Master Cell Research Articles

Optimized protocol for the isolation and expansion of menstrual blood-derived mesenchymal stem cells for combination with oncolytic adenoviruses in cancer treatment

Oncolytic viruses (OVs) are a promising therapeutic approach for cancer, although their systemic administration faces significant challenges. Mesenchymal stem cells have emerged as potential carriers to overcome these obstacles due to their tumor-tropic properties. This study investigates the use of menstrual blood-derived mesenchymal stem cells (MenSCs) as carriers for oncolytic viruses in cancer therapy, focusing on enhancing their efficacy through different culture conditions. MenSCs were isolated from donors of different ages and cultured under normoxic and hypoxic conditions, with varying adherence capacities. Hypoxic conditions significantly improved MenSCs proliferation and tumor migration capabilities, as demonstrated by proliferation assays and RNA-Seq analysis, which revealed upregulation of genes related to cell division and tumor tropism. In vivo studies using a lung adenocarcinoma mouse model confirmed that hypoxia-conditioned MenSCs had superior tumor homing abilities. The study also demonstrated the feasibility of establishing a master and working cell bank from a single menstrual blood donation. These findings suggest that hypoxia-conditioned MenSCs could be highly effective as OV carriers, potentially leading to better clinical outcomes in cancer treatment by enhancing tumor targeting and therapeutic efficacy.

Self-powered triboelectric-responsive microneedles with controllable release of optogenetically engineered extracellular vesicles for intervertebral disc degeneration repair

Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.

A cell bank paradigm for preclinical evaluation of an analogous cellular product for an allogeneic cell therapy

Toward the translation of allogeneic cell therapy products, cell banks are needed not only to manufacture the final human product but also during the preclinical evaluation of an animal-based analogous cellular product (ACP). These cell banks need to be established at both the master cell bank (MCB) level and the working cell bank (WCB) level. Inasmuch as most of the development of cell therapy products is at academic centers, it is imperative that academic researchers understand how to establish MCBs and WCBs within an academic environment. To illustrate this process, using articular cartilage as the model, a cell bank for an ACP was developed (MCBs at passage 2, WCBs at passage 5) to produce self-assembled neocartilage for preclinical evaluation (constructs at passage 7). The cell bank system is estimated to be able to produce between 160 000 and 400 000 constructs for each of the six MCBs. Overall, the ACP cell bank yielded constructs that are analogous to the intended human product, which is critical toward conducting preclinical evaluations of the ACP for inclusion in an Investigational New Drug application to the FDA.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Mechanical Properties of Selective Laser Melting‐Processed Multihierarchical Lattice Structure Based on Body‐Centered‐Cubic Unit Cell

Herein, three types of multihierarchical lattice structures (MHLSs) with different configurations are designed based on the body‐centered‐cubic (BCC) unit cell. The designed lattice structures are fabricated using Ti‐6Al‐4 V powder as feedstock material through selective laser melting (SLM) technology. The microstructure and surface morphology of the SLM‐formed samples are observed by optical microscopy and scanning electron microscopy, respectively. Theoretical calculation and quasistatic compression experiment are carried out to investigate their mechanical properties. The result shows that the size error of slave cell with small strut diameters is greater than that of master cell with larger strut diameters. All MHLSs exhibit superior mechanical properties compared to the BCC lattice structure. Moreover, the specific elastic modulus and specific yield strength of MHLSs with the best mechanical properties are 70.1% and 51.0% higher than that of BCC lattice structure, respectively. Meanwhile, theoretical calculation results of the elastic modulus and yield strength are consistent with the results of quasi‐static compression testing. The fracture morphology analysis indicates that the struts of the MHLSs samples exhibit a mixed brittle–plastic fracture mode, while the nodes exhibit a plastic fracture mode.

Identification of Age-Related Characteristic Genes Involved in Severe COVID-19 Infection Among Elderly Patients Using Machine Learning and Immune Cell Infiltration Analysis.

