Megakaryocyte Colony-forming Units Research Articles

Decreased Expression of IL-35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia.

Recent findings have shown that the level of interleukin-35 (IL-35) is abnormal in several autoimmune diseases. Nonetheless, whether IL-35 participates in the pathogenesis of immune thrombocytopenia (ITP) remains unclear. The current study investigates whether IL-35 modulates megakaryopoiesis. The results show that IL-35 receptors are progressively expressed on bone marrow megakaryocytes during the in vitro differentiation of CD34+ progenitors. IL-35 increases the number of megakaryocyte colony-forming units through the Akt pathway. The level of bone marrow IL-35 is reduced in ITP patients, and the decreased level of IL-35 may inhibit megakaryopoiesis. Then, the potential causes of decreased IL-35 in ITP patients are explored. The primary type of cell that secretes IL-35, known as IL-35-producing regulatory T cells (iTr35), is reduced in ITP patients. Bone marrow mesenchymal stem cells (MSCs) from ITP patients exhibit an impaired capability of inducing iTr35 due to enhanced apoptosis, which may contribute to the reduced level of bone marrow IL-35 in ITP patients. Iguratimod promotes megakaryocyte development and differentiation by elevating the expression of IL-35 receptors on megakaryocytes. Iguratimod improves response rates and reduces bleeding symptoms in corticosteroid-resistant ITP patients.

EFFECTS OF TRAUMA PLASMA-DERIVED EXOSOMES ON HEMATOPOIETIC PROGENITOR CELLS.

Background: Severe trauma disrupts bone marrow function resulting in persistent anemia and immunosuppression. Exosomes are extracellular vesicles implicated in disease, cellular functions, and immunomodulation. The effects of trauma plasma-derived exosomes on bone marrow hematopoiesis are unstudied; we hypothesized that trauma plasma-derived exosomes suppress bone marrow hematopoietic progenitor cell (HPC) growth and contribute to increased inflammatory cytokines and HPC mobilization. Methods: Plasma was collected from a prospective, cohort study of trauma patients (n = 15) with hip and/or femur fractures and an ISS > 15 and elective total hip arthroplasty (THA) patients (n = 15). Exosomes were isolated from both groups using the Invitrogen Total Exosome Isolation Kit. Healthy bone marrow was cultured with 2% plasma, 50 μg, 100 μg, or 200 μg of exosomal protein and HPC (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units [CFU-GEMM], erythroid burst-forming units [BFU-E], and macrophage colony-forming units [CFU-GM]) growth assessed. After culturing healthy bone marrow stroma with 100 μg of exosomal protein, expression of cytokines and factors influencing HPC mobilization were assessed by qPCR. Differences were compared using the ANOVA, with significance defined as P < 0.05. Results: The only demographic difference was age; trauma patients were significantly younger than THA (mean 44 vs. 63 years). In vitro exposure to trauma plasma significantly decreased growth of all HPCs. In vitro exposure to 100 μg or 200 μg of trauma exosomal protein significantly decreased growth of BFU-E and CFU-GM, whereas 50 μg had no effect. Culture of trauma exosomal protein with bone marrow stromal cells resulted in increased expression of IFN-γ, IL-1α, TNF-α, G-CSF, CXCR4, SDF-1, and VCAM-1 in bone marrow stroma. Conclusions: Both plasma and plasma-derived exosomes from trauma patients adversely affect bone marrow function. Plasma-derived exosomes may contribute to altered hematopoiesis after severe trauma; analysis of exosomal content may improve our understanding of altered bone marrow function.

Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs

AbstractCalreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

Tanshinone IIA Has a Potential Therapeutic Effect on Kawasaki Disease and Suppresses Megakaryocytes in Rabbits With Immune Vasculitis.

