Cord Blood CD34 Research Articles

Effects of Eupalinilide E and UM171, alone and in combination on cytokine stimulated ex-vivo expansion of human cord blood hematopoietic stem cells

Eupalinilide E was assessed for ex-vivo expansion activity on hematopoietic stem cells (HSCs) from human cord blood (CB) CD34+ cells in serum-free, SCF, TPO and FL stimulated 7 day cultures. Eupalinilide E ex-vivo enhanced phenotyped (p) HSCs and glycolysis of CD34+ cells isolated 7 days after culture as measured by extracellular acidification rate, but did not alone show enhanced NSG engrafting capability of HSCs as determined by chimerism and numbers of SCID Repopulating cells, a quantitative measure of functional human HSCs. This is another example of pHSCs not necessarily recapitulating functional activity of these cells. Lack of effect on engrafting HSCs may be due to a number of possibilities, including down regulation of CXCR4 or of the homing capacity of these treated cells. However, Eupalinilide did act in an additive to synergistic fashion with UM171 to enhance ex vivo expansion of both pHSCs, and functionally engrafting HSCs. While reasons for the disconnect between pHSC and function of HSCs with Eupalinilide E alone cultured CB CD34+ cells is yet to be determined, the data suggest possible future use of Eupalinilide and UM171 together to enhance ex vivo production of CB HSCs for clinical hematopoietic cell transplantation.

Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents.

The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug ) and triple-transgenic NOG-EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood-derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line-derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors. Basic Protocol 1: Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell-humanized mice Basic Protocol 2: Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell-humanized mice Support Protocol 1: Flow cytometry assessment of humanization in mice Support Protocol 2: Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models.

Preclinical evaluation of efficacy and cytokine release of bispecific T cell engagers in humanized NSG mice

Abstract Bispecific T cell engagers (BiTEs) are a growing class of cancer therapy that redirects effector T cells against target-expressing tumors. Mouse models to assess efficacy and cytokine release associated with T cell engagement are needed. JAX humanized NOD scid gamma (Hu-NSG™) mice are engrafted with cord blood-derived CD34+ HSC and have shown robust T cell reconstitution. In this report we evaluated two novel BiTEs, EGFR-CD3 and BCMA-CD3, in Hu-NSG™ mice implanted with MDA-MB-231 human breast cancer cells and NCI-H929 multiple myeloma, respectively. Following the initial dosing, we measured human cytokines and analyzed T cell activation by flow cytometry. Clinical observations and body weight change were also monitored. Treatment with EGFR-CD3 for 2 weeks resulted in complete tumor regression when the dosing was initiated around 100 mm3. 50% of the treated-tumors remained nonpalpable for 10 weeks after dosing ceased. EGFR-CD3 also inhibited tumor growth in mice bearing tumors of ~400 mm3 average in volume. In both cases human IFN-γ, IL-6, and IL-10 levels were elevated from the first dose before returning to the baseline and the frequency of CD69+ cells in T cell subsets increased after the treatment. BCMA-CD3 treatment also induced T cell activation and elevated levels of human IFN-γ, TNF-a, IL-6, and IL-10. NCI-H929 tumor growth inhibition by BCMA-CD3 showed donor to donor variability. We observed that significant growth inhibition in one donor while the other donor did not respond. Together, our Hu-NSG™ platform enables preclinical evaluation of drug efficacy and cytokine release induced by BiTEs and has the potential to be applied to other immunotherapies.

RUNX1 and TGF-β signaling cross talk regulates Ca2+ ion channels expression and activity during megakaryocyte development.

Thrombocytopenia is characterized by low platelet count and is typically observed among all preterm and low birthweight neonates admitted to the neonatal intensive care unit. Although the underlying cause for this predisposition is unclear, recent studies have proposed that the intrinsic inability of neonatal hematopoietic stem/progenitor cells to produce mature polyploid megakaryocytes (MKs) may result in delayed platelet engraftment. The developmental and molecular differences between neonatal and adult MKs are not yet fully understood. Previously, we had reported that the key MK transcription factor RUNX1, which is crucial for the regulation of MK specification and maturation, is down-regulated in neonatal MKs when compared with adult MKs. In humans, loss-of-function mutations in RUNX1 cause familial platelet disorder, which is characterized by thrombocytopenia, indicating its crucial role in MK development. However, information about its cross talk with developmentally regulated signaling pathways in MKs is lacking. In this study, we performed a differential gene expression analysis in MKs derived from human cord blood (CB) and adult peripheral blood (PB) CD34+ cells. Further, validation and correlation studies between RUNX1 and transforming growth factor beta (TGF-β) were performed in a differentiating megakaryocytic cell line model. The analysis revealed that TGF-β pathway was the main pathway affected between CB- and PB-MKs. RUNX1 is reported to be a modulator of TGF-β signaling in several studies. The correlation between RUNX1 and TGF-β pathway was analyzed in the PMA-induced megakaryocytic differentiating K562 cells, which exhibit mature megakaryocytic features. The RT2 profiler PCR array analysis revealed that TGF-β pathway components were up-regulated in the PMA-induced megakaryocytic differentiating cells. Furthermore, our study indicated that human TGF-β1 promotes cytosolic calcium (Ca2+ ) activity and MK maturation. We noticed that TGF-β1 increased intracellular free Ca2+ ([Ca2+ ]i) via reactive oxygen species-mediated activation of transient receptor potential (TRP) ion channels. Moreover, we observed that decreased cytosolic Ca2+ activity in the siRUNX1-transfected cells was associated with down-regulation of TRP ion channel expression. Finally, we demonstrated that TGF-β/SMAD signaling augments the development of MKs derived from CB-CD34+ . Present data suggest that RUNX1/TGF-β pathway cross talk is crucial for MK maturation.

A humanized mouse platform supporting in vivo maturation of human antibody responses

Abstract Mice are widely used models for research and drug discovery, yet provide limited predictive value due to their divergence from humans in immune elements and functions. This underscores the need for more faithful models of the in vivo human immune response. To address this shortcoming, we have generated a HuMice platform by grafting NSG KitW-41J mice with fresh human cord blood CD34+ stem cells via intracardiac injection. This yielded up to 98% human CD45+ cell reconstitution within all major lymphoid compartments, including GALT. Human leukocyte development and long-term persistence in CD34+ cell-engrafted HuMice revealed robust T and B cell maturation, leading to emergence of IgM, IgG, IgA and IgE forming cells. B cells from HuMice underwent in vitro CSR and plasma cell differentiation as efficiently as B cells isolated from healthy humans. Once conditioned with our immune potentiation treatment, HuMice supported T-dependent and T-independent antibody responses involving formation of germinal center-like structures, class-switching, somatic hypermutation, as well as plasma cell and memory B cell differentiation, this entailed further maturation of the B cell compartment resulting in high B2/B1 cell ratio. HuMice were amenable to hydrocarbon-induced autoimmunity leading to lupus-like disease and were susceptible to infection by and respond to human pathogens such as vaccinia virus and EBV. Thus, we have devised a novel in vivo HuMice platform, which allows for generation and maturation of human antibody responses to microbial agents and lends itself to in vivo modeling of autoimmunity, thereby offering opportunities for identification of therapeutic targets and vaccine development.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood.