Elderly patients infected with severe acute respiratory syndrome coronavirus 2 are at higher risk of severe clinical manifestation, extended hospitalization, and increased mortality. Those patients are more likely to experience persistent symptoms and exacerbate the condition of basic diseases with long COVID-19 syndrome. However, the molecular mechanisms underlying severe COVID-19 in the elderly patients remain unclear. Our study aims to investigate the function of the interaction between disease-characteristic genes and immune cell infiltration in patients with severe COVID-19 infection. COVID-19 datasets (GSE164805 and GSE180594) and aging dataset (GSE69832) were obtained from the Gene Expression Omnibus database. The combined different expression genes (DEGs) were subjected to Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Diseases Ontology functional enrichment analysis, Gene Set Enrichment Analysis, machine learning, and immune cell infiltration analysis. GO and KEGG enrichment analyses revealed that the eight DEGs (IL23A, PTGER4, PLCB1, IL1B, CXCR1, C1QB, MX2, ALOX12) were mainly involved in inflammatory mediator regulation of TRP channels, coronavirus disease-COVID-19, and cytokine activity signaling pathways. Three-degree algorithm (LASSO, SVM-RFE, KNN) and correlation analysis showed that the five DEGs up-regulated the immune cells of macrophages M0/M1, memory B cells, gamma delta T cell, dendritic cell resting, and master cell resisting. Our study identified five hallmark genes that can serve as disease-characteristic genes and target immune cells infiltrated in severe COVID-19 patients among the elderly population, which may contribute to the study of pathogenesis and the evaluation of diagnosis and prognosis in aging patients infected with severe COVID-19.

MASTer cell: chief immune modulator and inductor of antimicrobial immune response.

Mast cells have long been recognized for their involvement in allergic pathology through the immunoglobulin E (IgE)-mediated degranulation mechanism. However, there is growing evidence of other "non-canonical" degranulation mechanisms activated by certain pathogen recognition receptors. Mast cells release several mediators, including histamine, cytokines, chemokines, prostaglandins, and leukotrienes, to initiate and enhance inflammation. The chemical nature of activating stimuli influences receptors, triggering mechanisms for the secretion of formed and new synthesized mediators. Mast cells have more than 30 known surface receptors that activate different pathways for direct and indirect activation by microbes. Different bacterial strains stimulate mast cells through various ligands, initiating the innate immune response, which aids in clearing the bacterial burden. Mast cell interactions with adaptative immune cells also play a crucial role in infections. Recent publications revealed another "non-canonical" degranulation mechanism present in tryptase and chymase mast cells in humans and connective tissue mast cells in mice, occurring through the activation of the Mas-related G protein-coupled receptor (MRGPRX2/b2). This receptor represents a new therapeutic target alongside antibiotic therapy. There is an urgent need to reconsider and redefine the biological role of these MASTer cells of innate immunity, extending beyond their involvement in allergic pathology.

Abstract 5240: Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape

Abstract Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of hematologic malignancies. However, the durability of response of CAR T therapy can be impaired by heterogenous expression of the CAR-specific target antigen (1° Ag) on tumors. In combination with a CAR, T-cell engagers (TCEs) that target additional tumor antigen (2° Ag) can be a unique approach to address tumor heterogeneity and overcome antigen escape. Engineered induced pluripotent stem cell (iPSC) master cell lines are a renewable source material from which iPSC-derived CAR T (CAR-iT) cells can be manufactured and administered in an off-the-shelf manner. In developing allogeneic CAR-iT cells, the T-cell receptor (TCR) must be eliminated to prevent graft-versus-host disease. However, the absence of TCR expression leads to loss of surface CD3 and a corresponding loss of TCE compatibility. Here we discuss a novel CD3ε fusion receptor (CD3-FR) with a modified transmembrane and endodomain, which allows for CD3 surface expression on TCR-less T cells and effective combination of off-the-shelf allogeneic CAR-T cells and TCEs. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+) and absent of TCR. To demonstrate that CD3-FR+ CAR-iT cells can elicit TCE-dependent activity, 1° Ag- (CD19-)/2° Ag+ (EpCAM+) target cells were used in cytotoxicity assays. As shown, CD3-FR- CAR-iT cells failed to kill 1° Ag- target cells. In contrast, CD3-FR+ CAR-iT cells demonstrated potent activity toward the target cells (>80% cytolysis) but only in the presence of the TCE targeting 2° Ag. To test the ability of CD3-FR+ CAR-iT cells to mitigate antigen escape, we used a tumor cell population heterogenous for the CAR-specific target (50% 1° Ag-) but homogenous for TCE-specific target (100% 2° Ag+). In co-culture assays, CD3-FR- CAR-iT cells failed to prevent tumor cell growth due to 1° Ag- tumor escape. However, CD3-FR+ CAR-iT cells in the presence of TCE exhibited robust control (80% cytolysis) of the heterogenous tumor, mitigating 1° Ag- tumor escape via targeting of 2° Ag. Next, we tested the ability of CD3-FR+ CAR-iT cells to control heterogeneous tumor in vivo in a disseminated leukemia model. As expected, CD3-FR+ CAR-iT cells exhibited significantly better tumor growth inhibition in the presence of TCE (p-value <0.005) compared to controls lacking the CD3-FR modality. Ex vivo analysis of tumor in the bone marrow confirmed 1° Ag- tumor escape in the control treated mice, but not in mice treated with CD3-FR+ CAR-iT and TCE targeting 2° Ag. Taken together, these studies present a novel opportunity to leverage a novel CD3 fusion receptor and enable combination of TCEs with engineered off-the-shelf CAR T-cell products, uniquely harnessing two potent therapeutic modalities for improving cancer therapy. Citation Format: Dan Lu, Eigen Peralta, Hui-Yi Chu, Soo Park, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5240.

