Murine Bone Marrow Stromal Cells Research Articles

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

Human Endometrium Derived Induced Pluripotent Stem Cells Are Amenable to Directed Erythroid Differentiation

BACKGROUND:A protocol for using human endometrium derived induced pluripotent stem cells (iPSCs) to derive hematopoietic and erythroid lineages will be elaborated, through a two-phase culture system.METHODS:Discarded endometrial tissues were obtained from women receiving hysterectomy in their 4th to 5th decade due to benign uterine conditions. pCE-Sox2, Oct4, Klf4, L-Myc and Lin28 episomal vectors were used to electrotransfect the endometrial stromal cells. The first 8 days involves commitment to hematopoietic stem cells through embryoid body with robust expansion on murine bone marrow stromal cells. The second phase involves feeder free conditions with hydrocortisone, stem cell factor, interleukin-3, and recombinant EPO. After 22 days of feeder free culture, the expression profiles of CD235a+, CD34+, CD43+ and CD 71+ were analyzed by flow cytometry and Wright-Giemsa staining for differential counting. The oxygen carrying capacity of cultured RBCs was measured using a hemoxanalyser.RESULTS:As a result of inducing these cells via co-culture with murine stromal fibroblasts, all endometrium derived iPSCs were differentiated into erythroblasts with a stable yield of approximately 80% for polychromatic and orthochromatic normoblasts. The protocol for complete induction of erythroid lineage cells starting from human endometrial tissue via iPS cells has been optimized.CONCLUSION:Successful directed erythroid differentiation has occurred from human endometrium-derived iPS cells. A comprehensive process of actually deriving iPS cells using discarded surgical hysterectomy specimens to the erythroid fate has significance in that the scope of using human iPSC cell lines for tissue regeneration could be expanded in the future.

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures.

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Enhanced Alveolar Bone Regeneration by Promoting Angiogenesis.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we report the effects of a S1PR2 antagonist (JTE013) on bone regeneration. Murine bone marrow stromal cells (BMSCs) were treated with dimethylsulfoxide (DMSO) or JTE013 with or without infection by an oral bacterial pathogen Aggregatibacter actinomycetemcomitans. Treatment with JTE013 enhanced vascular endothelial growth factor A (VEGFA), platelet derived growth factor subunit A (PDGFA), and growth differentiation factor 15 (GDF15) gene expression and increased transforming growth factor beta (TGFβ)/Smad and Akt signaling. Eight-week-old male C57BL/6J mice were challenged with ligatures around the left maxillary 2nd molar for 15 days to induce inflammatory bone loss. After ligature removal, mice were treated with diluted DMSO or JTE013 in the periodontal tissues 3 times per week for 3 weeks. Calcein was also injected twice to measure bone regeneration. Micro-CT scanning of maxillary bone tissues and calcein imaging revealed that treatment with JTE013 enhanced alveolar bone regeneration. JTE013 also increased VEGFA, PDGFA, osteocalcin, and osterix gene expressions in the periodontal tissues compared to control. Histological examination of periodontal tissues revealed that JTE013 promoted angiogenesis in the periodontal tissues compared to control. Our findings support that inhibition of S1PR2 by JTE013 increased TGFβ/Smad and Akt signaling; enhanced VEGFA, PDGFA, and GDF15 gene expression; and subsequently promoted angiogenesis and alveolar bone regeneration.

Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.

Damaged Mesenchymal Cells Dampen the Inflammatory Response of Macrophages and the Formation of Osteoclasts.

Damage to mesenchymal cells occurs by dental implant drills as a consequence of shear forces and heat generation. However, how the damaged mesenchymal cells can affect the polarization of macrophages and their differentiation into osteoclastogenesis is not fully understood. To simulate cell damage, we exposed suspended ST2 murine bone marrow stromal cells to freeze/thawing or sonication cycles, followed by centrifugation. We then evaluated the lysates for their capacity to modulate lipopolysaccharide-induced macrophage polarization and RANKL-MCSF-TGF-β-induced osteoclastogenesis. We report that lysates of ST2, particularly when sonicated, greatly diminished the expression of inflammatory IL6 and COX2 as well as moderately increased arginase 1 in primary macrophages. That was confirmed by lysates obtained from the osteocytic cell line IDG-SW3. Moreover, the ST2 lysate lowered the phosphorylation of p65 and p38 as well as the nuclear translocation of p65. We further show herein that lysates of damaged ST2 reduced the formation of osteoclast-like cells characterized by their multinuclearity and the expression of tartrate-resistant phosphatase and cathepsin K. Taken together, our data suggest that thermal and mechanical damage of mesenchymal cells causes the release of as-yet-to-be-defined molecules that dampen an inflammatory response and the formation of osteoclasts in vitro.

