Human Cord Blood Cells Research Articles

Clinical safety in using unmatched allogeneic umbilical cord blood mononuclear cells transplantations in non-haematopoietic degenerative conditions.

Evaluation of safety in using unmatched human allogeneic umbilical cord blood cells for therapeutic use in individuals with non-haematopoietic degenerative conditions. The historical data and several recent immunological arguments suggest the therapeutic use of allogeneic Cord Blood Mononuclear Cells (CBMNCs), as these cells do not elicit immune response. Customarily, HLA matched cord blood MNCs are used along with prolonged immunosuppression in treatment of haematological conditions. Lately, unmatched CBMNCs are widely used in case of unavailability of HLA matched cord blood. There have been suggestions for using unmatched allogeneic cord blood MNCs for degenerative conditions without an immunoconditioning regimen. 49 patients with non-haematopoietic degenerative conditions were treated with HLA-unmatched allogeneic hUCB MNCs. Intrathecal/I.V injections (1-2 million cells/kg body weight) were given. Clinical, biochemical and haematological adverse events were evaluated. The haematological and biochemical parameters showed no major deviation from the normal. Clinically, no acute adverse effects or GVHD were observed with the used dosage. This study supports/suggests clinical safety in therapeutic medical use of unmatched allogeneic CBMNCs when used at low dosage in non-haematopoietic degenerative conditions.

Immunogenicity and therapeutic efficacy of a dual-component genetic cancer vaccine cotargeting carcinoembryonic antigen and HER2/neu in preclinical models.

Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety containing equal amounts of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu⁺ and CEA⁺ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clinical trials.

UM171 Is a Novel and Potent Agonist Of Human Hematopoietic Stem Cell Renewal

▪Various combinations of soluble factors have been reported to promote the ex vivo proliferation of human HSCs but few of them are capable of promoting self-renewal. Characterization of StemRegenin (SR1), an aryl hydrocarbon receptor (AhR) antagonist that promotes expansion of CD34+ cells ex vivo, provided a proof of principle that low molecular weight (LMW) compounds have the ability to modulate cellular pathways possibly leading to HSC expansion. In an attempt to identify novel putative agonists of HSC self-renewal, we tested a library comprising > 5,000 LMW molecules in a phenotypical screen based on in vitro expansion of CD34+CD45RA- cells. Eight hits were identified, the majority of which were AhR antagonists. Validation studies identified one hit synthesized in our institute which potently expanded human CD34+CD45RA- cord blood cells ex vivo. Importantly this molecule does not antagonize AhR. Structure activity relationship (SAR) generated a compound called UM171 which is 14-25 folds more potent than the starting molecule with an EC50 of 8-15 nM on CD34+ human cord blood (CB). Using the optimal dose of this analog, CD34+, CD34+CD45RA-, and CD34+CD38-CD90+CD45RA-CD49f+ were expanded 115±15, 62±3 and 121±6 fold respectively over the input values after 12 day culture in fed-batch system. The expansion of these phenotypic HSC populations was enhanced, in an additive manner by including SR1. Washout of UM171 led to a rapid loss of the CD34+CD45RA- population suggesting that its effect is reversible. UM171 is not a mitogen and cannot compensate for the absence of cytokines added to the media (Flt3L, SCF, and TPO). Our cell division studies revealed no effect of the compound on division rate compared to control indicating that UM171 cultured cells retain the CD34+CD45RA- phenotype. Apoptosis and cell phonotype analysis showed significantly less cell differentiation and cell death for UM171 cultured cells compared to controls or SR1 supplemented cultures suggesting that the compound acts by anti-differentiation/apoptosis. Similar to SR1, UM171 treated cells showed a 75±20 fold net increase in the numbers of multilineage colony forming cell (CFU-GEMM) and 150±20 fold when both compounds were present in the 12 day culture.To further understand the impact of UM171 on more primitive cell compartments, we transplanted 100, 500, 1000 and 10,000 uncultured CB CD34+ cells and their progeny after a 12-day culture (with or without UM171) in NSG mice and verified the human hematopoietic reconstitution in their bone marrow 20 weeks later. Mice transplanted with UM171 expanded cells showed a much higher level of human CD45 engraftment compared to uncultured or control expanded cells. The NSG Repopulating Cells (NRC) showed a net expansion ex vivo only in the presence of UM171 with values ranging from 3.1 to 5.5 fold increase over input value in 6 independent experiments. Interestingly, the co-treatment with SR1 did not significantly enhance the impact of UM171 on NRC expansion. In these fed-batch conditions SR1 treatment maintained the NRC population at input values. Importantly, we observed a linear relationship between the number of UM171-treated cells transplanted in vivo and level of human CD45 engraftment. Moreover, at limit dilution, these cells were capable of human myeloid and lymphoid differentiation in NSG BM environment suggesting that UM171 expanded cells remain multipotent. Together, our studies have identified a novel compound which truly expands human progenitors while amplifying the more primitive stem cell population. UM171 has successfully passed toxicity studies and is in the final phase of GMP production for a phase I study which will be launched shortly. In addition, preliminary evidence suggests that UM171 strikingly enhances gene transfer efficiency to primitive human CD34 CB cells. Disclosures:No relevant conflicts of interest to declare.

