Bone Marrow Proliferation Research Articles

T‐cell blast crisis of chronic myelogenous leukemia presented with coexisting p210 and p190 BCR‐ABL transcripts and t(10;11)(q11;p15)

BackgroundBlast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T‐LBL/ALL) is rare, and the molecular mechanism is still unclear.Case reportA 28‐year‐old woman who developed T‐ALL with coexpressing both p210 and p190 BCR‐ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t(9;22)(q34;q11) and t(10;11)(q25;p15). Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3.ConclusionIt is the first T‐cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T‐lymphocyte transformation depend on potential molecular and pathological mechanism.

Preparation and Characterization of Akermanite/Merwinite Scaffolds for Bone Tissue Repair

The ceramics in the system CaO–MgO–SiO2 has recently attracted a great deal of attention because they display a good in vitro bioactivity and have potential use as bone implants. Biphasic calcium-magnesium-silicate ceramics were prepared by a sol-gel method. The dried gel with chemical composition 3CaO.MgO.2SiO2 was thermally treated at 1200 °C for 2 hrs. The structural behavior of the synthesized ceramics was examined by means of X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Merwinite crystalline phase and akermanite phase were recognized. Then, porous akermanite/merwinite scaffolds were prepared to utilize polymer sponge method and evaluated by employing SEM. Furthermore, bone marrow stromal cells (BMSC) adhesion and proliferation on the scaffolds were evaluated by MTT assay test. Differentiation of the cells was assessed by measuring alkaline phosphatase (ALP) activity. The results demonstrated that BMSC adhered and spread well on akermanite scaffolds and proliferated with the increase in the culture time, and the differentiation rate of osteoblasts on scaffolds was comparable to that on blank culture plate control. Thus, the obtained results presented that the akermanite/merwinite scaffolds deserve attention for bone tissue engineering applications.

Chronic Hypoxia-Induced Microvessel Proliferation and Basal Membrane Degradation in the Bone Marrow of Rats Regulated through the IL-6/JAK2/STAT3/MMP-9 Pathway.

Chronic hypoxia (CH) is characterized by long-term hypoxia that is associated with microvessel proliferation and basal membrane (BM) degradation in tissues. The IL-6/JAK2/STAT3/MMP-9 pathway has been described in a variety of human cancers and plays an essential role in microvessel proliferation and BM degradation. Therefore, this study investigated the role of the IL-6/JAK2/STAT3/MMP-9 pathway in hypoxia-mediated microvessel proliferation and BM degradation in the rat bone marrow. Eighty pathogen-free Sprague Dawley male rats were randomly divided into four groups (20 per group)—control group, CH group (exposed to hypoxia in a hypobaric chamber at a simulated altitude of 5000 m for 28 d), CH + STAT3 inhibitor group (7.5 mg/kg/d), and CH + DMSO group. Microvessel density (MVD) and BM degradation in the bone marrow were determined by immunofluorescence staining and transmission electron microscopy. Serum IL-6 levels were assessed by enzyme-linked immunosorbent assay (ELISA), and the levels of P-JAK2, P-STAT3, and MMP-9 were assessed by western blot analysis and real-time reverse transcription PCR (RT-PCR). Hypoxia increased serum IL-6 levels, which in turn increased JAK2 and STAT3 phosphorylation, which subsequently upregulated MMP-9. Overexpression of MMP-9 significantly promoted the elevation of MVD and BM degradation. Inhibition of STAT3 using an inhibitor, SH-4-54, significantly downregulated MMP-9 expression and decreased MVD and BM degradation. Surprisingly, STAT3 inhibition also decreased serum IL-6 levels and JAK2 phosphorylation. Our results suggest that the IL-6/JAK2/STAT3/MMP-9 pathway might be related to CH-induced microvessel proliferation and BM degradation in the bone marrow.

