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

Cultures of human CD34(pos) 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. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) 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.

Mononuclear cells from a rare blood donor, after freezing under good manufacturing practice conditions, generate red blood cells that recapitulate the rare blood phenotype.

BACKGROUND: Cultured red blood cells (cRBCs) from cord blood (CB) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [AB]) may be used to generate cRBCs for transfusion has not been investigated. STUDY DESIGN AND METHODS: Erythroid progenitor cell content and numbers and blood group antigen profiles of erythroblasts (ERYs) and cRBCs generated in human erythroid massive amplification (HEMA) culture by CB (n = 7) and AB (n = 33, three females, three males, one AB with rare blood antigens cryopreserved using CB protocols) were compared. RESULTS: Variability was observed both in progenitor cell content (twofold) and number of ERYs generated (1 log) by CB and AB in HEMA. The average progenitor cell contents of the subset of AB and CB analyzed were similar. AB generated numbers of ERYs three times lower (p < 0.01) than CB in HEMA containing fetal bovine serum but similar to CB in HEMA containing human proteins. Female AB contained two times fewer (p < 0.05) erythroid progenitor cells but generated numbers of ERYs similar to those generated by male AB. Cryopreserved AB with a rare blood group phenotype and shipped to another laboratory generated great numbers of ERYs, 90% of which matured into cRBCs. Blood group antigen expression was consistent with the donor genotype for ERYs generated both by CB and AB but concordant with that of native RBCs only for cells derived from AB. CONCLUSION: Buffy coats from regular donors, including a donor with rare phenotypes stored under conditions established for CB, are not inferior to CB for the generation of cRBCs.

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.

Alternative blood products and clinical needs in transfusion medicine.

The primary focus of national blood programs is the provision of a safe and adequate blood supply. This goal is dependent on regular voluntary donations and a regulatory infrastructure that establishes and enforces standards for blood safety. Progress in ex vivo expansion of blood cells from cell sources including peripheral blood, cord blood, induced pluripotent stem cells, and human embryonic stem cell lines will likely make alternative transfusion products available for clinical use in the near future. Initially, alloimmunized patients and individuals with rare blood types are most likely to benefit from alternative products. However, in developed nations voluntary blood donations are projected to be inadequate in the future as blood usage by individuals 60 years and older increases. In developing nations economic and political challenges may impede progress in attaining self-sufficiency. Under these circumstances, ex vivo generated red cells may be needed to supplement the general blood supply.

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.

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.

Phenotypic definition of the progenitor cells with erythroid differentiation potential present in human adult blood.

In Human Erythroid Massive Amplification (HEMA) cultures, AB mononuclear cells (MNC) generate 1-log more erythroid cells (EBs) than the corresponding CD34(pos) cells, suggesting that MNC may also contain CD34(neg) HPC. To clarify the phenotype of AB HPC which generate EBs in these cultures, flow cytometric profiling for CD34/CD36 expression, followed by isolation and functional characterization (colony-forming-ability in semisolid-media and fold-increase in HEMA) were performed. Four populations with erythroid differentiation potential were identified: CD34(pos)CD36(neg) (0.1%); CD34(pos)CD36(pos) (barely detectable-0.1%); CD34(neg)CD36(low) (2%) and CD34(neg)CD36(neg) (75%). In semisolid-media, CD34(pos)CD36(neg) cells generated BFU-E and CFU-GM (in a 1 : 1 ratio), CD34(neg)CD36(neg) cells mostly BFU-E (87%) and CD34(pos)CD36(pos) and CD34(neg)CD36(low) cells were not tested due to low numbers. Under HEMA conditions, CD34(pos)CD36(neg), CD34(pos)CD36(pos), CD34(neg)CD36(low) and CD34(neg)CD36(neg) cells generated EBs with fold-increases of ≈9,000, 100, 60 and 1, respectively, and maturation times (day with >10% CD36(high)CD235a(high) cells) of 10-7 days. Pyrenocytes were generated only by CD34(neg)/CD36(neg) cells by day 15. These results confirm that the majority of HPC in AB express CD34 but identify additional CD34(neg) populations with erythroid differentiation potential which, based on differences in fold-increase and maturation times, may represent a hierarchy of HPC present in AB.

Recovery and Biodistribution of Ex Vivo Expanded Human Erythroblasts Injected into NOD/SCID/IL2Rγ mice.

Ex vivo expanded erythroblasts (EBs) may serve as advanced transfusion products provided that lodgment occurs in the macrophage-niche of the marrow permitting maturation. EBs expanded from adult and cord blood expressed the receptors (CXCR4, VLA-4, and P-selectin ligand 1) necessary for interaction with macrophages. However, 4-days following transfusion to intact NOD/SCID/IL2Rγ(null) mice, CD235a(pos) EBs were observed inside CD235a(neg) splenic cells suggesting that they underwent phagocytosis. When splenectomized and intact NOD/SCID/IL2Rγ(null) mice were transfused using retrovirally labeled human EBs, human cells were visualized by bioluminescence imaging only in splenectomized animals. Four days after injection, human CD235a(pos) cells were detected in marrow and liver of splenectomized mice but only in spleen of controls. Human CD235a(pos) erythrocytes in blood remained low in all cases. These studies establish splenectomized NOD/SCID/IL2Rγ(null) mice as a suitable model for tracking and quantification of human EBs in vivo.

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.

The dominant negative ß 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 GRß 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. GRß was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRß 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 ß-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ß-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 GRß. These data indicate that GRß 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.

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 (HEMA(ser)). 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 HEMA(ser) were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMA(def)). HEMA(def) sustained erythroid amplification as efficiently as HEMA(ser) for cord blood MNC and 10-fold higher than HEMA(ser) for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMA(def) 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.

