Human Cord Blood CD34 Research Articles

Effects of opioid and nonopioid analgesics on canine wheal formation and cultured human mast cell degranulation

Mast cell (MC) degranulation has been implicated in the side effect profile of a variety of clinically useful agents. Thus, after intrathecal delivery, formation of space-occupying, meningeally-derived masses may be related to local MC degranulation. We systematically characterized degranulating effects of opioid and nonopioid analgesics on cutaneous flares in the dog and in primary human MC (hMC) cultures. MethodsDogs were anesthetized with IV propofol and received intradermal (ID) injections (50μL). Flare diameters were measured at 30min. Drugs showing flare responses were tested after intramuscular (IM) cromolyn (10mg/kg), a MC stabilizer. Human primary MCs (human cord blood CD34+/CD45+ cells) were employed and β-hexosaminidase in cell-free supernatants were measured to assess degranulation. ResultsA significant skin flare for several classes of agents was observed including opioids, ziconotide, ketamine, ST-91, neostigmine, adenosine, bupivacaine, lidocaine, MK-801 and 48/80. Tizanidine, fentanyl, alfentanil, gabapentin and baclofen produced no flare. Flare produced by all ID agents, except adenosine, bupivacaine and lidocaine, was reduced by cromolyn. Naloxone had no effect upon opiate or 48/80 evoked flares. In hMC studies, 48/80 resulted in a concentration-dependent release of β-hexosaminidase. The rank order of drug-induced hMC β-hexosaminidase release was similar to that for flares. ConclusionsA variety of therapeutically useful drugs degranulate MCs. This action may account for side effects such as the intrathecal granuloma resulting from spinally-delivered opioids. This degranulating effect may be useful in predicting potential intrathecal toxicity in the development of novel agents.

Control of HIV Infection InVivo Using Gene Therapy with a Secreted Entry Inhibitor.

HIV entry inhibitors are highly effective in controlling virus replication. We have developed a lentiviral vector that expresses a secreted entry inhibitor, soluble CD4 (sCD4), which binds to the HIV envelope glycoproteins and inactivates the virus. We have shown that sCD4 was secreted from gene-modified CD4+ T cells, as well as from human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), and protected unmodified HIV target cells from infection in vitro. To investigate the in vivo application of our approach, we injected gene-modified HSPCs into NOD/SCID/γcnull (NSG) mice. NSG hosts supported multi-lineage differentiation of human gene-modified HSPCs. Upon challenge with HIV, humanized mice capable of secreting sCD4 demonstrated a reduction of viral load over time compared to control humanized mice. In contrast to gene therapy approaches that render only gene-modified HIV target cells resistant to infection, our approach also showed protection of unmodified CD4+ T cells in the peripheral blood and tissues. Our findings provide support for the continuous delivery of secreted entry inhibitors via gene therapy as an alternative to oral administration of antiretroviral drugs or injection of antiretroviral proteins, including antibodies.

The AF4-MLL fusion transiently augments multilineage hematopoietic engraftment but is not sufficient to initiate leukemia in cord blood CD34+ cells.

The translocation t(4;11)(q21;q23) is the hallmark genetic abnormality associated with infant pro-B acute lymphoblastic leukemia (B-ALL) and has the highest frequency of rearrangement in Mixed-lineage leukemia (MLL) leukemias. Unlike other MLL translocations, MLL-AF4-induced proB-ALL is exceptionally difficult to model in mice/humans. Previous work has investigated the relevance of the reciprocal translocation fusion protein AF4-MLL for t(4;11) leukemia, finding that AF4-MLL is capable of inducing proB-ALL without requirement for MLL-AF4 when expressed in murine hematopoietic stem/progenitor cells (HSPCs). Therefore, AF4-MLL might represent a key genetic lesion contributing to t(4;11)-driven leukemogenesis. Here, we aimed to establish a humanized mouse model by using AF4-MLL to analyze its transformation potential in human cord blood-derived CD34+ HSPCs. We show that AF4-MLL-expressing human CD34+ HSPCs provide enhanced long-term hematopoietic reconstitution in primary immunodeficient recipients but are not endowed with subsequent self-renewal ability upon serial transplantation. Importantly, expression of AF4-MLL in primary neonatal CD34+ HSPCs failed to render any phenotypic or hematological sign of disease, and was therefore not sufficient to initiate leukemia within a 36-week follow-up. Species-specific (epi)-genetic intrinsic determinants may underlie the different outcome observed when AF4-MLL is expressed in murine or human HSPCs.

