Ex Vivo Manufacture of Megakaryocytes and Platelets from Stem Cells: Recent Advances Toward Transfusion in Humans

Abstract

The generation of ex vivo functional megakaryocytes (MK) and platelets is an important issue in transfusion medicine as donor dependence implies in limitations, such as shortage of eligible volunteers. Indeed, platelet transfusion is still a procedure that saves the lives of patients with defective platelet production. Recent technological development has enabled the isolation and expansion of stem cells that can be used as a source for the production of functional platelets for transfusion. In this review, we discuss recent approaches of in vitro or ex vivo production of MK and platelets, suggesting that, in the near future, donor-independent sources may become a possibility. The feasibility of using these cells in the clinic may be safer, and in vitro manipulation could generate universally compatible products, solving problems related to platelet refractoriness. However, functionality and survival testing of these products in human beings are scarce; therefore, additional studies are needed to consolidate this purpose.

Introduction

Platelets are small anucleated and discoid cells with approximately 1–5 μm in diameter [1], which compose the human circulatory system. They have vital functions related mainly to hemostasis and thrombosis, being therefore responsible for containing bleeding and healing wounds by adhering to sites of vascular injury and forming a platelet plug [2]. These cells originate in vivo from the maturation of megakaryocytes (MK), in a process called thrombopoiesis, which in turn results from the differentiation of hematopoietic stem cells (HSC) from the bone marrow (BM), in a process known as megakaryopoiesis. Normally, healthy individuals produce platelets continuously and have 150–400 × 10⁹ platelets/L of blood, which remain in circulation for a maximum of ∼10 days [1,2].

The reduction in the number of circulating platelets to <100 × 10³ platelets/μL, a condition known as thrombocytopenia, may occur as a consequence of hematological diseases and as a result of chemotherapy treatment, cardiovascular surgery [3], and traumas resulting in hemorrhages, among others. Thrombocytopenia is associated with high risks of bleeding [4] and increased mortality [5], and is an independent risk factor for multiple organ failure, death, and other complications [6]. Thus, to avoid these undesirable outcomes, platelet concentrate units obtained only by means of voluntary donations, and usually by apheresis from random single donors, are transfused [7 –10].

Limitations and Problems Related to Obtaining Platelets for Transfusions from Random Voluntary Donors

Frequent platelet transfusions are required by a variety of thrombocytopenic patients, with BM failure-related diseases [11], such as those with aplastic anemia [12], or with hematological malignancies [4,13]. Patients undergoing chemotherapy [14] and HSC transplants [15,16], as well as surgical procedures like cardiovascular surgery [17] also benefit from platelets transfusions.

The obtainment of platelet concentrates is totally dependent on voluntary donations, creating difficulties and limitations in transfusion practice. Notably, a constant concern is obtaining enough compatible and fresh platelets to cover the high demands for transfusions and to renew stocks, due to their low shelf-life of 5–7 days at room temperature (RT) and gentle agitation, which does not allow effective long-term storage.

Cryopreserved platelets are usually stored for up to 2 years before use and can become an alternative for very specific situations [18]. Nevertheless, even today, there are no effective methodologies for platelet cryopreservation that fully preserves platelet functions. Currently, cryopreservation, mostly using dimethyl sulfoxide (DMSO), may damage a significant part of the platelets [19,20] and, in general, an average of about 28% may be lost during processing and cryopreservation [21]. In addition, reduction in the expression of glycoproteins GPIbα (a protein subunit involved in the formation of platelet plug) and GPIIb (receptor for fibrinogen and Von Willebrand factor) [22] and less aggregation capacity [23] were reported. After cryopreservation, platelets also show an increase in hemostatic effect in vivo in comparison to fresh cells, as well as a tendency to present a faster clearance of the circulation [24]. These characteristics, however, do not prevent the use of cryopreserved platelets in emergency situations, focused on bleeding control, such as in periods of unavailable fresh platelets for transfusion or in military operations [18]. However, thrombocytopenic patients would require fresh platelets with normal functions to obtain an effective treatment as fresh platelets have shown more efficiency in platelet increment after transfusion than cryopreserved platelets [25,26].

The transfusion of platelet concentrates may also present risk of bacterial and even viral infections, a problem that should be considered during these procedures. Platelet storage at RT, while helping to prevent the occurrence of early platelet activation, may contribute, in contrast, to increased probability of bacterial contamination [27]. Thus, the need to carry out tests to detect bacteria can reduce even further the time they are available for transfusion, since results may take up to 2 days to be released [27 –29].

Platelet Transfusion Refractoriness

In addition to these limitations, platelet refractoriness (PR) also represents a major challenge for health care professionals. In general, patients are considered refractory when they do not respond satisfactorily after receiving at least two equal transfusions [30]. More specifically, to define PR, it is necessary to quantify the post-transfusion platelet increment and this can be done from the calculation of corrected count increment and platelet percentage recovery, the most common methods. Both methods are based on the difference between platelet count, usually within one to 24 h after transfusion, and the platelet count before transfusion [31 –34].

In addition to rendering treatment based on platelet transfusion ineffective, PR can aggravate the patient's thrombocytopenic condition and thus their recovery. This increases the risk of bleeding, treatment duration, and costs [35–36].

In most cases, the occurrence of PR is multiple and linked to clinical and pharmacological causes, such as fever, infections, and antibiotic treatments [37], administration of amphotericin [38], disseminated intravascular coagulation, splenomegaly, and bleeding [30]. The incidence of PR due to nonimmunological factors alone has been reported in approximately 62%–67% of total refractory patients [33,37,39]. Moreover, problems linked to storage or platelet treatment, such as γ-irradiation [38], have been previously reported.

The remainder of refractory patients consists of refractoriness linked to immune factors or an association of these with nonimmune factors, constituting a proportion of ∼20% [40] to 37.5% [37], an important and significant number of patients. In addition, as this type of refractoriness is caused by a smaller number of known and more predictable factors, its resolution has a great potential to bring positive outcomes to these patients.

The most common causes of PR due to immunological events are more frequent in multitransfused patients [12], but can also occur after transplants and gestation [32] and involves prior exposure to Class I human leukocyte antigens (HLA Class I), which are the only ones presented by platelets [41]. PR can also occur due to exposure to the human platelet antigens (HPA) [1,33,42].

Frequent contact with blood elements from random donors, such as platelets, may induce the formation of alloantibodies against specific antigens in transfused patients, a process known as alloimmunization. The presence of these alloantibodies reactive to HLA Class I and HPA antigens in patient circulation is the fundamental cause of this type of PR [32]. To avoid the occurrence of PR, the most adopted strategy is the transfusion of platelets that do not have antigens reactive to the antibodies found in the patient's circulation, when they are available [40]. Despite the effectiveness of transfusion of HLA-A or HLA-B compatible platelets, which make up the HLA Class I system, and would help solve most of the PR issues, these are often unavailable [43].

In times of scarcity, the availability of compatible platelets for each patient can be compromised, especially for those who are alloimmunized, due to a greater variety of antibodies in their body and the greater difficulty and time spent to find compatible donors [42]. For this reason, the most affected patients are the multiple transfused ones [12].

Among these cases of refractoriness, those involving anti-HLA Class I alloantibodies are more frequent than the human platelet or anti-HPA antigens themselves, which occur very rarely [32,40,44]. A recent study has shown 108 patients with antibodies reactive for HLA Class I and 20 with anti-HPA [42], whereas another showed 110 patients for HLA and 2 patients for HPA [40]. Thus, HLA compatibility is essential as an effective treatment for refractory patients and should therefore be prioritized over HPA compatibility.

Taken together, these aspects make platelet transfusions an arduous and complex task, exposing the need to obtain platelets from more stable and safer sources, for this activity to be performed with greater specificity, and to avoid episodes of PR, lower risks of multiple immunization, and the transmission of infectious agents.

