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Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here,we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions,resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions,which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. View Publication

We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors,detected as long-term culture-initiating cells (LTC-IC),could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC,CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold,respectively,above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition,stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

High levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic,neural,and cancer stem cells have all been reported to have high ALDH activity,detected using Aldefluor,a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1,an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless,Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non-cell-autonomous manner,consistent with its role in cyclophosphamide metabolism in the liver. However,Aldh1a1 deficiency did not affect hematopoiesis,HSC function,or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally,Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice. View Publication

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies,particularly in view of the diverse methods for derivation and maintenance of these cell lines. However,characterization methods are generally not standardized and many currently used assays are subjective,making dependable and direct comparison of cell lines difficult. In order to address this problem,we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines,derived in two different laboratories using different derivation techniques,as pathogen free,genetically stable,and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control,a crucial element of successful hESC research and clinical translation. View Publication

Giant cell tumor of bone (GCTB) is a very rare tumor entity,which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis,emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells,stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation,differentiation,migration,MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,immunohistochemistry,antibody protein array,Alu in situ hybridization,FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies,differentiated to osteoblasts,migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4,indicating self-renewal,invasion and differentiation potential. Compared with adherent-growing cells,markers for pluripotency,stemness and cancer progression,including the CSC surface marker c-Met,were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib,an inhibitor of c-Met in phase II trials,eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB. View Publication

UNLABELLED This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs,(b) the delivery of cEPI-NCSCs into the spinal cord,and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study,we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord,may promote angiogenesis and tissue remodeling,and elicit a tissue response that may be beneficial in patients with spinal cord injury. SIGNIFICANCE It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities,clinically and pathologically,to the syndrome in people. For this reason,dogs provide a readily accessible,clinically realistic,spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI. View Publication

IntroductionAmid the persistent threat of future pandemics,the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions,highlighting the need for rapid and adaptable approaches to respond to immune escape variants.MethodsHere,we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.ResultsImmortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors,yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library,we identified 12 unique clones with neutralization activity for SARS-CoV-2,leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity,resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore,we engineered a bi-paratopic antibody combining KBA2401,a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD,and KBA2402,a broadly binding non-neutralizing antibody,resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.DiscussionOur findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats. View Publication

Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression,we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus,which codes for γ-globin protein,a component of HbF. Employing this reporter cell line,we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them,10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers,such as pomalidomide,lenalidomide,decitabine,idoxuridine,and azacytidine,which validate the translational nature of this screening platform. We identified avadomide,autophinib,triciribine,and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further,we demonstrated that pomalidomide and avadomide,but not idoxuridine,induced HbF expression through downregulation of several transcriptional repressors such as BCL11A,ZBTB7A,and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways,as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies. View Publication

Ankylosing spondylitis (AS) is an immune-mediated inflammatory disorder that primarily affects the axial skeleton,especially the sacroiliac joints and spine. This results in chronic back pain and,in extreme cases,ankylosis of the spine. Despite its debilitating effects,the pathogenesis of AS remains to be further elucidated. This study used single cell CITE-seq technology to analyze peripheral blood mononuclear cells (PBMCs) in AS and in healthy controls. We identified a number of molecular features associated with AS. CD52 was found to be overexpressed in both RNA and surface protein expression across several cell types in patients with AS. CD16+ monocytes overexpressed TNFSF10 and IL-18R$\alpha$ in AS,while CD8+ TEM cells and natural killer cells overexpressed genes linked with cytotoxicity,including GZMH,GZMB,and NKG7. Tregs underexpressed CD39 in AS,suggesting reduced functionality. We identified an overrepresented NK cell subset in AS that overexpressed CD16,CD161,and CD38,as well as cytotoxic genes and pathways. Finally,we developed machine learning models derived from CITE-seq data for the classification of AS and achieved an Area Under the Receiver Operating Characteristic (AUROC) curve of > 0.95. In summary,CITE-seq identification of AS-associated genes and surface proteins in specific cell subsets informs our understanding of pathogenesis and potential new therapeutic targets,while providing new approaches for diagnosis via machine learning. View Publication

Pancreatic cancer is one of the deadliest cancers,against which current immunotherapy strategies are not effective. Herein,we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically,NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However,tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression,but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer,its combination with radiation therapy hold greater therapeutic benefit. View Publication

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore,we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content,membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase,ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSC(lo)ALDH(br) cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall,9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison,primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover,we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We,therefore,conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly,the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity. View Publication