Background and ObjectiveIt is urgent to find out an alternative therapy for Kawasaki disease (KD) since around 20% patients are resistant to intravenous immunoglobulin (IVIG) or aspirin. Tanshinone IIA is the active component of the traditional Chinese medicine Danshen (Salvia miltiorrhiza), which has anti-inflammatory and anti-platelet properties; however, whether or not tanshinone IIA has a therapeutic effect on KD remains unclear. Therefore, the present study aimed to examine the effect of tanshinone IIA on KD patients and rabbits with immune vasculitis, and to identify the potential mechanisms with special emphasis on megakaryopoiesis and megakaryocytic apoptosis.MethodsKawasaki disease patients were recruited and prescribed with tanshinone IIA in the absence or presence of aspirin and IVIG, and the inflammatory responses and platelet functions were determined. Megakaryocytes (MKs) isolated from rabbits with immune vasculitis and human megakaryocytic CHRF-288-11 cells were treated with tanshinone IIA to examine the colony forming unit (CFU) and apoptosis, respectively. Microarray assay was conducted to identify potential targets of tanshinone IIA-induced apoptosis.ResultsTanshinone IIA reduced the serum levels of C-reactive protein (CRP), interleukin (IL)-1β, IL-6, and P-selectin in KD patients; such inhibitory effect was more significant compared to aspirin and IVIG. It also dose-dependently lowered the levels of tumor necrosis factor (TNF)-α and IL-8 in peripheral blood mononuclear cells isolated from KD patients. In rabbits with immune vasculitis, tanshinone IIA significantly reduced the serum levels of proinflammatory cytokines and platelet functions. In addition, tanshinone IIA significantly decreased the number of bone marrow MKs and inhibited the Colony Forming Unit-Megakaryocyte (CFU-MK) formation. In human megakaryocytic CHRF-288-11 cells, tanshinone IIA induced caspase-dependent apoptosis, probably through up-regulating TNF receptor superfamily member 9 (TNFRSF9) and the receptor (TNFRSF)-interacting serine/threonine-protein kinase 1 (RIPK1), which may contribute to its anti-platelet and anti-inflammatory properties.ConclusionTanshinone IIA exerts better anti-inflammatory and anti-platelet effects in treating KD patients than aspirin and IVIG. It attenuates immune vasculitis likely by inhibiting IL-mediated megakaryopoiesis and inducing TNFRSF9/RIPK1/caspase-dependent megakaryocytic apoptosis. The findings therefore suggest that tanshinone IIA may be a promising alternative therapy for the treatment of KD.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Atorvastatin May Reverse Corticosteroid-Resistant Immune Thrombocytopenia Via Improving Impaired Bone Marrow Endothelial Cells

Background: Immune thrombocytopenia (ITP) is generally considered to be an autoimmune disorder characterized by increased peripheral platelet destruction and reduced platelet production. The pathogenesis of corticosteroid-resistant ITP, a clinically challenging condition in which patients exhibit either no response to corticosteroids or corticosteroid-dependent, remains poorly understood. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) play a crucial role in regulating thrombopoiesis. We recently reported that the impaired BM EPCs, which could be quantitatively and functionally improved by atorvastatin in vitro, induced the occurrence of poor graft function following allo-transplant (Blood, 2016,128:2988-2999). However, little is known regarding the number and function of BM EPCs or how to improve impaired BM EPCs in corticosteroid-resistant ITP.Aims: To determine whether quantitative and/or functional abnormalities of BM EPCs are involved in the occurrence of corticosteroid-resistant ITP. Moreover, to investigate the effects of atorvastatin and N-Acetyl-L-cysteine (NAC, a reactive oxygen species (ROS) scavenger) on the number and function of cultivated BM EPCs derived from patients with corticosteroid-resistant ITP and its underlying molecular mechanisms. Finally, to evaluate the efficacy and safety of atorvastatin and NAC to adult patients with corticosteroid-resistant ITP.Methods: Forty patients with corticosteroid-resistant ITP, 40 patients with newly diagnosed ITP and 30 healthy donors (age 18-60) were enrolled from September 1, 2016 to May 1, 2017 at Peking University Institute of Hematology. BM EPCs were cultured as previous reported. The 5-day cultivated BM EPCs were administrated to the atorvastatin and NAC until tested on day 7. The BM EPCs were evaluated pre- and post-treatment by cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, cell proliferation, tube formation, levels of ROS and apoptosis or been cocultured with CD34+ cells for another 7 days. Proteins expressions for p38, ERK, JNK, Akt were measured by flow cytometry and western blot. Colony-forming unit megakaryocyte (CFU-MK) were counted and the quantity and maturation of megakaryocytes were determined by flow cytometry or cytology. Subsequently, a single-center pilot study was performed to evaluate the efficacy and safety of atorvastatin and/or NAC in corticosteroid-resistant ITP patients.Results: Human bone marrow EPCs were demonstrated as previous reported. Herein, reduced and dysfunctional BM EPCs, characterized by decreased capacities of proliferation, migration, and angiogenesis as well as higher levels of ROS and apoptosis, were observed in patients with corticosteroid-resistant ITP. Activation of phospho-p38 and deactivation of phospho-Akt were detected in BM EPCs from corticosteroid-resistant ITP patients. Furthermore, significant decreased and dysfunctional CD34+ cells with higher levels of ROS and apoptosis, while lower number of CFU-GEMM in patients of corticosteroid-resistant ITP. In vitro treatment with atorvastatin or NAC quantitatively and functionally improved BM EPCs derived from patients with corticosteroid-resistant ITP via down-regulating p38 MAPK and up-regulating Akt pathway and also partly rescued the impaired ability of BM EPCs to support megakaryocytopoiesis. In the single-center pilot study, a total of 13 corticosteroid-resistant ITP patients were recruited to receive either the combination of atorvastatin and NAC or alone. Although the results require further validation, the complete response (CR), response(R), and overall response (OR) rates were 23.1% (3/13), 46.2% (6/13) and 69.2% (9/13), respectively. In patients who achieved CR and R, the median (range) time to response(TTR) was 24 days (7-51 days), with no apparent adverse events.Summary / Conclusion: These findings suggest that impaired BM EPCs are involved in the pathogenesis of corticosteroid-resistant ITP. The effects of atorvastatin and NAC in patients with corticosteroid-resistant ITP are potentially mediated by improving the impaired ability of BM EPCs to support megakaryocytopoiesis. Further prospective multicenter randomized clinical trials are needed to validate the effects of atorvastatin and NAC, thereby providing a novel therapeutic avenue for corticosteroid-resistant ITP. DisclosuresNo relevant conflicts of interest to declare.