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

Deep Deconvolution of the Hematopoietic Stem Cell Regulatory Microenvironment Reveals a High Degree of Specialization and Conservation between Mouse and Human

Understanding the regulation of normal and malignant human hematopoiesis requires a comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Recent studies using scRNA-seq technologies have shed light on the organization of the hematopoietic regulatory microenvironment in the mouse. These studies have resolved some of the controversies regarding the overlap of stromal populations, the description of certain discrete stromal cells as professional, hematopoietic cytokine-producing populations, but also helped to delineate the relationship between specific stromal cell types in the murine BM.Nevertheless, these studies are limited by the number of cells sequenced, potentially hampering our ability to resolve the full spectrum of cellular states and differentiation stages that define the stromal BM microenvironment. Further, knowledge on the conservation of the cellular composition in the human BM stroma is in its infancy due to the difficulty of obtaining high-quality samples with sufficient stromal cell numbers from healthy individuals. This leaves us with two outstanding challenges; how to piece together such different fragments towards a comprehensive molecular atlas and to what extent such an atlas in mice is conserved in the human bone marrow.Here, we dissect the intrinsic organization and the heterogeneity within the endothelial (EC) and mesenchymal cell populations (MSC) governing the BM microenvironment in mouse and human. This was accomplished through customized bioinformatics integration of multiple scRNA-seq datasets along with the inclusion of over 50.000 murine and human bone marrow stromal cells. By these means, we were able to identify new subsets of MSC and EC, but more importantly, to define new molecular markers to identify highly specialized subpopulations of cells in the murine BM microenvironment. Pathway enrichment analysis unveiled multiple, potentially transient cell states defined by differential gene expression and the enrichment of specific functional characteristics. Importantly, 14 EC subsets were characterized by enrichment in pathways known to be essential for endothelial homeostasis maintenance, demonstrating a high degree of specialization in the endothelium. Similarly, 11 transient cell states in the MSC compartment were defined and characterized by their differentiation capacity. Importantly, our deep deconvolution of the heterogeneous mesenchymal and endothelial compartments became feasible only by integrating multiple datasets. Furthermore, based on the knowledge generated in the mouse, we were able to describe how much of the information and targets from the mouse can be of interest in human characterization. This analysis identified the expression of the human orthologs to the murine cluster-defining genes with different degrees of enrichment in the endothelium and mesenchyme. Moreover, some of these shared genes in mice and human stromal cells corresponded to the GO-defining genes of the different clusters identified in the mouse. These findings suggest a significant degree of conservation regarding the cellular states that define the stromal microenvironment in mouse and human. Although additional studies and improved processing of human samples will be required for deep characterization of the human BM microenvironment, these preliminary results validate our integrative cross-species approach.Taken together, our study provides a deeper understanding of the composition and specialization of the BM microenvironment and point towards a significant degree of conservation between species. Moreover, we demonstrate the usefulness of the multi-dataset integration and the customized clustering approach used in our study to improve the resolution of complex tissues and organs. This approach promises to aid in the construction of cell atlases by reducing the resources associated with sequencing that a single lab will need to invest in order to obtain meaningful depth in single-cell analysis. Future studies integrating genome, transcriptome, epigenome, proteome, and anatomical positioning together with functional assays to correlate descriptive phenotypes with functional data will help fully resolve the composition, regulation, and connectivity in the BM microenvironment in health and disease. [Display omitted] DisclosuresPaiva: Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. Saez: Magenta Therapeutics: Patents & Royalties. Prosper: BMS-Celgene: Honoraria, Research Funding; Janssen: Honoraria; Oryzon: Honoraria.

Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation

IntroductionSkeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation.ObjectiveThe current project sought to rigorously characterize the distinct metabolic processes and intracellular metabolite profiles in stromal cells throughout osteoblast differentiation using untargeted metabolomics.MethodsPrimary, murine bone marrow stromal cells (BMSCs) were characterized throughout osteoblast differentiation using standard staining protocols, Seahorse XFe metabolic flux analyses, and untargeted metabolomics.ResultsWe demonstrate here that the metabolic footprint of stromal cells undergoing osteoblast differentiation are distinct, and while oxidative phosphorylation drives adenosine triphosphate (ATP) generation early in the differentiation process, mature osteoblasts depend on glycolysis. Importantly, the intracellular metabolite profile supports these findings while also suggesting additional pathways critical for proper osteoblast function.ConclusionThese data are the first of their kind to characterize these metabolites in conjunction with the bioenergetic profile in primary, murine stromal cells throughout osteoblast differentiation and provide provocative targets for future investigation.

