Sijie Sun

In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art

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 may be 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 centers and patients. Because of the high utilization of platelets by patients undergoing chemotherapy or receiving stem cell transplants, platelet transfusions have 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 and drive donor recruitment efforts, and on occasion, platelet shortages can compromise the care of thrombocytopenic patients.

Distinct functional effects for dynamin 3 during megakaryocytopoiesis.

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.

Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders.

Three‐Dimensional Simulation of Mass Transfer in Artificial Kidneys

In this work, the three-dimensional velocity and concentration fields on both the blood and dialysate sides in an artificial kidney were simulated, taking into account the effects of the flow profiles induced by the inlet and outlet geometrical structures and the interaction between the flows of blood and dialysate. First, magnetic resonance imaging experiments were performed to validate the mathematical model. Second, the effects of the flow profiles induced by the blood and dialysate inlet and outlet geometrical structures on mass transfer were theoretically investigated. Third, the clearance of toxins was compared with the clearance value calculated by a simple model that is based on the ideal flow profiles on both the blood and dialysate sides. Our results show that as the blood flow rate increases, the flow field on the blood side becomes less uniform; however, as the dialysate flow rate increases, the flow field on the dialysate side becomes more uniform. The effect of the inlet and outlet geometrical structures of the dialysate side on the velocity and concentration fields is more significant than that of the blood side. Due to the effects of the flow profiles induced by the inlet and outlet geometrical structures, the true clearance of toxins is lower than the ideal clearance, especially when the dialysate flow rate is low or the blood flow rate is high. The results from this work are significant for the structural optimization of artificial kidneys and the accurate prediction of toxin clearance.

Expression of plasma membrane receptor genes during megakaryocyte development

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function.

A New Method to Increase the Adsorption of Protein‐Bound Toxins in Artificial Liver Support Systems

In this work, a new method, called the preconcentration method (PCM), is proposed to increase the adsorption of protein-bound toxins onto adsorbents in artificial liver support systems. In the PCM, a concentrator is installed before the inlet of the adsorbent cartridge. This method is validated in an experiment using activated carbon to remove albumin-bound bilirubin, and the mechanism of the increase in adsorption is theoretically explained with breakthrough curve and equilibrium adsorption analyses. Our results show that when this PCM is used, the mass transfer rate of bilirubin from solution to activated carbon is enhanced, the adsorbed bilirubin amount per unit mass of activated carbon is greatly increased, and more albumin-bound bilirubin molecules are quickly removed from the albumin solution. When the concentration ratio (the ratio of the inlet flow rate to the outflow rate of the concentrator) is 2.59, the adsorption efficiency of activated carbon at 120 min is increased by approximately 36%. Only approximately 60 min is required for the bilirubin concentration to decrease from 19.3 to 13.0 mg/dL; however, without the PCM, nearly 180 min is needed. In addition, by adjusting the concentration ratio, the adsorption of albumin-bound bilirubin onto activated carbon can be further increased.

Theoretical optimization of the removal of cryoprotective agents using a dilution-filtration system

BackgroundIn the cryopreservation of blood, removing cryoprotectants from the cryopreserved blood safely and effectively is always being focused on. In our previous work, a dilution-filtration system was proposed to achieve the efficient clearance of cryoprotectants from the cryopreserved blood.MethodIn this study, a theoretical method is presented to optimize the diluent flow rate in the system to further reduce the osmotic damage to red blood cells (RBCs) and shorten the washing time necessary to remove cryoprotective agents (CPAs), based on a discrete mass transfer concept. In the method, the diluent flow rate is automatically adjusted by a program code in each cycle to maximize the clearance of CPAs, whereas the volume of RBCs is always maintained below the upper volume tolerance limit.ResultsThe results show that the optimized diluent flow rate can significantly decrease the washing time of CPAs. The washing time under the optimized diluent flow rate can be reduced by over 50%, compared to the one under the fixed diluent flow rate. In addition, the advantage of our method becomes more significant when the blood flow rate is lower, the dilution region volume is larger, the initial CPA concentration is higher, or the cell-swelling limit set by the system is smaller.ConclusionThe proposed method for the dilution-filtration system is an ideal solution for not only guaranteeing the volume safety of RBCs but also shortening the washing time of CPAs. In practice, the optimization strategies provided here will be useful in the rapid preparation of cryopreserved blood for clinical use.

Statistical estimation of red blood cell osmotic damage during cryoprotective agent removal from cryopreserved blood

Statistical estimation of the osmotic damage of red blood cells (RBCs) during the removal of cryoprotective agents (CPAs) from cryopreserved blood has been a very difficult issue. In this paper, the discrete mass transfer model developed in our previous work is modified to study the volume variation of individual RBCs and thereby to estimate the osmotic damage of all RBCs statistically during CPA removal by the dilution-concentration method we proposed recently. The model is validated with respect to the experimental results either with or without RBCs. Then, it is used to investigate the effects of blood volume, hematocrit, blood and diluent flow rates on the osmotic damage of RBCs, as well as the washing time of CPAs. Our results show that both the increase of blood flow rates and the decrease of diluent flow rates can bring about a reduction in osmotic damage of RBCs; however, only the former can cause a decrease in the washing time of CPAs. The blood volume could also affect the osmotic damage of RBCs. For a given flow condition, there could exist an optimal blood volume range for the dilution-concentration system. The effect of blood volume could be alleviated by an increase in the dilution region volume. In addition, the osmotic damage of RBCs decreases as the hematocrit decreases. Therefore, in practice, the increase of blood flow rates is the best solution to reduce both the osmotic damage of RBCs and the washing time of CPAs simultaneously. A lower hematocrit in the cryopreserved blood and/or longer tubing in the dilution region are also recommended to achieve better performance for the dilution-concentration method.

Unloading of cryoprotectants from cryoprotectant-loaded cells on a microfluidic platform

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (CG), the survival rate of cells (SC) and the recovery rate of cells (RC) have been studied. Under the designed conditions, CG achieved ~80% and SC reached ~90%. However, RC was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high CG and SC, but low RC. For a low glycerin concentration, CG, SC, and RC were all high. For a high cell density, CG was high, but both SC and RC were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.

Fatigue damage to pig erythrocytes during repeated swelling and shrinkage.

During the removal of cryoprotectants from cryopreserved-thawed blood with the dialysis-based or dilution-filtration method, due to the change in the extracellular osmolality, erythrocytes usually undergo repeated swelling and shrinkage. However, the erythrocyte fatigue damage induced by this repeated volume change has not yet been studied. In this work, by successively loading hypotonic and hypertonic solutions, we mimicked the repeated swelling and shrinkage of pig erythrocytes and then examined the effect of the number of cycle loops on the steady-state volume and the mortality of the pig erythrocytes. The results suggest that because of cell leakage in the swelling process, the steady-state volume of the pig erythrocytes after one cycle is smaller than the volume before the cycle, even though the cell performs a self-protective regulatory procedure. If the number of cycle loops is increased, the repeated swelling and shrinkage will cause a continuous decrease in the steady-state volume, and the ability of the pig erythrocytes to resist osmotic damage will decrease; as a result, the mortality of the pig erythrocytes increases as the number of cycle loops increases. The viability of the cells is also affected by the hypotonic and isotonic processing times: a short processing time may contribute to a decrease in the mortality of the pig erythrocytes. This work is of significance to optimizing the process of removing cryoprotectants.