Acquired Amegakaryocytic Thrombocytopenic Purpura Associated with a Paroxysmal Nocturnal Hemoglobinuria Clone: A Case Report and Review of the Literature

Background: Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a rare bone marrow disorder characterized by a marked reduction in megakaryocytes with otherwise normal hematopoiesis. Both humoral and cell-mediated suppression of megakaryocytopoeisis have been postulated as mechanisms causing AATP. Herein we report a case of a 67-year-old man diagnosed with AATP with a co-existent paroxysmal nocturnal hemoglobinuria (PNH) clone and review the literature on AATP, focusing on proposed etiologies for this rare condition. Case: A 67-year-old man presented to clinic with a 1-week history easy bruising, petechiae and a platelet count of 6 x 109/L. He had a history of left elbow bursitis caused by S. pyogenes, treated with antibiotics 6-months prior to his presentation. His CBC was normal at that time. On assessment, HIV, HBV, HCV, and H. pylori serologies were negative; CMV and EBV serologies were positive for IgG and negative for IgM. ANA and RF were negative, and vitamin B12 level was normal. There was no hepatosplenomegaly on ultrasound. Bone marrow aspirate and biopsy demonstrated a normocellular marrow with severe megakaryocytic hypoplasia. Cytogenetics demonstrated normal male karyotype with loss of Y chromosome in 9/20 metaphases. Flow cytometry revealed a population of 3.99% GPI-deficient neutrophils by FLAER assay. Molecular testing for myeloid mutations and T-cell gene rearrangement is pending. The patient was initially treated with corticosteroids and IVIG, and showed no response with persistent isolated thrombocytopenia. He was managed with platelet transfusions which resulted in a normal platelet increment of 35 x 109/L 1-hour post-transfusion. A diagnosis of AATP was established and he was admitted for immunosuppressive therapy (IST) with ATG and cyclosporine. Methods: We conducted a narrative review of the literature on AATP, searching MEDLINE and EMBASE for articles on AATP published in English between 1946 and 2020. Reference lists of selected articles were reviewed to identify additional cases. We extracted data on presentation, bone marrow findings (including cytogenetics, molecular genetics, and flow cytometry), treatment regimens and outcomes. Results: We identified 47 cases of adult patients with thrombocytopenia attributed to AATP reported in the literature (Table 1). Three main mechanisms were proposed: (i) cell-mediated autoimmunity, (ii) humoral autoimmunity, and (iii) intrinsic stem cell defect. All three mechanisms were supported by in vitro studies, which demonstrated suppression of colony forming unit-megakaryocytes (CFU-M) by patients' T-lymphocytes (Gerwitz et al. 1986; Colovic et al. 2004) and serum (Hoffman et al. 1982) found to contain IgG antibodies inhibiting CFU-M formation, as well as intrinsic defects in CFU-M progenitor proliferation. Few studies reported cytogenetic abnormalities and only one documented molecular genetic testing. Response to IST was reported in several cases, most commonly ATG and cyclosporine. Four recent cases demonstrated remission following treatment with TPO agonists eltrombopag and romiplostim. Six cases progressed to aplastic anemia and 4 to myelodysplastic syndrome (MDS). Flow cytometry results were not reported in the majority of cases, and only 1 case reported coexistence of a PNH clone, identified in a pregnant patient with AATP (Zimmerman et al. 2019). Discussion: AATP is defined as severe thrombocytopenia with bone marrow showing marked decrease or absence of megakaryocytes with preservation of other cell lineages. This broad definition encompasses a range of causes, and our review of the literature highlights the heterogenous nature of AATP which has several proposed mechanisms and a number of therapeutic options. The best evidence suggests that AATP is often secondary to T-cell-mediated suppression of megakaryocytopoeisis, which has been demonstrated by in vitro studies, and is supported in vivo by a case of AATP following PD-1 inhibition (Iyama et al. 2020), and frequent response of AATP to T-cell-directed IST. The co-existence of a PNH clone in our case lends further support to a T-cell-mediated autoimmune process, analogous to the mechanism described in aplastic anemia and hypoproliferative MDS. The application of molecular diagnostics may help to further elucidate the role of clonal hematopoiesis and intrinsic stem cell defects versus humoral and cell-mediated autoimmunity in AATP. Disclosures No relevant conflicts of interest to declare.