Long-term storage does not alter functionality of in vitro generated human erythroblasts: implications for ex vivo generated erythroid transfusion products.

BACKGROUND: Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage. STUDY DESIGN AND METHODS: Using mononuclear cells from four adult blood donors, erythroblasts were generated ex vivo in expansion cultures supplemented with stem cell factor, interleukin-3, erythropoietin (EPO), dexamethasone, and estradiol. The viability and in vitro function of freshly expanded or short (1-2 months)- and long (8 years)-term-stored erythroblasts cryopreserved in dimethyl sulfoxide were compared. Erythroblast function was defined as ability to proliferate in expansion media and mature in response to EPO. Cell number was determined manually and expressed as fold increase. Viability was assessed by trypan blue and propidium iodide exclusion. Maturation was evaluated by morphologic analyses and CD36/CD235a expression profiling. Cytogenetic evaluation included karyotype and multicolor fluorescence in situ hybridization analyses. RESULTS: Equivalent numbers (>80%) of erythroblasts were viable after short- and long-term storage. Freshly expanded and short- and long-term-stored erythroblasts equally doubled in number (fold increase, 2.4) retaining an immature phenotype (23% of the cells were CD36(high)CD235a(neg)) when cultured for 4 days under expansion conditions. The numbers of freshly expanded and short-term-stored erythroblasts that matured when exposed for 4 days to EPO were also similar (approx. 22% of the cells became CD36(neg)CD235a(high)). In spite of the massive amplification, ex vivo generated erythroblasts demonstrated a normal (46,XY) karyotype with no obvious genomic rearrangements. CONCLUSION: Ex vivo expanded erythroblasts remain functional and genetically normal after long-term storage.

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

PURPOSE OF REVIEW: Red 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 FINDINGS: Knowledge 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 x 10 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 x 10 RBCs in the laboratory into a 'clinical grade production process'. SUMMARY: This 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.

Hormonal contraception, sickle cell trait, and risk for venous thromboembolism among African American women.

OBJECTIVE: We evaluated the effect of oral and other hormonal contraceptive (HC) use on venous thromboembolism risk among African American women and investigated whether the association was modified by the sickle cell trait. STUDY DESIGN: We report the findings of a case-control study that included 60 African American women with an idiopathic, first episode of venous thromboembolism and 196 African American controls. RESULTS: The odds of current HC use compared with noncurrent use contrasting cases and controls is 3.8 (95% confidence interval [CI], 1.7-8.1; P < .001). Among subjects with sickle cell trait, the odds ratio is higher (odds ratio [OR], 6.7; 95% CI, 1.0-43) than the odds ratio among subjects without sickle cell trait (OR, 2.6; 95% CI, 1.1-6.2), but the difference is not statistically significant. CONCLUSION: This study provides persuasive evidence that hormonal contraceptive use increases venous thromboembolism risk among African American women and that the increase in risk may be larger among women with sickle cell trait.

Sickle cell trait and the risk of venous thromboembolism among blacks.

People with sickle cell disease have a chronically activated coagulation system and display hemostatic perturbations, but it is unknown whether they experience an increased risk of venous thromboembolism. We conducted a case-control study of venous thromboembolism that included 515 hospitalized black patients and 555 black controls obtained from medical clinics. All subjects were assayed for hemoglobin S and hemoglobin C genotypes. The prevalence of the S allele was 0.070 and 0.032 for case patients and controls, respectively (P < .001). The odds that a patient had sickle cell trait were approximately twice that of a control, indicating that the risk of venous thromboembolism is increased approximately 2-fold among blacks with sickle cell trait compared with those with the wild-type genotype (odds ratio = 1.8 with 95% confidence interval, 1.2-2.9). The odds ratio for pulmonary embolism and sickle cell trait was higher, 3.9 (2.2-6.9). The prevalence of sickle cell disease was also increased among case patients compared with controls. We conclude that sickle cell trait is a risk factor for venous thromboembolism and that the proportion of venous thromboembolism among blacks attributable to the mutation is approximately 7%.

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. STUDY DESIGN AND METHODS: From August 15, 2001, to August 14, 2002, 29 sentinel sites provided daily reports of the number of units of RBCs in inventory, transfused, exported, and outdated by ABO and Rh, and platelets by random or apheresis donor. Days supply of each component category was calculated as the number of units in inventory reported on a day divided by the sum of units transfused, exported, and outdated on that day. Sites also provided daily responses to questions about threatened or actual shortages. RESULTS: The median of the days supply of RBCs at the 26 hospital transfusion services was 7.2 days. However, median days supply varied substantially by site and by day of the week. A+, O+, and O- units accounted for 30, 35, and 12 percent of total inventory and were maintained at a median supply of 7.4, 6.4, and 9.5 days, respectively. Reports of threatened RBC shortages peaked in early January 2002 and again in early July 2002. The July 2002 peak was about twice the January 2002 peak. Inventories at community-based centers were similar to those at hospital transfusion services. Hospitals maintained only a 1-day supply of platelets. Eight percent of random and 4 percent of apheresis platelets were outdated. There were 20 reports that surgery had to be postponed or canceled because platelets were unavailable. CONCLUSIONS: Inventories of RBCs maintained at the participating sites were sufficient, with only one brief exception, to meet local demand during the first year of this monitoring program. The weekly rate of threatened shortage reports was more sensitive than days inventory as a predictor of actual shortages of RBCs. Unlike RBCs, platelet days supply, reports of threatened or actual platelet shortages, and platelet outdate rates did not vary seasonally.