

The first report of the generation of human megakaryocytes and platelets in culture was published by Choi et al in 1995.18 This study showed that megakaryocytes, larger proplatelets, and smaller, platelet-sized particles could be generated in vitro from CD34+ peripheral blood progenitor cells in the presence of TPO and other cytokines. Since that report, multiple investigators have demonstrated the generation of megakaryocytes and platelets in culture starting with CD34+ progenitor cells derived either from umbilical cord blood,19 fetal liver,20 peripheral blood,21 or bone marrow.22 However, the generation of megakaryocytes and platelets from any of these sources still requires a continuous supply of donors due to the limited expansion potential from these primary, nonimmortalized hematopoietic cells, and it does not obviate alloreactivity. Additionally, use of CD34+ progenitor cells adds significant cost (apheresis procedure with CD34 selection) over the cost of collection of an apheresis platelet unit.

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)

One alternative new strategy for the large-scale production of megakaryocytes and platelets would be to use an immortalized progenitor cell that, upon appropriate stimulation, differentiates into mature megakaryocytes and platelets. The discovery and characterization of hESCs23,24 and more recently of hiPSCs25 provide potential renewable and unlimited sources of cells that can be expanded in culture and differentiated into megakaryocytes, eliminating donor dependency. Starting hESCs or hiPSCs can be fully characterized for specific surface molecules such as HLA implicated in alloimmunization, allowing banking of HLA-identical (or HLA-deficient26) cells that could be used in the setting of alloimmunization or matched to prevent alloimmunization in individual patients. Moreover, in patients completely refractory to platelet transfusions on the basis of alloimmunization, establishing personalized induced pluripotent stem cells (iPSCs) from skin fibroblasts or blood leukocytes may someday be feasible.

In 2006, the first report of using hESCs to generate megakaryocytes was published.23 In that report, 23 CD41+CD42+ human megakaryocytelike cells were generated, which expressed von Willebrand factor, a ligand normally limited in expression to megakaryocytes and endothelial cells. In addition, these cells reached ploidy levels of up to 32N and were responsive to agonist stimulation as measured by fibrinogen binding.23 The authors23 were only able to generate <1 megakaryocyte per input hESC, and the culture technique required serum. In 2011, a system amendable to a larger-scale production of hESC-derived megakaryocytes was reported.27 Variations in expansion frequencies were observed between starting embryonic stem cell (ESC) lines, but up to ∼60 megakaryocytes per starting hESC were noted. Theoretically, if each ex vivo–generated megakaryocyte could then shed 103 platelets, an input of 5 × 106 hESCs would be required to generate a single platelet transfusion unit of 3 × 1011 platelets (Table 1). Platelets generated were studied in vivo in an immunodeficient murine xenotransfusion model and shown to incorporate into a developing thrombus following a laser-induced injury. However, passive incorporation into a growing thrombus was not ruled out, and comparative functional analysis of these ex vivo–generated platelets to normal platelets in vivo was not performed.27 More rigorous tests of functionality, such as agonist threshold in platelet aggregation studies, in vivo half-life, ability to achieve a meaningful increase in platelet count, or correction of a bleeding diathesis were not tested, likely because sufficient numbers of platelets were not generated to enable this testing.

Table 1

Calculations of required cells to generate a unit of platelets for transfusion

There is concern that any cellular product derived from pluripotent ESCs or iPSCs could be oncogenic or teratogenic.28 Because platelets are anucleate, and any platelet product could be irradiated before infusion, this theoretically lessens concerns related to human in vivo use of cells generated from hESCs or hiPSCs. However, particularly with regard to hESC-derived products, potential ethical concerns about the source of these cells remain and, with both, technical hurdles still exist.