The human T lymphocyte compartment is highly dynamic over the course of a lifetime. Of the many changes, perhaps most notable is the transition from a predominantly naïve T cell state at birth to the acquisition of antigen-experienced memory and effector subsets following environmental exposures. These phenotypic changes, including the induction of T cell exhaustion and senescence, have the potential to negatively impact efficacy of adoptive T cell therapies (ACT). When considering ACT with CD4+CD25+CD127–/lo regulatory T cells (Tregs) for the induction of immune tolerance, we previously reported ex vivo expanded umbilical cord blood (CB) Tregs remained more naïve, suppressed responder T cells equivalently, and exhibited a more diverse T cell receptor (TCR) repertoire compared to expanded adult peripheral blood (APB) Tregs. Herein, we hypothesized that upon further characterization, we would observe increased lineage heterogeneity and phenotypic diversity in APB Tregs that might negatively impact lineage stability, engraftment capacity, and the potential for Tregs to home to sites of tissue inflammation following ACT. We compared the phenotypic profiles of human Tregs isolated from CB versus the more traditional source, APB. We conducted analysis of fresh and ex vivo expanded Treg subsets at both the single cell (scRNA-seq and flow cytometry) and bulk (microarray and cytokine profiling) levels. Single cell transcriptional profiles of pre-expansion APB Tregs highlighted a cluster of cells that showed increased expression of genes associated with effector and pro-inflammatory phenotypes (CCL5, GZMK, CXCR3, LYAR, and NKG7) with low expression of Treg markers (FOXP3 and IKZF2). CB Tregs were more diverse in TCR repertoire and homogenous in phenotype, and contained fewer effector-like cells in contrast with APB Tregs. Interestingly, expression of canonical Treg markers, such as FOXP3, TIGIT, and IKZF2, were increased in CB CD4+CD127+ conventional T cells (Tconv) compared to APB Tconv, post-expansion, implying perinatal T cells may adopt a default regulatory program. Collectively, these data identify surface markers (namely CXCR3) that could be depleted to improve purity and stability of APB Tregs, and support the use of expanded CB Tregs as a potentially optimal ACT modality for the treatment of autoimmune and inflammatory diseases.

Antioxidant Small Molecule Compound Chrysin Promotes the Self-Renewal of Hematopoietic Stem Cells.

There is an increasing demand for the expansion of functional human hematopoietic stem cells (hHSCs) for various clinical applications. Based on our primary screening of antioxidant small molecule compounds library, a small molecule compound C2968 (chrysin) was identificated to expand cord blood CD34+ cells in vitro. Then we further verified the optimum concentration and explored its effect on hHSCs phenotype and biological function. C2968 could significantly increase the proportion and absolute number of CD34+CD38−CD49f+ and CD34+CD38−CD45RA−CD90+ cells under 2.5 μM. Furthermore, the total number of colony-forming units and the frequency of LT-HSCs in C2968-treated group were significantly higher than control, indicating the multipotency and long-term activity of hematopoietic stem and progenitor cells were sustained. Additionally, C2968 treatment could maintain transplantable HSCs that preserve balanced multilineage potential and promote rapid engraftment after transplantation in immunodeficient (NOG) mice. Mechanistically, the activity of chrysin might be mediated through multiple mechanisms namely delaying HSC differentiation, inhibiting ROS-activated apoptosis, and modulating of cyclin-dependent kinase inhibitors. Overall, chrysin showed good ex vivo expansion effect on hHSCs, which could maintain the self-renewal and multilineage differentiation potential of hHSCs. Through further research on its antioxidant mechanism, it may become a promising tool for further fundamental research and clinical umbilical cord blood transplantation of hHSCs.

High‐yield production of hematopoietic growth factors by tobacco BY‐2 cells for ex vivo production of red blood cells

Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) represents a promising approach for overcoming the limitations associated with the use of donor’s blood, such as shortage of blood and risk of transfusion‐transmitted infections. Both expansion and differentiation of stem cells are highly reliant on growth and differentiation factors, with the activity and purity of these molecules being critical to successful research and clinical applications. The overarching goal of this project is to develop a novel plant cell‐based bioproduction platform for high‐quality (i.e. animal‐free and endotoxin‐free) erythropoietic growth factors (eGFs) that facilitate mass production of RBCs from HSCs at significantly reduced cost. In addressing the low productivity bottleneck of plant cell culture technology, this proposal aims to leverage a unique, plant‐specific, O‐glycosylation process called “hydroxyproline (Hyp)‐O‐glycosylation” for de novo design and engineering of Hyp‐O‐ glycosylated peptides (HypGPs”) that presumably function as a molecular carrier in boosting secretion of conjoined eGFs from cultured plant cells. This will dramatically increase the secreted yields of plant cell‐derived eGFs and simplify the product purification process. Three key eGFs essential for HSCs expansion and differentiation: erythropoietin (EPO), stem cell factor (SCF) and interleukin 3 (IL‐3), are expressed in tobacco BY‐2 cells as fusion with a strategically designed HypGP tag. The secreted protein yields, molecular structure, and Hyp‐O‐glycosylation of the HypGP‐tagged eGFs are characterized. The bioactivities of individual HypGP‐tagged eGF in stimulating proliferation and differentiation of relevant hematopoietic cell lines, includingTF1 erythroleukemic cell and umbilical cord blood‐derived CD34+ cell, are evaluated to establish a suitable design of HypGP tag (size and orientation) for each eGF. A scalable cell culture system fortified sorely with plant cell‐produced eGFs is developed for production of RBCs from CD34+ HSC cells. This project provides a proof‐of‐concept for ex vivo manufacturing of stem cell‐derived RBCs with plant cell‐produced eGFs.Support or Funding InformationNational Science Foundation Directorate for Engineering [1605564]; National Institutes of Health‐Arkansas INBRE [P20GM103429]

Heparanase Facilitates PMA-Induced Megakaryocytic Differentiation in K562 Cells via Interleukin 6/STAT3 Pathway.

Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan sulfate and hence participates in remodeling of the extracellular matrix, leading to release of cytokines that are immobilized by binding to heparan sulfate proteoglycans (HSPGs), and consequently activating signaling pathways. This function of HPSE is correlated to its expression level that is normally very low in majority of the tissues. Exceptionally, human platelets express high level of HPSE, suggesting a unique physiological role in this cell. Using K562 cell line, we found a progressive increase of HPSE during the megakaryocytic differentiation. Analysis of a series of megakaryocytic differentiation-related heparin-binding proteins (HBPs) in the cell culture medium revealed an exclusive positive correlation between the level of interleukin 6 (IL-6) and HPSE expression. IL-6 modulated megakaryocytic differentiation through activation of STAT3. Further, we demonstrated that overexpression of HPSE potentiates megakaryocytic differentiation, whereas elimination of HPSE led to a delayed differentiation. This function of HPSE is associated with its activity, as overexpression of inactive HPSE had no effect on IL-6 production and megakaryocytic differentiation. The role of HPSE is further supported by the observation in an umbilical cord blood CD34+ cells megakaryocytic differentiation model. Our data propose a novel role for HPSE in platelets production by a HPSE/IL-6/STAT3 positive feedback loop that specifically regulates megakaryocytes maturation.

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development.

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.

Purifying stem cell-derived red blood cells: a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration.

Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.

Impact of maternal and neonatal factors on umbilical cord CD34+ cells.

The achievement of optimal number of CD34+ umbilical cord stem cells is essential for successful umbilical cord stem cell transplantation. So the aim of this study was to assess the potential effect of both maternal and neonatal factors on the umbilical cord blood CD34+ cell count. The study was done on umbilical cord blood samples obtained from 20 mothers during labor. Their ages ranged from 22 to 34 years and were subjected to history taking, physical examination of the baby and assessment of the CD34+ cells count in umbilical cord blood. Number of previous live births and weight of the baby had a significant effect on CD34+ cells count while the sex of the baby, delivery route, maternal age and gestation period had no significant effect on CD34+ cells count. Umbilical cord blood-derived CD34+ cell count is better with good weight and first babies and decreased with subsequent babies.

