Whirling Platelets Away for Transfusion.

With a growing demand for platelet transfusions, large-scale ex vivo platelet production would alleviate the reliance on donors. Now, Ito et al. report that turbulence is an important physical regulator of platelet generation in vivo and can be exploited in a bioreactor to enable clinical scale production of functional platelets starting from human iPSCs.

The glucocorticoid receptor elicited proliferative response in human erythropoiesis is BCL11A-dependent.

Prior evidence indicates that the erythroid cellular response to glucocorticoids (GC) has developmental specificity, namely, that developmentally more advanced cells that are undergoing or have undergone fetal to adult globin switching are more responsive to GC-induced expansion. To investigate the molecular underpinnings of this, we focused on the major developmental globin regulator BCL11A. We compared: a) levels of expression and nuclear content of BCL11A in adult erythroid cells upon GC stimulation; b) response to GC of CD34+ cells from patients with BCL11A microdeletions and reduced BCL11A expression, and; c) response to GC of two cellular models (HUDEP-2 and adult CD34+ cells) before and after reduction of BCL11A expression by shRNA. We observed that: a) GC-expanded erythroid cells from a large cohort of blood donors displayed amplified expression and nuclear accumulation of BCL11A; b) CD34+ cells from BCL11A microdeletion patients generated fewer erythroid cells when cultured with GC compared to their parents, while the erythroid expansion of the patients was similar to that of their parents in cultures without GC, and; c) adult CD34+ cells and HUDEP-2 cells with shRNA-depleted expression of BCL11A exhibit reduced expansion in response to GC. In addition, RNA-seq profiling of shRNA-BCL11A CD34+ cells cultured with and without GC was similar (very few differentially expressed genes), while GC-specific responses (differential expression of GILZ and of numerous additional genes) were observed only in controls cells with unperturbed BCL11A expression. These data indicate that BCL11A is an important participant of certain aspects of the stress pathway sustained by GC.

IL-13/IL-4 signaling contributes to fibrotic progression of the myeloproliferative neoplasms.

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor ß and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

The Variation in the Traits Ameliorated by Inhibitors of JAK1/2, TGF-ß, P-Selectin, and CXCR1/CXCR2 in the Gata1low Model Suggests That Myelofibrosis Should Be Treated by These Drugs in Combination.

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-ß (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-ß) signaling and TGF-ß trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-ß content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-ß or CXCL1).

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Novel targets to cure primary myelofibrosis from studies on Gata1low mice.

In 2002, we discovered that mice carrying the hypomorphic Gata1low mutation that reduces expression of the transcription factor GATA1 in megakaryocytes (Gata1low mice) develop myelofibrosis, a phenotype that recapitulates the features of primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms (MPNs). At that time, this discovery had a great impact on the field because mutations driving the development of PMF had yet to be discovered. Later studies identified that PMF, as the others MPNs, is associated with mutations activating the thrombopoietin/JAK2 axis raising great hope that JAK inhibitors may be effective to treat the disease. Unfortunately, ruxolitinib, the JAK1/2 inhibitor approved by FDA and EMEA for PMF, ameliorates symptoms but does not improve the natural course of the disease, and the cure of PMF is still an unmet clinical need. Although GATA1 is not mutated in PMF, reduced GATA1 content in megakaryocytes as a consequence of ribosomal deficiency is a hallmark of myelofibrosis (both in humans and mouse models) and, in fact, a driving event in the disease. Conversely, mice carrying the hypomorphic Gata1low mutation express an activated TPO/JAK2 pathway and partially respond to JAK inhibitors in a fashion similar to PMF patients (reduction of spleen size but limited improvement of the natural history of the disease). These observations cross-validated Gata1low mice as a bona fide animal model for PMF and prompted the use of this model to identify abnormalities that might be targeted to cure the disease. We will summarize here data generated in Gata1low mice indicating that the TGF-ß/P-selectin axis is abnormal in PMF and represents a novel target for its treatment.

Concise Review: Advanced Cell Culture Models for Diamond Blackfan Anemia and Other Erythroid Disorders.

In vitro surrogate models of human erythropoiesis made many contributions to our understanding of the extrinsic and intrinsic regulation of this process in vivo and how they are altered in erythroid disorders. In the past, variability among the levels of hemoglobin F produced by adult erythroblasts generated in vitro by different laboratories identified stage of maturation, fetal bovine serum, and accessory cells as "confounding factors," that is, parameters intrinsically wired in the experimental approach that bias the results observed. The discovery of these factors facilitated the identification of drugs that accelerate terminal maturation or activate specific signaling pathways for the treatment of hemoglobinopathies. It also inspired studies to understand how erythropoiesis is regulated by macrophages present in the erythroid islands. Recent cell culture advances have greatly increased the number of human erythroid cells that can be generated in vitro and are used as experimental models to study diseases, such as Diamond Blackfan Anemia, which were previously poorly amenable to investigation. However, in addition to the confounding factors already identified, improvement in the culture models has introduced novel confounding factors, such as possible interactions between signaling from cKIT, the receptor for stem cell factor, and from the glucocorticoid receptor, the cell proliferation potential and the clinical state of the patients. This review will illustrate these new confounding factors and discuss their clinical translation potential to improve our understanding of Diamond Blackfan Anemia and other erythroid disorders. Stem Cells 2018;36:172-179.

Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient.

Congenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient's peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients' clinical and phenotypic features, and elucidate the dominant character of the mutation.

P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-ß release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-ß content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-ß reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-ß-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-ß content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Tribute to Donald Metcalf.

A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of "the father of hematopoietic cytokines".

Activation of non-canonical TGF-ß1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-ß1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-ß1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-ß1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-ß1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-ß1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.

Characterization of the TGF-ß1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-ß1 release. To clarify the role of TGF-ß1 in the pathogenesis of this disease, the TGF-ß1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-ß1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-ß1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-ß1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-ß1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield.

Expansion of erythroblasts from human peripheral blood mononuclear cells is 4- to 15-fold more efficient than that of CD34(+) cells purified from peripheral blood mononuclear cells. In addition, purified CD34(+) and CD34(-) populations from blood do not reconstitute this erythroid yield, suggesting a role for feeder cells present in blood mononuclear cells that increase hematopoietic output. Immunodepleting peripheral blood mononuclear cells for CD14(+) cells reduced hematopoietic stem and progenitor cell expansion. Conversely, the yield was increased upon co-culture of CD34(+) cells with CD14(+) cells (full contact or transwell assays) or CD34(+) cells re-constituted in conditioned medium from CD14(+) cells. In particular, CD14(++)CD16(+) intermediate monocytes/macrophages enhanced erythroblast outgrowth from CD34(+) cells. No effect of CD14(+) cells on erythroblasts themselves was observed. However, 2 days of co-culturing CD34(+) and CD14(+) cells increased CD34(+) cell numbers and colony-forming units 5-fold. Proliferation assays suggested that CD14(+) cells sustain CD34(+) cell survival but not proliferation. These data identify previously unrecognized erythroid and non-erythroid CD34(-) and CD34(+) populations in blood that contribute to the erythroid yield. A flow cytometry panel containing CD34/CD36 can be used to follow specific stages during CD34(+) differentiation to erythroblasts. We have shown modulation of hematopoietic stem and progenitor cell survival by CD14(+) cells present in peripheral blood mononuclear cells which can also be found near specific hematopoietic niches in the bone marrow.

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.

A3669G polymorphism of glucocorticoid receptor is a susceptibility allele for primary myelofibrosis and contributes to phenotypic diversity and blast transformation.

The frequency of A3669G single nucleotide polymorphism (SNP) of human glucocorticoid receptor has been reported increased in polycythemia vera. We investigated the frequency of A3669G SNP and its impact on disease phenotype and progression in 499 patients with primary myelofibrosis (PMF). The distribution of the A3669G allele differed between PMF patients and 2 healthy control populations (odds ratio, 1.6 and 1.8). The variant allele at the homozygous state (G/G) was associated with higher white blood cell count, larger spleen index, and higher frequency of circulating CD34(+) cells at diagnosis. The latter association remained significant after correction for the JAK2V617F genotype. In patients JAK2V617F mutated, the G/G genotype was associated with shorter overall survival (77.6 months vs 298 months, P = .049) and blast transformation (BT)-free survival (76.7 months vs 261 months; P = .018). The latter association remained significant after correction for the known BT risk factors, such as age, sex, white blood cell count, percentage of blasts, IPSS prognostic score, and homozygosity for JAK2V617F (hazard ratio = 3.3; P = .006). In conclusion, the glucocorticoid receptor A3669G is a susceptibility allele for PMF: it contributes to confer the phenotype of excess myeloproliferation, and it cooperates with the JAK2V617F mutation in determining BT.

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.

Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.

Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1(low) mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1(low) mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1(low) mice were crossed with P-sel(null) mice according to standard genetic protocols and Gata1(low)P-sel(wt), Gata1(low)P-sel(null) and Gata1(WT)P-sel(null) or Gata1(wt)P-sel(wt) (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1(low) mice. Moreover, platelet microparticles are reduced in Gata1(low) mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1(low) mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1(low) mice significantly higher frequency of thrombotic events was seen in adult and old Gata1(low) mice compared to Gata1(low)P-sel(null) mice. Thus, presence of the P-selectin null trait rescued Gata1(low) mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1(low) mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.

Erythropoietin: A Personal Alice in Wonderland Trip in the Shadow of the Giants.

The identification of the hormone erythropoietin (EPO), which regulates red blood cell production, and its development into a pharmaceutical-grade product to treat anemia has been not only a herculean task but it has also been the first of its kind. As with all the successes, it had "winners" and "losers", but its history is mostly told by the winners who, over the years, have published excellent scientific and divulgate summaries on the subject, some of which are cited in this review. In addition, "success" is also due to the superb and dedicated work of numerous "crew" members, who often are under-represented and under-recognized when the story is told and often have several "dark sides" that are not told in the polished context of most reviews, but which raised the need for the development of the current legislation on biotherapeutics. Although I was marginally involved in the clinical development of erythropoietin, I have known on a personal basis most, if not all, the protagonists of the saga and had multiple opportunities to talk with them on the drive that supported their activities. Here, I will summarize the major steps in the development of erythropoietin as the first bioproduct to enter the clinic. Some of the "dark sides" will also be mentioned to emphasize what a beautiful achievement of humankind this process has been and how the various unforeseen challenges that emerged were progressively addressed in the interest of science and of the patient's wellbeing.