Recent lessons learned for ex-vivo platelet production.

Abstract

Purpose of review:

Platelet transfusion can be life-saving but carries a risk of infection or alloimmunization and is limited by insufficient donor sources and restricted unit shelf life. Generating sufficient platelets in vitro to replace a unit of collected blood remains a challenge. Here we examine the latest advances in the regulation of megakaryocyte maturation and expansion along with platelet formation and survival. We also discuss alternative therapies investigated to induce platelet production.

Recent findings:

Recent studies examined candidate niche cells in the bone marrow microenvironment for promoting platelet formation and developed an explant-based bioreactor to enhance platelet production ex-vivo. Chemical inhibitors were examined for their ability to promote megakaryocyte maturation and expansion. Microparticles from megakaryocytes or platelets were found to improve megakaryocyte maturation and platelet formation. Membrane budding was identified as a novel mode of platelet formation. Lastly, a chemical inhibitor to improve cold-stored platelets was identified.

Summary:

Recent advances in the regulation of megakaryocyte expansion and platelet production provide exciting promise for the development of improved approaches to generate platelets in vitro. These findings bring the field one step closer to achieving the ultimate goal of creating a unit of platelets without the need for donation.

INTRODUCTION

Every year, more than 2 million units of allogeneic platelets are transfused in the United States for indications such as thrombocytopenia, cancer or genetic disorders[1]. Finding sufficient sources of donor platelets is challenging due to an increasing need for units as a result of a growing population of aged adults[2] along with a limited unit shelf-life of 5–7 days when stored at room temperature[3]. Additionally, units carry the risk of viral or bacterial infection[3, 4] necessitating costly safety testing[5]. Identification of new techniques to generate platelets ex-vivo would greatly enhance the ability to meet clinical demands, assist in reducing the cost of safety testing, enhance transfusion medicine, and support patient treatment. Furthermore, the development of designer platelets could reduce the risk of alloimmunization or, in the case of genetic disorders, gene editing of cells could be conducted to correct the defect.

Many groups have investigated methods of generating platelets in vitro, using cell sources including human umbilical cord blood stem cells, induced pluripotent stem cells (iPS), embryonic stem cells (ES), megakaryocyte cell lines, and adipose tissue derived mesenchymal stem cells [6–12]. Using in vitro culture systems [13, 14] and live cell imaging in vivo[15–17], it was demonstrated that megakaryocytes can produce thin cytoplasmic proplatelet extensions containing beads resembling platelets. Furthermore, limited numbers of platelets can be shed under static culture, which can be improved with use of hydrodynamic forces[15]. Thus, various bioreactor designs were developed including microfluidic polydimethylsiloxane (PDMS) perfusion devices, 3D printed devices, 3D silk biomimetic models, along with a recent turbulence inducing bioreactor[18–24]. Nevertheless, the goal of achieving clinical numbers of generated platelets with full functionality and low baseline activation levels has yet to be fully realized. Here we present recent advances in the regulation of megakaryocyte maturation and expansion, as well as platelet formation and survival. We also discuss alternative therapies, which have been found to promote platelet production.

Role of the bone marrow microenvironment in platelet formation and activation

Within the bone marrow, the process of platelet formation begins with hematopoietic stem cells (HSC), that differentiate into megakaryocyte progenitor cells, which in turn further differentiate into immature megakaryocytes (Figure 1). While the signals that induce the differentiation of megakaryocytes remain incompletely understood, thrombopoietin (TPO) secreted systemically from the liver and locally by mesenchymal stromal cells (MSCs) was shown to be a key player in promoting this process[25–31]. MSCs, which line the outside of the vascular structures in the bone marrow secrete CXCL12, which promotes the migration of megakaryocyte progenitor cells towards the sinusoids[32]. Our group recently showed that these MSCs can also improve the maturation of megakaryocytes, leading to increased numbers of platelets, and assist in lowering the baseline activation levels of the produced platelets[10]. Other cytokines and chemokines were found to enhance megakaryopoiesis and platelet production but are reviewed elsewhere[33]. While precise mechanisms by which platelets are released into the circulation is unclear, high resolution in vivo imaging studies have demonstrated that some megakaryocytes are situated on the outer wall of the sinusoid and release proplatelets into the circulation, which are later broken down into individual platelets[15]. Megakaryocytes were also found to be present in the interstitial space of the lungs and locally release proplatelets into the circulation[16]. The relative release of platelets from the bone marrow versus that which occurs in the lungs remains to be further explored. Additional studies are necessary to understand the exact cellular players and associated signals involved in both the bone marrow and lungs that promote proplatelet formation and release.