Intracellular density is a novel indicator of differentiation stages of murine osteoblast lineage cells.

Osteoblasts are primary bone-making cells originating from mesenchymal stem cells (MSCs) in the bone marrow. The differentiation of MSCs to mature osteoblasts involves an intermediate stage called preosteoblasts, but the details of this process remain unclear. This study focused on the intracellular density of immature osteoblast lineage cells and hypothesized that the density might vary during differentiation and might be associated with the differentiation stages of osteoblast lineage cells. This study aimed to clarify the relationship between intracellular density and differentiation stages using density gradient centrifugation. Primary murine bone marrow stromal cell cultures were prepared in an osteogenic induction medium, and cells were separated into three fractions (low, intermediate, and high-density). The high-density fraction showed elevated expression of osteoblast differentiation markers (Sp7, Col1a1, Spp1, and Bglap) and low expression of MSC surface markers (Sca-1, CD73, CD105, and CD106). In contrast, the low-density fraction showed a high expression of MSC surface markers. These results indicated that intracellular density increased during differentiation from preosteoblasts to committed osteoblasts. Intracellular density may be a novel indicator for osteoblast differentiation stages. Density gradient centrifugation is a novel technique to study the process by which preosteoblasts transform into bone-forming cells.

Warfarin Impairs the Bone Marrow Microenvironment and Hematopoiesis Via the Periostin/Integrin β3 Pathway

The coagulation system is a complex system comprised of various proteases and other factors which result in the formation of a blood clot, and proteases in general are known to regulate the mobilization of normal hematopoietic stem cells (HSC) from the bone marrow microenvironment (BMM). However, whether the proteases of the coagulation system are involved in the mobilization or maintenance of hematopoietic stem and progenitor cells (HSPC) is unknown.Hypothesizing that the proteases of the coagulation system play a role in the normal physiology of the BMM and that modulation of coagulation factors may have an effect on HSPC, we tested the effects of the anticoagulant warfarin, used in 1% of people worldwide for prophylactic or therapeutic anticoagulation. Indeed, treatment of wildtype mice with warfarin led to a decrease in myeloid cells in peripheral blood, decreased HSC function and mobilization from the BMM and an almost 8 fold reduction of functional HSC. Intrafemoral co-transplantation of warfarin-treated, ex-vivo expanded primary murine bone marrow stromal cells with untreated HSPC led to decreased engraftment of HSPC compared to co-transplantation of vehicle-treated stroma cells suggesting that warfarin may compromise bone marrow stroma. We have implicated periostin, a vitamin K dependent factor produced by bone marrow stroma - binding to integrin β3 and signaling via AKT in HSC - as being the cause of impairment of hematopoiesis during treatment with warfarin.In addition, warfarin treatment of NOD SCID interleukin-2 receptor gamma knockout (NSG) mice or intrafemoral cotransplantation of human warfarin-treated stroma cells and untreated human CD34+ HSC led to reduced engraftment of human CD45+ leukocytes. In corroboration of our murine data we showed that the total leukocyte and monocyte count, as well as the percentage of basophils and eosinophils were significantly reduced in 282 patients receiving warfarin and 89 patients receiving another vitamin K antagonist, fluindione, for anticoagulation compared to untreated healthy controls.In summary, warfarin or vitamin K antagonism treatment impairs HSC function in humans and mice via modification of the BMM and, specifically, by the impairment of the periostin/integrin β3 axis, which we, thereby, implicate also for the maintenance of normal HSC. [Display omitted] DisclosuresFontenay:Celgene: Research Funding.

CAR T Cell Relapse CD19 Negative ALL Cells Express Integrins and Integrin Blockade Can Prolong Survival In Vivo