Off-Tumor Target Expression Levels Do Not Predict CAR-T Cell Killing: A Foundation For The Safety Of CD44v6-Targeted T Cells

IntroductionOff-tumor expression of the target antigen raises justified safety concerns about newly designed chimeric antigen receptors (CARs). We have recently developed a CAR targeting the tumor-promoting antigen CD44v6 and demonstrated potent antitumor effects against acute myeloid leukemia (AML) and multiple myeloma (MM) both in vitro and in vivo. Despite promising activity against epithelial tumors, the administration of the CD44v6-specific mAb used for deriving our CAR (bivatuzumab) showed reversible myelosuppression and mucositis when conjugated with radioisotopes, and severe skin toxicity when conjugated with the potent cytotoxic drug mertansine. Preclinically evaluating the potential off-tumor toxicities of CD44v6-targeted T cells is therefore crucial before they can be safely translated to the clinic. AimTo profile the off-tumor expression of CD44v6 and to verify the susceptibility of expressing cells to CAR-T cell killing. ResultsQuantitative RT-PCR analysis on a wide panel of cDNA from normal tissues revealed restricted CD44v6 expression on flat stratified epithelia, like the skin, albeit at considerably lower levels compared with primary leukemic blasts. We therefore addressed the issue of keratinocyte recognition in co-culture experiments. Strikingly, at the E:T ratios allowing the potent antitumor effects of CD44v6-targeted T cells, keratinocytes were not killed and there was no cytokine production. Interestingly, comparative analysis of accessory molecules showed that, differently from leukemic blasts, keratinocytes expressed significant lower levels of adhesion/costimulatory molecules, including (ICAM-1, LFA-3 and B7.2), but higher levels of the critical checkpoint molecule PD-L1. Of the different cells of the hematopoietic system analyzed, only circulating CD14+ monocytes expressed CD44v6 and were killed by CD44v6-targeted T cells. Interestingly, by immunohistochemistry, we found no CD44v6 expression on bone-marrow monocytes, lymph-node macrophages, brain microglia, liver Kuppfer cells and dermal macrophages, suggesting a low risk for by-stander toxicity against these tissues. Moreover, CD44v6-targeted T cells did not interfere with the generation of virus-specific CTLs by antigen-specific stimulation in vitro. Importantly, both RT-qPCR and FACS demonstrated lack of CD44v6 expression on hematopoietic stem cells (HSCs) and progenitors. Accordingly, CD44v6-targeted T cells did not interfere with their clonogenic potential in vitro and, in co-culture experiments with whole bone marrow from MM patients, were able to selectively eliminate tumor cells, while sparing HSCs and progenitors. Finally, we tested the potential hematological toxicities of CD44v6-targeted T cells in NSG mice transgenic for human IL-3, SCF and GM-CSF (NSG-3GS). NSG-3GS mice transplanted with human CD34-selected cord blood cells showed enhanced myeloid reconstitution compared to NSG mice, including CD44v6+ monocytes. The infusion of CD44v6-targeted T cells in reconstituted NSG-3G mice resulted in the selective elimination of monocytes, but in the preservation of other cell subsets. Importantly, after in vivo exhaustion of CD44v6-targeted T cells, NSG-3G mice reconstituted monocytes de novo, indicating preservation of the HSC pool. For enabling rapid and conditional ablation of CD44v6-targeted T cells, we have finally co-expressed the CD44v6-CAR with TK or the inducible caspase-9 and validated the suicide gene approach in hyperacute xenogeneic GVHD surrogating maximal toxicity. ConclusionsOur results indicate that off-tumor target expression levels do not automatically predict the susceptibility to CAR T-cell killing. Moreover they suggest that, differently from mAb-derived pharmaceuticals, therapeutic doses of suicidal CD44v6-targeted T cells might associate with acceptable and/or reversible toxicities. Disclosures:Bordignon:MolMed SpA: Employment.