Good Manufacturing Practice–compliant human induced pluripotent stem cells: from bench to putative clinical products

Background aimsFew human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. MethodsOur standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). ResultsTwo MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). ConclusionsWe successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Biological basis of extensive pleiotropy between blood traits and cancer risk.

The immune system has a central role in preventing carcinogenesis. Alteration of systemic immune cell levels may increase cancer risk. However, the extent to which common genetic variation influences blood traits and cancer risk remains largely undetermined. Here, we identify pleiotropic variants and predict their underlying molecular and cellular alterations. Multivariate Cox regression was used to evaluate associations between blood traits and cancer diagnosis in cases in the UK Biobank. Shared genetic variants were identified from the summary statistics of the genome-wide association studies of 27 blood traits and 27 cancer types and subtypes, applying the conditional/conjunctional false-discovery rate approach. Analysis of genomic positions, expression quantitative trait loci, enhancers, regulatory marks, functionally defined gene sets, and bulk- and single-cell expression profiles predicted the biological impact of pleiotropic variants. Plasma small RNAs were sequenced to assess association with cancer diagnosis. The study identified 4093 common genetic variants, involving 1248 gene loci, that contributed to blood-cancer pleiotropism. Genomic hotspots of pleiotropism include chromosomal regions 5p15-TERT and 6p21-HLA. Genes whose products are involved in regulating telomere length are found to be enriched in pleiotropic variants. Pleiotropic gene candidates are frequently linked to transcriptional programs that regulate hematopoiesis and define progenitor cell states of immune system development. Perturbation of the myeloid lineage is indicated by pleiotropic associations with defined master regulators and cell alterations. Eosinophil count is inversely associated with cancer risk. A high frequency of pleiotropic associations is also centered on the regulation of small noncoding Y-RNAs. Predicted pleiotropic Y-RNAs show specific regulatory marks and are overabundant in the normal tissue and blood of cancer patients. Analysis of plasma small RNAs in women who developed breast cancer indicates there is an overabundance of Y-RNA preceding neoplasm diagnosis. This study reveals extensive pleiotropism between blood traits and cancer risk. Pleiotropism is linked to factors and processes involved in hematopoietic development and immune system function, including components of the major histocompatibility complexes, and regulators of telomere length and myeloid lineage. Deregulation of Y-RNAs is also associated with pleiotropism. Overexpression of these elements might indicate increased cancer risk.