A comparative study of the osteogenic performance between the hierarchical micro/submicro-textured 3D-printed Ti6Al4V surface and the SLA surface

Three-dimensional (3D) printed titanium and its alloys have broad application prospect in the field of biomedical implant materials, although the biological performance of the original surface should be improved. Learning from the development experience of conventional titanium implants, to construct a hierarchical hybrid topological surface is the future direction of efforts. Since the original 3D-printed (3D hereafter) Ti6Al4V surface inherently has micron-scale features, in the present study, we introduced submicron-scale pits on the original surface by acid etching to obtain a hierarchical micro/submicro-textured surface. The characteristic and biological performance of the 3D-printed and acid-etched (3DA hereafter) surface were evaluated in vitro and in vivo, compared with the conventional sandblasted, large-grit, acid-etched (SLA hereafter) surface. Our results suggested the adhesion, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stromal cells (BMSCs), as well as the in vivo osseointegration on 3DA surfaces were significantly improved. However, the overall osteogenic performance of the 3DA surface was not as good as the conventional SLA surface.

Effects of Granulocyte Colony-Stimulating Factor on Proliferation and Apoptosis of B Cells in Bone Marrow of Healthy Donors

Background and ObjectiveThe aim of this study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on the proliferation and apoptosis of bone marrow (BM) B cells from healthy donors and its mechanism. Materials and MethodsThe proliferation ability and apoptosis of BM cells from healthy donors before and after in vivo G-CSF application were determined by multiparameter flow cytometry. The gene expression of B cells was detected by RNA-Seq. In vitro experiments were performed to investigate the effects of G-CSF on the proliferation and apoptosis of BM B cells through which gene. ResultsTreating healthy donors with G-CSF significantly decreased proliferation and increased apoptosis of BM B cells. The proliferation of CD19+CD27- B cell subgroup and CD19+CD24hiCD38hi B cell subset were also decreased. G-CSF also significantly altered proapoptotic genes, cell cycle arrest genes, and DNA replication and cell cycle genes, especially significantly increased SOCS1 expression of BM B cells. In vitro experiments showed that SOCS1 overexpression did not affect B cell proliferation ability and apoptosis. ConclusionsOur results suggest that extensive effects of G-CSF on BM B cells, such as inhibiting proliferation, inducing apoptosis, and altering a series of gene expression.

Hepatosplenic T Cell Lymphoma: A Rare Disease

Hepatosplenic T cell lymphoma (HSTCL) is an uncommon neoplasm comprising 5% of peripheral T-cell lymphomas. We report an uncommon case of Peripheral T-cell lymphoma that is characterized by primary extranodal disease with malignant T cell proliferation in spleen, liver and bone marrow. 19 year old male patient presented with fever, weakness and pain abdomen for 2 months. On clinical examination he was pale and had massive hepatosplenomegaly. The diagnosis was quite challenging as thorough clinical, hematological and immunophenotypic correlation was required.

TI-6AL-4V alloy and β-tricalcium phosphate-based systems for in vitro study of mesenchymal stem cell functions at implant–tissue interface

Hip replacement is one of the most effective surgical procedures in medicine. Despite the fact that the clinical results of arthroplasty are usually excellent, some implants loosen with time and require revision. To help resolve this problem, research has focused on providing conditions that have a positive biological effect on bone building elements, resulting in a fast, seamless proliferation and differentiation of mesenchymal stem cells (MSC) into osteoblasts. That is why developing an in vitro model for studying MSC functions at the implant–tissue contact zone is essential. The aim of this study was to investigate the proliferation and differentiation capacity of human bone-marrow derived MSC in contact with medical implant-grade Ti-6Al-4V alloy and β-tricalcium phosphate (β-TCP). Human MSC were isolated from bone marrow aspirates obtained from the femoral canal during hip replacement surgeries and seeded on Ti-6Al-4V alloy squared specimens and β-TCP granules. The results indicate that MSC were able to effectively adhere to both biomaterial surfaces and, at the same time, retain their proliferative and osteogenic/adipogenic differentiation potential. We suggest that both Ti-6Al-4V alloy and β-TCP surfaces provide suitable conditions for the proliferation and differentiation of bone marrow MSC and can be used as in vitro models to explore MSC functions at the bone–implant interface.

Preparation and biological properties of silk fibroin/nano-hydroxyapatite/graphene oxide scaffolds with an oriented channel-like structure.