EPCR expression marks UM171-expanded CD34+ cord blood stem cells

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.

Targeted release of transcription factors for human cell reprogramming by ZEBRA cell-penetrating peptide

Transcription factors (TFs) are key actors of the control of gene expression and consequently of every major process within cells, ranging from cell fate determination, cell cycle control and response to environment. Their ectopic expression has proven high potential in reprogramming cells for regenerative medicine; ontogenesis studies and cell based modelling. Direct delivery of proteins could represent an alternative to current reprogramming methods using gene transfer but still needs technological improvements. Herein, we set-up an efficient cellular penetration of recombinant TFs fused to the minimal transduction domain (MD) from the ZEBRA protein. We show that ZEBRA MD-fused TFs applied on primary human fibroblasts and cord blood CD34+ hematopoietic stem cells route through the cytoplasm to the nucleus. The delivery of Oct4, Sox2 and Nanog by MD leads to the activation of mRNA transcripts from genes regulated by these TFs. Moreover, the expression of genes involved in the pluripotency network but not directly bound by these TFs, is also induced. Overall, the repeated application of MD-Oct4, MD-Sox2, MD-Nanog TFs and the post-transcriptional regulator RNA-binding protein MD-Lin28a, triggers the rejuvenation of human fibroblasts and CD34+ cells. This study provides powerful tools for cell fate reprogramming without genetic interferences.

Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells.

The ex vivo generation of human red blood cells on a large scale from hematopoietic stem and progenitor cells has been considered as a potential method to overcome blood supply shortages. Here, we report that functional human erythrocytes can be efficiently produced from cord blood (CB) CD34+ cells using a bottle turning device culture system. Safety and efficiency studies were performed in murine and nonhuman primate (NHP) models. With the selected optimized culture conditions, one human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1% ± 6.2% and 50% ± 5.7% (mean ± SD) for CD235a+ cells and enucleated cells, respectively. The yield of erythrocytes from one CB unit (5 million CD34+ cells) could be, in theory, equivalent to 500 blood transfusion units in clinical application. Moreover, induced human erythrocytes had normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model. In NHP model, xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia. These findings suggested that the ex vivo‐generated erythrocytes could be an alternative blood source for traditional transfusion products in the clinic. Stem Cells Translational Medicine 2017;6:1698–1709

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells

BackgroundHumanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points.ResultsHuman cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time.ConclusionsOverall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

Splicing factor SF3B1K700E mutant dysregulates erythroid differentiation via aberrant alternative splicing of transcription factor TAL1.

More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of β-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.

HIF-1α-stabilizing agent FG-4497 rescues human CD34+ cell mobilization inresponse to G-CSF in immunodeficient mice.

Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxia-inducible factor (HIF)-1α, synergize with G-CSF invivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg-/- mice by transplanting them with human umbilical cord blood CD34+ HSPCs and then treating them with G-CSF with and without co-administration of the PHD inhibitor FG-4497. We observed that combination treatment with G-CSF and FG-4497 resulted in significant mobilization of human lineage-negative (Lin-) CD34+ HSPCs and more primitive human Lin-CD34+CD38- HSPCs into blood and spleen, whereas mice treated with G-CSF alone did not mobilize human HSPCs significantly. These results suggest that the PHD inhibitor FG-4497 also increases human HSPC mobilization in a xenograft mouse model, suggesting the possibility of testing PHD inhibitors to boost HSPC mobilization in response to G-CSF in humans.

Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models

Recently, NOD-SCID IL2Rγ-/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties.To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). Invitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34+ cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. Invivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34+ cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis.

Generation of non-transgenic iPS cells from human cord blood CD34+ cells under animal component-free conditions

Recently, many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells, reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation, cultivation, reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components.

Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells).

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

An effective ex-vivo approach for inducing endothelial progenitor cells from umbilical cord blood CD34+ cells

BackgroundTransplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. We established an effective approach to stimulate the expansion and differentiation of EPCs for potential therapeutic applications.MethodsCD34+ cells isolated from human cord blood were cultured in a two-step system for 21 days. The generated adherent cells were characterized via flow cytometry and immunofluorescent staining. Moreover, single-cell clonogenic and tube-forming assays were carried out to evaluate their potential to proliferate and form vessel networks. Furthermore, these cells were transplanted into a mouse model of hepatic sinusoidal endothelium injury by hepatic portal vein injection to investigate their in-vivo behavior.ResultsThe two-step culture protocol promoted the expansion and differentiation of human cord blood CD34+ cells efficiently, resulting in a large number of adherent cells within 3 weeks. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. Moreover, these EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. More significantly, tissue examination of mice after transplantation revealed that the injected EPCs/ECs migrated and integrated into the liver, reconstituting the sinusoidal endothelial compartment.ConclusionsWe developed an approach for the generation of cord blood-derived EPCs/ECs on a large scale, characterized them phenotypically, and demonstrated their in-vivo functional capacity. Our approach provides an excellent source of healthy EPCs/ECs for use in cell therapy in a clinical setting.

Loss of Forkhead box M1 promotes erythropoiesis through increased proliferation of erythroid progenitors

Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.

Evaluations of Intravenous Administration of CD34+ Human Umbilical Cord Blood Cells in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34⁺ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term “neonatal encephalopathy,” their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Transplantation of human umbilical cord blood CD34+ cells into the liver of newborn NOD/SCID/IL-2Rγ null (NSG) mice after busulfan conditioning.

Transplantation of human umbilical cord blood CD34+ cells into the liver of newborn NOD/SCID/IL-2Rγ null (NSG) mice after busulfan conditioning.

Evidence for the in vivo safety of insulated foamy viral vectors.

Retroviral vector mediated stem cell gene therapy is a promising approach for the treatment of hematopoietic disorders. However, genotoxic side effects from integrated vector proviruses are a significant concern for the use of retroviral vectors in the clinic. Insulated foamy viral (FV) vectors are potentially safer retroviral vectors for hematopoietic stem cell gene therapy. We evaluated two newly identified human insulators, A1 and A2 for use in FV vectors. These insulators had moderate insulating capacity and higher titers than previously developed insulated FV vectors. The A1 insulated FV vector was chosen for comparison with the previously described 650cHS4 insulated FV vector in human cord blood CD34+ repopulating cells in an immunodeficient mouse model. To maximize the effects of the insulators on the safety of FV vectors, FV vectors containing a highly genotoxic spleen focus forming virus (SFFV) promoter was used to elicit differences in genotoxicity. In vivo, the A1 insulated FV vector showed an approximate 50% reduction in clonal dominance compared to either the 650cHS4 insulated or control FV vectors, although the transduction efficiency of the A1 insulated vector was higher. This data suggests that the A1 insulated FV vector is promising for future pre-clinical and clinical studies.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as ‘the storage lesion’. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