M2 Macrophages, but Not M1 Macrophages, Support Megakaryopoiesis Via up-Regulating PI3K-AKT Pathway

Background Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×109/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated. Aims To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients. Methods This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway. Results Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily. Summary/Conclusion The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients. Disclosures No relevant conflicts of interest to declare.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

Imetelstat inhibits growth of megakaryocyte colony-forming units from patients with essential thrombocythemia

Key Points Clinically relevant imetelstat concentrations significantly inhibit CFU-MEG formation from MNCs of ET patients and reduce hTERT expression. However, similar concentrations of imetelstat do not inhibit cytokine-induced CFU-MEG from MNCs of healthy donors.

CD9 Marks Flt3+ Multipotent Hematopoietic Progenitors within Lsk Cells

Hematopoietic Stem Cells (HSCs) that give rise to all lineages of the blood are defined as a lineage-negative (Lin-), c-Kit+, Sca-1+(LSK) population in mouse. The expression of Flt3 on LSK cells is accompanied by loss of self-renewal capacity, and transition to lymphoid-primed multipotent progenitors (LMPPs). The magnitude of Flt3 surface abundance has been shown to be correlated with lineage potential, where megakaryocyte/erythroid (MegE) potential is incompatible with high Flt3 levels (Buza-Vidas et al. 2011). However, it is unclear whether MegE capacity is marked by Flt3 itself or restricted to a specific population within the Flt3 spectrum. Interestingly, it has been shown that commitment to MegE lineages is an early event in HSC differentiation and that MegE progenitors share several cell-surface markers with HSCs. One such marker is CD9, which is highly expressed on megakaryocyte progenitors (Nakorn, Miyamoto, and Weissman 2003)as well as on the most primitive stem cells in mouse (Karlsson et al. 2013). In fact, we have recently shown that CD9 captures all long-term reconstitution activity of the murine LSK population, even in cell fractions that lacks some of the most established HSC markers .To compare CD9- and Flt3 expression we performed FACS analysis of the LSK compartment and plotted CD9 high- and low cells on top of the CD34/Flt3 expression profile. Interestingly we found that even if CD9 expression were predominantly high in LT-(CD34-Flt3-) and ST-HSCs (CD34+Flt3-), CD9highcells reached all the way into the Flt3+population but were lost in LMPPs, as defined by the 25% highest Flt3 expressing cells. To addresswhether CD9highcells represent a distinct subpopulation within Flt3intcells, LSKCD34+Flt3int(Flt3int) and LSKCD34+FLT3intCD9high(CD9high) populations, were subjected to single-cell RNA-Sequencing analysis. Interestingly,our results demonstratethatCD9highcells are enriched in the most primitive cluster of the Flt3intpopulation. This was further validated in transplantation assays where only CD9highFlt3intcells reconstituted irradiated recipients. However, long-term reconstitution was limited to lymphoid lineages, while myeloid engraftment was only observed at early time points and self-renewal activity was absent. To address the clonal capacity of CD9highcells, we designed a protocol to assess the potential of single-cells to generate B-, Myeloid (My)- and Erythroid (Er) cells in vitro.In these conditions 68% of clones generated from CD9highcells were bipotent (55% My/B