Knockdown Of The Piwi - Like Protein 4 (PIWIL4) Delays Leukemic Growth and Is Associated With Gross Changes In The Global Histone Methylation Marks In Human MLL - Rearranged AML

Piwi proteins belong to a class of proteins which were shown to be critically involved in the maintenance of the self-renewal property of stem cells in lower organisms. Furthermore, it was shown that they preserve genomic integrity through epigenetic silencing of transposable elements via CpG methylation and repressive histone modifications such as H3K9me3 in close interaction with a novel class of non-coding RNA called piRNA. So far there are neither precise data on the function of Piwi proteins in human acute myeloid leukemia, nor are there reports on expression of piRNAs in this disease. In a first step we tested PIWIL gene expression levels in normal human hematopoietic cells and leukemic patient samples by qRT-PCR. Among the family of human PIWI genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in normal hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 showed aberrantly high expression in more than 72% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ bone marrow (BM) and total BM cells (n=3). Notably, in nine of the ten MLL-AF9 rearranged AML patients, PIWIL4 was 64-fold higher expressed compared to normal CD34+ BM (p<0.0001) and 8-fold higher compared to inv(16), PML-RARa or cytogenetically normal AML patients (p<0.0001). To further validate this finding we analysed gene expression data performed on CD34+ human cord blood cells transduced with MLL-AF9 (n=9) vs AML-ETO (n=6) vs MYH11 (n=3): of note, PIWIL4 showed a 6 fold increase in expression in the MLL-AF9 transduced cells compared to the other experimental arms. Stable knockdown of PIWIL4 in the MLL rearranged AML cell lines MV4-11 (MLL-AF4) and THP-1 (MLL-AF9) significantly impaired growth in vitro (n=3) reducing proliferation and clonogenic growth by 83%/93% and 91%/93%, respectively. In addition, depletion of PIWIL4 delayed onset of leukemia in NSG mice transplanted with MV4-11/ THP-1 cells transduced with shPIWIL4 compared to the scrambled control (shRNA: AML onset 48/62d after transplantation vs. 30/30 days in the scrambled control; n=4/8 per arm; p< 0.0001/p<0.001). ChIP-seq analysis revealed that depletion of PIWIL4 in the THP1 cell line results in a marked global reduction in repressive H3K9me3 marks and in an increase in activating H3K4me3 marks as compared to cells transduced with the scrambled control. RNA-seq analyses revealed over 2500 differentially expressed genes upon PIWIL4 depletion with 60% of the genes being upregulated compared to the scrambled control (p<0.05). Among them genes involved in cell cycle such as RB1, P21, TGFB1 as well as epigenetic modifiers such as SETDB1, HDAC1,2 and demethylating enzyme TDG were differentially expressed. RB1 and EED, a protein necessary for PRC2 complex function, displayed an increase in expression and loss of H3K9me3 modifications on their promoters upon knockdown of PIWIL4. To prove piRNA expression in human AML and to test any association between PIWIL4 expression and piRNA signatures, microarray analyses covering 23,677 piRNAs was performed on the MLL-AF9 rearranged THP-1 cell line, of which 14193 piRNAs showed expression levels higher than 4 (arbitrary log2 scale). PIWIL4 knockdown induced differential expression of 981 piRNAs (p≤0.01, fold change ≥2), of which 527 were downregulated and 454 upregulated. Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML, affects leukemic growth, shapes epigenetic marks and impacts piRNA expression in this disease. Disclosures:No relevant conflicts of interest to declare.

Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection

Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71(+) erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71(+) cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71(+) cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71(+) cell-mediated susceptibility to infection is counterbalanced by CD71(+) cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71(+) cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.

