Erythroid Culture Based on Differentiation of Progenitor Cells from Leftover Buffy Coats Produced in the Reveos Automated Blood Processing System

Background: Efforts to culture red blood cells (RBCs) for transfusion purposes are ongoing in numerous research laboratories worldwide. In addition, smaller-scale erythroid culture is one of many approaches toward better understanding of erythropoiesis, enucleation and RBC biology. Our center recently changed from traditional blood component separation to the Reveos(R) automated blood- processing system. Most buffy coats (BCs) after whole- blood donations were previously used for platelet production but leftover BCs are now discarded. BCs are still popular among scientists, mainly as a source of peripheral leukocytes but we asked the question if these leftover bags could be useful for erythroid culture. Aims: We aimed to set up and evaluate a protocol for in vitro erythroid culture of CD34+ progenitor cells extracted from Reveos(R) BCs. Methods: Total CD34+ cells in Reveos(R) BCs were measured using flow cytometry and the BD Cell Enumeration Kit. Following pre-enrichment and fractionation on a density gradient, the mononuclear cells were labeled with anti-CD34 and separated by magnetic particles using an EasySep™ magnet. Culture conditions for the extracted CD34+ cells were divided into phases I-III, each lasting for 7 days. Phase I is an expansion phase, phase II an erythroid differentiation phase and phase III a terminal differentiation phase. StemSpan™ SFEM II culture medium containing bovine serum albumin, human insulin and iron-saturated human transferrin was used in all phases. In phase I, SFEM II was supplemented with SCF, IL-3, FLT3L, TPO and dexamethasone, in phase II with SCF, IL-3, EPO and dexamethasone and in phase III with 30% serum, EPO and holo-transferrin. Fetal bovine serum (FBS) or human serum (HS) were used initially in parallel cultures, to compare their influence on proliferation rate and terminal differentiation. In all cultures, the total number of cells and cell viability were determined by cell counting in a Bürker chamber after trypan blue dye staining. The cultures were carefully monitored and diluted to 0.4 × 106/ml when entering phase II. Erythroid differentiation was analyzed on day 19, 21 and 23 by flow cytometry with anti-GPA-APC, anti-Band3-PE and anti- CD49d-PE-Cy7. The frequency of erythroblasts and enucleated cells present in the cultures were counted on May-Grünwald-Giemsa stained cytospin samples. Results: We found the CD34+ cell content by flow cytometry in BCs (n = 11, volume of 9.5-13.5 ml) to be 0.2-1.5 × 106 with a mean of 93% viability. Thereafter progenitor cells were isolated and subjected to a 3-phase erythroid culture procedure to evaluate their potential to expand and differentiate. Cells cultured in 30% HS in phase III were less viable but showed slightly higher co-expression of CD49d and Band 3 compared to cultures grown in 30% FBS (86% vs 75% on day 19, and 95% vs 86% on day 21), as well as a higher proportion Band 3-positive, CD49d-negative cells, indicating an increased proportion of late erythroblasts and reticulocytes. In order to promote end-stage maturation, HS cultures were favored. After 23 days in culture with HS in phase III, 8-21% of the cells were enucleated and the cultures contained ∼60% erythroblasts. Summary/Conclusions: Extraction of CD34+ hematopoietic progenitors at acceptable viability from leftover Reveos(R) BCs is feasible and convenient. Despite their small volume and the processing cycle, these BCs provide suitable starting material for small-scale, experimental erythroid cultures directly from single donors. Conditions in this culture model can be fine-tuned further to focus on different aspects of erythropoiesis

A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA

Recombinant α 1-Microglobulin (rA1M) Protects against Hematopoietic and Renal Toxicity, Alone and in Combination with Amino Acids, in a 177Lu-DOTATATE Mouse Radiation Model.

177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α 1-microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse 177Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post- 177Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177Lu-DOTATATE

Human radical scavenger α1-microglobulin protects against hemolysis in vitro and α1-microglobulin knockout mice exhibit a macrocytic anemia phenotype

During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α1-microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M-/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific

Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells

Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.status: publishe

Yippee like 4 (Ypel4) is essential for normal mouse red blood cell membrane integrity

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity