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.

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.

GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder with limited therapeutic options. Insufficient understanding of driver mutations and poor fidelity of currently available animal models has limited the development of effective therapies. Since GATA1 deficient megakaryocytes sustain myelofibrosis, we hypothesized that they may also induce fibrosis in lungs. We discovered that lungs from IPF patients and Gata1low mice contain numerous GATA1negative immune-poised megakaryocytes that, in mice, have defective RNA-seq profiling and increased TGF-ß1, CXCL1 and P-selectin content. With age, Gata1low mice develop fibrosis in lungs. Development of lung fibrosis in this model is prevented by P-selectin deletion and rescued by P-selectin, TGF-ß1 or CXCL1 inhibition. Mechanistically, P-selectin inhibition decreases TGF-ß1 and CXCL1 content and increases GATA1positive megakaryocytes while TGF-ß1 or CXCL1 inhibition decreased CXCL1 only. In conclusion, Gata1low mice are a novel genetic-driven model for IPF and provide a link between abnormal immune-megakaryocytes and lung fibrosis.

A Phase Ib Trial of AVID200, a TGFß 1/3 Trap, in Patients with Myelofibrosis.

PURPOSE: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by systemic symptoms, cytopenias, organomegaly, and bone marrow fibrosis. JAK2 inhibitors afford symptom and spleen burden reduction but do not alter the disease course and frequently lead to thrombocytopenia. TGFß, a pleiotropic cytokine elaborated by the MF clone, negatively regulates normal hematopoiesis, downregulates antitumor immunity, and promotes bone marrow fibrosis. Our group previously showed that AVID200, a potent and selective TGFß 1/3 trap, reduced TGFß1-induced proliferation of human mesenchymal stromal cells, phosphorylation of SMAD2, and collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PC) with wild-type JAK2 rather than JAK2V617F. PATIENTS AND METHODS: We conducted an investigator-initiated, multicenter, phase Ib trial of AVID200 monotherapy in 21 patients with advanced MF. RESULTS: No dose-limiting toxicity was identified at the three dose levels tested, and grade 3/4 anemia and thrombocytopenia occurred in 28.6% and 19.0% of treated patients, respectively. After six cycles of therapy, two patients attained a clinical benefit by IWG-MRT criteria. Spleen and symptom benefits were observed across treatment cycles. Unlike other MF-directed therapies, increases in platelet counts were noted in 81% of treated patients with three patients achieving normalization. Treatment with AVID200 resulted in potent suppression of plasma TGFß1 levels and pSMAD2 in MF cells. CONCLUSIONS: AVID200 is a well-tolerated, rational, therapeutic agent for the treatment of patients with MF and should be evaluated further in patients with thrombocytopenic MF in combination with agents that target aberrant MF intracellular signaling pathways.

Single cell analysis of the localization of the hematopoietic stem cells within the bone marrow architecture identifies niche-specific proliferation dynamics.

Introduction: Hematopoietic stem cells (HSC) reside in the bone marrow (BM) in specialized niches which provide support for their self-replication and differentiation into the blood cells. Recently, numerous studies using sophisticated molecular and microscopic technology have provided snap-shots information on the identity of the BM niches in mice. In adults, HSC are localized around arterioles and sinusoids/venules whereas in juvenile mice they are in close to the osteoblasts. However, although it is well recognized that in mice the nature of the hematopoietic niche change with age or after exposure to inflammatory insults, much work remains to be done to identify changes occurring under these conditions. The dynamic changes occurring in niche/HSC interactions as HSC enter into cycle are also poorly defined. Methods: We exploit mice harboring the hCD34tTA/Tet-O-H2BGFP transgene to establish the feasibility to assess interactions of the HSC with their niche as they cycle. In this model, H2BGFP expression is driven by the TET trans-activator under the control of the human CD34 promoter which in mice is active only in the HSC. Since Doxycycline inhibits TET, HSC exposed to this drug no longer express H2BGFP and loose half of their label every division allowing establishing the dynamics of their first 1-3 divisions. To this aim, we first validated user-friendly confocal microscopy methods to determine HSC divisions by hemi-decrement changes in levels of GFP expression. We then tracked the interaction occurring in old mice between the HSC and their niche during the first HSC divisions. Results: We determined that in old mice, most of the HSC are located around vessels, both arterioles which sustain quiescence and self-replication, and venules/sinusoids, which sustain differentiation. After just 1 week of exposure to Doxycycline, great numbers of the HSC around the venules lost most of their GFP label, indicating that they had cycled. By contrast, the few HSC surrounding the arterioles retained maximal levels of GFP expression, indicating that they are either dormant or cycle at very low rates. Conclusion: These results reveal that in old mice, HSC cycle very dynamically and are biased toward interactions with the niche that instructs them to differentiate.

Inhibition of CXCR1/2 reduces the emperipolesis between neutrophils and megakaryocytes in the Gata1low model of myelofibrosis.