In Vitro Manufactured Platelets

The use of renewable cell sources as stem cells (SC), with customizable characteristics, capable of originating specific cellular products through cell differentiation, consists of a promising alternative for the production of functional, donor-independent platelets aimed at transfusions in the future. As SC are capable of both self-renewal, that is, remaining as an undifferentiated population and differentiating to become specialized, they may be used to constitute cell banks, which, different from the platelets themselves, can be stored for long periods of time, being submitted to differentiation protocols for platelet manufacture when necessary.

Platelets are produced through megakaryopoiesis followed by thrombopoiesis, two complex and carefully regulated biological processes. Megakaryopoiesis consists of the terminal maturation of MK, differentiated from HSC, displaying the CD41- and CD42-specific surface markers and for carrying out endomitosis, a chromosomal replication process, resulting in polyploidy. Thrombopoiesis represents the final stage of platelet production, with the formation of proplatelets in the sinusoid vessels of BM and the release of platelets into the bloodstream.

In general, the production of MK and platelets in vitro is an attempt to mimic the environment and conditions found in BM in a static manner, providing cells in differentiation with cytokines or cellular interaction stimuli, usually by the contact with specific formulation culture media and feeder cells.

Isolated and cloned during the early 1990s, the thrombopoietin (TPO) in complex with its receptor, c-MPL, is the main regulatory factor for in vitro and in vivo megakaryopoiesis, or the terminal maturation of MK from HSC and thrombopoiesis that comprises the formation of proplatelets and platelet release [45,46]. However, other cytokines such as human recombinant IL-3 [47], IL-6 [45], and stem cell factor (SCF) [48] act in the proliferation of MK, and IL-6 and IL-11 are related to MK maturation, including endomitosis [45,47,49,50].

Recently, new mechanisms of cell signaling under stress states are being elucidated, such as inflammatory conditions and acute loss of platelets, which hold potential for application in in vitro differentiation. They are based on molecules that display MK-biased platelet biogenesis actions.

The sphingosine-1-phosphate (S1P), for example, found abundantly in the circulation of mice and humans during inflammation, acts in the final stages of thrombopoiesis. S1P acts by guiding the platelets along BM's sinusoid blood vessels, until they reach the bloodstream, being subjected to the shear stress, resulting in the release of platelets [51]. The interaction of S1P with its receptor, S1pr1, present in MK, proved to be essential for normal thrombopoiesis in mice. However, the effects of S1P-S1pr1 signaling on human cells are not yet known, nor has the use of S1P been shown to stimulate the formation of proplatelets in human cell cultures. Nevertheless, it is likely that other proteins or cytokines not yet identified could perform this function in the human BM.

Recently, an aminoacyl transfer RNA (tRNA) synthetase, usually active in protein synthesis, apparently evolved acquiring a nontranslational function of regulating megakaryopoiesis and thrombopoiesis. More specifically, the activated form of the tyrosil-tRNA synthetase (YRS^ACT) could enhance platelet production from murine BM cells in vitro and also accelerate platelet recovery of thrombocytopenic murine models. The YRS^ACT was able to induce HSC differentiation directly to specific MK progenitors (Sca1⁺ F4/80) capable of producing platelets and also by activating the TLR/MyD88 pathway in monocytic cells, which induces the production of cytokines IL-6 and Interleukin IL-1α, stimulating platelet production. On the other hand, YRS^ACT thrombopoietic activity is relevant for inducing the expansion and maturation of MK from human cells such as peripheral HSC CD34⁺ collected after donor stimulation with G-CSF and also pluripotent stem cells (iPSC)-derived human CD34⁺, through an indirect stimulation. Human cells only respond to this treatment when in contact with the supernatant from cultures of human peripheral blood mononuclear cells treated with YRS^ACT. This suggests that YRS^ACT induces megakaryopoiesis by stimulating the production of cytokines by monocytic cells.

The increase in circulatory levels of cytokine IL-1α, also associated with acute platelet loss and inflammatory stimuli, induces rupture-dependent thrombopoiesis with rapid release of platelets into the circulation. Activated IL-1α originates from the cleavage mediated by thrombin of pro-interleukin IL-1 α present in macrophages and active platelets, ultimately activating caspase-3, which induces instability in the MK plasma membrane, reduction in tubulin expression, and inhibition in formation of proplatelets, resulting in platelet release [52,53]. The rupture-dependent release of platelets has not yet been confirmed to occur in human cells, either in vivo or in vitro, although active IL-1 α has been detected in humans during sepsis episodes. The confirmation of the occurrence of this mechanism in humans must still be carried out so that its potential in the production of platelets in vitro can be explored, with more precise control in the release of platelets from MK, or in the general increase in efficiency of in vitro thrombopoiesis.

More research is needed to be able to accurately assess the potential benefits of using these molecules for the production of human platelets in vitro, although the results in animal models are promising. Each of these mechanisms reveals the complexity of signals that control the different functions that can be performed by cells and molecules in vivo, indicating that more discoveries in this field can help differentiation systems in vitro to get closer to what occurs in vivo.

Recapitulation of the complex environment found in the BM is still a major challenge to be overcome to make the manufacture of blood cells from SC feasible. Not only the discovery of signaling molecules and mediators are part of this microenvironment but also parameters, including concentration of gases, temperature, and protein composition. Furthermore, the effects of blood circulation and shear stress have recently been proven to be essential for thrombopoiesis in vivo [54,55]. Recent results obtained from the differentiation in xenogeneic BM, using the cellular niche of a swine thigh bone, further suggest that essential factors exist in the BM environment [56].

Indeed, bioreactors and nonstatic systems demonstrated a greater capacity for scaling up, producing more platelets for each differentiated MK (Table 1). Although there is no consensus on the number of platelets released by mature MK in vivo, there are estimates indicating that no current method of differentiation compares to the natural efficiency of the human organism, whose estimated values are from 1,000 [9] to 4,000 or 5,000 platelets per MK [57,58].

Table 1.

Studies of In Vitro Platelet Production

Research/year [Ref.]	Cell source	Approximately maximum number of platelets/MK	Differentiation system: megakaryopoiesis/thrombopoiesis and platelet release
Avanzi et al. (2016) [59]	HSC (UCB)	<100	Static culture of HSC in defined growth medium/MK trapped on a pseudo-3D membrane, subjected to a shear flow induced by the passage of a defined culture medium.
Choi et al. (1995) [67]	HSC (PB)	<112	Static culture of HSC in defined growth medium.
De Bruyn et al. (2005) [68]	HSC (UCB)	<1	Static culture of HSC in defined growth medium.
Feng et al. (2014) [88]	iPS	<6	Static culture of iPS in defined growth medium/culture of iPS-derived MK in defined medium in ultra-low attachment plates.
Fujiyama et al. (2020) [56]	MK from HSC (UCB)	<5	Static culture of HSC in defined growth medium/infusion of generated MK in a 3D xenogeneic BM (porcine thighbone). After incubation in vivo, a perfusion medium was pressed through the system in one side, collecting the platelets released by the MK on the other side.
Gaur et al. (2006) [81]	ESC	<1	Static co-culture on OP9 cells and defined growth medium.
Ito et al. (2018) [60]	iPS	<80	Bioreactor VerMES and DOX regulated system: Dox-On (MK proliferation)/Dox-Off (MK differentiation).
Ito et al. (2018) [60]	iPS	<20	Static culture of iPS in defined growth medium.
Lu et al. (2011) [82]	ESC	<7	Hemangioblasts differentiated from ESC with culture in defined medium/static culture of MK in defined growth medium
Matsunaga et al. (2006) [69]	HSC (UCB)	<22	Static three-phase co-culture over hTERT stroma/static culture with cytokines.
Mattia et al. (2002) [50]	HSC (PB and UCB)	<2	Static culture in defined growth medium.
Moreau et al. (2016) [93]	ESC and iPS	<6	Static culture of ESC or iPS with the exogenous expression of GATA1, FLI1, and TAL1 transcription factors/co-culture in C3H10T1/2 and defined medium cells.
Nakagawa et al. (2013) [118]	iPS derived HSC	<1	Static co-culture of iPS-derived HSC in C3H10T1/2 cells with defined growth medium/MK differentiated in constructed bioreactor with porous scaffold and hydrodynamic flow.
Nakamura et al. (2014) [91]	ESC and iPS	<10	Static culture of ESC or iPS with a DOX-regulated system.
Norol et al. (1998) [70]	HSC (BM)	<50	Static culture of HSC in defined growth medium.
Ono-Uruga et al. (2016) [96]	ASC	<15	Static culture of ASC in defined growth medium.
Pallotta et al. (2011) [55]	HSC (UCB)	<250	Static culture of HSC in defined growth medium/MK grown in a bioreactor developed in house, based on silk microtubes, imitating BM environment where MK migrate and hydrodynamic forces.
Suzuki et al. (2020) [98]	iPS	<5	Culture of immortalized iPS-derived MK (Seo et al. 2018), with a DOX-regulated system, in shaking conditions and defined growth medium.
Takayama et al. (2008) [119]	ESC	<50	Static co-culture of ESC on C3H10T1/2 or OP-9 cells in defined growth medium.
Thon et al. (2014) [120]	iPS	<30	Static culture of iPS in defined growth medium for MK production and maturation (Feng et al. 2014)/MK infusion in a microfluidic bioreactor.