Abstract A75: Efficacy of novel immunogene combinations for Kras and LKB1 mutant NSCLC in a humanized mouse model

Abstract Due to lack of suitability of current preclinical models for immunotherapy research, we recently developed an improved humanized mouse by reconstituting a human immune system in NSG mice by transplanting fresh human cord blood-derived CD34+ stem cells (Hu-mice). The Hu-mice show functional representation of human T, B, natural killer (NK), dendritic cells (DC), myeloid-derived suppressor cells (MDSC), and responsiveness to checkpoint blockade. TUSC2 has recently been recognized as a novel immunogene that induces apoptosis in tumor cells and promotes a wide spectrum of tumor-specific innate and adaptive immune responses. We previously reported that TUSC2 delivered systemically by nanovesicles downregulates PD-L1 expression in NSCLC and synergizes with anti-PD1 in inhibiting tumor growth in Kras-mutant syngeneic mouse models through upregulating NK and cytotoxic T cells. In this study, we aimed to evaluate the antitumor efficacy of TUSC2 in combination with standard immunotherapy on highly metastatic Kras and LKB1 mutant human lung cancer in Hu-mice. Hu-mice were challenged with A549 cells (Krasmt/LKB1-) and lung metastases were treated with TUSC2, nivolumab, or the combination. The results showed a synergistic antitumor effect with the combination. A significantly increased antitumor effect was found when TUSC2 was combined with pembrolizumab in Hu-mice. Pembrolizumab alone significantly reduced tumor burden as compared with an untreated control, whereas no antitumor effect was observed in non-Hu-mice implanted with A549 cells. The antitumor effect was correlated with significantly higher levels of CD8+ T and CD8+CD69+ active T and significantly lower levels of MDSC and regulatory T cells in the combination group. A significantly higher percentage of CD56+ NK and CD56+CD59+ active NK cells was found in the TUSC2 alone and combination groups, indicating TUSC2 related NK activation. We tested whether TUSC2 enhances efficacy to carboplatin+pembrolizumab in Hu-mice implanted with A549-luc metastatic cells. The results showed that the level of antitumor effect of carboplatin+pembrolizumab was similar to that of TUSC2 alone, but when TUSC2 was combined with carboplatin+pembrolizumab, metastases regression was significantly greater than either TUSC2 alone or carboplatin+pembrolizumab treatments. Significantly fewer or no visible tumor nodules were found in dissected lungs in the TUSC2 combination as compared with other groups. In conclusion, TUSC2 immunogene therapy in combination with pembrolizumab and carboplatin+pembrolizumab showed strong antitumor efficacy in metastatic human NSCLC in a clinically relevant humanized mouse model, supporting a clinical trial. Citation Format: Ismail M. Meraz, Mourad Majidi, Meng Feng, RuPing Shao, Min Jin Ha, Jeffrey Morris, Elizabeth J. Shpall, Jack A. Roth. Efficacy of novel immunogene combinations for Kras and LKB1 mutant NSCLC in a humanized mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A75.

Humanized Culture Medium for Clinical-Grade Generation of Erythroid Cells from Umbilical Cord Blood CD34+ Cells.

Purpose: Transfusion of red blood cells (RBCs) is a supportive and common treatment in surgical care, trauma, and anemia. However, in vivo production of RBC seems to be a suitable alternative for blood transfusions due to the limitation of blood resources, the possibility of disease transmission, immune reactions, and the presence of rare blood groups. Cell cultures require serum-free or culture media supplemented with highly expensive animal serum, which can transmit xenoviruses. Platelet lysate (PL) can be considered as a suitable alternative containing a high level of growth factors and a low production cost. Methods: Three-step culture media supplemented with PL or fetal bovine serum (FBS) were used for proliferation and differentiation of CD34+ umbilical cord blood stem cells to erythrocytes in co-culture with bone marrow mesenchymal stem cells (BM-MSCs). The cells were cultivated for 15 days and cell proliferation and expansion were assessed using cell counts at different days. Erythroid differentiation genes, CD71 and glycophorin A expression levels were evaluated. Results: Maximum hematopoietic stem cells (HSCs) proliferation was observed on day 15 in PL-containing medium (99±17×103-fold). Gene expression and surface markers showed higher differentiation of cells in PL-containing medium. Conclusion: The results of this study indicate that PL can enhance erythroid proliferation and differentiation of CD34+ HSCs. PL can also be used as a proper alternative for FBS in the culture medium and HSCs differentiation.