Enhancing bone marrow regeneration by SALL4 protein

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient’s morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.

A novel STAT inhibitor, OPB-31121, has a significant antitumor effect on leukemia with STAT-addictive oncokinases

Signal transduction and activator of transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate cell proliferation, apoptosis, differentiation, development and the immune response. Aberrant signals of STAT induce uncontrolled cell proliferation and apoptosis resistance and are strongly involved in cancer. STAT has been identified as a promising target for antitumor drugs, but to date most trials have not been successful. Here, we demonstrated that a novel STAT inhibitor, OPB-31121, strongly inhibited STAT3 and STAT5 phosphorylation without upstream kinase inhibition, and induced significant growth inhibition in various hematopoietic malignant cells. Investigation of various cell lines suggested that OPB-31121 is particularly effective against multiple myeloma, Burkitt lymphoma and leukemia harboring BCR–ABL, FLT3/ITD and JAK2 V617F, oncokinases with their oncogenicities dependent on STAT3/5. Using an immunodeficient mouse transplantation system, we showed the significant antitumor effect of OPB-31121 against primary human leukemia cells harboring these aberrant kinases and its safety for normal human cord blood cells. Finally, we demonstrated a model to overcome drug resistance to upstream kinase inhibitors with a STAT inhibitor. These results suggested that OPB-31121 is a promising antitumor drug. Phase I trials have been performed in Korea and Hong Kong, and a phase I/II trial is underway in Japan.

Analysis of the clonal growth and differentiation dynamics of primitive barcoded human cord blood cells in NSG mice

AbstractHuman cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD34+ CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rγ−/− mice, each transplanted with ∼105 of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lympho-myeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo.Key Points

MiR-125b regulates cell proliferation and survival in neonatal megakaryocytes

Dear Editor, We read with interest the research article by Emmrich et al. [1] on role of miRNAs in the ex vivo generation of platelets from megakaryocytes (MKs). Differentiation protocol was validated using cord blood (CB)and peripheral blood (PB)-derived human hematopoietic stem and progenitor cells. miR-125b plays pivotal role in proliferation and self-renewal of megakaryocyte progenitors[2]. Overexpression of miR-125b increased the number of colony-forming units during in vitro \culture and also produced more platelets (2.5-fold) per MK, showing its effect as oncomiR on megakaryopoiesis [3]. Ailing newborn infants are uniquely predisposed to developing thrombocytopenia. They are affected by disorders such as TAR syndrome and transientmyeloproliferative disorder (TMD). Neonatal MK progenitors are hyperproliferative and generate more MKs per colony but are smaller and less polyploid compared to adult progenitors that generate less platelets per MK [4–7]. The molecular mechanisms for this variation are unknown and thought to be related to the biological differences between them. Recent reports indicate that miRNAs play a crucial role in the regulation of megakaryocytopoiesis. To test the role of miR-125b in megakaryopoiesis, we cultured CD34+ human CB and PB cells in defined serum free medium. After 14 days of culture, >90% of the cells wereMKs (CD41+). miRNA was prepared from these cells and miR-125b levels were measured by qRT-PCR on days 0, 7, 11 and 14 (n=3); the data was analysed by ΔΔ Ct method. Western blot was performed by standard method. miR-125b levels in CB were significantly higher compared to PB-derived MKs, and these differences were consistent at all stages of MK development (0, 7, 11 and 14 days, p <0.05, Fig. 1a). We observed significant (p <0.01) downregulation of miR-125b during different stages of differentiation in CBMKs (Fig. 1b). miR-125b was reported to be overexpressed in TMD and AMKL samples compared to normal MKs [4]. To further support our hypothesis, we checked miR-125b levels in two MK cell lines of different developmental origin, CMK (child) and MEG-01 (adult). Consistent with their developmental origin, we found that miR-125b levels in CMK cells were significantly higher than in MEG-01 cells (∼7.5-fold, Fig. 1c). How exactly miR-125b might contribute to the hyperproliferative phenotype of neonatal MK progenitors is unclear. miRNAs regulate the target gene expression on a post-transcriptional level by binding to the target mRNAs which, in turn, leads to translational arrest. Web-based computational approaches (TargetScan, PicTar and MiRanda) can be used widely in predicting potential targets. Recent report suggests that miR-125b downregulates apoptosisassociated proteins in p53 pathway including BAK1 and TP53INP1, and it blocks the myeloid differentiation in part by targeting CBFβ [1]. miR-125b has been shown to target key proteins regulating apoptosis, innate immunity, inflammation, and hematopoietic differentiation [8]. Here, we show that miR-125b downregulates p53, BAK1 and CDK6 in CB-MKs (Fig. 1d). The high levels of miR-125b expression in CB-MKs might contribute in their rapid proliferation, resulting in increased megakaryocyte numbers; thus, it could be a potential therapeutic target in blood disorders. R. Kandi :R. Undi : R. K. Gutti (*) Hematologic Oncology, Stem Cells and Blood Disorders Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, (PO) Gachibowli, Hyderabad 500046, AP, India e-mail: guttiravi@gmail.com Ann Hematol (2014) 93:1065–1066 DOI 10.1007/s00277-013-1928-5