Constructing an ideal bone tissue engineering scaffold has been one of the research hotspots in the biomedical field. Silk fibroin (SF), nano-hydroxyapatite (nHAp) and graphene oxide (GO) are excellent biomaterials, and have been studied and explored extensively. To better mimic natural bone, we fabricated a SF/nHAp/GO hybrid scaffold with an oriented channel-like structure by using directional temperature field freezing technology. A comparative analysis was carried out for the SF, SF/nHAp, unoriented SF/nHAp/GO and oriented SF/nHAp/GO scaffolds. The physical and chemical properties were studied by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and universal mechanical testing. The data showed that the oriented channel-like SF/nHAp/GO porous scaffold expressed high interconnectivity, suitable pore diameter and porosity and anisotropic mechanical properties. Cytocompatibility tests indicated that the oriented channel-like SF/nHAp/GO porous scaffold was more favorable for stimulating bone marrow mesenchymal stem cells (BMSCs) adhesion and proliferation. Additionally, human umbilical vein endothelial cells (HUVECs) grew unimpeded along the channel, indicating it had advantages for vascularization. For further testing in vitro, osteogenic induction was carried out on BMSCs inoculated on the above scaffolds, and then alkaline phosphatase (ALP) activity was tested and cell mineralization was observed. The results indicated that the oriented channel-like SF/nHAp/GO porous scaffold was more conducive to osteogenic differentiation of BMSCs. Hence, the material may prove to be a promising scaffold for bone tissue engineering.

The effect of pore size within fibrous scaffolds fabricated using melt electrowriting on human bone marrow stem cell osteogenesis

Limitations associated with current bone grafting materials has necessitated the development of synthetic scaffolds that mimic the native tissue for bone repair. Scaffold parameters such as pore size, pore interconnectivity, fibre diameter, and fibre stiffness are crucial parameters of fibrous bone tissue engineering (BTE) scaffolds required to replicate the native environment. Optimum values vary with material, fabrication method and cell type. Melt electrowriting (MEW) provides precise control over extracellular matrix (ECM)-like fibrous scaffold architecture. The goal of this study was to fabricate and characterise poly-ε-caprolactone (PCL) fibrous scaffolds with 100, 200, and 300 μm pore sizes using MEW and determine the influence of pore size on human bone marrow stem cell (hMSC) adhesion, morphology, proliferation, mechanosignalling and osteogenesis. Each scaffold was fabricated with a fibre diameter of 4.01 ± 0.06 μm. The findings from this study highlight the enhanced osteogenic effects of controlled micro-scale fibre deposition using MEW, where the benefits of 100 μm square pores in comparison with larger pore sizes are illustrated, a pore size traditionally reported as a lower limit for osteogenesis. This suggests a lower pore size is optimal when hMSCs are seeded in a 3D ECM-like fibrous structure, with the 100 μm pore size optimal as it demonstrates the highest global stiffness, local fibre stiffness, highest seeding efficiency, maintains a spread cellular morphology, and significantly enhances hMSC collagen and mineral deposition. Similarly, this platform represents an effective in vitro model for the study of hMSC behaviour to determine the significant osteogenic benefits of controlling ECM-like fibrous BTE scaffold pore size using MEW.

In vitro proliferation and differentiation of canine bone marrow derived mesenchymal stem cells over hydroxyl functionalized CNT substrates

Nanotopography of culture substrate acts as a positive cue in cell-biomaterial based tissue regeneration. Considering the potentiality of carbon nanotubes (CNTs) this study was designed to evaluate its two functionalized form by an in vitro culture condition using canine mesenchymal stem cells as cellular model. Cells were isolated and its behaviour, proliferation and differentiation processes were elucidated onto CNT substrates. Beside the variations in cellular behaviour it was remarkably noted that even though proliferation was reduced but osteogenic and chondrogenic differentiation was enhanced over multi-walled CNTs, whereas neuronal differentiation was better supported by single walled CNTs as evidenced by our cytochemical, immunocytochemical, gene expression and flow cytometry assays. The former one was noticed more cytocompatible by our different apoptosis studies. The outcome of these experiments collectively indicated that hydroxylated functionalized CNTs could be a potential scaffold constituent for future experimentations as well as for the application in regenerative medicine.

Apolipoprotein AI) Promotes Atherosclerosis Regression in Diabetic Mice by Suppressing Myelopoiesis and Plaque Inflammation.

Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering. Aortic arches containing plaques developed in Ldlr-/- mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined. Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus. Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.