and 13% My/Er). In contrast, the majority of CD9lowcells were unipotent (32% My- and 52% B cells).Moreover,the colony forming capacity for the CD9highpopulation was significantly higher compared to CD9lowcells and when assayed for colony-forming unit-megakaryocyte (CFU-MK) capacity, Meg potential was exclusively observed in LT-HSCs, ST-HSCs as well as the CD9highFlt3intpopulation, while neither CD9lownor LMPPs generated any megakaryocyte colonies. In an attempt to improve the efficiency of MegE differentiation, single-cell clonogenic assays were performed in suspension cultures. Here,multipotency including MegE potential was gradually down regulated with increasing Flt3 cell surface expression, and decreasing CD9 expression. Together our results shows thatCD9 captures the most primitive cells within the LSKFlt3+population, including multipotent cells with MegE differentiation potential, thus resolving the heterogeneity of Flt3+hematopoietic progenitors and identifying a multipotent LSKCD9+Flt3+population distinct from LMPPs. Disclosures No relevant conflicts of interest to declare.

A novel thrombopoietin mimetic RWJ-800088 increases megakaryopoiesis without causing malignant proliferation in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

e18527 Background: In patients (pts) with AML and MDS, thrombocytopenia is a source of morbidity/mortality and there is a need for safer treatment options. RWJ-800088 (RWJ) is a novel pegylated thrombopoietin (TPO) mimetic peptide that increases megakaryopoiesis preclinically and in humans. Before using a TPO mimetic agent in AML and MDS pts, it is important to evaluate if the agent has potential stimulatory effects on malignant myeloid cells. To assess whether RWJ is likely to stimulate malignant myeloid cells, we evaluated its effects in vitro on primary human samples from healthy controls and AML/MDS pts. Methods: We studied the effect of various RWJ doses on proliferation of AML/MDS cell lines; clonogenic capacity for leukemic colony forming units (CFU) and megakaryocytic CFU (CFU-Mk) of mononuclear cells (MNC) from controls and several MDS and AML pts; and expression of differentiation markers in MNC cultures. Results: RWJ treatment had no effect on proliferation of THP1, MOLM13, MV411 and MDSL cell lines at doses up to 1000 ng/ml. A slight increase in proliferation of CMK, a megakaryocytic cell line, was seen at doses &gt; 5xEC50 (mean 12%, p = 0.03). Primary clonogenic assays from 18 AML/MDS pts did not show increased growth of leukemic CFU at RWJ concentrations up to 10 ng/ml and RWJ had a nonsignificant inhibitory effect in 4 pts. Notably, RWJ significantly increased normal CFU-Mk formation from MNC in 6 of 8 AML and all MDS pts (mean 2.6-fold, p = 0.02), as well as from healthy CD34+ stem cells and healthy MNC controls (mean 1.7-fold, p = 0.03). FACS analysis of primary cultures treated with RWJ showed no increase in the malignant blast compartment, no change in differentiation markers of granulocyte/monocyte or erythrocyte lineages but a significant increase in the number of CD41a+ megakaryocytic cells (mean 1.8-fold, p &lt; 0.01). RWJ treatment increased phosphorylation of STAT5 but not STAT3, demonstrating on target stimulation of the TPO receptor in hematopoietic cells. Conclusions: Our results show that the novel TPO mimetic RWJ stimulates megakaryopoiesis without causing proliferation of the malignant myeloid clone in MDS and AML.

Cyanidin-3-O-β-glucoside, a Natural Polyphenol, Exerts Proapoptotic Effects on Activated Platelets and Enhances Megakaryocytic Proplatelet Formation.