Direct reprogramming

In 2012, investigators reported reprogramming megakaryocytes directly from adipocytes29 or fibroblasts30 without first generating iPSCs. Ono et al30 showed that NF-E2 transcriptional complex expression in fibroblasts is sufficient to drive megakaryocyte differentiation and platelet release ex vivo. These platelets were shown to be functional both in vitro and in vivo. While these cells potentially offer a less complicated and safer alternative to the generation of megakaryocytes from hESCs or hiPSCs, caution is appropriate, because transcription factor dysregulation can have pleiotropic and unpredictable effects on megakaryocyte and platelet biology, resulting, for instance, in macrothrombocytes with alterations in both surface receptors and intracellular signaling pathways.31

Self-renewing megakaryocyte progenitors

An alternative strategy to generate ex vivo–derived megakaryocytes is to develop an intermediate self-renewing progenitor cell line from ESCs or iPSCs that, upon appropriate stimulation, rapidly differentiates into megakaryocytes. Such a progenitor cell line might reduce the lag time in ex vivo–platelet production and simplify the process. A model32 for such a strategy has been described in the mouse. Starting with a GATA1-deficient mouse ESC line,32 investigators cultured the cells under differentiation conditions in the presence of TPO and noted rapid outgrowth of self-replicating progenitor cells, termed G1ME.33 With re-introduction of GATA1 expression into these proliferating cells, approximately half the cells differentiated into erythrocytes and the other half into megakaryocytes. G1ME cells provided a relatively homogeneous source of megakaryocytes that could potentially be used to generate platelets or serve as a model for studying platelet generation and release; however, this concept has not yet been tested in a parallel human system.

The challenges of large-scale platelet generation

There are 2 quantitative roadblocks to the development of ex vivo–generated, donor-independent platelets for therapeutic use: first, generating sufficient numbers of megakaryocytes from a reasonable number of starting cells, and second, generating sufficient numbers of viable and functional platelets per megakaryocyte. Starting with hematopoietic progenitor CD34+ cells, investigators have been able to generate between 100 and 102 megakaryocytes per input cell (Table 1). The yield of ex vivo–generated platelets per megakaryocyte is low, although actual numbers per megakaryocyte have not been reported. In part, this may be due to the fact that a significant portion of the platelets shed in culture may actually be larger fragments of cytoplasm (proplatelets) potentially able to eventually generate multiple platelets, and in part this may be due to the nonsynchronous release of platelets over several days. While plateletlike structures generated to date using ex vivo–culture techniques resemble peripheral blood platelets in terms of structure by electron microscopy34 and can participate in clotting in vivo,27 no one has yet harvested sufficient numbers of platelets to perform traditional aggregometry tests to assess classical platelet function, which requires ∼108 platelets per study. Nor have there been reports of sufficient numbers of ex vivo–derived platelets for in vivo xenotransfusion studies in immunosuppressed mice to measure increases in platelet counts, even when recipient mice are severely thrombocytopenic (1% to 10% of baseline) after irradiation.27 The detection of infused platelets has been limited to video and/or flow cytometric studies for very rare events.27 The basis for this inefficiency remains unknown, but several lines of investigation have begun to identify new strategies for overcoming limited platelet yield.

Altering the physical environment

We still do not understand what “triggers” platelet production and release from mature megakaryocytes. In culture, only a small percentage of cells generate and release proplatelets. This means that ex vivo–generated platelets are released in a nonsynchronized fashion that results in inefficient platelet yield over several days in culture. Perhaps large-scale, continuous harvesting techniques using bioreactor technologies, outside the scope of most research laboratories, are needed to address this challenge. In the meantime, several groups have begun exploring replication of the physical nature of the bone marrow niche, including altering the physical rigidity of the culture environment,35 decreasing oxygen tension,36 increasing culture temperature,37 or simulating blood flow shear conditions38 to enhance platelet yield.

Shin et al35 hypothesized that a soft matrix for cell culture as well as sustained inhibition of nonmuscle myosin-II would maximize megakaryocyte maturation and platelet release. Using blebbistatin, a small molecule inhibitor of myosin II, and culturing megakaryocytes on a soft collagenous gel matrix, Shin et al35 attempted to mimic the microenvironment of the perisinusoidal niche within the bone marrow. These investigators35 were able to increase ploidy of megakaryocytes by as much as 50% and increase proplatelet formation. Transplantation of treated megakaryocytes into nonobese diabetic/severe combined immunodeficiency mice showed an approximately fourfold increase in platelet generation from blebbistatin-treated megakaryocytes.