Human Umbilical Cord Blood Mononuclear Cell-Conditioned Media Inhibits Hypoxic-Induced Apoptosis in Human Coronary Artery Endothelial Cells and Cardiac Myocytes by Activation of the Survival Protein Akt

We have previously demonstrated in acute myocardial infarctions that human umbilical cord blood mononuclear cells (HUCBCs), which contain hematopoietic, endothelial, and mesenchymal stem cells, reduce acute myocardial infarction size by ≥50% and preserve LV contractility. We hypothesize that the beneficial effects of HUCBCs are due to secretion of biologically active factors that activate in cardiac endothelial cells and myocytes the cell survival protein Akt. We determined by protein microarrays the growth factors and anti-inflammatory cytokines secreted by HUCBCs into culture media during 12 h of hypoxia (1% O2). We then determined by Western blots the effects of cell-free media from hypoxic-conditioned HUCBCs (HUCM) on activation of the cell survival protein Akt in human coronary artery endothelial cells and cardiac myocytes in culture during 24 h of 1% O2. We also determined in separate experiments endothelial cell and myocyte apoptosis by caspase-3 and Annexin V. In the present experiments, HUCBCs secreted multiple growth factors, anti-inflammatory cytokines, and inhibitors of metalloproteinase during normoxia and hypoxia. Human cord blood cells increased the concentration in culture media of angiopoietin, hepatocyte growth factor, interleukin-4, insulin-like growth factor, placental growth factor, vascular endothelial cell growth factor, angiogenin, and stem cell factor by 100 to >10,000% during 12 h of 1% O2 (p<0.001). HUCM, which contained these biological factors, significantly increased Akt phosphorylation/activation in coronary artery endothelial cells and cardiac myocytes subjected to 24 h of 1% O2 by more than 60% (p<0.05) and increased the antiapoptotic protein Bcl-2 expression by 34-50% in comparison with endothelial cells and myocytes treated without HUCM in 1% O2(p<0.05). HUCM also significantly decreased caspase-3 activity and decreased hypoxic endothelial cell and cardiac myocyte apoptosis by more than 40% in comparison with cells cultured without HUCM (p<0.05). Inhibition of Akt activation in endothelial cells and myocytes by the sensitive and specific antagonist API-1 during 24 h of hypoxia nearly completely prevented the beneficial effects of HUCM on inhibiting caspase-3 activity and apoptosis. We conclude that HUCBCs secrete biologically active factors during hypoxia that activate survival proteins in endothelial cells and myocytes that significantly limit apoptosis.

Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells

RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.

Protective role of functionalized single walled carbon nanotubes enhance ex vivo expansion of hematopoietic stem and progenitor cells in human umbilical cord blood

In this study, carboxylic acid functionalized single walled carbon nanotubes (f-SWCNT-COOH) was shown to support the viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood–mononucleated cells (UCB-MNC). Our in vitro experiments showed that f-SWCNT-COOH increased the viability of the CD45+ cells even without cytokine stimulation. It also reduced mitochondrial superoxides and caspase activity in CD45+ cells. f-SWCNT-COOH drastically reduced the proportions of CD45− cells in the non-enriched UCB-MNC. Phenotypic expression analysis and functional colony forming units (CFU) showed significant ex vivo expansion of HSPC, particularly of CD45+CD34+CD38− population and granulocyte-macrophage (GM) colonies, in f-SWCNT-COOH augmented cultures supplemented with basal cytokines. In vivo data suggested that f-SWCNT-COOH expanded UCB-MNC could repopulate immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease (GVHD) and f-SWCNT-COOH dependent toxicity. From the Clinical EditorIn this paper a novel method is presented by using single wall functionalized carbon nanotubes to enhance viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells in human umbilical cord blood -mononucleated cells. Detailed data is presented about enhanced viability, including improved repopulation of immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease.

