Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo

Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

The effect of exosomes derived from unrestricted somatic stem cells on murine model of sepsis

Sepsis is a systemic infection mainly caused by bacterial infections. Despite all efforts and advances in treatment of sepsis, it's still considered as one of the leading causes of death in the hospitalized patients. Today we have to use novel therapies and one of the most important is cell free therapy. Exosomes have been introduced to have all cell contents without compatible tissue complex proteins which is a good candidate for transplantation. Unrestricted somatic stem cells (USSC) also known as mesenchymal stem cell progenitors due to their high proliferative capacity and low immune response, which is a novel therapy for sepsis. In this study, the effect of USSC-derived exosomes on sepsis was investigated using a mouse model. USSCs were isolated from human cord blood and characterized by flow cytometry and multilineage differentiation. The exosomes were then harvested from USSCs and characterized by transmission electron microscopy, Western blotting, and dynamic light scattering. The harvested exosomes were injected into the mouse model of sepsis. Biochemical, histological, molecular, and survival studies were performed in different groups. Our observation showed that USSC-derived exosomes can reduce inflammation in septic mice. Histopathological and biochemical findings in the sham group obviously showed multiorgan involvement, but these changes disappeared after seven days of exosome administration. Moreover, the expression of IRAK-1 and TRAF-6 (main adapter molecules in signaling pathways of inflammation) was decreased through negative regulation by miR-146a after 72 h of exosome administration; finally, it leads to a 2-fold increase in the level of IL-10 and a 2-fold decrease in the levels of IL-6 and TNF-α . In conclusion, we reported that direct injection of USSC- derived exosomes can be one of the important methods for the treatment of various aspects of sepsis due to their immunomodulatory properties.

Mitofusin2 (MFN2) Preserves Mitochondrial Integrity and Function in Megakaryocytes and Platelets

Genome wide association studies (GWAS) have associated mitochondria related loci with platelet numbers, function, and CVD. However, causality has not been established for many of these variants, and their mechanism and functional consequences are unknown. One such variant, MFN2 eQTL rs1474868 (T/T), has been associated with reduced platelet counts and reduced expression (5 fold) of MFN2 RNA in platelets. We show here that the MFN2 T/T variant corresponds with significantly reduced MFN2 protein in platelets. This difference contributes to a significant correlation between MFN2 RNA levels and mitochondrial load and potential in platelets. MFN2 RNA is also reduced by T/T in human cord blood derived megakaryocytes resulting in unfused mitochondria and impaired megakaryopoiesis. Using platelet/megakaryocyte specific Mfn2-/- (Mfn2 KO) mice, we show that Mfn2 impacts platelet numbers, activation and function by regulating mitochondrial energetics. Platelets without Mfn2 had reduced mitochondrial membrane potential and significantly reduced platelet lifespan (P<0.01) that was attributed to an increased rate of phosphatidylserine (PS) flipping (P=0.01). Increased RNA expression with conversely reduced protein expression of Ndufb8 (P<0.01), an index nuclear encoded complex I subunit that is stable only in a fully assembled complex I, suggested a defect in complex I assembly in Mfn2 KO platelets. Furthermore, complex I activity was reduced in Mfn2 KO platelets compared to WT platelets (P<0.01). Both basal and thrombin triggered mitochondrial oxygen consumption rate as assessed by Seahorse analyzer was significantly reduced in Mfn2 KO platelets (1.28 pmol/min/µg protein) compared to WT control platelets (3.06 pmol/min/µg protein). Platelet activation was subtly, yet significantly, decreased in Mfn2 KO platelets compared to WT platelets as assessed by surface expression of activated integrin alpha2b/beta3 and P-selectin. In addition, Mfn2 KO platelets had impaired Ca 2+ signaling, ROS generation, and procoagulant platelet formation (PS +ve platelets), and formed fewer platelet-neutrophil aggregates (PNAs) compared to WT platelets (P=0.01). Consistent with this, we observed significantly prolonged bleeding times in Mfn2 KO mice compared to their WT control littermates (P=0.001). Finally, mice with loss of platelet Mfn2 exhibited a modest reduction in ischemic stroke infarct size after cerebral ischemia-reperfusion that was statistically significant (P<0.01). Taken together these results suggest that MFN2 preserves mitochondrial functions necessary for platelet survival and activity, and that loss of MFN2 leads to accelerated platelet death, dysfunction, and altered hemostasis and thrombosis. Disclosures Rondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.