Effect of recombinant human thrombopoietin on endotoxin-induced thrombocytopenia in mice

To investigate the effect of recombinant human thrombopoietin (rhTPO) on thrombocytopenia (TCP) induced by endotoxin lipopolysaccharide (LPS) in mice. Sixty male C57BL/6 mice were divided into normal saline (NS) control group (NS group), sepsis-induced TCP model group (LPS group) and rhTPO treatment group (LPS+rhTPO group) by random number table with 20 mice in each group. Sepsis-induced TCP model was reproduced by one intraperitoneal injection of LPS 30 mg/kg, and the mice in NS group were given the same amount of NS. In LPS+rhTPO group, 2.7 kU/kg rhTPO was subcutaneously injected into mice immediately after intraperitoneal injection of LPS, once every 24 hours. The mice in NS group and LPS group were injected subcutaneously with the same amount of NS. The observation period of each group lasted for 72 hours. The inner canthus blood was harvested before and every 24 hours after modeling, and the platelet count (PLT) was measured by animal blood cell counter. The eyeball blood of mice was harvested at 72 hours after modeling, and the proportion of CD61+CD62p+ cells in platelet-rich plasma was detected by flow cytometry, by which the platelet activation was reflected. Lung and spleen tissues of mice were harvested, and the positive expression of CD41 was determined by immunohistochemistry, by which the platelet sequestration in organs was reflected. Bone marrow cells from unilateral femur of mice were harvested, and the proportion of CD41+CD61+ cells was determined by flow cytometry to reflect the proliferation of bone marrow megakaryocytes. There was no significant difference in PLT among the groups before modeling. With the extension of the time after modeling, PLT in LPS group was decreased continuously, and increased slightly at 72 hours, but it was still significantly lower than that in NS group (×109/L: 308.60±21.70 vs. 1 152.72±50.27, P < 0.05); PLT in LPS+rhTPO group was increased continuously with the extension of modeling time, and it was significantly higher at 72 hours than that in LPS group (×109/L: 926.78±48.85 vs. 308.60±21.70, P < 0.05). At 72 hours after modeling, the proportion of CD61+CD62p+ cells in platelet-rich plasma of LPS group was significantly higher than that of NS group [(25.07±2.55)% vs. (4.17±0.38)%, P < 0.05], while the value in LPS+rhTPO group was significantly lower than that of LPS group [(15.92±1.26)% vs. (25.07±2.55)%, P < 0.05]. The proportion of CD41+CD61+ cells in bone marrow megakaryocytes of LPS group was significantly higher than that of NS group [(11.84±0.80)% vs. (3.60±0.42)%, P < 0.05], and the proportion of CD41+CD61+ cells in LPS+rhTPO group was significantly higher than that in LPS group [(30.96±2.49)% vs. (11.84±0.80)%, P < 0.05]. Immunohistochemistry showed that the positive expressions of CD41 in lung and spleen tissues of LPS group increased significantly than NS group [A value: 828.94±119.30 vs. 447.09±16.19 in lung tissue, (280.15±16.71)×103 vs. (0.65±0.26)×103 in spleen tissue, both P < 0.05], while the positive expressions of CD41 in lung and spleen tissues of LPS+rhTPO group decreased significantly than LPS group [A value: 542.78±2.95 vs. 828.94±119.30 in lung tissue, (129.40±13.49)×103 vs. (280.15±16.71)×103 in spleen tissue, both P < 0.05]. The rhTPO in endotoxin-induced TCP may stimulate the proliferation of bone marrow megakaryocytes, inhibit platelet activation and affect platelet sequestration in organs, so as to increase platelet levels.

Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated with Reduced Graphene Oxide for Rapid Bone Ingrowth and Repair.

Hydroxyapatite (HA), the traditional bone tissue replacement material was widely used in the clinical treatment of bone defects because of its excellent biocompatibility. However, the processing difficulty and poor osteoinductive ability greatly limit the application of HA. Although many strategies have been reported to improve the machinability and osteointegration ability, the performance including mechanical strength, porosity, cell adhesion, etc. of material still can not meet the requirements. In this work, a soft template method was developed and a porous scaffold with hierarchical pore structure, nano surface morphology, suitable porosity and pore size, and good biomechanical strength was successfully prepared. The hierarchical pore structure is beneficial for cell adhesion, fluid transfer, and cell ingrowth. Moreover, the loaded reduced graphene oxide (rGO) can improve the adhesion and promote the proliferation and spontaneous osteogenic differentiation bone marrow mesenchymal stem cells. The scaffold is then crushed, degraded and wrapped by the newly formed bone and the newly formed bone gradually replaces the scaffold. The degradation rate of the scaffold well matches the rate of the new bone formation. The hierarchical porous HA/rGO composite scaffolds can greatly accelerate the bone ingrowth in the scaffold and bone repair in critical bone defects, thus providing a clinical potential candidate for large segment bone tissue engineering.