HSC CD34⁺ obtained from PB, UCB, BM.

3D, three dimensional; ASC, human adipose tissue-derived stem cells; BM, bone marrow; DOX, doxycycline; ESC, embryonic stem cells; HSC, hematopoietic stem cells; iPS, induced pluripotent stem cells; MK, megakaryocytes; PB, peripheral blood; UCB, umbilical cord blood.

In a recent study, a pseudo-three-dimensional (3D) culture system could simulate the perivascular space of the BM to accommodate a greater number of MK for differentiation and performed the separation of MK and platelets using a double chamber system. It consists of two chambers separated by a nanofiber or a PVC membrane, and the pressure exerted by a syringe was responsible for the flow induction in the system. Initially, megakaryopoiesis was stimulated by cytokines TPO and SCF, with the induction of polyploidization using latrunculin or Rho-kinase inhibitor Y27632. The movement of the culture medium from one chamber to another allowed the retention of mature MK on the membrane in one of the chambers with the extension of proplatelets and the collection of platelets on the other side, due to the action of shear stress [59].

More efficient cell culture systems involve the inclusion of factors such as turbulence and shear stress, key events of thrombopoiesis that are considered fundamental for scale-up in production [60]. A greater number of platelets released per MK is associated with these factors [61], although overuse is also responsible for the inhibition of productivity [59], therefore constituting additional aspects that must be carefully regulated to make this complex manufacture more effective.

Further development of culture systems could help us to understand the details responsible for megakaryopoiesis and thrombopoiesis, as well as reach, more efficiently, production levels that would allow clinical applications.

Hematopoietic stem cells

HSC are multipotent SC that are responsible for the continuous production of all the multiple mature cells that make up the blood. They can also proliferate, to maintain a population of undifferentiated daughter cells through the process of self-renewal [62]. As HSC have these two characteristics concomitantly, they are classified as SC. However, even among these cells, identified mainly by the CD34⁺ surface marker, some are not able to originate all blood cells since it is a heterogenous population with different capabilities [63]. Many of the CD34⁺ cells are actually hematopoietic progenitors, that is, they are cells derived from HSC, which have limited self-renewal capacities, and are already committed to differentiation into specific mature cell lines [64]. In fact, evidence shows that more stringent subpopulations like CD34⁺CD38⁻ are associated with long-term HSC, and production of lymphoid and myeloid cells and recovery of hematopoiesis [65]. Nevertheless, even with a greater restricted selection such as CD34⁺ CD38⁻ CD90⁺ Lin⁻ cells, the population is heterogeneous and with different self-renewal and differentiation potentials [63]. Therefore, these cells are difficult to identify and isolate, and the only way to identify them as bona fide long-term HSC is still the in vivo test, with transfusion in properly prepared murine models and the reconstitution of their hematopoiesis.

Within the hematopoietic system, HSC are the only cells that have the capacity for both self-renewal and differentiation, and it is the only one that can reconstitute hematopoiesis for long term in organisms with extensive cellular damage due to exposure to radiation and chemical elements [63]. Therefore, HSC obtained from healthy BM through donation are the basis for the treatment of leukemias, lymphomas and cancers of patients who have lost the ability to produce healthy blood cells from hematopoiesis [66].

Since they have these remarkable differentiation capabilities, HSC are also considered to be potential starting points for the production of platelets in vitro and ex vivo for transfusion. They were the first type of SC used for the production of human platelets in vitro, demonstrating the entire differentiation process up to the production of functional platelets using human plasma and TPO [67].

Functional platelets could be obtained by differentiating HSC from: mobilized peripheral blood [68], umbilical cord blood (UCB) [69], and BM [70].

However, HSC are rare, making up about 0.01%–0.1% of the nucleated cells in the peripheral circulation [71], although HSC can be mobilized from the BM by increasing this amount considerably, [72] and 1%–3% in the BM [71,73] in healthy individuals. An unit of UCB can provide ∼1 × 10⁶ HSC, but they have limited proliferation capacity in vitro and undergo changes of a phenotypic and functional nature when expanded in culture, since the complex regulation that occurs in vivo, in the hematopoietic niches, is not yet completely reproduced in vitro [74]. For this reason, dependency on recurrent donations of HSC CD34⁺ would yet be necessary, with subsequent selection of these cells to differentiate into platelets, which would be less practical and more expensive than a platelet collection by apheresis [75]. Even though it has been demonstrated that it is possible to obtain the minimum number of platelets necessary for a clinical application scale [59,69], that is around 1–5 × 10¹¹ platelets [76], there will still be dependence on HSC donations.

Pluripotent stem cells

Embryonic stem cells (ESC) are pluripotent cells that constitute the internal mass of blastocysts, an embryonic stage of development through which embryos of mammals, including humans, pass, starting from the totipotent cells. Because they are pluripotent, they can form cells derived from the three embryonic tissues, endoderm, mesoderm, and ectoderm, having the potential to originate all the functional cells of an adult organism. This ability was demonstrated in vitro for the first time in 1998 by Thomson, as well as their proliferation capacity, while remaining in an undifferentiated state by means of self-renewal [77].

The iPS [78,79] have the same properties as ESC regarding pluripotency, but without presenting the ethical issues surrounding the destruction of blastocysts for its obtention. The iPS are the result of cell reprogramming experiments, that is, the reversion of the differentiated state of an adult cell to the pluripotent state of ESC. This can be achieved by inducing ectopic expression of specific transcription factors, notably Oct4, Sox2, Klf4, and c-MYC among others, in target cells. Although the mechanisms are not completely elucidated, it is known that these factors are related to the maintenance of the pluripotent state and to the self-renewal of cells of the initial embryonic development, such as the ESC themselves. As they compose and feed an autoregulatory loop or circuit of pluripotency, different combinations of these factors may work together to maintain a stable expression of pluripotency genes [80] as well as similar properties as ESC [79].

The generation of MK from pluripotent stem cells (PSC) was performed primarily from ESC, with the induction of megakaryopoiesis from co-culture in OP9 feeder cells, fetal bovine serum, and TPO. Despite the low yield of <1 MK per input ESC, endomitosis was observed with 2N to 32N CD41⁺ cells, a typical marker of MK lineage [81]. Subsequent research was carried out, demonstrating the production of MK from differentiated ESC-generated hemangioblasts, with the production of functional platelets in large scale, in the absence of serum and stroma of animal origin. In this study, evidence of platelet functionality in vivo was also presented for the first time [82].

ESC and iPS can also form embryoid bodies (EB), using the method of Spin EB differentiation [83,84]. The forced cell aggregation forms a 3D structure, which is capable of differentiating and originating cells from the three embryonic tissues at random, but with the use of specific human recombinant cytokines such as BMP4, VEGF, and SCF can be directed to the production of hematopoietic progenitors. The subsequent addition of TPO, SCF, and IL-3 promotes megakaryopoiesis from EB cells, and thrombopoiesis occurs under the influence of the same cytokines, from these mature MK in collagen-based colony-forming assays [84]. The employment of a complex VerMES bioreactor, capable of simultaneously including turbulence and shear stress in the differentiation process, determined as important thrombopoiesis regulatory processes, allowed the obtaining of platelets on a clinical scale from iPS [60].