Generation of a bank of clinical-grade, HLA-homozygous iPSC lines with high coverage of the Spanish population

BackgroundInduced pluripotent stem cell (iPSC)-derived cell therapies are an interesting new area in the field of regenerative medicine. One of the approaches to decrease the costs of iPSC-derived therapies is the use of allogenic homozygous human leukocyte antigen (HLA)-matched donors to generate iPSC lines and to build a clinical-grade iPSC bank covering a high percentage of the Spanish population.MethodsThe Spanish Stem Cell Transplantation Registry was screened for cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes. Seven donors were selected with haplotypes covering 21.37% of the haplotypes of the Spanish population. CD34-positive hematopoietic progenitors were isolated from the mononuclear cell fraction of frozen cord blood units from each donor by density gradient centrifugation and further by immune magnetic labeling and separation using purification columns. Purified CD34 + cells were reprogrammed to iPSCs by transduction with the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit.ResultsThe iPSCs generated from the 7 donors were expanded, characterized, banked and registered. Master cell banks (MCBs) and working cell banks (WCBs) from the iPSCs of each donor were produced under GMP conditions in qualified clean rooms.ConclusionsHere, we present the first clinical-grade, iPSC haplobank in Spain made from CD34 + cells from seven cord blood units homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes within the Spanish population. We describe their generation by transduction with Sendai viral vectors and their GMP-compliant expansion and banking. These haplolines will constitute starting materials for advanced therapy medicinal product development (ATMP).

Dynamic Expression of Erg Controls Fetal-to-Adult Maturation of the Hematopoietic System

Many blood diseases are biased in their age of onset toward infancy, childhood, or mature adulthood. This occurs in concert with underlying changes in the properties of hematopoietic stem and progenitor cells (HSPCs) over the course of development, maturation, and aging that tailor the output of effector blood cells to age-specific physiology. Understanding how blood formation changes over the course of a lifetime would form a foundation upon which to investigate age-related determinants of manifestations of blood diseases. A growing body of work has shown that the heterochronic Lin28b/ let-7 switch defines fetal versus adult definitive HSPC states. In previous work, we found that the Polycomb repressive complex 1 component Cbx2 is a downstream target of let-7 that functions to specify the temporal maturation state of the hematopoietic system. In our current study, we find that Cbx2 represses the master hematopoietic stem cell (HSC) transcription factor (TF) Erg in fetal HSPCs, providing a mechanism by which the Lin28b/ let-7 axis integrates with the core HSC TF circuitry. During developmental maturation, Erg is upregulated during the fetal-to-adult HSC transition in humans (5.5-fold), mice (3.8-fold), and zebrafish (4.9-fold). Moreover, by analyzing existing ATAC-seq datasets, we find that Erg motifs are highly enriched in chromatin regions specifically active in adult relative to fetal HSPCs (P &amp;lt; 0.0001). Together, these findings suggest age-biased role(s) for Erg in hematopoiesis. We therefore hypothesized that since Erg is a key regulator of self-renewal and lineage output in HSCs, Erg likely serves stage-specific roles in HSCs. To test this, we either suppressed Erg expression in adult HSPCs or ectopically activated Erg in fetal HSPCs to determine consequences on HSPC function. We found that ectopic expression of Erg in HSPCs reprogrammed to the fetal state by expression of LIN28B resulted in blunting of fetal-like lineage biases. In adult mice, loss of a gene dose of Erg resulted in persistence of hallmarks of the fetal HSC state among blood effector cells and fetal-like HSPC distributions into maturity. During the normal process of fetal-to-adult maturation, HSCs transition from a highly proliferative to a quiescent state. We find that early postnatal Erg +/- HSCs do not effectively transition to quiescence and maintain active fetal-like self-renewal, which is leads to impairment in HSC function and susceptibility to replicative exhaustion later in adulthood. Mechanistically, we find that adult Erg +/- HSPCs ectopically maintain fetal-like gene expression profiles and patterns of promoter-enhancer looping. This is associated with failure to repress self-renewal promoting TFs that are normally expressed only in fetal HSPCs. Together, these findings demonstrate that master hematopoietic TFs and their associated networks are tightly titrated to implement age-appropriate rates self-renewal and mature cell output in HSPCs. Our results confirm heterochronic roles for Erg in defining age-tailored hematopoiesis on schedule with normal development and maturation and form a basis for applying this understanding of temporal hematopoietic maturation to disease models.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (&amp;gt;98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

TIM-3/Gal-9 interaction affects glucose and lipid metabolism in acute myeloid leukemia cell lines.