Abstract 301: Bone Marrow Niche-Mediates Cardiac Repair is Associated With Adiponectin in Retnla Deficiency

Background: Resistin like α (Retnla) is known to be upregulated in anti-inflammatory macrophages and to modulate lipid profile in atherosclerosis. However, the role of Retnla is unknown in the cardiac pathology. Methods: Myocardial infarction (MI) was induced in wild type, Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiomyocytes, cardiac fibroblasts, macrophages and bone marrow cells were treated with Retnla protein for mechanistic studies. For an adoptive transfer, bone marrow cells were isolated from KO mice to transplant to the infarcted heart. Results: TUNEL staining showed the number of apoptotic cardiomyocytes was higher in the Retnla KO mice than in the TG mice 1 day after MI. Cardiac function and angiogenesis were significantly better in the Retnla KO group, while were worsen in the TG group. Retnla is an anti-inflammatory marker in macrophages, but the expressions of inflammation-related genes were not different between both groups. Interestingly, however, the numbers of macrophages isolated from bone marrow, peritoneum, and cardiac tissue of Retnla KO mice were higher than in the wild type mice. Moreover, phospho-histone H3-positive proliferating cells in the bone marrow were much more abundant in the Retnla KO mice than in the wild type mice. Adiponectin was highly expressed in bone marrow of KO mice, and we transplanted bone marrow cells from KO mice to recipient wild type mice. Two weeks after MI, recipient group transplanted with bone marrow cells from KO mice showed better angiogenesis, reserved cardiac function, and more anti-inflammatory macrophages. In Retnla-treated cardiomyocytes, bax, phopho-p38, and p21 were notably upregulated, while bcl2 and adiponectin were significantly reduced. To examine whether Retnla acted negatively on adiponectin, we administrated a synthetic adiponectin receptor agonist AdipoRon to Retnla-treated cardiomyocytes. AdipoRon blunted the effect of Retnla on cardiomyocytes. These data showed better cardiac recovery was associated with bone marrow-derived adiponectin in the Retnla KO group. Conclusion: Our study suggested that substantial cardiac recovery was strongly associated with adiponectin derived from highly proliferating bone marrow cells in Retnla KO mice.

SMUG1 Promotes Telomere Maintenance through Telomerase RNA Processing

Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3'-polyadenylated and 3'-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.

Limitations of neutrophil depletion by anti-Ly6G antibodies in two heterogenic immunological models

Neutrophil-depleting antibodies, such as anti-GR1 (RB6-8C5) and anti-Ly6G (1A8), are commonly used to study the in vivo function of neutrophils in murine disease models. Anti-Ly6G antibodies became the standard, because in contrast to anti-GR1, these do not bind Ly6C. The efficiency of the depletion needs to be carefully analysed as flow cytometry plots may be misinterpreted. For example, the staining intensity of GR1 on neutrophils (CD11b+ GR1hi) drops upon anti-Ly6G administration. We show that this drop is due to competition between anti-GR1 and anti-Ly6G antibodies. Neutrophil depletion with anti-Ly6G in naive mice was organ- and strain-specific. Furthermore, an incomplete anti-Ly6G-dependent neutrophil depletion was obtained in two immune-mediated mouse models, i.e. in malaria-infected C57BL/6 mice and in complete Freund’s adjuvant (CFA)-challenged BALB/c mice. BrdU-incorporation studies show a slight increase in proliferating bone marrow neutrophils upon depletion in naive mice. Strikingly, depletion with anti-Ly6G in CFA-challenged BALB/c mice resulted in a significant increase in proliferating splenic neutrophils, causing a fast rebound of new immature neutrophils. In conclusion, our results emphasize the importance of careful panel design, gating strategies and duration of neutrophil depletion and highlight the context-dependent Ly6G depletion efficiency. It furthermore underlines the need for new tools to understand the in vivo role of neutrophils in immunological models.