Because the marrow is thought to be particularly hypoxic as a watershed area of blood flow, recent investigations have focused on the role of oxygen tension in the self-renewal and differentiation of hematopoietic cells.39 Low oxygen tension (1%) increases megakaryocyte and erythrocyte proliferation,37 resulting in a several-fold increase in the number of megakaryocytes produced compared with controls grown at ambient oxygen tension. Incorporation of low oxygen tension during the early phases of megakaryopoiesis also increased the numbers of mature megakaryocytes.36

To simulate a complex marrow niche, a woven polyester surgical fabric in a 3-dimensional single-pass perfusion bioreactor system was developed, which allowed the continuous collection of platelets for over 30 days with yields of up to 36 × 106 platelets per day per bioreactor.40 Subsequently, the same group was able to start with ∼2 × 106 CD34+ cells, vary oxygen tension in this system, and produce approximately 300 platelets per starting cell over 30 days.41 However, the platelets produced in this system responded to the strong agonist collagen but not to the weak agonists, adenosine 5′-diphosphate or epinephrine, suggesting that the harvested platelets may already be partially activated. The in vivo half-life of the platelets was not tested. Thus, this system appears to face the challenge of balancing enhanced yield and platelet preactivation.

Another complex bioreactor system that attempted to mimic both the osteoblastic niche and the vascular niche has been described, and it involved a chemokine stromal-derived factor-1 (SDF-1) chemotactic gradient between the niches to encourage cell migration.42 “Vascular” tubes were made of porous silk fibers and coated in Matrigel, von Willebrand factor, fibrinogen, and SDF-1. Media were perfused through the silk tubes, and platelets were collected from the media. Researchers calculated that approximately 200 platelets were collected per megakaryocyte that had extended proplatelets into the vascular tubes, but only ∼7% of the cultured megakaryocytes extended proplatelets.42 These platelets responded to the strong agonist thrombin by binding the activated anti-CD41 monoclonal antibody PAC-1, and they expressed increased surface P selectin; however, they spread poorly on collagen. Clearly, this ambitious bioreactor system needs further modification to enhance efficacy and improve the quality of the resulting platelets.

Human CD34+ cells have also been placed into a collagen type 1–containing Matrigel portion of a microbioreactor that also contains a microvessel network.42 Megakaryocytes migrated out of this “osteoblastic niche” to a “perivascular niche” and even into an intravascular space, representing shedded platelets to megakaryocytes. These bioreactor systems offer an exciting opportunity to better understand the regulation of megakaryopoiesis and thrombopoiesis. Whether they can be scaled up to yield functional platelets of clinical relevance remains a challenge.

Coculturing with other cells

Hematopoietic cells do not exist in isolation within the bone marrow. Multiple other cell lineages coexist within the intramedullary space, resulting in a complex 3-dimensional structure involving both extracellular matrices and a variety of cell-cell interactions. Investigators have explored recapitulating the complex in vivo environment using coculture techniques: growing hematopoietic and mesenchymal stem cells and/or osteoblasts together.43 Coculturing CD34+ cells in long-term cultures with mesenchymal cell lines has been reported,44 as have ESC culture systems using stromal cell lines for support of hematopoietic cell development.45 Because infused platelets can be irradiated before infusion, contaminated products with live supportive cells can be avoided, although other complications remain from contaminating support cells and irradiating products.

