Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors.
ABSTRACT: Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7?×?107 MKs per 50?ml medium. Laminin-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion. In vitro generated MKs represent a promising alternative to donor PLTs and open the possibility for the development of innovative MK-based cell therapies.
Project description:Platelet (PLT) transfusion is indispensable to maintain homeostasis in thrombocytopenic patients. However, PLT transfusion refractoriness is a common life-threatening condition observed in multi-transfused patients. The most frequent immune cause for PLT transfusion refractoriness is the presence of alloantibodies specific for human leukocyte antigen (HLA) class I epitopes. Here, we have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source for the generation of low immunogenic cell products. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In this study, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder-free and xeno-free conditions. On day 19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. Thus in vitro produced low immunogenic MKs and PLTs may become an alternative to PLT donation in PLT-based therapies and an important component in the management of severe alloimmunised patients.
Project description:Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin "in vivo" and "in vitro" not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1.
Project description:In-vitro-derived platelets (PLTs) could potentially overcome problems associated with donated PLTs, including contamination and alloimmunization. Although several groups have produced functional PLTs from stem cells in vitro, the challenge of developing this technology to yield transfusable PLT units has yet to be addressed. The asynchronous nature of in vitro PLT generation makes a single harvest point infeasible for collecting PLTs as soon as they are formed. The current standard of performing manual centrifugations to separate PLTs from nucleated cells at multiple points during culture is labor-intensive, imprecise, and difficult to standardize in accordance with current Good Manufacturing Practices (cGMP). In an effort to develop a more effective method, we adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs from nucleated cells and recover immature megakaryocytes (MKs), the precursor cells to PLTs, for continued culture. Processing a mixture of in-vitro-derived MKs and PLTs on the adapted device yielded a pure PLT population and did not induce PLT pre-activation. MKs recovered from the separation process were unaffected with respect to viability and ploidy, and were able to generate PLTs after reseeding in culture. Being able to efficiently harvest in-vitro-derived PLTs brings this technology one step closer to clinical relevance.
Project description:Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.
Project description:An Aberration in megakaryopoiesis and thrombopoiesis, 2 important processes that maintain hemostasis, leads to thrombocytopenia. Though platelet transfusions are used to treat this condition, blood banks frequently face a shortage of platelets. Therefore, methods to generate platelets on a large scale are strongly desirable. However, to generate megakaryocytes (MKs) and platelets (PLTs) in numbers sufficient for clinical application, it is essential to understand the mechanism of platelet production and explore efficient strategies accordingly. We have earlier reported that the N-6 and N-3 poly-unsaturated fatty acids (PUFAs), Arachidonic acid (AA)/Docosahexanoic acid (DHA) have beneficial effect on the generation of MKs and PLTs from umbilical cord blood derived CD34+ cells. Here we tested if a similar effect is observed with peripheral blood derived CD34+ cells, which are more commonly used in transplantation settings. We found a significant enhancement in cell numbers, surface marker expression, cellular ploidy and expression of cytoskeletal components during PLT biogenesis in cultures exposed to media containing AA/DHA than control cultures that were not exposed to these PUFAs. The test cells engrafted more efficiently in NOD/SCID mice than control cells. AA/DHA appears to have enhanced MK/PLT generation through upregulation of the NOTCH and AKT pathways. Our data show that PUFAs could be valuable additives in the culture system for large scale production of platelets for clinical applications.
Project description:In vitro-derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture.
Project description:BACKGROUND:Platelet (Plt)-derived extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular EC permeability, similar to fresh Plts. To investigate this hypothesis, we used in vitro and in vivo models of vascular endothelial compromise and bleeding. METHODS:In the vitro model, Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function were assessed by transendothelial electrical resistance measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts was assessed by multiple electrode Plt aggregometry. Using an in vivo model, the effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by an established Miles assay of vascular permeability and a tail snip bleeding assay. RESULTS:In the in vitro model, Plt-EVs displayed exosomal size distribution and expressed Plt-specific surface markers. Platelets and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced thrombin receptor-activating peptide-mediated aggregation of whole blood, whereas Plts enhanced thrombin receptor-activating peptide-, arachidonic acid-, collagen-, and adenosine diphosphate-mediated aggregation. In the in vivo model, Plt-EVs are equivalent to Plts in attenuating vascular endothelial growth factor (VEGF)-A-induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. CONCLUSION:Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability, and mitigate the endotheliopathy of trauma.
Project description:Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs.
Project description:BACKGROUND:Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS:Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22°C), cold (4°C, n = 6), or cryopreservation (-80°C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS:Platelet aggregation of cold-stored AS-PCT PLTs was 44%?±?11% compared to 57%?±?14% for cold-stored standard PLTs and 58%?±?21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53%?±?9% compared to 77%?±?6% for cold-stored standard PLTs and 69%?±?13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836?±?140?sec) compared to cold-stored standard PLTs (960?±?192?sec) and RT-stored AS-PCT PLTs (1134?±?220?sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297?±?47?nmol/L thrombin compared to 159?±?33?nmol/L for cold-stored standard PLTs and 83?±?25?nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23%?±?10%) was not significantly different from cryopreserved standard PLTs (25%?±?8%). CONCLUSION:This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation.
Project description:The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.