Figure 1. Platelet formation process in the bone marrow.

Hematopoietic stem cells within the bone marrow undergo myeloid differentiation to form megakaryocyte erythroid progenitor (MEP) cells and immature megakaryocytes, followed by their maturation into multi-nucleated mature megakaryocytes. Mature megakaryocytes attach to the sinusoid wall and generate platelets via membrane budding and proplatelets which are released into the circulation. Neighboring mesenchymal stem cells provide both TPO and CXCL12 local signaling to enhance the maturation and migration of megakaryocytes towards the sinusoids. Endothelial cells lining the sinusoids are also a source of CXCL12 and contribute to the process of platelet production, which is reviewed elsewhere[62].

To further harness the role of the bone marrow microenvironment for improving platelet production in vitro, Fujiyama, S. et al examined the utility of a bone explant containing a central marrow cavity for serving as a novel platelet production bioreactor[34*]. In this study, dissected porcine thighbones were drilled at two points to create a perfusion inlet and outlet. Megakaryocytes were introduced into the bioreactor and allowed to incubate for 3 hours, followed by perfusion of the system and collection of the platelets for quantification. Interestingly, the bioreactor culture led to improved platelet yields per input megakaryocyte (4.41×10³) compared to megakaryocytes in standard culture (0.12 ×10³), and the platelets produced were able to activate in response to agonist stimulation[34*]. This bioreactor design differs from other attempts, in that continuous perfusion was not required to improve platelet production, suggesting that the improvement was due to signals from the local bone marrow microenvironment rather than the effects of mechanical stimulation due to perfusion.

Regulation of megakaryocyte maturation and expansion

Many studies have attempted to generate megakaryocytes in vitro from stem cells using approximations of the conditions natively found in vivo and these approaches have yielded limited numbers of platelets. Previous studies demonstrated that Bromodomain-containing proteins can promote murine HSC stem cell maintenance[35, 36] but whether they could affect megakaryopoiesis from human HSCs was previously unknown. Recently, the bromodomain and extra-terminal motif inhibitor CPI203 was found to improve the expansion of both human cord blood derived HSCs and megakaryocytes, and enhance megakaryocyte maturation[37*]. However, it remains to be seen whether platelet production in CPI203 treated megakaryocytes is altered, and whether produced platelets have changes in their baseline activation levels and functionality.

Megakaryocytes increase their size as they mature before generating proplatelets[38]. This expansion process is correlated with the formation of a demarcation membrane system, created through the invagination of the megakaryocyte plasma membrane along with the incorporation of released vesicles from the golgi apparatus and endoplasmic reticulum[13, 15, 39]. Phosphatidylinositol 3-monophosphate (PI3P) is important for regulating vesicle trafficking[40], although it was previously unknown whether PI3P could regulate megakaryocyte maturation. A recent study identified that PI3P was expressed in the late endosomes and lysosomes of mature megakaryocytes. These vesicles were found in the outer regions of the cell that localize together with the plasma membrane marker phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and contribute to membrane expansion[41]. Furthermore, inhibition or deletion of PI3P led to a marked decrease in proplatelet formation[41]. Whether PI3P mediated signaling can be pharmacologically targeted to enhance proplatelet formation in vitro remains to be explored.

Platelet derived microparticles, released during thrombosis and under pathologic conditions, contain cytokines, chemokines or RNA which is transferred to effector cells such as endothelial cells, immune cells or HSCs[42, 43]. Qu, M. et al recently showed that in response to acute liver injury, hematopoietic stem and progenitor cells can internalize platelet derived microparticles, which promote their differentiation into the megakaryocyte lineage[44**]. This process was found to be mediated by miR-1915–3p, which when internalized into the target cell, leads to the suppression of RHOB, and in turn promotes megakaryopoiesis in a TPO independent manner[44**]. Interestingly, the same process could be replicated by IV injection of platelet-derived microparticles into wild-type irradiated mice[44**], which opens up a novel avenue to pursue for modulating platelet production as a cell therapy product.

Megakaryocyte cell product and megakaryocyte derived microparticles for inducing platelet formation in vivo.