Another concern with such cocultures is that the platelets generated in the presence of stromal cells may be functionally impaired and have shortened circulating half-lives due to the loss of the extracellular domains of glycoprotein Ibalpha, glycoprotein V, glycoprotein VI., as was observed when differentiating hESCs on stromal cells.46 The investigators demonstrated that pretreatment of cultures with TAPI-1 or GM6001 (metalloproteinase inhibitors) was able to restore the function of hESC-derived platelets.46 TAPI-1 or GM6001 inhibit megakaryopoiesis so that these treatments have to be carefully timed in relation to platelet harvest.46 Subsequent studies examining the ability of hESC- or hiPSC-derived platelets to incorporate into growing thrombi27 have not suggested similar surface receptor issues, so the etiology for the observed metalloproteinase effect in this report remains unclear.

Drug modification of megakaryopoiesis and/or thrombopoiesis

Several groups are investigating the modification of cultured megakaryocytes using various compounds (other than cytokines) to determine if this might enhance platelet formation. The addition of nicotinamide (vitamin B3) to mobilized peripheral blood–derived megakaryocyte cultures increased final ploidy and produced more-elaborate cytoplasmic extensions, and presumably proplatelets.47 Others have reported that while nicotinamide increased the ploidy of cord blood–derived megakaryocytes, it decreased cell viability and final ploidy, such that adding nicotinamide in their system did not actually increase total platelet production.48 Src (sarcoma) kinase has been reported to play a role in megakaryopoiesis as a downstream kinase in TPO signaling.49 Addition of the Src kinase inhibitor SU6656 to bone marrow hematopoietic progenitor cells increased the ploidy and size of the megakaryocytes.50 These investigators50 did not examine the effect on platelet formation, and it is not clear if Src kinase inhibition promotes endomitosis and whether it increases the yield of platelets. To understand the different roles of the MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinase) pathways downstream of TPO signaling, others have studied the effect of a MAPK inhibitor, PD908059, and an inhibitor of mTOR (mammalian target of rapamycin), rapamycin. They found that MAPK inhibition increased megakaryocyte ploidy but decreased cell proliferation, while rapamycin treatment of cells decreased cell proliferation without increasing polyploidization.51 Data from Liu et al52 suggest that, at least for fetal megakaryopoiesis, high ploidy is not a requisite for efficient platelet production, and so it does not necessarily follow that increasing ploidy alone will increase platelet production. These studies demonstrate that the complex nature of megakaryopoiesis may be difficult to control in culture by targeting a single signaling pathway statically, and that the timing of drug exposure to a particular phase of megakaryocyte differentiation may be critical. In addition, studies that focus on the effects of pharmacologic modifiers on thrombopoiesis and final platelet functionality are needed.

An alternative strategy to generating platelets: infusion of ex vivo–generated megakaryocytes

An alternative approach to making platelets from ex vivo–generated megakaryocytes is one in which mature megakaryocytes are directly infused and mature platelets are formed and released in vivo.53 This strategy is based on reports that megakaryocytes might physiologically shed platelets in the pulmonary vasculature.14,15 Whether this is a physiologic mechanism of platelet production remains a matter of controversy. Two-photon calvarial microscopy of thrombopoiesis in mice demonstrated that, in vivo, large cytoplasmic fragments and even whole megakaryocytes are released from the marrow space into the vasculature (unlike in vitro culture, where proplatelets are shed as beads on a string).13 The larger fragments must be processed to platelets, and it is not unreasonable to assume that this process might occur in the pulmonary vasculature. Indeed, infusion of large, polyploid murine megakaryocytes (either fetal liver– or bone marrow–derived) resulted in the release of a minimum of 160 platelets into the circulation per large megakaryocyte (unpublished data), and this release was completed in less than 90 minutes.53 The number of platelets derived in vivo was robust. The first demonstration of a clearly detectable rise in the platelet count following treatment of irradiated, thrombocytopenic mice was reported, and it lasted for up to 48 hours. This rise in platelet count was better than that observed using infused syngeneic murine platelets. Thrombocytopenia did not have to be induced in the recipient mice to see a rise in platelet count.53 Platelet size and surface receptor density of these in vivo–generated platelets were normal, and the platelets could be incorporated into growing thrombi using in situ studies. Moreover, hemostatic correction was demonstrated in the recipient mouse using a ferric chloride carotid injury model in a mouse with a bleeding diathesis (eg, a mild form of Glanzmann thrombasthenia with a deficiency in the number of alphaIIb beta3 receptors per platelet53). The half-life of these platelets as measured by flow cytometry was slightly shorter than that of infused platelets. The basis for this relatively short half-life is not clear, but it could be related to megakaryocyte damage from growth in plastic dishes.46