PS996 TCF4 IS IMPORTANT FOR MUTANT RUNX1‐MEDIATED CELL IMMORTALIZATION AND PREDICTS OUTCOME IN ACUTE MYELOID LEUKEMIA

Background:Aberrations of the RUNX1 transcription factor, either mutations (RUNX1 mut) or translocations t(8;21) creating a RUNX1‐RUNXT1 fusion gene, are frequently observed in AML. However, the pathomechanisms underlying RUNX1 mut AML and explaining its poor prognosis in contrast to t(8;21) cases are still poorly understood.Aims:As recent data suggested deregulation of the transcription factor 4 (TCF4) gene to play a potential role, we studied the importance of TCF4 in the context of RUNX1 mut and t(8;21).Methods:Chip‐sequencing data was analyzed to identify binding of RUNX1 on the TCF4 promoter and Luciferarse reporter assays were used to assay impact of RUNX1 on TCF4 promoter activity.CD34+ UCB cells were transduced with a pRRL construct overexpressing the RUNX1 R201Q mutant (RUNX1mut) or an empty vector (EV). Simultaneously, TCF4‐RNAi (TCF4kd) or a non‐targeting control (NT) was co‐transduced. These double transduced CD34+ UCB cells were cultured on MS5 bone marrow stromal layer and proliferation assays, colony forming unit assays and long‐term culture‐initiating cells assays were performed. TCF4 expression values were derived from a previously reported cohort of 436 AML patients of which 330 had a known RUNX1 status. Survival curves were calculated by the Kaplan‐Meier method and compared using the logrank test. Multivariate survival analysis was carried out using the Cox proportional hazards model, and covariates included were RUNX1 mutation and white blood cell count (WBC &gt; 100 ∗109/L) with and without TCF4 expression (lowest 75% vs highest 25%).Results:While TCF4 is found upregulated in RUNX1 mut in contrast to t(8;21) AML, Chip‐seq and transactivation assays revealed that RUNX1 binds and regulates the TCF4 promoter activity. RUNX1 wild type (RUNX1wt) and the RUNX1‐RUNX1T1 fusion protein repressed TCF4 promoter activity, which was lost in RUNX1mut AML. In CD34+ cells RUNX1 mut enhanced colony‐formation and increased long‐term culture initiating cells, whereas RNA interference based TCF4 downregulation in RUNX1 mut cells abolished this effect. Being required for stem cell maintenance activity in RUNX1 mut AML, TCF4 upregulation accounted for the poor prognostic outcome of RUNX1 mut in AML patients, as RUNX1 mut lost its significance in multivariate analysis if TCF4 expression was included.Summary/Conclusion:In summary, TCF4 is an important downstream target of RUNX1mut AML contributing to leukemogenesis and poor response to standard AML therapy.

PB1990 THE CHANGE OF BONE MARROW MICROENVIRONMENT OF SECONDARY POOR GRAFT FUNCTION AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION AND ITS EFFECT ON PROGNOSIS

Background:Secondary poor graft functionis a serious life‐threatening complication after allogeneic hematopoietic stem cell transplantation, which pathogenesis, evolution and prognosis are still unclear.Aims:To explore the role of adhesion related indicators in secondary poor graft functionafter allogeneic hematopoietic stem cell transplantationMethods:In this study, clinical data and post‐transplantation status of 106 patients with hematological tumour who were enrolled in our center and underwent allo‐HSCT from Jan. 1, 2016 to Jan. 31, 2017 was counted.The patients were divided into good graft function (GGF) group and secondary poor graft function (sPGF) group according to the implantation status after transplantation. The differences of age, primary disease, stem cell source, HLA matching, sex matching, ABO blood group matching, degree of bone marrow hyperplasia before transplantation, recurrence rate and mortality between the two groups were compared.29 patients with sPGF after transplantation were selected as the experimental group while 29 patients with GGF were selected as the control group.Results:The changes of adhesion related indicators in the two groups were detected.There were no significant differences in age, primary disease, stem cell origin, HLA matching, sex matching, ABO blood group matching and the degree of bone marrow proliferation before transplantation between the two groups (P &gt; 0.05). The infection rate of CMV in sPGF group was significantly higher than that in GGF group, and the remission rate before transplantation in sPGF group was significantly lower than that in GGF group (P &lt; 0.05).Cox regression analysis showed that the survival rate of patients with sPGF after allo‐HSCT was significantly lower than that of patients without sPGF (P &lt; 0.05, HR = 3.598).The expression of CD44, CD29, ICAM‐1 and VCAM‐1 in sPGF group was not significantly different from that in control group (P &gt; 0.05), but the expression of CD105 was higher than that in control group, and the expression of Cx43 was lower than that in control group (P &lt; 0.05).Summary/Conclusion:We believe that the survival rate of patients with sPGF after allo‐HSCT will decrease significantly, and the occurrence of sPGF after transplantation may be related to the increase of CD105 expression and the decrease of Cx43 expression in bone marrow stromal cells before transplantation.