An alternative approach to overcome the shortcomings of platelet donation is the transfusion of non-platelet derived biologicals such as microparticles or megakaryocytes. Human megakaryocytic microparticles (huMkMPs) are CD41+CD42b+CD62P-, represent the most prevalent microparticles in the circulation, and differ from platelet-derived microparticles[45*]. Recently, huMkMPs were demonstrated to target human CD34+ HSCs, promote their differentiation into the megakaryocyte lineage, and lead to platelet production, without the need for TPO supplementation[45*]. Interestingly, IV injection of 2×10⁶ huMkMPs in mice let to a 50% increase in their platelet levels after 24 hours, though this level dropped to 27% after 72 hours[45*]. While the effect of huMkMPs in vivo on human HSCs has yet to be demonstrated, this approach may serve as an alternative therapy to quickly increase platelet counts among thrombocytopenic individuals when platelet units are scarce or unavailable.

A scaled-up umbilical cord blood CD34+ derived megakaryocyte product was developed yielding 3 × 10⁷ CD41+ megakaryocytes per starting unit of umbilical cord blood[46]. When infused into NOD/SCID IL2Rγnull mice (NSG), this megakaryocytic cell product led to the formation of functional human platelets persisting for 8 weeks[46]. Importantly, this product can be cryopreserved and the cryorecovered cells are able to maintain their phenotype and functionality[46]. While the total cellularity of the product was reduced after cryorecovery, no significant difference in the platelet yield was detected in vivo after infusion when compared to the fresh product.

Small molecule inhibitors consisting of Src kinase inhibitor SU6656, Rho-associated kinase inhibitor Y27632, and aurora B kinase inhibitor AZD1152 were examined for their ability to increase in vivo platelet yield per CD34+ derived megakaryocyte[47]. Following in vitro treatment of CD34+ derived megakaryocytes with one of these inhibitors, the treated megakaryocytes were IV infused into NSG mice[47]. While all inhibitors led to increased megakaryocyte ploidy, size and granularity, only SU6656 led to increased platelet yield per starting number of CD34+ cells, which was fourfold higher compared to untreated megakaryocytes. The effectiveness of SU6656 treated megakaryocytes in increasing platelet yield in humans remains to be demonstrated.[47]

Regulation of proplatelet formation and platelet survival

A number of key findings were recently uncovered in the biological regulation of proplatelet formation and platelet survival. The microRNA miR-125a-5p was recently demonstrated to regulate proplatelet formation both in vitro in human megakaryocytes and in vivo in genetic mouse models[48*]. MiR-125a-5p targets LCP1, which reduces the expression of L-plastin, responsible for regulating actin-bundling. While L-plastin expression regulates megakaryocyte progenitor cell migration, its expression was associated with decreased proplatelet formation and platelet numbers[48*]. Therefore, inhibiting L-plastin or miR-125a-5p may lead to increased platelet production in vitro.

G protein-coupled receptors are imperative for mediating platelet activation, and their signaling can be enhanced by regulators of G protein signaling (RGS)[49]. Interestingly, constitutive deletion of two RGS family signaling members RGS10 and RGS18 in mice led to increased baseline activation of platelets. Importantly, in addition to activation effects, deletion of both RGS10 and RGS18 led to decreased platelet counts and platelet lifespan [50*]. Whether the same effects can be phenocopied in human megakaryocytes remains to be explored. Small molecule agonists to enhance RGS 10 and RGS18 signaling may serve as an unexplored avenue for augmenting platelet production in vitro and survival in vivo.

Previous studies have demonstrated differences in proplatelet morphology and regulatory mechanisms between the process which occurs in vivo and that which occurs in the culture dish. A recent study further elucidated the genetic basis of these differences by examining live cell imaging in calvarial bone marrow of genetically modified mouse models compared to cultured platelets from these mice[51]. Using Myh9 −/− mice, myosin IIA was found to be important in vivo for maintaining the non-continuous process of proplatelet elongation involving pause and retraction phases, whereas in vitro it limited the number and complexity of proplatelets. Interestingly β-Tubulin knockout mice (Tubb1 −/−) could not extend proplatelet extrusions in vitro[51]. However in vivo, these knockout mice exhibited increased numbers of megakaryocytes with a 45% reduction in the number of protrusions and decreased circulating platelets compared to wild-type mice. Additionally, differences in microtubule organization and distribution were observed among in vivo formed proplatelets compared to those generated in vitro[51]. To further investigate the importance of microtubules for proplatelet extension, Myh9 −/− mice were treated with vincristine to induce microtubule depolymerization. Surprisingly, proplatelet size and elongation was not affected, suggesting this process is independent of microtubules and may be due to hemodynamic forces. These findings further highlight the complexity of the bone marrow microenvironment and the importance of shear stress in proplatelet formation. Other unidentified signals may resolve differences between in vivo mechanisms of platelet formation from that which occurs in vitro.