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

A3.14 PD-1 Signalling and Low RORC mRNA Content Influence the TREG/TH17 Balance in Human Cord Blood

<h3>Background and Objectives</h3> In neonates, the immune system is less responsive than in adults. Explanations for this involve differences in T helper (Th) 1 versus Th2 cell numbers and altered cytokine profiles after stimulation. We hypothesised that neonatal immune deviation may also involve FOXP3⁺ regulatory T cells (Treg) and Th17 cells. <h3>Materials and Methods</h3> In order to investigate this, we compared CD4⁺CD25^–CD45RO^– naïve T cells from human cord blood (CB) with cells from adult peripheral blood (APB). Cells were activated in vitro by anti-CD3 in the presence of viable antigen presenting cells (APB). The induction of regulatory T cells and Th17 cells was analysed by flow cytometry. Cytokine production was measured by multiplex immunoassays and RORC mRNA content by PCR. Specific interactions between APC and T cells were blocked with monoclonal antibodies during cell culture. <h3>Results</h3> CB cells show lower numbers of Treg ex vivo than APB. However, upon activation, high percentages of functionally suppressive FOXP3⁺ Treg are induced in CB. Replacement of viable APC by irradiated APC or anti-CD28 abrogated this effect, suggesting that live APC from CB in particular may imprint a Treg phenotype. Indeed, CB APC were able to induce high numbers of FOXP3⁺ cells in alloreactive immune reactions with APB T cells as well. Addition of blocking antibodies against CD80, CD86 or CTLA-4 to CB cultures did not affect the number of Tregs induced. Blockade of the PD-1/PD-Ligand (PD-L)1 interaction however showed a marked decrease in FOXP3⁺ cell numbers. CB cells showed a consistently reduced protein kinase B (PKB)/c-Akt phosphorylation upon activation. On top of that, in contrast to APB, low concentrations of APC derived pro-inflammatory cytokines were detected in CB cultures and no Th17 cells were induced. Addition of Th17 inducing cytokines reduced FOXP3⁺ cell numbers in CB, but did not induce IL-17 production. PCR of the Th17 defining transcription factor RORC showed a significantly reduced concentration of this protein in CB. <h3>Conclusions</h3> Human cord blood cells have an increased propensity to become FOXP3⁺ Treg as compared to APB. This effect is due to low production of inflammatory cytokines and PD-1/PD-L1 interaction. Inability to induce Th17 cells in CB could not be abrogated by Th17 inducing cytokines, but was due to reduced RORC mRNA content. This study shows mechanisms involving the human Th17/Treg induction switch that can also be important in inflammation and auto-immune disorders.

Enhanced normal short-term human myelopoiesis in mice engineered to express human-specific myeloid growth factors

AbstractBetter methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients. We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34+ human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection.

Characterization of cardiac-resident progenitor cells expressing high aldehyde dehydrogenase activity.

High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDHbr) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDHbr cells in the heart has not been evaluated so far. We have characterized ALDHbr cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDHbr. ALDHvery-br cells were more frequent in neonatal hearts than adult. ALDHbr cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDHvery-br cells, intermediate in ALDHbr cells, and lowest in ALDHdim cells. ALDH1A2 expression was highest in ALDHvery-br cells, intermediate in ALDHdim cells, and lowest in ALDHbr cells. ALDH1A3 and ALDH2 expression was detectable in ALDHvery-br and ALDHbr cells, unlike ALDHdim cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDHbr cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDHbr cells, unlike ALDHdim cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α-actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDHbr cells declined with cell passage. In conclusion, the cardiac-derived ALDHbr population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDHbr cells remains to be evaluated.

Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions

Endothelial progenitor cells (EPCs) may be incorporated into local vessels to enhance angiogenesis within ischemic tissue. Recently, EPC transplantation has become a potential therapy for improving tissue function in cardiovascular disease. However, the mechanisms of proliferation, differentiation, and survival of EPCs in a hypoxic microenvironment remain unclear. In this study, CD34(+)VEGFR-2(+) EPCs were isolated from mononuclear cells of human umbilical cord blood, and differentiation to endothelial cells was induced with VEGF. When EPC autophagy was inhibited with 3-methyladenine (3-MA) under normoxic conditions, proliferation and viability of the cells were decreased, and the cells failed to differentiate into endothelial cells. Under hypoxic conditions (1% O(2)), Beclin-1 expression of the cells was upregulated and both MDC-labeled and LC3-positive puncta and autophagic ultrastructures in the cells increased significantly. The number of lysosomes also increased in hypoxia-exposed cells. When autophagy was inhibited with 3-MA under hypoxic conditions, the number of apoptotic cells increased, and the number and size of lysosomes decreased. Conversely, apoptosis of the hypoxic EPCs was reduced when autophagy was induced by pretreatment with rapamycin. These results demonstrate that autophagy is involved in proliferation and differentiation of EPCs. Furthermore, hypoxia activates autophagy, promoting EPC survival by inhibiting apoptosis. Enhancing autophagy with hypoxic preconditioning may be beneficial for survival of the transplanted EPCs in a local hypoxic environment.

Comparison of effects of fentanyl, sufentanil and remifentanil on immune function of dendritic cells in human umbilical cord blood

Objective To compare the effects of fentanyl,sufentanil and remifentanil on the immune function of dendritic cells in human umbilical cord blood.Methods Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation and seeded in 24-well plates with a density of 1 × 106/ml (2ml/hole).The cells were randomly divided into 7 groups (n =15 each):control group (group C),fentanyl 1.0 ng/ml group (group F1),fentanyl 5.0 ng/ml group (group F5),sufentanil 0.1 ng/ml group (group S1),sufentanil 0.5 ng/ml group (group S5),remifentanil 1.0 ng/ml group (group R1),and remifentanil 5.0 ng/ml group (group R5).The cells were incubated for 10 days in serum-free culture medium containing 50 ng/ml recombinant human granulocyte colony stimulating factor,10 ng/ml recombinant human interleukin-4 or the corresponding concentration of fentanyl,sufentanil or remifentanil,and then 50 ng/ml recombinant human tumor necrosis factor alpha was added to the culture medium and the cells were incubated for another 4 days in the seven groups.Three holes in each group were chosen and the cell morphology was examined with inverted microscope.Six holes in each group were chosen for determination of the concentration of IL-12 in the supernatant and expression of CD80/CD86.Six holes in each group were chosen for measurement of the cell viability.Results Compared with group C,the concentration of IL-12 and cell viability were significantly decreased and the expression of CD80/CD86 was down-regulated in groups F5,S1,S5,R1 and R5 (P ＜ 0.05).The concentration of IL-12,cell viability and expression of CD80/CD86 were significantly lower in groups S1 and R1 than in group F1 (P ＜ 0.05).Compared with group F5,the concentration of IL-12 was significantly decreased in group S5,and the concentration of IL-12 and cell viability were significantly decreased and the expression of CD80/CD86 was down-regulated in group R5 (P ＜ 0.05).The concentration of IL-12 and cell viability were significantly lower in group R1 than in group S1 (P ＜ 0.05).The concentration of IL-12,cell viability and expression of CD80/CD86 were significantly lower in group R5 than in group S5 (P ＜ 0.05).Conclusion Remifentanil has stronger inhibitory effect on the immunological function of dendritic cells in human umbilical cord blood than sufentanil,and the inhibitory effect of sufentanil is stronger than that of fentanyl. Key words: Fentanyl; Sufentanil; Piperidines; Immunity, cellular; Dendritic cells

Short Term Signalling Responses of the Most Primitive Subsets of Human Hematopoietic Cells Stimulated in Vitro Correlate with Their Subsequent Self-Renewal Behaviour.