Alternatively, it is possible to produce HSC-like cells in vitro through the direct differentiation of iPS, to obtain blood cells, including platelets. iPS may originate the hegemonic endothelium, under specific culture conditions, such as through the forced expression of transcription factors [85,86], or by using a cytokine approach in defined culture medium [87] to obtain MK and functional platelets [88]. In vivo, the initial production of long-term HSC starts from the hemogenic endothelium, from which endothelial cells arise and by the endothelial-to-hematopoietic transition, hematopoietic progenitors are generated, colonizing niches that support definitive hematopoiesis [89].

The ESC, iPS, and derived cells, together with the promise of generating platelet independent of donors, may accumulate oncogenic mutations while in culture [90], present tumorigenic potential and genetic instability concerns, and hence, for their employment in the clinic. The possibility of the presence of undifferentiated cells among mature cells represents the main reason for this concern. Further research is needed to attest to the safety of platelets generated in vitro from ESC and iPS, including tests for platelet infusion irradiated in vivo, as well as whether contamination of nucleated cells would be eliminated with this process.

Immortalized MK

There is also the possibility of immortalizing MK, cells that can be cryopreserved and expanded in vitro, which would represent the possibility of producing platelets more quickly. MK progenitors (imMKCL) produced from ESC and iPS were obtained for the first time from the overexpression of c-MYC, BMI1, and BCL-XL genes. They act in conjunction with the regulated expression of c-MYC, inhibiting apoptosis and senescence pathways and promoting MK proliferation for up to 5 months. A vector controlled by the exogenous concentration of doxycycline is used to suppress this overexpression, which results in the induced final maturation of MK and thrombopoiesis, with the production of functional platelets by the imMKCL [91]. The imMKCL strain showed to be capable of responding to stimuli from other molecules, alternatives as agonists chemically synthesized to the TPO receptor, c-MPL. In a recent study, the compound TA-316 was shown to be the most effective, producing twice as many platelets in vitro in comparison to human recombinant TPO [92].

Based on the exogenous expression of the transcription factors GATA1, FLI1, and TAL1 in differentiating ESC and iPS, another group was able to obtain expandable MK cells. The proliferation period lasts for 120–132 days and a drop in cell viability was observed after 90 days. However, they obtained a high degree of MK purity (>90%) and a great expansion potential (2 × 10⁵ per differentiated PSC approximately). The induction of thrombopoiesis for the production of platelets occurred with the co-culture of mature MK in murine C3H10T1/2 feeder cells in the presence of TPO, IL-6, IL-11, SCF, and heparin [93]. However, the genetic modifications necessary for the generation of immortalized MK strains cause some concerns, as reported by these authors, such as the possibility of chromosomal translocations after a few months of cultivation. This factor, together with the use of co-cultivation as well as serum of animal origin, may pose difficulties in their employment in clinical tests.

Human adipose tissue-derived stem cells

It has been recently shown that progenitor cells from subcutaneous adipose tissue are also capable of originating MK and functional platelets in vitro. The population of human adipose tissue-derived stem cells (ASC) is multipotent and easily obtained in large quantities from the disposal of liposuction surgery [94] and can also be expanded into bioreactors to increase the scale of their production [95]. Furthermore, ASC do not depend on an external supply of TPO, which is necessary to perform megakaryopoiesis in vitro starting from the other cell types. ASC are therefore capable of producing endogenous TPO, by means of activation of the CD71 membrane receptors using transferrin, with positive ASC CD71 producing more endogenous TPO as well as MK cells than CD71-negative ASC [96].

HLA-Universal Platelets

Platelet production alone would represent a breakthrough and a great potential help for thrombocytopenic patients. However, alloimmunization and refractoriness-associated problems would still be present. Therefore, obtaining donor-independent HLA-universal platelets would be even more beneficial, since they could be used for treatment of a greater number of patients.

Transfusion of HLA-compatible platelets is an effective way to manage refractory patients [40], and for this reason, the use of HLA-universal platelets also has great potential for the treatment of these patients, without requiring a specific donor or platelet selection.

Although research evaluating generated β2-microglobulin (B2M)-knockout (KO) and HLA Class I-KO platelets in humans has not yet been described, there are examples of MK and platelets with these characteristics that have been evaluated in the circulation of humanized mouse models. In these models, B2M-KO MK and HLA Class I-KO platelets generated from iPS were able to survive [97] and avoid rejection by natural killer cells [98]. Furthermore, the fact that transfusions of HLA-universal platelets were also able to prevent the occurrence of refractoriness in animal models [99] strengthens the potential of using these cells in cell therapies in the future.

Class I HLA antigens (HLA-A, B, and C) are composed of two polypeptide chains, the largest, responsible for the serological detection of these antigens, for carrying the polymorphisms that distinguish them, and the smallest, corresponding to B2M [100]. B2M expression consists of one of the levels of regulation of the expression of HLA antigens on the cell surface, as even if the larger chain is produced, the smaller one is necessary for the exteriorization of the antigens. This has been observed in studies of Daudi cell lineage, originating from a patient with Burkitt's lymphoma, who have no HLA class I antigens on their cell membrane, but recovered this expression after being transfected with the murine B2M gene [101,102]. Thus, the relationship between the expression of HLA class I and B2M antigens became evident and has become the main target for the viability of HLA-universal platelets.

Some studies have shown a reduction in the expression of HLA class I proteins in platelets produced in vitro, by means of silencing the B2M protein in precursor cells. A reduction of up to 82% in the expression of these HLA molecules was obtained by silencing the B2M gene in iPS strains. The silencing of this gene was carried out by transducing the iPS with lentiviral vectors encoding interference RNA (RNAi), in this case, short double-stranded molecules in the form of a clamp or short hairpin RNA (shRNA) [103]. Functional platelets with a reduction of 85% in HLA expression were obtained from CD34⁺ HSC, using the same strategy [104]. The knockout of the B2M gene, obtained using the CRISPR/Cas9 technique, was capable of eliminating the expression of both B2M and HLA Class I molecules [98]. In these cases, until the end of the cell differentiation and maturation experiments, for the verification of their functionality, the expression of HLA remained reduced at the reported levels and silencing or knockout procedures of cells did not affect platelet differentiation or functionality at all.

With the possibility of generating precursor cells with permanent modifications in B2M, a renewable cell bank with cells capable of being differentiated into functional HLA-universal platelets would be closer to feasibility.

It is important to note that HLA-negative platelets would still have HPA antigens; however, the possibility of editing these antigens has been demonstrated recently. The allele PIA1 form of GPIIIa, the most frequently related to alloimmune bleeding disorders as the neonatal alloimmune thrombocytopenia (NAIT) and post-transfusion purpura (PTP), was converted in the PIA2 form. This conversion was performed in DAMI cells, a megakaryocytic strain, and in iPS from which megakaryocytic progenitors were obtained, correctly expressing the modified antigens [105]. The possibility of converting HPA would allow, in theory, the customization of manufactured platelets and would have applicability in diagnostics, research, and therapies. The production of HLA-universal platelets associated with HPA edition has not yet been made possible; nevertheless, it holds also great potential for application in the clinic and in research in the future.

Clinical Application Feasibility

Platelet production in vitro/ex vivo may constitute a permanent source of platelets for transfusion in the future, mainly helping to replenish stocks. Cell banks could be also maintained with HPA antigen variables, associated with HLA-null, for differentiation and obtaining very specific platelets. The mean number of platelets in a concentrate unit for transfusion has been reported as being between 5.5 × 10¹⁰ [106] and 9.69 × 10¹⁰ [107] each. However, the time to manufacture enough platelets to compose a transfusion unit is long and can reach 14 [59], 15 [55], or even 26 days [60] with current technology. This could be an impediment when a patient requires a large number of platelets at once or multiple transfusions in a short period of time, such as in emergency situations [108]. However, this would not represent an impediment in planned or predictable situations, such as in managing alloimmunized patients who require periodical transfusions of specific platelets.