Infused mature megakaryocytes almost exclusively localize to the lungs, where the cells shed their cytoplasm within the first 30 minutes and the nuclei disappear within 3 hours. There was no untoward mortality in mice following megakaryocyte infusions, and indeed the number of infused megakaryocytes needed to generate a clinically relevant rise in platelet counts was estimated to represent occupancy of 1% to 3% of the microvessels in the pulmonary bed. In clinical practice today, following bone marrow transplantation, infused nucleated cells also initially localize to the pulmonary bed, and the number of cells given per body weight of these cells is 10 times the number of megakaryocytes infused into mice in our studies,53 suggesting that megakaryocyte infusion may be well tolerated in many clinical settings.

As mentioned, 1 of the advantages of isolating platelets from ex vivo–derived megakaryocytes is the possibility of modifying these megakaryocytes so that the resulting platelets can be used to deliver targeted therapy to sites of vascular injury. The first demonstration of transfused ex vivo–derived megakaryocytes for platelet-targeted drug delivery was performed using hematopoietic progenitor cells from a mouse that transgenically expressed urokinase specifically within its α-granules.53 These urokinase-laden platelets derived from ex vivo–grown megakaryocytes prevented thrombosis in the ferric chloride carotid artery injury model.

Conclusions

In summary, important concepts and methodologies are in place for generating large numbers of human megakaryocytes in culture, but a number of hurdles have been identified that need to be overcome to generate a sufficient number of functional human platelets ex vivo or in vivo following megakaryocyte infusion. While hESC and hiPSC technologies have improved the likelihood of generating platelets for transfusion from ex vivo–grown megakaryocytes, daunting inefficiencies in this strategy may be challenging for economically generating the number of required megakaryocytes and platelets to have clinical impact. Enhancing the efficiency of ex vivo megakaryopoiesis and/or thrombopoiesis may contribute to a solution, or establishing a self-proliferative intermediate line derived from hESCs/iPSCs or some other tissues, such as adipocytes that can be induced to differentiate into mature megakaryocytes, may be a necessary component of a final strategy toward the generation of sufficient numbers of functional and longer-lasting platelets. Using bioreactors in which the physical nature, cocultured cells, 3-dimensional organization, and supplemental drugs can be controlled and that allow continuous harvest of platelets may also contribute to the eventual development of therapeutic platelet products. A more complete understanding of the signals that activate platelet production is vital for optimizing these systems. In vivo platelet generation from infused megakaryocytes is encouraging and may become an alternative to ex vivo thrombopoiesis, but this approach is limited by the time delay in achieving a maximal rise in platelet counts and the potential concerns with the infusion of whole cells, particularly in patients with actual or potential pulmonary disease.

Authorship

Contribution: M.P.L. generated the earliest draft, reviewed the literature, revised the work, and submitted the manuscript. S.K.S. revised drafts and reviewed the literature. R.F. revised drafts and contributed the section on in vivo generation of platelets from ex vivo–generated megakaryocytes. D.L.F. provided scientific direction, substantial revision of the manuscript, and expertise in the field. M.P. provided scientific direction, substantial revision of the manuscript, and expertise in the field as well as providing the original idea for the review.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Michele P. Lambert, The Children’s Hospital of Philadelphia, 3615 Civic Center Blvd, ARC, Room 316G, Philadelphia, PA 19104; e-mail: lambertm{at}email.chop.edu.

  • Submitted September 13, 2012.
  • Accepted January 2, 2013.

References

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