MAF Induces Inflammatory Mediators Involved in the Pathogenesis of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm characterized by the presence of hyperplastic/dysplastic megakaryocytes and the development of myelofibrosis and osteosclerosis. This disruption of the bone marrow (BM) structure results from a deregulated release of growth factors by malignant hematopoietic cells, particularly monocytes and megakaryocytes, that activate BM stromal cells (Desterke-C, Mediators Inflamm, 2015). Therefore, it remains of primary importance to identify novel therapeutic strategies to restore BM microenvironment homeostasis in PMF patients.In order to better characterize the molecular mechanisms underlying PMF pathogenesis, we have recently compared the gene expression profile (GEP) of CD34+ hematopoietic progenitor cells (HPCs) from PMF patients and healthy donors, and we observed the upregulation of the transcription factor MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) (Norfo-R, Blood, 2014).With the aim to investigate the role of MAF in PMF pathogenesis and to reproduce the condition observed in PMF CD34+ cells, we studied the effects of MAF overexpression on HPCs differentiation. Human cord blood-derived CD34+ cells were transduced with a retroviral vector carrying the coding sequences for the two MAF transcript variants (LMAFVAR1IDN and LMAFVAR2IDN). The megakaryocyte lineage-committed CD41+ population was increased in LMAFVAR1IDN (38.2±2.3%) and LMAFVAR2IDN (39.4±2.3%) samples compared with the empty vector LXIDN (16.1±2.7%) at day 3 after NGFR+ cells purification. Values are reported as mean ± SEM from 3 independent experiments, p<.05. Moreover, MAF overexpression determined a remarkable increase of the CD14+ monocyte population in LMAFVAR1IDN (45.5±5.7%) and LMAFVAR2IDN (43.1±8.7%) compared to LXIDN control (8.0±0.4%) at t0 post-purification. Data represent mean ± SEM from 3 independent experiments, p<.05.Next, we analyzed the GEP of MAF-overexpressing CD34+ cells and observed the upregulation of genes involved in megakaryopoiesis, such as MERTK and RCAN1, and in monocyte/macrophage differentiation (e.g. EGR2 and KLF4). In particular, GEP revealed the upregulation of genes related to processes deregulated in PMF, such as fibrosis (i.e. ENPP2, ADAM9) and inflammation (i.e. TNF, CCL2 and CCL3). Noteworthy, several upregulated transcripts encoded for secreted proteins.Based on their role in inflammation and fibrosis, we selected a set of secreted molecules (IL8, MMP9, CCL2, SPP1, LGALS3 and PLAUR) and assessed them by quantitative enzyme-linked immunoassay (ELISA) in the culture supernatants from LMAFVAR1IDN-, LMAFVAR2IDN- and LXIDN-transduced cells. The levels in culture supernatants of all these molecules were increased in MAF-overexpressing samples compared to control. In order to correlate the expression of these molecules with MAF upregulation in PMF, we assessed them in plasma samples of PMF patients (n=30) and healthy donors (n=10). All the selected molecules displayed higher plasma levels in PMF patients than in healthy controls (Figure 1).Since the increase of SPP1 and LGALS3 has never been described before in PMF, we investigated which cell type could be responsible for SPP1 and LGALS3 production. Real Time qRTPCR performed on healthy donors peripheral blood-derived cells showed that the highest SPP1 expression levels were observed in CD34+ cells-derived megakaryocytes and granulocytes, whereas LGALS3 was expressed at higher levels in monocytes compared to the other cell types.Because the increased proliferation of fibroblasts is involved in BM fibrosis and is a PMF feature, we investigated the effects of LGALS3 and SPP1 on normal human primary dermal fibroblasts. The treatment with recombinant human LGALS3 (1000 ng/ml) or SPP1 (500 ng/ml) caused an increase in fibroblasts cell count of 20.9±2.7% (p<.001) and 14.9±4.1% (p<.01) compared to the untreated control, respectively. Values are reported as mean ± SEM from 7 independent experiments.Finally, we observed that increased SPP1 plasma levels in PMF patients correlate with a more severe fibrosis and lower overall survival (p<.05).In conclusion, our data unveil the involvement of MAF in PMF pathogenesis through the induction of a pro-fibrotic and pro-inflammatory phenotype in the malignant myeloid differentiated progeny. [Display omitted] DisclosuresVannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Human Hematopoietic Stem Cell Homing and Engraftment Promoted By Glucocorticoid Induced Chromatin Remodeling

Hematopoietic stem cells (HSC) which give rise to cells of all the blood lineages throughout the lifetime are key cells in hematopoietic cell transplantation (HCT). HCT has been used to treat patients with malignant and non-malignant hematological disorders. Cord blood (CB) is an increasingly used source of HSC for HCT, but one considered limitation to its greater use is the limited numbers of HSC in single CB collections. Thus, in this situation, the homing efficiency of HSC can be a pivotal factor for a successful HCT based clinical therapy. One means to enhance single CB unit HCT is to enhance the homing efficiency of CB HSC. The SDF-1/CXCR4 chemotactic axis is a major pathway directing migration and homing of HSC from peripheral blood to bone marrow niches. To get enhanced insight into achieving better homing efficiency of human CB HSC, we performed a compound screen to search for small molecules which could enhance surface expression of CXCR4 and SDF-1/CXCR4 axis mediated chemotaxis. From a nuclear hormone ligand library including 74 chemical compounds, we found that treatment of CB CD34+ cells with Dexamethasone, a synthetic glucocorticoid (GC), greatly promoted surface expression of CXCR4. Expression of CXCR4 on CB CD34+ cells was also increased after treating cells with other glucocorticoids (not existing in the library) including Flonase, Cortisol, and Medrol. We focused on use of Flonase, the compound which forms the most stable activated complex with glucocorticoid receptor (GR) (Högger, P. & Rohdewald, P. Steriods, 1994), and which enhanced CXCR4 expression at concentrations as low as 10 nM. Pretreament of CB CD34+ cells with Flonase significantly increased surface CXCR4 expression and promoted SDF-1/CXCR4 axis mediated chemotaxis of human CB HSC. Further more, Flonase pretreatment rigorously enhanced homing of phenotypically-defined human CB HSC and progenitor cells (HPC) (by ~5 fold, p<0.01). To assess long-term reconstituting ability of GC pretreated human CB CD34+ cells, we performed limiting dilution analysis to calculate the frequency of in vivo SCID Repopulating Cells (SRC). Poisson distribution analysis revealed an SRC frequency of 1/5237 in Vehicle-treated CB CD34+ cells and an enhanced SRC frequency of 1/1078 in Flonase-treated CB CD34+ cells, suggesting the respective presence of 191 SRCs and 928 SRCs in Vehicle- and Flonase-pretreated and administered cells (~4.8 fold, p<0.001). GC pretreatment significantly increased mRNA and protein levels of CXCR4 in human CB CD34+ cells. A search for the consensus GR binding sequence AGAACAnnnTGTNCN (Watson, L.C. et al. Nat. Struct. Mol. Biol. 2013), found that a glucocorticoid response element (GRE), AGAACATTCTGTGCA existed in the CXCR4 promoter region (from -1662 to -1648). Chromatin immunoprecipitation analysis suggested that GR was remarkably enriched in this region of the CXCR4 promoter upon administration of GC. CXCR4 promoter activity was enhanced (by ~2.5 fold, p<0.001) by Flonase stimulation, and this was abrogated by deletion of the GRE. Co-immuoprecipitation analysis demonstrated that the SRC1/p300 complex which has histone acetyltransferase activity was recruited to the CXCR4 promoter by GR upon Flonase treatment. Acetylation levels of CXCR4 associated Histone 4 on Lysine 5 and Lysine 16 (H4K5 and H4K16) were dramatically enhanced by Flonase treatment, while acetylation levels of Histone 3 (H3K9 and H3K14) remained unchanged. Enhanced chemotaxis and homing of Flonase treated CB HSC and HPC was totally blocked by administration of p300 inhibitor (c646). Our findings indicate that short-term pretreatment with glucocorticoid hormone dramatically improves CB HSC homing and engraftment by selective chromatin remodeling. This suggests a simple, new, and easily adaptable means to enhance clinical efficacy of CB HCT. DisclosuresBroxmeyer:CordUse: Other: SAB Member .