Mimicking megakaryopoiesis in vitro using biomaterials: Recent advances and future opportunities.

Presently donor-derived platelets used in the clinic are associated with concerns about adequate availability, expense, risk of bacterial contamination and complications due to immunological reaction. To prevail over our dependence on transfusion of donor-derived platelets, efforts are being made to generate them in vitro. Development of biomaterials that support or mimic bone marrow niche micro-environmental cues could improve the in vitro production of platelets from megakaryocytes (MKs) derived from various stem cell sources. In spite of significant advances in the production of MKs from various stem cell sources using 2D as well as 3D culture approaches in vitro and the development of biomaterials-based platelet systems, yield and quality of these platelets remains unsuitable for clinical use. Thus, in vitro production of clinically useful platelets on a large scale remains an unmet target to date. This review summarizes the most frequently used 2D and 3D approaches to generate MKs and platelets in vitro, emphasizing the importance of mimicking in vivo micro-environment. Further, this review proposes the use of interpenetrating network (IPN) biomaterial-based approach as a promising strategy for improving the generation of MK and platelets in sufficient numbers in vitro. STATEMENT OF SIGNIFICANCE: Thrombocytopenia is one of the major global health and socio-economic problems. Transfusion with donor-derived platelets (PLTs) is the only effective treatment for this condition. However, this approach is limited by factors like short shelf-life of PLTs, PLT activation, alloimmunization, risk of bacterial contamination, infection etc. In vitro generated MKs and PLTs derived from non-donor-dependent sources may help to overcome the platelet transfusion concerns. Here we have reviewed various 2D and 3D strategies used for in vitro generation of MKs and PLTs, with special emphasis on various biomaterial platforms and different physico/chemical cues being used for the purpose. We have also proposed a biomaterial-based approach of using interpenetrating network (IPN) for generating clinically relevant numbers of MKs and PLTs.

Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice.

Heterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor α (TNF-α) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy.

Scalable generation of universal platelets from human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the ß2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Polyunsaturated fatty acids confer cryoresistance on megakaryocytes generated from cord blood and also enhance megakaryocyte production from cryopreserved cord blood cells.

BACKGROUND AIMS: Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated. METHODS: MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34(+) cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5-3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2-5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples. CONCLUSIONS: Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.

Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium.

BACKGROUND AIMS: Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. METHODS: Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. RESULTS: The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. CONCLUSIONS: Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.