This study investigated whether the anthocyanin cyanidin-3-O-β-glucoside (Cy-3-g) could affect platelet apoptosis and proplatelet formation in vitro. Thrombin-stimulated or resting human platelets and Meg-01 megakaryocytes were incubated with Cy-3-g (0, 0.5, 5, or 50 μM). We found that the percentage of the platelet mitochondrial membrane potential treated with 5 and 50 μM Cy-3-g was significantly higher than control (15.50% ± 3.24% and 29.77% ± 4.06% versus 2.76% ± 1.33%, respectively; P < 0.05). Treatment with 5 and 50 μM Cy-3-g significantly increased phosphatidylserine exposure compared with control (40.56% ± 10.53% and 76.62% ± 8.28% versus 15.43% ± 3.93%, respectively; P < 0.05). Moreover, Cy-3-g significantly increased the expression of Bax, Bak, and cytochrome c while markedly decreasing Bcl-xL and Bcl-2 expression as well as stimulating caspase-3, caspase-9, caspase-8, Bid, and gelsolin cleavage in thrombin-activated platelets in a dose-dependent manner ( P < 0.05). However, no significant differences were observed in the apoptosis of resting platelets when treated with Cy-3-g ( P > 0.05). Furthermore, Cy-3-g significantly ( P < 0.05) enhanced cell viability (50 μM versus control, 1.34 ± 0.01 versus 0.35 ± 0.02), the number of colony-forming unit-megakaryocytes (50 μM versus control, 38 ± 3 versus 8 ± 3), CD41 expression (50 μM versus control, 96.80% ± 2.55% versus 25.57% ± 2.86%), DNA ploidy (16N) (50 μM versus control, 19.73% ± 2.34% versus 4.42% ± 1.96%), and proplatelet formation (50 μM versus control, 27.5% ± 3.77% versus 7.67% ± 2.25%) in Meg-01 cells. In conclusion, Cy-3-g promotes activated platelet apoptosis and enhances megakaryocyte proliferation, differentiation, and proplatelet formation in vitro.

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells

Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

CD106 is a novel mediator of bone marrow mesenchymal stem cells via NF-\u03baB in the bone marrow failure of acquired aplastic anemia

BackgroundAcquired aplastic anemia (AA) is characterized by deficiency or dysfunction of the bone marrow (BM) microenvironment. However, little is known about the impairment of BM-derived mesenchymal stem cells (MSCs) in AA patients.MethodsWe used Illumina HiSeqTM 2000 sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry (FCM), and Western blotting to test the expression of CD106 gene (vascular cell adhesion molecule 1 (VCAM1)) and CD106 protein of BM-MSCs. Furthermore, we used hematoxylin and eosin (H&E) and histochemical staining analysis, immunofluorescence, and the formation of capillary-like structures to analyze capillary tube-like formation in vitro; we also used the Matrigel plug assay to test in vivo vasculogenesis, and an assay of colony forming units (CFUs) and colony-forming unit-megakaryocyte (CFU-MK) to detect the support function of MSCs in vitro. The in vivo engraftment of CD34+ cells and MSCs in NOD/SCID mice was tested by FACS and survival assay; the expression of NF-κB was tested by NanoPro analysis and immunofluorescence. NF-κB-regulated CD106 gene (VCAM1) was confirmed by tumor necrosis factor alpha (TNF-α)-stimulated and lipopolysaccharide (LPS)-stimulated MSCs, blockade assay, and immunofluorescence.ResultsHere, we report that BM-MSCs from AA patients exhibited downregulation of the CD06 gene (VCAM1) and low expression of CD106 in vitro. Further analysis revealed that CD106+ MSCs from both AA patients and healthy controls had increased potential for in vitro capillary tube-like formation and in vivo vasculogenesis compared with CD106– MSCs, and the results were similar when healthy MSCs were compared with AA MSCs. CD106+ MSCs from both AA patients and healthy controls more strongly supported in vitro growth and in vivo engraftment of CD34+ cells in NOD/SCID mice than CD106– MSCs, and similar results were obtained when healthy MSCs and AA MSCs were compared. The expression of NF-κB was decreased in AA MSCs, and NF-κB regulated the CD106 gene (VCAM1) which supported hematopoiesis.ConclusionsThese results revealed the effect of CD106 and NF-κB in BM failure of AA.