T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in acute myeloid leukemia and master cell survival and proliferation. In this study, we aimed to explore the effect of TIM-3 interaction with its ligand galectin-9 (Gal-9) on glucose and lipid metabolism in AML cell lines. HL-60 and THP-1 cell lines, representing M3 and M5 AML subtypes, respectively, were cultured under appropriate conditions. The expression of TIM-3 on the cell surface was ascertained by flow cytometric assay. We used real-time PCR to examine the mRNA expression of GLUT-1, HK-2, PFKFB-3, G6PD, ACC-1, ATGL, and CPT-1A; colorimetric assays to measure the concentration of glucose, lactate, GSH, and the enzymatic activity of G6PD; MTT assay to determine cellular proliferation; and gas chromatography-mass spectrometry (GC-MS) to designate FFAs. We observed the significant upregulated expression of GLUT-1, HK-2, PFKFB-3, ACC-1, CPT-1A, and G6PD and the enzymatic activity of G6PD in a time-dependent manner in the presence of Gal-9 compared to the PMA and control groups in both HL-60 and THP-1 cell lines (p > 0.05). Moreover, the elevation of extracellular free fatty acids, glucose consumption, lactate release, the concentration of cellular glutathione (GSH) and cell proliferation were significantly higher in the presence of Gal-9 compared to the PMA and control groups in both cell lines (p < 0.05). TIM-3/Gal-9 ligation on AML cell lines results in aerobic glycolysis and altered lipid metabolism and also protects cells from oxidative stress, all in favor of leukemic cell survival and proliferation.

Prevalence and risk factors of geohelminths in primary schools children aged 5 to 15 years in the city of Moundou, southwestern Chad

Geohelminthiases are endemic in Chad and constitute a serious public health problem. This study aimed at determing the prevalence and risk factors of intestinal geohelminthiasis in children aged 5–15 years in the city of Moundou, Chad. This was a cross-sectional and descriptive study carried out in the city of Moundou. A total of 333 pupils participated in this study and it included children aged from 5 to 15 years attending three public primary schools in Moundou. A questionnaire was administered to each student after obtaining Informed Consent from either parent. Stool samples were collected in a sterile container and, the formalin-ethyl ether concentration technique was used to identify parasite. Parasitic load was assessed using the Mc Master cell method. The collected data were analyzed using Excel; Word 2016 and SPSS 20 software. An overall prevalence of 16.52% was obtained, 9.3% for Trichuris trichiura, 6.9% for Ascaris lumbricoides, and 1.2% for Hookworms. Male participants were more infected (67.24%) than females (32.76%). The age group]9–13] was the most infected (53.44%), followed by the age group [5–9](44.83%) and finally the age group]13–15] (1.73%). The Ouhoud school was the most infected (55.17%) followed by the Adoum Dallah school (39.66%) and finally the Centre school (5.17%). However, no statistically significant difference between gender and geohelminthiasis infection was recorded (p > 0.05). Regarding risk factors, statistical analysis showed that age group]9–13] (OR = 1.997 at 95% CI at [1.085–3.677]), Central Public School (OR = 1.55 at 95% CI at [0.63–2.46]), tap water (OR = 29 at 95% CI at [20.89–38.70]), not maintaining latrines (OR = 2.37 at 95% CI at [0.62–3.78]), and maintenance of latrines by pupils (OR = 1.5 at 95% CI at [0.63–2.46]) were risk factors. This study shows a high prevalence of geohelmenthiasis among children of three primary schools in Moundou, Chad. Although males were more infected than female there was no significant difference between gender and geohelminth infections (p = 0.114). was no gender difference. Identified risk factors of geohelmenthiasis infections among the study population were: age between 9 and 13 years, school water consumption, the use of unmaintained latrines and latrines maintained by students. Surveillance of geohelminthiases and hygiene should be intensified to reduce the pathological risk related to these parasites in Chad.

