Carolyn Whitsett

Erythroid cells in vitro: from developmental biology to blood transfusion products.

Purpose of reviewRed blood cells (RBCs) transfusion plays a critical role in numerous therapies. Disruption of blood collection by political unrest, natural disasters and emerging infections and implementation of restrictions on the use of erythropoiesis-stimulating agents in cancer may impact blood availability in the near future. These considerations highlight the importance of developing alternative blood products. Recent findingsKnowledge about the processes that control RBC production has been applied to the establishment of culture conditions allowing ex-vivo generation of RBCs in numbers close to those (2.5 × 1012 cells/ml) present in a transfusion, from cord blood, donated blood units or embryonic stem cells. In addition, experimental studies demonstrate that such cells protect mice from lethal bleeding. Therefore, erythroid cells generated ex vivo may be suitable for transfusion provided they can be produced safely in adequate numbers. However, much remains to be done to translate a theoretical production of approximately 2.5 × 1012 RBCs in the laboratory into a ‘clinical grade production process’. SummaryThis review summarizes the state-of-the-art in establishing ex-vivo culture conditions for erythroid cells and discusses the most compelling issues to be addressed to translate this progress into a clinical grade transfusion product.

The Potential of Stem Cells as an In Vitro Source of Red Blood Cells for Transfusion

Recent advances have increased excitement about the potential for therapeutic production of red blood cells (RBCs) in vitro. However, generation of RBCs in the large numbers required for transfusion remains a significant challenge. In this article, we summarize recent progress in producing RBCs from various cell sources, and discuss the hurdles that remain for translation into the clinical arena.

Humanized culture medium for clinical expansion of human erythroblasts.

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.

The dominant negative β isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Use of sentinel sites for daily monitoring of the US blood supply

BACKGROUND : This report describes the first year of a government‐sponsored program that uses daily reports from 29 sentinel sites to monitor the capacity of the US blood supply to meet demand.

Concise Review: Stem Cell-Derived Erythrocytes as Upcoming Players in Blood Transfusion

Blood transfusions have become indispensable to treat the anemia associated with a variety of medical conditions ranging from genetic disorders and cancer to extensive surgical procedures. In developed countries, the blood supply is generally adequate. However, the projected decline in blood donor availability due to population ageing and the difficulty in finding rare blood types for alloimmunized patients indicate a need for alternative red blood cell (RBC) transfusion products. Increasing knowledge of processes that govern erythropoiesis has been translated into efficient procedures to produce RBC ex vivo using primary hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Although in vitro‐generated RBCs have recently entered clinical evaluation, several issues related to ex vivo RBC production are still under intense scrutiny: among those are the identification of stem cell sources more suitable for ex vivo RBC generation, the translation of RBC culture methods into clinical grade production processes, and the development of protocols to achieve maximal RBC quality, quantity, and maturation. Data on size, hemoglobin, and blood group antigen expression and phosphoproteomic profiling obtained on erythroid cells expanded ex vivo from a limited number of donors are presented as examples of the type of measurements that should be performed as part of the quality control to assess the suitability of these cells for transfusion. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements. Stem Cells2012;30:1587–1596

Assessment of blood administration procedures:problems identified by direct observationand administrative incident reporting

BACKGROUND: Adverse events in blood administration frequently involve the identification of transfusion recipients or components. This report details the results of an investigation of the efficacy of direct observation and that of a hospital‐wide incident‐reporting system in detecting standard operating procedures (SOPs) for deviations in blood administration.

Ex-vivo expansion of red blood cells: How real for transfusion in humans?

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5×10(12) cells vs 10(7) cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion

Cultures of human CD34pos cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169pos macrophages established multiple rapid ‘loose’ interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169neg macrophages established ‘tight’ interactions with mature erythroblasts and phagocytosed these cells. ‘Loose’ interactions of CD169pos macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death☆

The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.