15. ヒト臍帯血巨核球コロニー形成細胞CFU-Megの高凍結感受性(平成11年度第45回低温生物工学会研究報告)

15. ヒト臍帯血巨核球コロニー形成細胞CFU-Megの高凍結感受性(平成11年度第45回低温生物工学会研究報告)

Dual role of IL-21 in megakaryopoiesis and platelet homeostasis.

Gene profiling studies have indicated that in vitro differentiated human megakaryocytes express the receptor for IL-21 (IL-21R), an immunostimulatory cytokine associated with inflammatory disorders and currently under evaluation in cancer therapy. The aim of this study was to investigate whether IL-21 modulates megakaryopoiesis. We first checked the expression of IL-21 receptor on human bone marrow and in vitro differentiated megakaryocytes. We then investigated the effect of IL-21 on the in vitro differentiation of human blood CD34+ progenitors into megakaryocytes. Finally, we analyzed the consequences of hydrodynamic transfection-mediated transient expression of IL-21, on megakaryopoiesis and thrombopoiesis in mice. The IL-21Rα chain was expressed in human bone marrow megakaryocytes and was progressively induced during in vitro differentiation of human peripheral CD34+ progenitors, while the signal transducing γ chain was down-regulated. Consistently, the STAT3 phosphorylation induced by IL-21 diminished during the later stages of megakaryocytic differentiation. In vitro, IL-21 increased the number of colony-forming unit megakaryocytes generated from CD34+ cells and the number of megakaryocytes differentiated from CD34+ progenitors in a JAK3- and STAT3-dependent manner. Forced expression of IL-21 in mice increased the density of bi-potent megakaryocyte progenitors and bone marrow megakaryocytes, and the platelet generation, but increased platelet clearance with a consequent reduction in blood cell counts. Our work suggests that IL-21 regulates megakaryocyte development and platelet homeostasis. Thus, IL-21 may link immune responses to physiological or pathological platelet-dependent processes.

Signals of the Inflammatory Microenvironment Promote a Mutation-Associated Functional Dysregulation of the Circulating Megakaryocyte Progenitors of Myelofibrosis

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis.Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining.Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients.The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients.Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. DisclosuresMartinelli:BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.

The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood.Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors.Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice.Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Ex-vivo expansion of nonhuman primate CD34+ cells by stem cell factor Sall4B.

BackgroundHematopoietic CD34+ stem cells are widely used in the clinical therapy of complicated blood diseases. Stem cell factor Sall4B is a zinc finger transcription factor that plays a vital role in hematopoietic stem cell expansion. The purpose of our current study is to further evaluate how Sall4B might affect the expansion of CD34+ cells derived from nonhuman primates.MethodsSall4B was overexpressed in nonhuman primate bone marrow-derived CD34+ cells via a lentiviral transduction system. The granulocyte–erythrocyte–macrophage–megakaryocyte colony-forming unit (CFU) assay evaluated the differentiation potential of primate CD34+ cells that were expanded with Sall4B. Furthermore, an in-vivo murine system was employed to evaluate the hematopoietic potential of primate Sall4B-expanded CD34+ cells.ResultsOverexpression of Sall4B promoted ex-vivo nonhuman primate CD34+ cell expansion by 9.21 ± 1.94-fold on day 9, whereas lentiviral transduction without Sall4B expanded cells by only 2.95 ± 0.77-fold. Sall4B maintained a significant percentage of CD34+ cells as well. The CFU assay showed that the Sall4B-expanded CD34+ cells still possessed multilineage differentiation potential. A study using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice in vivo revealed that Sall4B led to an increase in the number of repopulating cells and the 9-day-old Sall4B-transduced CD34+ cells still possess self-renewal and multilineage differentiation capacity in vivo, which are similar stemness characteristics to those in freshly isolated primate bone marrow-derived CD34+ cells.ConclusionsWe investigated the expansion of nonhuman primate bone marrow-derived CD34+ cells using the Sall4B lentiviral overexpression approach; our findings provide a new perspective on mechanisms of rapid stem cell proliferation. The utilization of Sall4B to expand CD34+ cells on a large scale through use of suitable model systems would prove helpful towards preclinical trials of autologous transplantation.