Alternative ways to increase the efficiency of platelet production have yet to be developed and improved in order for manufactured platelets to be feasible for clinical use. This effort is observed, in addition to the development of more efficient cell culture systems, including more comprehensive reproductions of the BM environment, and in the discovery of compounds that act in thrombopoiesis. Recently, a drug testing platform based on the immortalized MK imMKCL lineage, modified for the expression of fluorescent reporter associated with β1-tubulin, was developed, allowing large-scale compound testing, culminating in the discovery of two compounds involved in the maturation of MK ex vivo [109].

In addition to the production capacity of platelets in vitro, other aspects must be considered to make these cells closer to their use in practice, including quality and especially regarding functionality. Fortunately, some important advances have been established in this matter, contributing to the possibility of using these platelets in clinical practice.

Platelets produced in vitro from ASC [96] and iPS [98] demonstrated the ability to agglutinate in vitro, in a similar way to natural platelets. The binding capacity of stimulated platelets with agonists such as thrombin and epinephrine, among others, to labeled fibrinogen [110] is also comparable with natural platelets. Other tests, including immunophenotyping for CD62P marker expression and PAC-1 binding, another marker antibody that only binds to activated platelets, have been used to demonstrate functionality similar to natural platelets [55,110]. In quantitative aggregation tests, different results were observed, where the capacity is comparable to [60] or slightly reduced, reaching a maximum of 80% of what is seen in natural platelets [111]. Although this capacity may vary slightly, in general, these studies show that different cells can originate platelets capable of activating and aggregating in a similar way to natural platelets. These tests also demonstrated that it is possible to control the timing of platelet activation, which did not aggregate spontaneously, a fundamental aspect for in vivo and clinical application.

The in vitro platelet production from ESC and iPS specifically involves the ectodomain shedding of the GPIbα glycoprotein, which is essential for the platelet adhesion to the injured vessel wall and also determinant to its in vivo circulation lifetime. The temperature-dependent activation of the disintegrin and metalloproteinase ADAM17, at 37°C, removes GPIbα from the surface, making the platelet dysfunctional. However, it was possible to discover an ADAM17 inhibitor, the KP-457, which allows the retention of GPIbα by platelets and does not present health risks, unlike other currently known inhibitors, also bringing these cells closer to be used in the clinic [112].

In vivo functionality tests also reveal promising results (Table 2), as bleeding interruption tests were performed on thrombocytopenic mice, with similar results from in vitro produced platelets and natural platelets on reduction of blood loss [111]. Furthermore, platelets generated from ESC and iPS were able to incorporate into the thrombi in formation, resulting from vascular injuries produced in mice [82,112].

Table 2.

Research Involving Assessment of Cultured Megakaryocyte or Platelet Functionality in Vivo

Author	Cell source	Cell type infused	Organism	Results/achievements
Do Sacramento et al. (2020) [111]	Human CD34⁺	Cultured platelets	NSG mice	Recirculation after infusion
Do Sacramento et al. (2020) [111]	Human CD34⁺	Cultured platelets	Thrombocytopenic NSG mice	Bleeding interruption in vivo
Hirata et al. (2017) [112]	ESC and iPS	Cultured platelets	NOG mice	Hemostatic functionality in vivo
Ito et al. (2018) [60]	iPS	Cultured platelets	NOG mice	Hemostatic functionality in vivo
Lu et al. (2011) [82]	Human ESC	Cultured platelets	Laser-induced vascular injury C57BL/6 mice	Clot formation in vivo
Wang et al. (2015) [116]	Human BM CD34⁺ and mononuclear cells from human fetal liver	Human MK	NSG mice	Incorporation of platelets into thrombi
Wang et al. (2015) [116]	CD34⁺ from BM, mononuclear cells from human fetal livers or iPS	Human MK	NSG mice	Release of platelets within the lung's vasculature
Xi et al. (2013) [117]	Human UCB mononuclear cells	Human megakaryocytic progenitor cells (MP)	Patients with advanced hematological malignancies	Safety and tolerability of infusion MP in humans

BM, bone marrow; ESC, embryonic stem cells; Human CD34⁺, HSC enriched from leukofilters; iPS, induced pluripotent stem cells; MK, megakaryocytes; MP, megakaryocyte progenitors; NOG, NOD/Shi-scid IL2rgamma(null); NSG, NOD scid gamma mouse; UCB, umbilical cord blood.

There is still no proof of principle for platelets generated in vitro/ex vivo and infused in humans as was done with red blood cells [113], where erythroblasts and erythrocytes generated ex vivo were demonstrated to be capable of surviving and of remaining functional after infusion in humans. However, some studies involving the infusion of MK in mice and humans have already been carried out with promising results [114]. The in vivo production of functional and viable platelets, for example, from MK infused in animal models suggests the possibility of using these cells, instead of the platelets generated ex vivo, to achieve the recovery of the number of platelets in patients [115,116]. MK obtained from the differentiation of UCB mononuclear cells were infused in human patients with hematological malignancies and undergoing chemotherapy treatment, with no major adverse effects. From a total of 24 patients, 19 showed platelet recovery of 60–100 × 10⁹ cells/L, in variable periods ranging from one to 16 days. The remaining five patients did not demonstrate platelet recovery and required transfusion of platelet units, but the causes were not assessed [117].

In addition, it has been already shown that MK produced ex vivo, without HLA class I antigens, can scape antibody-mediated complement-dependent cytotoxicity in vitro and also in mice [99], suggesting that transfusions of these cells in humans will not be impaired by the action of the nonspecific immune system [98,103].

Conclusion

In summary, the production of platelets in vitro or ex vivo is therefore an alternative still under development, but with great potential for donor-independent platelet supply. However, new studies must be developed, not only to make the process cheaper or to find alternatives to scale-up production but also to develop nonimmunogenic platelets, which prevent alloimmunization and refractoriness, focusing on in vivo functionality and safety, before clinical tests could be viable in humans.

Footnotes

Author Disclosure Statement

The authors declare that they have no conflicts of interest relevant to the article submitted to Stem Cells and Development.

Funding Information

This study was funded by grant number 2017/21801-2, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); and grant number 168179/2017-2, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

References

Forest

and Hod

. (2016). Management of the platelet refractory patient. Hematol Oncol Clin North Am, 30:665–677.

Curtis

and McFarland

. (2014). Human platelet antigens-2013. Vox Sang, 106:93–102.

Warkentin

and Greinacher

. (2003). Heparin-induced thrombocytopenia and cardiac surgery. Ann Thorac Surg, 76:638–648.

Webert

, Cook

, Sigouin

, Rebulla

and Heddle

. (2006). The risk of bleeding in thrombocytopenic patients with acute myeloid leukemia. Haematologica, 91:1530–1537.

Williamson

, Lesur

, Tétrault

J-P

, Nault

and Pilon

. (2013). Thrombocytopenia in the critically ill: prevalence, incidence, risk factors, and clinical outcomes. Can J Anesth, 60:641–651.

Nydam

, Kashuk

, Moore

, Johnson

, Burlew

, Biffl

, Barnett

and Sauaia

. (2011). Refractory postinjury thrombocytopenia is associated with multiple organ failure and adverse outcomes. J Trauma, 70:401–407.

Estcourt

, Stanworth

and Murphy

. (2011). Platelet transfusions for patients with haematological malignancies: who needs them?. Br J Haematol, 154:425–440.

Holcomb

, Tilley

, Baraniuk

, Fox

, Wade

, Podbielski

, Del Junco

, Brasel

, Bulger

, et al. (2015). Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma. The PROPPR randomized clinical trial. JAMA, 313:471–482.

Thon

, Medvetz

, Karlsson

and Italiano

. (2015). Road blocks in making platelets for transfusion. J Thromb Haemost, 13:S55–S62.

10.

Estcourt

, Birchall

, Allard

, Bassey

, Hersey

, Kerr

, Mumford

, Stanworth

and Tinegate

. (2017). Guidelines for the use of platelet transfusions. Br J Haematol, 176:365–394.

11.