AbstractAbstract 2341 Background:Growth factors (GFs) that stimulate the proliferation of human hematopoietic stem cells (HSCs) in vitro have been identified, but maintenance of the original stem cell potential has remained a challenge. We have confirmed the observation that UG26–1B6 cells produce factors that enhance the maximum yield in vitro of HSCs achievable with SCF, FLT3-L, IL-3, IL-6, and G-CSF (5 GFs) alone (∼5-fold increase in lympho-myeloid repopulating cells by limiting dilution transplant analysis in NOD/SCID-IL-2Rγc null mice assessed 20 weeks post-transplant). It was therefore of interest to determine whether these different outcomes could be correlated with specific signalling events. Methods:CD34+cell-enriched human cord blood (CB) cells were stimulated with 5 GFs ± UG26 conditioned medium (CM) for defined periods, and then fixed in paraformaldehyde, stained for surface markers, alcohol permeabilized and stained with antibodies specific for multiple intracellular signalling intermediates. Samples were then analyzed using either a CyTOF mass cytometer (34 unique parameters) or a LSR Fortessa cell analyzer (11 parameters). Data analysis was performed using a combination of Cytobank, SPADE, and R using the ’flowCore’, ’flowType’, ’lattice’, ’SamSpectral’, and ’gplots’ packages. Results:Analysis by CyTOF mass cytometry of 106 CD34+ CB cells after 15 minutes of their stimulation ± 5 GFs ± CM showed that the HSC-enriched subset (Lin−CD34+CD38−CD90+CD49f+cells) exhibited similar responses to the 5 GFs and 5 GFs+CM cocktails (average shifts in median intensity [Δasinh (stimulated-unstimulated)] of 0.32 [-0.14 to 1.96] and 0.37 [-0.02 to 1.78]) with increased phosphorylation of S6, STAT3, STAT5, CREB, IκBa (total protein), AKT, Syk/ZAP70, and ERK1/2. However, the addition of CM did selectively reduce the level of pS6 and pAKT seen with 5 GFs alone, and also caused an increase in pCREB. In addition, activation of pSyk/ZAP70 was detected only in HSCs stimulated with 5 GFs + CM. Phosphoflow analysis of the same subset of cells from a second CB pool confirmed the CyTOF results for pAKT, pSTAT5, pCREB and pERK1/2 (variably) following a 15 minute stimulation but not pSyk/ZAP70. After 2 hours of stimulation, the activation patterns were sustained for most targets for cells exposed to 5 GFs + CM but were reduced in 5 GFs alone - particularly pCREB which was decreased to levels in unstimulated cells. After 24 hours, pAKT and pSTAT5 showed continued activation in response to 5 GFs ± CM, whereas pCREB and pERK1/2 signal were reduced to below starting levels.Hartigans’ Dip Test for Unimodality revealed significant deviations from unimodality for the mass cytometry HSC data. Principal component analysis (PCA) of the HSC data followed by subsequent clustering also revealed a number of differentially responsive sub-populations including a broadly cytokine non-responsive, non-apoptotic (cleaved PARP−) population, as well as a minority population with high levels of pSrc and pPLCγ, but reduced activation of other pathways. Further analysis using SPADE revealed decreased cytokine responsiveness with higher CD49f intensity. Weak but significant negative correlations between CD49f and pSTAT3, pCREB, and pS6 and positive correlations with pSrc and pPLCγ (holm-adjusted p values <0.05; r values −0.12 – 0.38) were also observed.In the CyTOF experiment, multipotent progenitors (MPP; Lin−CD34+CD38−CD90−CD49f– cells) showed broadly similar patterns and levels of activation as HSCs when CM was added (p=0.44), but the median activation was attenuated in 5 GFs alone (p=0.048). MPPs also showed lower levels of multiple phosphorylation events, despite both HSC and MPP populations being Ki67−. This was consistent regardless of cell stimulation status, time point or measurement platform. Conclusions:We have discovered heterogeneity in the signal responses of highly purified fractions of very primitive subsets of human CB cells to culture conditions that affect their self-renewal differentially, that distinguish HSCs from MPPs, and that further subdivide the HSC subset. Among the events surveyed, differences in the timing and intensity of signal activation down common pathways were identified rather than any evidence of selective pathway utilization. These findings hold promise for the identification of how external factors modulate the fates of normal and transformed human HSCs when they are stimulated to divide. Disclosures:No relevant conflicts of interest to declare.