Adult bovine platelet lysate-derived serum “NeoSERA” is safe, less ethical and powerful alternative to fetal bovine serum for the culture of mesenchymal stem cells

Background & Aim Fetal bovine serum (FBS) is a common component of culture media and usually used for cellular research, as well as recent cell-based medical products. However, due to the high risk of contaminations and the variation from batch to batch, FBS might influence the outcome of research or cellular manufacturing. FBS also contains moral concerns because it harvested from bovine fetuses taken from pregnant cows. In addition, FBS is most expensive part of cell culture. To overcome several problems regarding FBS, we developed a new serum, adult bovine platelet lysate-derived cell culture supplement (NeoSERA), and investigate its usefulness as an alternative to FBS. Methods, Results & Conclusion NeoSERA is produced in Japan, considered to be a negligible bovine spongiform encephalopathy (BSE) risk country. We obtain adult donor bovine blood from animal less than 36 months old according to the regulation of EMA for BSE (EMA/410/01 rev.3). Using apheresis medical devices with closed disposable kits, sterile platelet-rich plasma (PRP) is collected from healthy donor bovine receiving a regular veterinary check. After stimulation and removal of coagulated fibrin by centrifugation, NeoSERA is collected in a completely closed system. To meet the scope of directives that apply to produce medicinal products from the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/743/00) and the United States Department of Agriculture (9CFR§113.450), NeoSERA is finally g-irradiated at a dose of over 30 kGy. NeoSERA completely meets the standard for biological ingredients in Japan (Ministry of Health, Labour and Welfare Notification No. 375 2014) and also obtained the certificate of eligibility for the raw material of regenerative medicine from Pharmaceuticals and Medical Devices Agency (PMDA, No. 0417002, 2017. 4.17). To test whether NeoSERA is useful for the expansion of mesenchymal stem cells (MSCs), a cell culture experiment was performed. 3 to 5 days after NeoSERA treatment, the proliferation of human bone marrow-, adipose tissue-, umbilical cord- and amnion-derived MSCs was significantly increased (p Our results confirm that safe, less ethical and economical bovine NeoSERA profoundly enhances the proliferation of MSCs for regenerative medicine and several cell lines for vaccine production.

Salt‐Assisted Toughening of Protein Hydrogel with Controlled Degradation for Bone Regeneration

AbstractRecently, strong polymer‐based hydrogels have been intensively investigated. However, the development of tough protein hydrogels with controlled degradation for bone regeneration has rarely been reported. Here, regenerated silk fibroin/gelatin (RSF/G) hydrogels with both strength and controlled degradation are prepared via physically and chemically double‐crosslinked networks. As a representative example, the 9%RSF/3%G hydrogel shows approximately 80% elongation and a compressive and tensile modulus of up to 0.25 and 0.21 MPa, respectively. It also shows a degradation rate that can be adjusted to approximately three months in vivo, a value between that of the rapidly degrading gelatin hydrogel and the slowly degrading RSF hydrogel. The 9%RSF/3%G hydrogel has good biocompatibility and promotes the proliferation and differentiation of bone marrow–derived stem cells compared with the control and pure RSF hydrogels. At 12 weeks after implantation of the gel in a calvarial defect, micro‐computed tomography shows greater bone volume and bone mineral density in the 9%RSF/3%G group. More importantly, histology reveals more mineralization and enhancements in the quality and rate of bone regeneration with less of a tissue response in the 9%RSF/3%G group. These results indicate the promising potential of this tough protein hydrogel with controlled degradation for bone regeneration applications.