Endothelial Cell Niche Promotes Leukemic Transformation of Human Cord Blood Stem/Progenitor Cells Expressing CBFA2T3-GLIS2 Oncogenic Fusion

The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo. Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells. The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs. To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs. Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E). To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs. Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Regulatory association of long noncoding RNAs and chromatin accessibility facilitates erythroid differentiation

Erythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and lncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that associate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin and interacting with cytoskeleton network during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the associated network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.

Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SC) through the Galectin-9-Dependent Cell Contact Mechanism

Background: We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SC) on different compartments of immune cells such as T cells and monocytes/macrophages in diabetes and other autoimmune diseases. However, the effects of CB-SC on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SC, we explored the modulations of CB-SC on human B cells. Methods: CB-SC were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMC). Next, the activated B cells were treated in the presence or absence of coculture with CB-SC for 7 days before undergoing flow cytometry analysis of phenotypic change with different markers. RT-PCR was utilized to evaluate the levels of galectin expressions with or without treatment of activated B cells in order to find the key galectin contributing to the B-cell modulation. Results: Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SC, leading to the down-regulation of immunoglobulin productions from the activated B cells. Phenotypic analysis revealed that treatment with CB-SC increased the percentage of IgD+CD27- naive B cells, but decreased the percentage of IgD-CD27+ switched B cells. Transwell assay showed that the immune suppression of CB-SC on B cells was dependent on the manner of cell-cell contact via Gal-9 molecule, as confirmed by the blocking experiment with the anti-Gal-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were down-regulated after the treatment with CB-SC, causing the decline of mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SC. Conclusions: CB-SC displayed multiple immune modulations on B cells through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advances current understanding about the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.

Developmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish

The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to neurotoxic chemicals can result in neurobehavioral disorders. In this study, we uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits using zebrafish as a vertebrate model. We investigated the effects of the abundant non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. Morphological and biochemical data demonstrate that even though PCB153-induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling appears to contribute to PCB-induced motor deficits. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and increased selection pressures in vertebrate wildlife.

The Role of Cord Blood in the Regulation of the Cellular and Humoral Link of Immunity in Experimental Atopic Dermatitis

Background. Atopic dermatitis (AD) as one of the most common diseases of autoimmune genesis in the structure of dermatological practice, is characterized by itching, dryness, thickening of the skin, characteristic rashes. The drugs of choice in the treatment of AD are steroidal anti-inflammatory drugs. However, the development of unwanted side effects is a serious problem attributed to using hormone therapy. The search for effective methods of treating AD is an urgent task of medicine and in particular dermatology. At the same time, there is an obvious need for the participation in the solution of this problem also of specialists-immunologists working in the field of application of cell therapy drugs, acting on various pathogenetic links of the disease. The development of new or optimization of existing methods of treating AD is the urgent task facing them. Objective. Evaluation of the immunocorrective effect of lyophilized (lHCBL) and cryopreserved human cord blood leucoconcetrate (cHCBL) on a AD model. Methods. The experiments were carried out on 6-month-old Wistar rats. Upon induction of AD, the inflammation focus was formed on the rat's back (9–10 cm2) by daily rubbing in a 5% alcohol-acetone solution of dinitrochlorobenzene (DNCB) for 21 days. cHCBL and lHCBL were injected intraperitoneally, 0.5 ml at a dose of 5´106 cells in one day after the final DNCB treatment. The adhesive and phagocytic activity of the cells of the peritoneal cavity, the level of circulating immune complexes, the population and subpopulation of lymphocytes (CD3+, CD4+, CD8+, CD16+, CD4+CD25+), the immunoregulatory index of lymphocytes, the concentration of immunoglobulins in the blood serum were determined. Results. For AD induced by DNCB, systemic changes in the immune status are characteristic, which is expressed by changes in the parameters of cellular and humoral immunity. The most fundamental changes in cell subpopulations in spleen of rats with AD were revealed: a decrease in the number of total T-lymphocytes and their two main subpopulations (CD4+ and CD8+ cells). Against this background, changes were noted in the monocytic-phagocytic and humoral systems of immunity. The paper shows the effectiveness of the use of cHCBL and lHCBL in the correction of pathological manifestations of experimental AD. On the background of treatment, the features of the immunocorrective effect of each of the drugs were noted. Thus, when assessing intergroup values, a more pronounced increase in T-reg was revealed in rats of the 5th group – 3.9 [3.8; 4.0] versus 3.2 [3.0; 3.3] in the 4th group (P < 0.01); IgA level – 1.6 [1.5; 1.7] versus 1.3 [1.2; 1.4] (P < 0.01). Conclusions. Thus, lHCBL exhibits immunocorrective activity in the treatment of experimental AD, surpassing in some parameters the activity of сHCBL, which is promising for its use in clinical practice.