Using high resolution imaging of fetal and adult tissues, Potts, K.S. et al identified the formation of membrane buds on the surface of megakaryocytes, challenging the existing model of proplatelet formation serving as the major source of circulating platelets[52**]. Nuclear factor, erythroid 2 (NF-E2) was previously shown to be an important regulator of platelet formation in vivo. Fetal liver derived mouse megakaryocytes from Nfe2 −/− cannot form pro-platelets in vitro. Since Nfe2 −/− do not survive past early neonatal life, transplanted adult wild-type mice with Nfe2 knockout cells were previously found to lack circulating platelets[53–56]. Recently, Potts, K.S. et al re-examined these knock-out mice and found that platelet membrane budding was significantly reduced during development, whereas proplatelet formation was unchanged[52**]. This suggests that platelet budding largely contributes to the overall platelet volume in circulation. Furthermore, adult GFP+ mice transplanted with Nfe2 −/− fetal liver cells were found to have limited numbers of proplatelets in the bone marrow and spleen but importantly, displayed significantly fewer membrane buds. The lungs in the chimeric mice were found to mainly contain pro-platelet forming megakaryocytes and lacked the presence of membrane buds. Thus, abrogation of NFE2 leads to a drastic reduction in platelets formed by membrane budding which contributes to decreased numbers of circulating platelets [52**]. This study further highlights differences in the microenvironment for regulating platelet formation, where the in vivo bone marrow favors platelet budding compared to the lungs and in vitro cultures, which favor proplatelet formation.

Cold storage of platelets was previously investigated to overcome the restrictive shelf-life, risk of bacterial contamination, and accumulated storage lesion[57] associated with room temperature storage. However, after 24 hours at 4°C, platelets exhibit alterations in morphology, increased activation state, and rapid clearance in vivo [58, 59], limiting their suitability to trauma or severe bleeding cases where rapid platelet activation is beneficial. Platelet storage lesion and cold-storage of platelets were found to induce activation of p38 mitogen activated protein kinase (p38MAPK). Inhibition of p38MAPK in prolonged room temperature stored platelets led to improved maintenance of their functional, structural, and metabolic properties[60]. Recently, the p38MAPK chemical inhibitor VX-702 was investigated for its effect on prolonging the functionality of cold-stored platelets[61*]. Treatment of platelets with VX-702 led to decreased expression of baseline activation markers, and increased circulation time in vivo, while maintaining their ability to activate in response to agonist stimulation[61*]. Additional studies are necessary to fully evaluate the treatment’s effect on their in vivo functionality.

CONCLUSION

Recent advances have provided the field with invaluable mechanistic insight into the regulation of megakaryocyte maturation and expansion along with platelet formation and survival. Nevertheless, the microenvironment and process of platelet formation in vivo is complex, and to date, these processes remain incompletely understood. The relative distribution of megakaryocytes in the bone marrow versus that in the lungs and the respective contribution from each source to platelet formation remains unresolved. Identification of improved in vivo imaging methods for capturing platelet release from proplatelets or membrane buds, will help to characterize the relative contribution of each platelet form to the total composition of circulating platelets. Determining the optimal method of storing donated platelets to preserve their functionality and prevent activation will greatly improve transfusion medicine. Identification of these parameters will unveil a more comprehensive view of the process of platelet formation, and help to identify the ideal conditions for culturing platelets in vitro to improve platelet yields and quality.

Key points:

• Signaling from the bone marrow microenvironment is vital for promoting platelet production, even in the absence of added fluid stimulation.

• Both platelet-derived and megakaryocyte-derived microparticles can induce megakaryopoiesis and modulate platelet production.

• Platelet membrane budding may serve as an important source of circulating platelets.

• Improved understanding of the platelet formation process in vivo and mechanisms of platelet release from proplatelets or budded platelets could be essential for developing effective in vitro methods to increase platelet yield and functionality.