Osmotic parameters of red blood cells from umbilical cord blood

The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (L_p), permeability to cryoprotectant glycerol (P_glycerol), and corresponding Arrhenius activation energies (E_a). For L_p and P_glycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. L_p and P_glycerol were characterized at 4 °C, 20 °C, and 35 °C using Jacobs and Stewart equations with the E_a calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher E_a for L_p and a lower E_a for P_glycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.     

Osmotic tolerance limits of red blood cells from umbilical cord blood

Effective methods for long-term preservation of cord red blood cells (RBCs) are needed to ensure a readily available supply of RBCs to treat fetal and neonatal anemia. Cryopreservation is a potential long-term storage strategy for maintaining the quality of cord RBCs for the use in intrauterine and neonatal transfusion. However, during cryopreservation, cells are subjected to damaging osmotic stresses during cryoprotectant addition and removal and freezing and thawing that require knowledge of osmotic tolerance limits in order to optimize the preservation process. The objective of this study was to characterize the osmotic tolerance limits of cord RBCs in conditions relevant to cryopreservation, and compare the results to the osmotic tolerance limits of adult RBCs. Osmotic tolerance limits were determined by exposing RBCs to solutions of different concentrations to induce a range of osmotic volume changes. Three treatment groups of adult and cord RBCs were tested: (1) isotonic saline, (2) 40% w/v glycerol, and (3) frozen–thawed RBCs in 40% w/v glycerol. We show that cord RBCs are more sensitive to shrinkage and swelling than adult RBCs, indicating that osmotic tolerance limits should be considered when adding and removing cryoprotectants. In addition, freezing and thawing resulted in both cord and adult RBCs becoming more sensitive to post-thaw swelling requiring that glycerol removal procedures for both cell types ensure that cell volume excursions are maintained below 1.7 times the isotonic osmotically active volume to attain good post-wash cell recovery. Our results will help inform the development of optimized cryopreservation protocol for cord RBCs.     

K(+) transport in red blood cells from human umbilical cord

The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.     

Results of unrelated umbilical cord blood hematopoietic stem cell transplantation

The number of umbilical cord blood transplants is increasing worldwide. The purpose of Eurocord is to evaluate the results and to compare the outcome of umbilical cord blood transplants with allogeneic bone marrow transplants. Data have been reported to Eurocord by multiple transplant centers. Close links have been established with the cord blood banks through Netcord. Bone marrow transplant data have been provided by transplant centers and also through the European Group for Blood and Marrow Transplantation (EBMT) and International Bone Marrow Transplant Registries (IBMTR). Eurocord has analyzed the outcome of unrelated umbilical cord blood transplants from 121 transplant centers and 29 countries. The results showed that survival with unrelated mismatched umbilical cord blood transplants was comparable to that with unrelated bone marrow transplants. Engraftment with cord blood was delayed, resulting in an increased incidence of early transplant complications. The incidence of acute and chronic graft-vs.-host disease was reduced with cord blood grafts even in human leukocyte antigen (HLA)-mismatched transplants and in adults. In patients with leukemia, the rate of relapse was similar to the rate of relapse after bone marrow transplant. The overall event-free survival with umbilical cord blood transplantation was not statistically different when compared to bone marrow transplants. This large registry study confirms the potential benefit of using umbilical cord blood hematopoietic stem cells for allogeneic transplants.     

Quality comparison of umbilical cord blood cryopreserved with conventional versus automated systems

Umbilical cord blood (CB) banks usually freeze and store CB for clinical transplantation using conventional controlled-rate freezer or the automated BioArchive system. The aim of this study is to compare the quality of CB cryopreserved with conventional and automated methods and to make clear the cause of the quality difference between the two methods. The experiment used 80 CB units: 40 were conventionally cryopreserved and the remainder were cryopreserved with a BioArchive. After thawing, the following measures of CB quality were compared: recovery rates of cell count, cell viability of total nucleated cells (TNCs), mononuclear cells (MNCs), and CD34+ cells, as well as colony-forming unit-granulocyte/macrophage (CFU-GM) content. Additionally, processing and storage records were reviewed to quantify the number of exposures of CB units at room temperature (transient warming event, TWE), which was analyzed in relation to CB quality. MNC and CD34+ cell viability were as follows: MNC, 78.2% ± 6.8% (conventional), 81.7% ± 7.2% (automated); CD34+ cell, 90.6% ± 6.9% (conventional), 94.7% ± 3.5% (automated). The absolute CFU-GM content per CB unit was 7.1 × 10⁵ ± 5.9 × 10⁵ with conventional cryopreservation and 12.3 × 10⁵ ± 12.0 × 10⁵ with automated cryopreservation. CBs cryopreserved with BioArchive showed significantly higher MNC and CD34+ cell viability, and CFU-GM content than those conventionally cryopreserved. The CB quality comparison depending on the amount of TWEs showed no significant quality difference between groups that were more exposed to TWEs and groups that were less exposed. CBs cryopreserved with BioArchive were of higher quality than conventionally cryopreserved CBs, and the cause of quality difference might be due to the difference of freezing conditions rather than the TWE effect.     