Identification of osteolineage cell-derived extracellular vesicle cargo implicated in hematopoietic support.

Osteolineage cell‐derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non‐stimulatory osteolineage EVs by next‐generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV‐derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood‐derived CD34+ HSPCs with stimulatory EVs‐altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell‐HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.

Expansion and preservation of the functional activity of adult hematopoietic stem cells cultured ex vivo with a histone deacetylase inhibitor.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum‐free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA‐CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA‐treated cultures were able to engraft in immune‐deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA‐mediated ex vivo HSC expansion for gene modification of adult HSCs.

P460 Evaluation of potential mechanisms underlying the safety observations of filgotinib in clinical studies in rheumatoid arthritis

Abstract Background Inhibitors of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway have demonstrated efficacy in the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Differences in selectivity of JAK inhibitors for JAK1, JAK2, JAK3 and TYK2 may influence their respective safety profiles, and the mechanisms responsible are not currently known. Filgotinib (FIL), a JAK1 inhibitor, did not negatively impact haemoglobin, LDL:HDL ratios or natural killer (NK) cell counts in clinical trials. Here, we compare the in vitro mechanistic profiles of four JAK inhibitors at clinically relevant doses. Methods JAK inhibitors (FIL, FIL metabolite [GS-829845], baricitinib [BARI], tofacitinib [TOFA], and upadacitinib [UPA]) were evaluated in vitro in human-cell-based assays. Growth of erythroid progenitors from human cord blood CD34+ cells was assessed using a HemaTox™ liquid expansion assay, NK cell proliferation was induced by IL-15 and LXR agonist-induced cholesteryl ester transfer protein (CETP) expression was assessed in the hepatic cell line, HepG2. Using assay-generated IC50 values and the reported human plasma concentrations from clinical studies, we calculated the target coverage for each JAK inhibitor at clinically relevant doses. The activity of FIL in humans was based on PK/PD modelling of FIL + GS-829845. Results Inhibition of cellular activity was calculated for each JAK inhibitor based on in vitro dose-response data, human exposure data and modelled PK/PD relationships. At clinically relevant doses, FIL resulted in lower calculated inhibition of NK cell proliferation compared with other JAK inhibitors. FIL 100 mg and 200 mg also reduced CETP expression, whereas other JAK inhibitors had no effect. There was no difference in the effect of FIL vs. other JAK inhibitors on erythroid progenitor cell differentiation or maturation. Conclusion FIL, a JAK1 inhibitor, resulted in less inhibition of NK cell proliferation compared with BARI, TOFA, and UPA. FIL also reduced LXR agonist-induced CETP expression, while the other inhibitors did not alter these levels. These results provide a potential mechanistic link between the observed reduction of CETP concentration following FIL treatment and the previously observed reduction in the LDL:HDL ratio in RA patients.

Maternal Type 1 Diabetes Reduces Autoantigen-Responsive CD4+ T Cells in Offspring

Autoimmunity against pancreatic β-cell autoantigens is a characteristic of childhood type 1 diabetes (T1D). Autoimmunity usually appears in genetically susceptible children with the development of autoantibodies against (pro)insulin in early childhood. The offspring of mothers with T1D are protected from this process. The aim of this study was to determine whether the protection conferred by maternal T1D is associated with improved neonatal tolerance against (pro)insulin. Consistent with improved neonatal tolerance, the offspring of mothers with T1D had reduced cord blood CD4+ T-cell responses to proinsulin and insulin, a reduction in the inflammatory profile of their proinsulin-responsive CD4+ T cells, and improved regulation of CD4+ T cell responses to proinsulin at 9 months of age, as compared with offspring with a father or sibling with T1D. Maternal T1D was also associated with a modest reduction in CpG methylation of the INS gene in cord blood mononuclear cells from offspring with a susceptible INS genotype. Our findings support the concept that a maternal T1D environment improves neonatal immune tolerance against the autoantigen (pro)insulin.