Stroncek

and Rebulla

. (2007). Platelet transfusions. Lancet, 370:427–438.

12.

Pai

S-C

, Lo

S-C

, Lin Tsai

S-J

, Chang

J-S

, Lin

D-T

, Lin

K-S

and Lin

L-I

. (2010). Epitope-based matching for HLA-alloimmunized platelet refractoriness in patients with hematologic diseases. Transfusion, 50:2318–2327.

13.

Gmur

, Burger

, Schanz

, Fehr

and Schaffner

. (1991). Safety of stringent prophylactic platelet transfusion policy for patients with acute leukaemia. Lancet, 338:1223–1226.

14.

Weycker

, Hatfield

, Grossman

, Hanau

, Lonshteyn

, Sharma

and Chandler

. (2019). Risk and consequences of chemotherapy-induced thrombocytopenia in US clinical practice. BMC Cancer, 19:1–8.

15.

Wandt

, Schaefer-Eckart

, Frank

, Birkmann

and Wilhelm

. (2006). A therapeutic platelet transfusion strategy is safe and feasible in patients after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant, 37:387–392.

16.

Schiffer

, Bohlke

, Delaney

, Hume

, Magdalinski

, McCullough

, Omel

, Rainey

, Rebulla

, et al. (2018). Platelet transfusion for patients with cancer: American Society of Clinical Oncology clinical practice guideline update. J Clin Oncol, 36:283–299.

17.

Thiele

and Greinacher

. (2020). Platelet transfusion in perioperative medicine. Semin Thromb Hemost, 46:50–61.

18.

Cohn

, Dumont

, Lozano

, Marks

, Johnson

, Ismay

, Bondar

, T'Sas

, Yokoyama

APH

, et al. (2017). Vox Sanguinis International Forum on platelet cryopreservation. Vox Sang, 112:e69–e85.

19.

Gläfke

, Akhoondi

, Oldenhof

, Sieme

and Wolkers

. (2012). Cryopreservation of platelets using trehalose: the role of membrane phase behavior during freezing. Biotechnol Prog, 28:1347–1354.

20.

Kelly

and Dumont

. (2019). Frozen platelets. Transfus Apher Sci, 58:23–29.

21.

Slichter

, Jones

, Ransom

, Gettinger

, Jones

, Christoffel

, Pellham

, Bailey

, Corson

and Bolgiano

. (2014). Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets. Transfus Med Rev, 28:212–225.

22.

Johnson

, Reade

, Hyland

, Tan

and Marks

. (2015). In vitro comparison of cryopreserved and liquid platelets: potential clinical implications: cryopreserved vs. liquid-stored PLTs. Transfusion, 55:838–847.

23.

Dumont

, Cancelas

, Dumont

, Siegel

, Szczepiorkowski

, Rugg

, Pratt

, Worsham

, Hartman

, et al. (2013). A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxide-frozen autologous platelets in healthy volunteers. Transfusion, 53:128–137.

24.

Johnson

, Tan

, Wood

, Davis

and Marks

. (2016). Refrigeration and cryopreservation of platelets differentially affect platelet metabolism and function: a comparison with conventional platelet storage conditions. Transfusion, 56:1807–1818.

25.

Slichter

, Dumont

, Cancelas

, Jones

, Gernsheimer

, Szczepiorkowski

, Dunbar

, Prakash

, Medlin

, et al. (2018). Safety and efficacy of cryopreserved platelets in bleeding patients with thrombocytopenia. Transfusion, 58:2129–2138.

26.

Bohonek

, Kutac

, Landova

, Koranova

, Sladkova

, Staskova

, Voldrich

and Tyll

. (2019). The use of cryopreserved platelets in the treatment of polytraumatic patients and patients with massive bleeding. Transfusion, 59:1474–1478.

27.

Albertoni

, Andrade

, Araújo

PRB

, Carvalho

, M Girão

JBC

and Barreto

. (2011). Evaluation of two detection methods of microorganisms in platelet concentrates. Transfus Med, 21:408–416.

28.

Loza-Correa

, Kou

, Taha

, Kalab

, Ronholm

, Schlievert

, Cahill

, Skeate

, Cserti-Gazdewich

and Ramirez-Arcos

. (2017). Septic transfusion case caused by a platelet pool with visible clotting due to contamination with Staphylococcus aureus . Transfusion, 57:1299–1303.

29.

Martini

, Horner

, de Abreu Rodrigues

, Kempfer

, Tizotti

and Ratzlaff

. (2012). Bacteriological analysis of platelets and cases of septic reactions associated with transfusion of contaminated samples. Transfus Apher Sci, 47:313–318.

30.

Bishop

, McGrath

, Wolf

, Matthews

, De Luise

, Holdsworth

, Yuen

, Veale

, Whiteside

, Cooper

and Szer

. (1988). Clinical factors influencing the efficacy of pooled platelet transfusions. Blood, 71:383–387.

31.

Rebulla

. (2005). A mini-review on platelet refractoriness. Haematologica, 90:247–253.

32.

Stanworth

, Navarrete

, Estcourt

and Marsh

. (2015). Platelet refractoriness – practical approaches and ongoing dilemmas in patient management. Br J Haematol, 171:297–305.

33.

Murphy MF. (2014). Managing the platelet refractory patient. ISBT Sci Ser, 9:234–238.

34.

Chenna

, Shastry

and Baliga

. (2020). Evaluation and monitoring of response to platelet transfusion therapy: experience from a tertiary care center. Acta Clin Belg, 22; 1–4.

35.

Schmidt

, Refaai

and Coppage

. (2019). HLA-Mediated Platelet Refractoriness. Am J Clin Pathol, 151:353–363.

36.

Meehan

, Matias

, Rathore

, Sandler

, Kallich

, LaBrecque

, Erder

and Schulman

. (2000). Platelet transfusions: utilization and associated costs in a tertiary care hospital. Am J Hematol, 64:251–256.

37.

Legler

, Fischer

, Dittmann

, Simson

, Lynen

, Humpe

, Riggert

, Schleyer

, Kern

, Hiddemann

and Kohler

. (1997). Frequency and causes of refractoriness in multiply transfused patients. Ann Hematol, 74:185–189.

38.

Slichter SJ. (2005). Factors affecting posttransfusion platelet increments, platelet refractoriness, and platelet transfusion intervals in thrombocytopenic patients. Blood, 105:4106–4114.

39.

Doughty

, Murphy

and Metcatfe

. (1994). Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sang, 66:200–205.

40.

Wang

, Xia

, Deng

, Xu

, Shao

, Ding

, Chen

, Liu

, Chen

, Ye

and Santoso

. (2017). Analysis of platelet-reactive alloantibodies and evaluation of cross-match-compatible platelets for the management of patients with transfusion refractoriness. Transfusion Med, 28:40–46.

41.

Brown

and Navarrete

. (2011). Clinical relevance of the HLA system in blood transfusion. Vox Sang, 101:93–105.

42.

Kiefel

, Konig

, Kroll

and Santoso

. (2001). Platelet alloantibodies in transfused patients. Transfusion, 41:766–770.

43.

Petz

, Garratty

, Calhoun

, Clark

, Terasaki

, Gresens

, Gornbein

, Landaw

, Smith

and Cecka

. (2002). Selecting donors of platelets for refractory patientson the basis of HLA antibody specificity. Transfusion, 40:1446–1456.

44.

Godeau

, Fromont

, Seror

, Duedari

and Bierling

. (1992). Platelet alloimmunization after multiple transfusions: a prospective study of 50 patients. Br J Haematol, 81:395–400.

45.

Nagasawa

, Orita

, Matsushita

J-I

, Tsuchiya

, Neichi

, Imazeki

, Imai

, Ochi

, Kanma

and Abe

. (1990). Thrombopoietic activity of human interleukin-6. FEBS Lett, 260:176–178.

46.

Matsumura

and Kanakura

. (2002). Molecular control of megakaryopoiesis and thrombopoiesis. Int J Hematol, 75:473–483.

47.

Broudy

, Lin

and Kaushansky

. (1995). Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood, 85:1719–1726.

48.