EVALUATING THE QUALITY OF VERO 76 CELL BANK NATIONAL REFERENCE STANDARD USED FOR QUALITY CONTROL OF VACCINES AND BIOLOGICALS PRODUCTS USED IN TESTING VACCINES AND BIOLOGICALS PRODUCTS

The Master cell bank (Vero 76 NICVB/MCB01-18) and Working cell bank (Vero 76 NICVB/WCB01-19) are produced from international standard Vero cells ATCC 76 (ATCC CRLM- 1587TM) was studied and evaluated for quality according to the standards of the World Health Organization (WHO) including criteria such as (identification; sterility; homogeneity; viability; Mycoplasma; Retrovirus; tuberculosis bacteria; extraneous agents in vitro and in vivo). The research results show that the cell banks Vero 76 NICVB/MCB01-18 and Vero 76 NICVB/WCB01-19 meet the quality requirements according to WHO TRS 978, part 3, 2013 and WHO TRS 932 part 1, 2006 for National Reference Standard used for research and quality control of vaccines and biological products.

Multi-cell trap developments towards the accumulation and confinement of large quantities of positrons

The multi-cell Penning–Malmberg trap concept has been proposed as a way to accumulate and confine unprecedented numbers of antiparticles, an attractive but challenging goal. We report on the commissioning and first results (using electron plasmas) of the World's second prototype of such a trap, which builds and improves on the findings of its predecessor. Reliable alignment of both ‘master’ and ‘storage’ cells with the axial magnetic field has enabled confinement of plasmas, without use of the ‘rotating wall’ (RW) compression technique, for over an hour in the master cell and tens of seconds in the storage cells. In the master cell, attachment to background neutrals is found to be the main source of charge loss, with an overall charge-confinement time of 8.6 h. Transfer to on-axis and off-axis storage cells has been demonstrated, with an off-axis transfer rate of$50\,\%$of the initial particles, and confinement times in the storage cells in the tens of seconds (again, without RW compression). This, in turn, has enabled the first simultaneous plasma confinement in two off-axis cells, a milestone for the multi-cell trap concept.

Abstract 18 Leveraging the BMDI Cord Blood Bank to Create a GMP-Compliant Cord Blood (CB)–Derived Induced Pluripotent Stem Cell (iPSC) Master Cell Bank (MCB) for Potential Allogeneic Therapies

Abstract Introduction The BMDI Cord Blood Bank (CBB) in Melbourne is a public cord blood bank that operates under a Good Manufacturing Practice (GMP) cell-manufacturing licence from the Therapeutic Goods Administration (TGA) and is internationally accredited by the Foundation for the Accreditation of Cellular Therapy (FACT). An iPSC MCB derived from cord blood (CB) could form the basis of future cell-based therapies. Banked CB is an ideal source of starting material for the creation of iPSCs, with a host of advantages not afforded by other cell sources. Objectives We have previously described the technical aspects of creating “GMP-like” iPSCs from banked CB (https://www.frontiersin.org/articles/10.3389/fcell.2022.835321/full). Our objective is to leverage the CBB to transition our “GMP-like” protocols, developed in the research lab, into a fully GMP-compliant environment. Methods A small lab within the CBB was seconded for the purpose of establishing an Institutional Biosafety Committee (IBC) – approved PC2 laboratory to create GMP-compliant iPSC lines from banked cord blood. CB donors have been re-consented to use a portion of their banked HLA homozygous CB to create iPSC lines (https://doi.org/10.1093/stcltm/szac060). The CBB Quality Framework was replicated as a side module, mimicking all key components. Results * Established an IBC-approved PC2 laboratory within the GMP-compliant CBB facility. * Developed a Quality Systems framework in-line with the FACT Common Standards for Cellular Therapy, leveraging off that in place for the CBB. * Tested and validated the process in its entirety, including robustness of the Quality Systems, by the creation of new CB-derived “GMP-mock” iPSC lines. * Established collaborations for pre-clinical studies to use our CB-derived iPSC lines for therapies directed towards retinal and neurological repair, NK and CAR-NK immunotherapies. Discussion A PC2 GMP-compliant laboratory has been established. Challenges, and solutions, will be discussed. Quality systems have been developed that mimic those in place for the CBB. The robustness of the Quality framework and GMP manufacturing have been confirmed by a full mock-run to create new CB-derived iPSC lines. Lower grade reagents used for the mock-run have now been swapped out for more expensive, fully GMP-certified (CTS) reagents and creation of our first fully GMP CB-derived iPSC line is underway.

Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart

Abstract Introduction Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration. Objectives The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study. Methods After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing. Results Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT). Discussion Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.