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1.  In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art 
Transfusion medicine reviews  2010;24(1):33-43.
The exciting and extraordinary capabilities of stem cells to proliferate and differentiate into numerous cell types not only offers promises for changing how diseases are treated, but may also impact how transfusion medicine is practiced in the future. The possibility of growing platelets in the laboratory to some day supplement and/or replace standard platelet products has clear advantages for blood bank centers and patients. Due to the high utilization of platelets by patients undergoing chemotherapy or receiving stem cell transplants, platelet transfusion has steadily increased over the past decades. This trend is likely to continue as the number of adult and pediatric patients receiving stem cell transplants is also continuously rising. As a result of increased demand coupled with the short shelf-life of platelet concentrates, providing platelets to patients can stretch the resources of most blood centers, drive donor recruitment efforts, and on occasion platelet shortages can compromise the care of thrombocytopenic patients.
The purpose of this article is to review current scientific progress to develop in vitro strategies to manufacture platelets, with an emphasis on efforts to produce functional platelets in quantities that would be required for clinical transfusion. There are a number of publications indicating that human platelets can be obtained in vitro from the controlled differentiation of hematopoietic stem cells. However, the hemostatic quality of such manufactured platelets has not been confirmed and current technologies are inadequate to ensure satisfactory expansion and platelet biogenesis on an industrial scale. Nonetheless, these studies provide proof-of-principle that developing in vitro strategies to manufacture platelets is feasible and also provide a foundation for developing more sophisticated approaches to achieve this goal.
PMCID: PMC2790431  PMID: 19962573
Megakaryocyte; Stem cells; Megakaryocytopoiesis; Thrombocytopoiesis; ex vivo cultures; Platelet; Platelet biogenesis; Hematopoiesis
2.  Dynamin 3 Participates in the Growth and Development of Megakaryocytes 
Experimental hematology  2008;36(12):1714-1727.
High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture derived megakaryocytes (MKs). As previously published, 3 replicate microarray data sets from 3 different sources of organ donor marrow were analyzed using the software program Rosetta Resolver® [1]. After setting a stringent p-value of ≤0.001 with a fold change cut-off of ≥3 in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2±2.1 (mean±S.D.). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2 & DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time qRT-PCR confirmed that DNM3 increased by 20.7±3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in pro-platelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from cultured derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Over expression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony forming cells (CFCs) and colony forming unit-megakaryoyctes (CFU-MKs), and a concomitant increase in the expression of NFE-2 and β1 tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.
PMCID: PMC2728587  PMID: 19007685
3.  Distinct Functional Effects for Dynamin 3 During Megakaryocytopoiesis 
Stem Cells and Development  2011;20(12):2139-2151.
Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41+ and CD61+ MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.
PMCID: PMC3225063  PMID: 21671749
4.  A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures 
Biopreservation and Biobanking  2011;9(4):355-362.
Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem–Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (Vb=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, −5°C, −10°C, and −20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, −10, −20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10−3 cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C–12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.
PMCID: PMC3247705  PMID: 22232706
5.  A Steady-State Mass Transfer Model of Removing CPAs from Cryopreserved Blood with Hollow Fiber Modules 
Hollow fiber modules are commonly used to conveniently and efficiently remove cryoprotective agents (CPAs) from cryopreserved cell suspensions. In this paper, a steady-state model coupling mass transfers across cell and hollow fiber membranes is theoretically developed to evaluate the removal of CPAs from cryopreserved blood using hollow fiber modules. This steady-state model complements the unsteady-state model which was presented in our previous study. As the steady-state model, unlike the unsteady-state model, can be used to evaluate the effect of ultrafiltration flow rates on the clearance of CPAs. The steady-state model is validated by experimental results and then is compared with the unsteady-state model. Using the steady-state model, the effects of ultrafiltration flow rates, NaCl concentrations in dialysate, blood flow rates and dialysate flow rates on CPA concentration variation and cell volume response are investigated in detail. According to the simulative results, the osmotic damage of red blood cells (RBCs) can easily be reduced by increasing ultrafiltration flow rates, increasing NaCl concentrations in dialysate, increasing blood flow rates or decreasing dialysate flow rates.
PMCID: PMC2882658  PMID: 20524740
Mass Transfer; Cryoprotective Agent; Ultrafiltration; Cell Volume; Hollow Fiber

Results 1-5 (5)