Briddell

, Bruno

, Cooper

, Brandt

and Hoffman

. (1991). Effect of c-kit ligand on in vitro human megakaryocytopoiesis. Blood, 78:2854–2859.

49.

Navarro

, Debili

, Le Couedic

, Klein

, Breton-Gorius

, Doly

and Vainchenker

. (1991). Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication. Blood, 77:461–471.

50.

Mattia

, Vulcano

, Milazzo

, Barca

, Macioce

, Giampaolo

and Hassan

. (2002). Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34⁺ cells are correlated with different levels of platelet release. Blood, 99:888–897.

51.

Zhang

, Orban

, Lorenz

, Barocke

, Braun

, Urtz

, Schulz

, M-Lv Bruhl, Tirniceriu

, et al. (2012). A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis. J Exp Med, 209:2165–2181.

52.

Nishimura

, Nagasaki

, Kunishima

, Sawaguchi

, Sakata

, Sakaguchi

, Ohmori

, Manabe

, Italiano

Jr. , et al. (2015). IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs. J Cell Biol, 209:453–466.

53.

Burzynski

, Humphry

, Pyrillou

, Wiggins

, Chan

JNE

, Figg

, Martin

, Mr Bennett and Clarke

MCH

. (2019). The coagulation and immune systems are directly linked through the activation of interleukin-1α by thrombin. Immunity, 50:1033.e6–1042.e6.

54.

Junt

, Schulze

, Chen

, Massberg

, Goerge

, Krueger

, Wagner

, Graf

, Italiano

Jr. , Shivdasani

and von Andrian

. (2007). Dynamic visualization of thrombopoiesis within bone marrow. Science, 317:1767–1770.

55.

Pallotta

, Lovett

, Kaplan

and Balduini

. (2011). Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes. Tissue Eng C Methods, 17:1223–1232.

56.

Fujiyama

, Hori

, Sato

, Enosawa

, Murata

and Kobayashi

. (2020). Development of an ex vivo xenogeneic bone environment producing human platelet-like cells. PloS One, 15:e0230507.

57.

Kaufman

, Airo

, Pollack

and Crosby

. (1965). Circulating megakaryocytes and platelet release in the lung. Blood, 26:720–731.

58.

Lee

, Godara

and Haylock

. (2014). Biomanufacture of human platelets for transfusion: rationale and approaches. Exp Hematol, 42:332–346.

59.

Avanzi

, Oluwadara

, Cushing

, Mitchell

, Fisher

and Mitchell

. (2016). A novel bioreactor and culture method drives high yields of platelets from stem cells. Transfusion, 56:170–178.

60.

Ito

, Nakamura

, Sugimoto

, Shigemori

, Kato

, Ohno

, Sakuma

, Ito

, Kumon

, et al. (2018). Turbulence activates platelet biogenesis to enable clinical scale ex vivo production. Cell, 174:636.e18–648.e18.

61.

Pietrzyk-Nivau

, Poirault-Chassac

, Gandrille

, Derkaoui

S-M

, Kauskot

, Letorneur

, Le Visage

and Baruch

. (2015). Three-dimensional environment sustains hematopoietic stem cell differentiation into platelet-producing megakaryocytes. PloS One, 10:e0136652.

62.

Wilkinson

, Igarashi

and Nakauchi

. (2020). Haematopoietic stem cell self-renewal in vivo and ex vivo. Nat Rev Genet, 21:541–554.

63.

Seita

and Weissman

. (2010). Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med, 2:640–653.

64.

Sieburg

, Cho

, Dykstra

, Uchida

, Eaves

and Muller-Sieburg

. (2006). The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets. Blood, 107:2311–2316.

65.

Huang

and Terstappen

. (1994). Lymphoid and myeloid differentiation of single human CD34⁺, HLA-DR⁺, CD38⁻ hematopoietic stem cells. Blood, 83:1515–1526.

66.

Chabannon

, Kuball

, Bondanza

, Dazzi

, Pedrazzoli

, Toubert

, Ruggeri

, Fleischhauer

and Bonini

. (2018). Hematopoietic stem cell transplantation in its 60s: a platform for cellular therapies. Sci Transl Med, 10:eaap9630.

67.

Choi

, Nichol

, Hokom

, Hornkohl

and Hunt

. (1995). Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood, 85:402–413.

68.

De Bruyn

, Delforge

, Martiat

and Bron

. (2005). Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood. Stem Cells Dev, 14:415–424.

69.

Matsunaga

, Tanaka

, Kobune

, Kawano

, Tanaka

, Kuribayashi

, Iyama

, Sato

, et al. (2006). Ex vivo large-scale generation of human platelets from cord blood CD34⁺ cells. Stem Cells, 24:2877–2887.

70.

Norol

, Vitrat

, Cramer

, Guichard

, Burstein

, Vainchenker

and Debili

. (1998). Effects of cytokines on platelet production from blood and marrow CD34⁺ cells. Blood, 91:830–843.

71.

Sutherland

, Anderson

, Keeney

, Nayar

and Chin-Yee

. (1996). The ISHAGE guidelines for CD34⁺ cell determination by flow cytometry. J Hematother, 5:213–226.

72.

Bendall

and Bradstock

. (2014). G-CSF: from granulopoietic stimulant to bone marrow stem cell mobilizing agent. Cytokine Growth Factor Rev, 25:355–367.

73.

Körbling

and Anderlini

. (2001). Peripheral blood stem cell versus bone marrow allotransplantation: does the source of hematopoietic stem cells matter?. Blood, 98:2900–2908.

74.

Mayani

. (2019). Human hematopoietic stem cells: concepts and perspectives on the biology and use of fresh versus in vitro–generated cells for therapeutic applications. Curr Stem Cell Rep, 5:115–124.

75.

Lambert

, Sullivan

, Fuentes

, French

and Poncz

. (2013). Challenges and promises for the development of donor-independent platelet transfusions. Blood, 121:3319–3324.

76.

Slichter

, Kaufman

, Assmann

, McCullough

, Triulzi

, Strauss

, Gernsheimer

, Ness

, Brecher

, et al. (2010). Dose of prophylactic platelet transfusions and prevention of hemorrhage. N Engl J Med, 362:600–613.

77.

Thomson JA. Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147.

78.

Takahashi

and Yamanaka

. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.

79.

Takahashi

, Tanabe

, Ohnuki

, Narita

, Ichisaka

, Tomoda

and Yamanaka

. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.

80.

Omole

and Fakoya

AOJ

. (2018). Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications. PeerJ, 6:e4370.

81.

Gaur

, Kamata

, Wang

, Moran

, Shattil

and Leavitt

. (2006). Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function. J Thromb Haemost, 4:436–442.

82.

S-J

, Li

, Yin

, Feng

, Kimbrel

, Hahm

, Thon

, Wang

, Italiano

, Cho

and Lanza

. (2011). Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Cell Res, 21:530–545.

83.

, Davis

, Hatzistavrou

, Stanley

and Elefanty

. (2008). Directed differentiation of human embryonic stem cells as spin embryoid bodies and a description of the hematopoietic blast colony forming assay. Curr Protoc Stem Cell Biol, 4:1D.3.1–1D.3.23.

84.

Pick

, Azzola

, Osborne

, Stanley

and Elefanty

. (2013). Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium. PLoS One, 8:e55530.

85.

Doulatov

, Vo

, Chou

, Kim

, Arora

, Li

, Hadland

, Bernstein

, Collins

, Zon

and Daley

. (2013). Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell Stem Cell, 13:459–470.

86.

Blaser

and Zon

. (2018). Making HSCs in vitro: don't forget the hemogenic endothelium. Blood, 132:1372–1378.

87.

Lange

, Hoffmann

, Schwarzer

, Ha

T-C

, Philipp

, Lenz

, Morgan

and Schambach

. (2020). Inducible forward programming of human pluripotent stem cells to hemato-endothelial progenitor cells with hematopoietic progenitor potential. Stem Cell Rep, 14:122–137.

88.

Feng

, Shabrani

, Thon

, Huo

, Thiel

, Machlus

, Kim

, Brooks

, Li

, et al. (2014). Scalable generation of universal platelets from human induced pluripotent stem cells. Stem Cell Rep, 3:817–831.