脐带血移植的应用进展及脐带血库建设

脐带血(umbilical cord blood)作为公认的造血干细胞重要来源之一,已经被广泛地用于治疗儿童和成人的良恶性血液系统疾病以及中枢神经系统疾病、实体瘤、缺血性下肢血管病和组织再生等。相对于骨髓移植和外周血来源的造血干细胞移植,脐带血移植(UCBT)在细胞收集使用、干细胞增殖能力以及移植物抗宿主反应等方面都具有明显的优势。目前的数据显示,因为HLA配型等原因而无法进行骨髓移植的患者应该尽早进行UCBT。此外,UCBT的增多促进了脐带血库的快速建设。本文针对UCBT和脐带血库的最新进展进行了综述。     

Fatty acid values in the plasma of neonates, umbilical cord and maternal blood

The proportion of a wide range of fatty acids was studied in the plasma of 15 healthy newborn infants following a physiological pregnancy and delivery. The same measurements were done in seven healthy mothers (immediately after parturition) and the proportion of fatty acids was analysed in mixed umbilical cord blood (n = 7). The fatty acids were identified by gas chromatography as their methyl esters (FAME). In newborn infants the proportion of saturated fatty acids was found to be 42.3%, of monoene fatty acids 31.3% and of polyene fatty acids 25.4%; type n-6 fatty acids formed 13.9% and n-3 11.1%. The proportion of the various fatty acids in healthy maternal plasma was 37.9% (saturated), 34.4% (monoene) and 25.0% (polyene) respectively; type n-6 fatty acids formed 21.6% and type n-3 only 3.6%. The values in mixed cord blood were 44% (saturated FA), 34.8% (monoene FA) and 20% (polyene FA); group n-6 FA accounted for 17.4% and n-3 for only 2.1%. In the above three series we also described the sequence of the fatty acids present in the largest amounts. This is part of an extensive study, in a large series, of the commonest perinatal risks. We particularly draw attention to the high proportion of long-chain fatty acids (C 22 - C 26) in the plasma of healthy newborn infants.     

人脐血血浆外泌体提取方法的比较北大核心CSCD

为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。     

Fucosylated umbilical cord blood hematopoietic stem cell expansion on selectin-coated scaffolds

Despite the advantages of transplantation of umbilical cord blood's (UCB's) hematopoietic stem cells (uHSCs) for hematologic malignancy treatment, there are two major challenges in using them: (a) Insufficient amount of uHSCs in a UCB unit; (b) a defect in uHSCs homing to bone marrow (BM) due to loose binding of their surface glycan ligands to BM's endothelium selectin receptors. To overcome these limitations, after poly l -lactic acid (PLLA) scaffold establishment and incubation of uHSCs with fucosyltransferase-VI and GDP-fucose, ex vivo expansion of these cells on selectin-coated scaffold was done. The characteristics of the cultured fucosylated and nonfucosylated cells on a two-dimensional culture system, PLLA, and a selectin-coated scaffold were evaluated by flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming unit (CFU) assay, and CXCR4 expression at the messenger RNA and protein levels. According to the findings of this study, optimized attachment to the scaffold in scanning electron microscopy micrograph, maximum count of CFU, and the highest 570 nm absorption were observed in fucosylated cells expanded on selectin-coated scaffolds. Furthermore, real-time polymerase chain reaction showed the highest expression of the CXCR4 gene, and immunocytochemistry data confirmed that the CXCR4 protein was functional in this group compared with the other groups. Considered together, the results showed that selectin-coated scaffold could be a supportive structure for fucosylated uHSC expansion and homing by nanotopography. Fucosylated cells placed on the selectin-coated scaffold serve as a basal surface for cell–cell interaction and more homing potential of uHSCs. Accordingly, this procedure can also be considered as a promising technique for the hematological disorder treatment and tissue engineering applications.     

Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.     

Biochemical stabilization enhances red blood cell recovery and stability following cryopreservation

Glycerolized red blood cells (RBC) are approved for long-term cryopreservation. However, the need to remove the glycerol cryoprotectant prior to transfusion has limited the usefulness of this cryopreservation method. This report describes using non-cryoprotectant biochemical stabilization techniques to substitute for the standard glycerol cryoprotectant. The glycerolized RBC method was compared to a newly developed LC-V method that combines transfusable cryoprotectants (hydroxyethyl starch and dextran) and specific non-cryoprotectant biochemical stabilizers (nicotinamide, nifedipine, and flurbiprofen). Results demonstrate that the biochemical stabilizers significantly reduce cryopreservation-induced hemolysis compared to cryopreservation in their absence and that thaw hemolysis levels approach those of standard 40% (w/v) glycerolized RBC (3.1+/-0.2% for 40% glycerol compared to 8.7+/-0.9% for the LC-V protocol). Furthermore, LC-V cryopreserved RBC exhibit a significantly enhanced post-thaw stability compared to glycerolized RBC as determined by osmotic fragility index (0.557+/-0.034 for 40% glycerol compared to 0.478+/-0.016 for the LC-V protocol). Analysis of biochemically stabilized RBC proteins revealed a transient translocation of carbonic anhydrase to the membrane fraction. However, the enhanced RBC recovery and stability could not be attributed to this event. Finally, DSC analysis demonstrated that the biochemical stabilizers of the LC-V process were not functioning as surrogate cryoprotectants in that they did not affect the quantity or quality of ice formed. Overall, this work demonstrates that cryopreservation-induced RBC damage may be corrected or prevented through specific biochemical stabilization and represents a significant step toward a directly transfusable cryopreserved RBC product.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.