Emperipolesis between neutrophils and megakaryocytes was first identified by transmission electron microscopy. Although rare under steady-state conditions, its frequency greatly increases in myelofibrosis, the most severe of myeloproliferative neoplasms, in which it is believed to contribute to increasing the transforming growth factor (TGF)-ß microenvironmental bioavailability responsible for fibrosis. To date, the challenge of performing studies by transmission electron microscopy has hampered the study of factors that drive the pathological emperipolesis observed in myelofibrosis. We established a user-friendly confocal microscopy method that detects emperipolesis by staining with CD42b, specifically expressed on megakaryocytes, coupled with antibodies that recognize the neutrophils (Ly6b or neutrophil elastase antibody). With such an approach, we first confirmed that the bone marrow from patients with myelofibrosis and from Gata1low mice, a model of myelofibrosis, contains great numbers of neutrophils and megakaryocytes in emperipolesis. Both in patients and Gata1low mice, the emperipolesed megakaryocytes were surrounded by high numbers of neutrophils, suggesting that neutrophil chemotaxis precedes the actual emperipolesis event. Because neutrophil chemotaxis is driven by CXCL1, the murine equivalent of human interleukin 8 that is expressed at high levels by malignant megakaryocytes, we tested the hypothesis that neutrophil/megakaryocyte emperipolesis could be reduced by reparixin, an inhibitor of CXCR1/CXCR2. Indeed, the treatment greatly reduced both neutrophil chemotaxis and their emperipolesis with the megakaryocytes in treated mice. Because treatment with reparixin was previously reported to reduce both TGF-ß content and marrow fibrosis, these results identify neutrophil/megakaryocyte emperipolesis as the cellular interaction that links interleukin 8 to TGF-ß abnormalities in the pathobiology of marrow fibrosis.

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.

Preclinical studies on the use of a P-selectin-blocking monoclonal antibody to halt progression of myelofibrosis in the Gata1low mouse model.

The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-ß (TGF-ß) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-ß and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients.

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 CXCR1/CXCR2 Inhibitor Reparixin Alters the Development of Myelofibrosis in the Gata1 low Mice.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-ß, the megakaryocytes from the bone marrow of the Gata1 low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1 low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-ß1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1 low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-ß1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice.

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-ß1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

A Novel Megakaryocyte Subpopulation Poised to Exert the Function of HSC Niche as Possible Driver of Myelofibrosis.

Careful morphological investigations, coupled with experimental hematology studies in animal models and in in vitro human cultures, have identified that platelets are released in the circulation by mature megakaryocytes generated by hematopoietic stem cells by giving rise to lineage-restricted progenitor cells and then to morphologically recognizable megakaryocyte precursors, which undergo a process of terminal maturation. Advances in single cell profilings are revolutionizing the process of megakaryocytopoiesis as we have known it up to now. They identify that, in addition to megakaryocytes responsible for producing platelets, hematopoietic stem cells may generate megakaryocytes, which exert either immune functions in the lung or niche functions in organs that undergo tissue repair. Furthermore, it has been discovered that, in addition to hematopoietic stem cells, during ontogeny, and possibly in adult life, megakaryocytes may be generated by a subclass of specialized endothelial precursors. These concepts shed new light on the etiology of myelofibrosis, the most severe of the Philadelphia negative myeloproliferative neoplasms, and possibly other disorders. This perspective will summarize these novel concepts in thrombopoiesis and discuss how they provide a framework to reconciliate some of the puzzling data published so far on the etiology of myelofibrosis and their implications for the therapy of this disease.

The Glucocorticoid Receptor Polymorphism Landscape in Patients With Diamond Blackfan Anemia Reveals an Association Between Two Clinically Relevant Single Nucleotide Polymorphisms and Time to Diagnosis.

NR3C1, the gene encoding the glucocorticoid receptor, is polymorphic presenting numerous single nucleotide polymorphisms (SNPs) some of which are emerging as leading cause in the variability of manifestation and/or response to glucocorticoids in human diseases. Since 60-80% of patients with Diamond Blackfan anemia (DBA), an inherited pure red cell aplasia induced by mutations in ribosomal protein genes became transfusion independent upon treatment with glucocorticoids, we investigated whether clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. The eight SNPs rs10482605, rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, rs6196, and rs33388/rs33389 were investigated in a cohort of 91 European DBA patients. Results were compared with those observed in healthy volunteers (n=37) or present in public genome databases of Italian and European populations. Although, cases vs. control analyses suggest that the frequency of some of the minor alleles is significantly altered in DBA patients with respect to healthy controls or to the Italian or other European registries, lack of consistency among the associations across different sets suggests that overall the frequency of these SNPs in DBA is not different from that of the general population. Demographic data (47 females and 31 males) and driver mutations (44 S and 29 L genes and eight no-known mutation) are known for 81 patients while glucocorticoid response is known, respectively, for 81 (36 responsive and 45 non-responsive) and age of disease onsets for 79 (55 before and 24 after 4months of age) patients. Neither gender nor leading mutations were associated with the minor alleles or with disease manifestation. In addition, none of the SNPs met the threshold in the response vs. non-responsive groups. However, two SNPs (rs6196 and rs860457) were enriched in patients manifesting the disease before 4months of age. Although the exact biomechanistical consequences of these SNPs are unknown, the fact that their configuration is consistent with that of regulatory regions suggests that they regulate changes in glucocorticoid response during ontogeny. This hypothesis was supported by phosphoproteomic profiling of erythroid cells expanded ex vivo indicating that glucocorticoids activate a ribosomal signature in cells from cord blood but not in those from adult blood, possibly providing a compensatory mechanism to the driving mutations observed in DBA before birth.

hGATA1 Under the Control of a µLCR/ß-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced by the Gata1 low Mutation in Mice.

