Key Points Abnormal signatures in TGF-β1 signaling gene expression were identified in spleen and marrow from the Gata1low model of MF. These signatures include abnormalities in individual gene (Id2, Stat1, mTOR) in spleen and of gene pathways (Smads and BMPs) in marrow.
Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6–12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10−6 M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMAser were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMAdef). HEMAdef sustained erythroid amplification as efficiently as HEMAser for cord blood MNC and 10-fold higher than HEMAser for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMAdef by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.
© F e r r a t a S t o r t i F o u n d a t i o nIn this study we showed that human PBMC-derived CD14 + Methods Cell sortingCD3, CD19, CD14 and CD34 MicroBeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were used for magnetic-activated cell sorting (MACS) from PBMC (manufacturer's protocol). Prior to sorting, monocytes/macrophages were purified from PBMC by counterflow centrifugal elutriation (JE-6B Beckman-Coulter centrifuge, Beckman Instruments Inc.; Palo Alto, CA, USA). Monocyte/macrophage subsets and hematopoietic precursors were sorted on a FACS-Aria II/III (BD Biosciences; Oxford, UK). Cell cultureHuman cells were cultured in StemSpan (Stem Cell Technologies; Grenoble, France) supplemented with stem cell factor (SCF; supernatant equivalent to 100 ng/mL), erythropoietin (2 U/mL, ProSpec; East Brunswick, NJ, USA), dexamethasone (1 μM, Sigma; St. Louis, MO, USA) and cholesterol-rich lipids (40 μg/mL, Sigma) as described elsewhere.14,15 Informed consent was given in accordance with the Declaration of Helsinki and Dutch national and Sanquin internal ethic boards. Conditioned media were collected from CD14 + cells cultured for 2 days at 5-10x10 6 cells/8 mL, filtered (0.22 μm) and stored at 4°C. Isolated CD34 + cells were cultured with conditioned media diluted 1:2 with fresh culture medium. The media were replenished every 2 days. Co-culture experiments CD34+ cells were co-cultured with purified hematopoietic effector cells using ratios found in PBMC (1:100 CD14 Transwell assays CD14+ and CD34 + cells were seeded into transwells (0.4 µm polyester membrane, Corning; NY, USA) with CD34 + cells inside the transwell and CD14 + cells in the well (at a ratio of 1:100). Cells were analyzed after 2-8 days on the flow cytometer. Colony assaysColony assays were started with freshly purified, sorted, or cultured cells mixed with methacult (Medium ColonyGel ™ Cell Systems; Troisdorf, Germany). After 14 days, total colony numbers and colony-forming units were manually scored twice using a wide-field microscope (Axiovert 200M, Carl Zeiss Inc.; Thornwood, NY, USA). Proliferation assays CD34+ cells were washed in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA, Sigma; PSA). Cells were labeled with 0.5 μM carboxyfluoroscein succinimidyl ester (CFSE, Molecular Probes; Leiden, The Netherlands) for 8 min with gentle shaking at 37°C. Cells were washed in 1% PSA and cultured with or without CD14+ cells (at a ratio of 1:100) for 2 days. CFSE dilution was measured by flow cytometry. Flow cytometryCells were washed in PBS and re-suspended in 1% PSA. Cells were incubated with primary antibodies for 30 min at 4°C, washed in PBS and measured on a FACS Canto II or LSR Fortessa (both BD Biosciences) and analyzed using FlowJo software (FlowJo v7.6.4/v10; Ashland, OR, USA). The antibodies are detailed in the Online Supplementary Methods. For apoptosis experiments, cells were stained in 100 μL binding buffer with annexin V-APC (BD Biosciences; 1:200) at 4°C. After 30 min, 100 μL binding buffer and...
Coronavirus disease 2019 (COVID-19), a viral respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been recently recognized as a systemic disorder inducing a prothrombotic state. The molecular mechanisms underlying the hypercoagulable state seen in patients with COVID-19 is still incompletely understood, although it presumably involves the close link between inflammatory and hemostatic systems. The laboratory coagulation monitoring of severely ill COVID-19 patients is mandatory to identify those patients at increased thrombotic risk and to modulate thromboprophylaxis accordingly. In this review, we summarize the current understanding on the pathogenesis, epidemiology, clinical and laboratory features and management of coagulopathy associated with COVID-19.
Key Points Elevation of HbF in 3 patients heterozygous for distinct 2p15-p16.1 syndrome microdeletions affecting BCL11A. Identification of novel, putative regulatory elements downstream of BCL11A that govern its expression in erythroid cells.
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