89.

Chen

and Zon

. (2009). Zebrafish blood stem cells. J Cell Biochem, 108:35–42.

90.

Merkle

, Ghosh

, Kamitaki

, Mitchell

, Avior

, Mello

, Kashin

, Mekhoubad

, Ilic

, et al. (2017). Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature, 545:229–233.

91.

Nakamura

, Takayama

, Hirata

, Seo

, Endo

, Ochi

, Fujita

K-I

, Koike

, Harimoto

K-I

, et al. (2014). Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell, 14:535–548.

92.

Aihara

, Koike

, Abe

, Nakamura

, Sawaguchi

, Nakamura

, Sugimoto

, Nakauchi

, Nishino

and Eto

. (2017). Novel TPO receptor agonist TA-316 contributes to platelet biogenesis from human iPS cells. Blood Adv, 1:468–476.

93.

Moreau

, Evans

, Vasquez

, Tijssen

, Yan

, Trotter

, Howard

, Colzani

, Arumugam

, et al. (2016). Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat Commun, 7:11208.

94.

Matsubara

, Saito

, Suzuki

, Watanabe

, Murata

and Ikeda

. (2009). Generation of megakaryocytes and platelets from human subcutaneous adipose tissues. Biochem Biophys Res Commun, 378:716–720.

95.

Haack-Sørensen

, Juhl

, Follin

, Harary Sondergaard

, Kirchhoff

, Kastrup

and Ekblond

. (2018). Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate. Scand J Clin Lab Invest, 78:293–300.

96.

Ono-Uruga

, Tozawa

, Horiuchi

, Murata

, Okamoto

, Ikeda

and Matsubara

. (2016). Human adipose tissue-derived stromal cells can differentiate into megakaryocytes and platelets by secreting endogenous thrombopoietin. J Thromb Haemost, 14:1285–1297.

97.

Norbnop

, Ingrungruanglert

, Israsena

, Suphapeetiporn

and Shotelersuk

. (2020). Generation and characterization of HLA-universal platelets derived from induced pluripotent stem cells. Sci Rep, 10:8472.

98.

Suzuki

, Flahou

, Yoshikawa

, Stirblyte

, Hayashi

, Sawaguchi

, Akasa

, Nakamura

, Higashi

, et al. (2020). iPSC-derived platelets depleted of HLA class I are inert to anti-HLA class I and natural killer cell immunity. Stem Cell Rep, 14:49–59.

99.

Gras

, Schulze

, Goudeva

, Guzman

, Blasczyk

and Figueiredo

. (2013). HLA-universal platelet transfusions prevent platelet refractoriness in a mouse model. Hum Gene Ther, 24:1018–1028.

100.

Arce-Gomez

, Jones

, Barnstable

, Solomon

and Bodmer

. (1978). The genetic control of HLA-A and B antigens in somatic cell hybrids: requirement for β2 microglobulin. Tissue Antigens, 11:96–112.

101.

D'Urso

, Wang

, Cao

, Tatake

, Zeff

and Ferrone

. (1991). Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m gene expression. J Clin Invest, 87:284–292.

102.

Seong

, Clayberger

and Krensky

. (1988). Rescue of daudi cell HLA expression by transfection of the mouse B2-Microglobulin gene. J Exp Med, 167:288–299.

103.

Börger

A-K

, Eicke

, Wolf

, Gras

, Aufderbeck

, Schulze

, Engels

, Eiz-Vesper

, Schambach

, et al. (2016). Generation of HLA-universal iPSC-derived megakaryocytes and platelets for survival under refractoriness conditions. Mol Med, 22:274–285.

104.

Figueiredo

, Goudeva

, Horn

, Eiz-Vesper

, Blasczyc

and Seltsam

. (2010). Generation of HLA-deficient platelets from hematopoietic progenitor cells: generation of HLA-deficient PLTs. Transfusion, 50:1690–1701.

105.

Zhang

, Zhi

, Curtis

, Rao

, Jobaliya

, Poncz

, French

, Newman

. (2016). CRISPR/CAS9-mediated conversion of human platelet alloantigen allotypes. Blood, 127:675–680.

106.

Murphy

, Rebulla

, Bertolini

, Holme

, Moroff

, Snyder

and Stromberg

. (1994). In vitro assessment of the quality of stored platelet concentrates. Transfus Med Rev, 8:29–36.

107.

Kelley

, Fegan

, Ng

, Kennedy

, Blanda

, Chambers

, Kennedy

and Lasky

. (1997). High-yield platelet concentrates attainable by continuous quality improvement reduce platelet transfusion cost and donor exposure. Transfusion, 37:482–486.

108.

Martínez-Botía

, Acebes-Huerta

, Seghatchian

and Gutiérrez

. (2020). In vitro platelet production for transfusion purposes: where are we now?. Transfus Apher Sci, 59:102864.

109.

Seo

, Chen

, Hashimoto

, Endo

, Nishi

, Ohta

, Yamamoto

, Hotta

, Sawaguchi

, et al. (2018). A b1-tubulin–based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production. Blood Adv, 2:2262–2272.

110.

Tozawa

, Ono-Uruga

, Yazawa

, Mori

, Murata

, Okamoto

, Ikeda

and Matsubara

. (2019). Megakaryocytes and platelets from a novel human adipose tissue–derived mesenchymal stem cell line. Blood, 133:633–643.

111.

Do Sacramento

, Mallo

, Freund

, Eckly

, Hechler

, Mangin

, Lanza

, Gachet

and Strassel

. (2020). Functional properties of human platelets derived in vitro from CD34⁺ cells. Sci Rep, 10:914.

112.

Hirata

, Murata

, Suzuki

, Nakamura

, Jono-Ohnishi

, Hirose

, Sawaguchi

, Nishimura

, Sugimoto

and Eto

. (2017). Selective inhibition of ADAM17 efficiently mediates glycoprotein Ibα retention during ex vivo generation of human induced pluripotent stem cell-derived platelets: way for GPIbα retention on iPSC-derived platelets. Stem Cells Transl Med, 6:720–730.

113.

Giarratana

M-C

, Rouard

, Dumont

, Kiger

, Safeukui

, Le Pennec

P-Y

, François

, Trugnan

, Peyrard

, et al. (2011). Proof of principle for transfusion of in vitro-generated red blood cells. Blood, 118:5071–5079.

114.

Heazlewood

, Nilsson

, Cartledge

, Be

, Vinson

, Gel

and Haylock

. (2017). Progress in bio-manufacture of platelets for transfusion. Platelets, 28:649–656.

115.

Fuentes

, Wang

, Hirsch

, Wang

, Rauova

, Worthen

, Kowalska

and Poncz

. (2010). Infusion of mature megakaryocytes into mice yields functional platelets. J Clin Invest, 120:3917–3922.

116.

Wang

, Hayes

, Jarocha

, Sim

, Harper

, Fuentes

, Sullivan

, Gadue

, Torok-Storb

, Marks

, French

, Poncz

. (2015). Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale. Blood, 125:3627–3636.

117.

, Zhu

, Liu

, Nan

, Zheng

, Liu

, Shi

, Chen

, Lv

, et al. (2013). Infusion of megakaryocytic progenitor products generated from cord blood hematopoietic stem/progenitor cells: results of the phase 1 study. PloS One, 8:e54941.

118.

Nakagawa

, Nakamura

, Nakajima

, Endo

, Dohda

, Takayama

, Nakauchi

, Arai

, Fukuda

, Eto

. (2013). Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes. Experimental Hematology, 41:742–748.

119.

Takayama

, Nishikii

, Usui

, Tsukui

, Sawaguchi

, Hiroyama

, Eto

, Nakauchi

. (2008). Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood, 111,(11)::5298–5306.

120.

Thon

, Mazutis

, Wu

, Sylman

, Ehrlicher

, Machlus

, Feng

, Lu

, Lanza

, Neeves

, Weitz

, Italiano Jr

. (2014). Platelet bioreactor-on-a-chip. Blood, 124:1857–1867.