The phenotype of mice carrying the Gata1 low mutation that decreases expression of Gata1 in erythroid cells and megakaryocytes, includes anemia, thrombocytopenia, hematopoietic failure in bone marrow and development of extramedullary hematopoiesis in spleen. With age, these mice develop myelofibrosis, a disease sustained by alterations in stem/progenitor cells and megakaryocytes. This study analyzed the capacity of hGATA1 driven by a µLCR/ß-globin promoter to rescue the phenotype induced by the Gata1 low mutation in mice. Double hGATA1/Gata1 low/0 mice were viable at birth with hematocrits greater than those of their Gata1 low/0 littermates but platelet counts remained lower than normal. hGATA1 mRNA was expressed by progenitor and erythroid cells from double mutant mice but not by megakaryocytes analyzed in parallel. The erythroid cells from hGATA1/Gata1 low/0 mice expressed greater levels of GATA1 protein and of α- and ß-globin mRNA than cells from Gata1 low/0 littermates and a reduced number of them was in apoptosis. By contrast, hGATA1/Gata1 low/0 megakaryocytes expressed barely detectable levels of GATA1 and their expression of acetylcholinesterase, Von Willebrand factor and platelet factor 4 as well as their morphology remained altered. In comparison with Gata1 +/0 littermates, Gata1 low/0 mice contained significantly lower total and progenitor cell numbers in bone marrow while the number of these cells in spleen was greater than normal. The presence of hGATA1 greatly increased the total cell number in the bone marrow of Gata1 low/0 mice and, although did not affect the total cell number of the spleen which remained greater than normal, it reduced the frequency of progenitor cells in this organ. The ability of hGATA1 to rescue the hematopoietic functions of the bone marrow of the double mutants was confirmed by the observation that these mice survive well splenectomy and did not develop myelofibrosis with age. These results indicate that hGATA1 under the control of µLCR/ß-globin promoter is expressed in adult progenitors and erythroid cells but not in megakaryocytes rescuing the erythroid but not the megakaryocyte defect induced by the Gata1 low/0 mutation.

Role of ß1 integrin in thrombocytopoiesis.

Thrombocytopoiesis is a complex process beginning at the level of hematopoietic stem cells, which ultimately generate megakaryocytes, large marrow cells with a distinctive morphology, and then, through a process of terminal maturation, megakaryocytes shed thousands of platelets into the circulation. This process is controlled by intrinsic and extrinsic factors. Emerging data indicate that an important intrinsic control on the late stages of thrombopoiesis is exerted by integrins, a family of transmembrane receptors composed of one α and one ß subunit. One ß subunit expressed by megakaryocytes is the ß1 integrin, the role of which in the regulation of platelet formation is beginning to be clarified. Here, we review recent data indicating that activation of ß1 integrin by outside-in and inside-out signaling regulates the interaction of megakaryocytes with the endosteal niche, which triggers their maturation, while its inactivation by galactosylation determines the migration of these cells to the perivascular niche, where they complete their terminal maturation and release platelets in the bloodstream. Furthermore, ß1 integrin mediates the activation of transforming growth factor ß (TGF-ß), a protein produced by megakaryocytes that may act in an autocrine fashion to halt their maturation and affect the composition of their surrounding extracellular matrix. These findings suggest that ß1 integrin could be a therapeutic target for inherited and acquired disorders of platelet production.

TGF-ß1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-ß plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-ß1 than TGF-ß2 and TGF-ß3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-ß1/TGF-ß3 protein trap, to block the excessive TGF-ß signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-ß1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-ß1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-ß1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

An Outline of the Outset of Thrombopoiesis in Human Embryos At Last.

By single-cell transcriptome profiling of human yolk sacs and fetal livers, Wang et al. (2021) (in this issue of Cell Stem Cell) track two alternative routes for differentiation of megakaryocytes. The authors have shown that these megakaryocytes have hemostatic- and HSC-supporting functions, and that hESC-derived thrombospondin1-positive endothelial cells are capable of generating megakaryocytes in vitro.

The Role of Megakaryocytes in Myelofibrosis.

Megakaryocytes give rise to platelets, which have a wide variety of functions in coagulation, immune response, inflammation, and tissue repair. Dysregulation of megakaryocytes is a key feature of in the myeloproliferative neoplasms, especially myelofibrosis. Megakaryocytes are among the main drivers of myelofibrosis by promoting myeloproliferation and bone marrow fibrosis. In vivo targeting of megakaryocytes by genetic and pharmacologic approaches ameliorates the disease, underscoring the important role of megakaryocytes in myeloproliferative neoplasms. Here we review the current knowledge of the function of megakaryocytes in the JAK2, CALR, and MPL-mutant myeloproliferative neoplasms.