Relapsed and refractory B-cell precursor acute lymphoblastic leukemia (BCP ALL) remains difficult to treat, despite advances in upfront therapy and improved survival for de novo ALL. Chimeric antigen receptor T (CART) cell therapy is a major chemotherapy-independent approach, achieving disease-free remission that functions through an immunologic mechanism. However, even with this novel therapy, it is becoming evident that relapses occur with appreciable frequency. In some cases, anti-CD19 CART cell treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Based on our in-vivo relapse model using relapse ALL post-CART therapy lacking CD19 surface expression, we found that relapse occurs in the bone marrow. Therefore, we hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CART-cell resistance (CAM-CART-R) can occur resulting in relapse of ALL. To test our hypothesis, primary ALL cells were co-cultured with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures, and anti-CD19 targeting CAR T cells were added to the culture. We observed that CAR T cells had less activity against ALL cells when in OP9-coculture, compared to media only co-cultures, supporting the notion of CAM-CART-R. Interestingly, three cases of primary ALL with CD19 knocked out using CAS9-CRISPR, showed significantly more adhesion to OP9 cells compared to CD19 competent ALL cells. Phenotypic analysis revealed high expression of integrins after knockout of CD19 in all three primary ALL cells. Phenotypic profiling of three cases of primary ALL relapse after anti-CD19 CAR T cell therapy also showed high expression of adhesion molecules including integrin alpha4 and alpha6. We showed that blocking integrins can de-adhere these primary ALL relapses from their respective counter-ligands in vitro. As a proof of principle that CD19-negative ALL cells post-CAR T cell relapse can benefit from integrin blocking therapy in vivo, we used the humanized anti-human integrin alpha4 antibody, Natalizumab. NSG mice were injected with one case of CD19 negative ALL cells post-CART cell relapse and treated intraperitoneally (n=6/group): with control IgG or Natalizumab. Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control IgG group (MST =85 days vs. MST= 66 days, p<0.005). Further combination treatments with chemotherapy are in progress. In summary, our data indicates that targeting adhesion molecules may be a new approach to treat anti-CD19 CAR T cell relapsed ALL. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia depletes contaminating CD45+ hematopoietic cells from murine bone marrow stromal cell (BMSC) cultures: Methods for BMSC culture purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSCs continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation is widely based on adherence to tissue culture plastic; however, high hematopoietic contamination is a significant impediment in murine models. Remarkably, when cultured at a physiological oxygen tension of 1% O2, a 10-fold reduction in CD45+ hematopoietic cells associated with a concomitant increase in PDGFRα+ stromal cells occur. This is due, in part, to a differential response of the two populations to hypoxia. In standard tissue culture conditions of 21% O2, CD45+ cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O2. In contrast, PDGFR α+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR α+ stromal cell population in murine BMSC cultures.

A comparative study on biological properties and chemical profiles of different solvent extracts from Centaurea bingoelensis, an endemic plant of Turkey

This study focused on the biological evaluation and chemical characterization of Centaurea bingoelensis, an endemic plant to Turkey. Different solvent extracts (hexane, dichloromethane (DCM), ethyl acetate (EA), methanol, hydro-methanol, and water extracts) were prepared. The antioxidant, enzyme inhibitory, antimicrobial and cytotoxic activities were investigated. The hydro-methanol extract, possessing high phenolic content (41.57 mg gallic acid equivalent (GAE)/g extract), showed highest reducing capacity (136.87 and 82.16 mg Trolox equivalent[TE]/g extract, for cupric reducing antioxidant capacity and ferric reducing antioxidant power, respectively) and radical scavenging potential (70.72 and 76.53 mg TE/g extract, against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), respectively). Ultra performance liquid chromatography - tandem mass spectrometer (UHPLC/MS/MS) analysis revealed the presence of 67 and 62 compounds in the methanolic and water extracts, respectively. According to the broth microdilution results, DCM and EA extracts showed significant antimicrobial activity with Sarcina lutea being the most sensitive bacterium to the extracts (MIC = 0.19 (DCM) and 0.097 mg/mL (EA)). Most extracts showed promising antifungal activity against Candida albicans. The methanol extract was most toxic against human hepatocarcinoma (HepG2) cells, and also significantly reduced the cell viability of the non-tumoral murine bone marrow stromal (S17) cells. This study has provided valuable scientific data on this endemic plant which can be considered as a promising candidate for the development of novel phyto-pharmaceuticals.

Nanoengineered shear-thinning and bioprintable hydrogel as a versatile platform for biomedical applications

The development of bioinks based on shear-thinning and self-healing hydrogels has recently attracted significant attention for constructing complex three-dimensional physiological microenvironments. For extrusion-based bioprinting, it is challenging to provide high structural reliability and resolution of printed structures while protecting cells from shear forces during printing. Herein, we present shear-thinning and printable hydrogels based on silicate nanomaterials, laponite (LA), and glycosaminoglycan nanoparticles (GAGNPs) for bioprinting applications. Nanocomposite hydrogels (GLgels) were rapidly formed within seconds due to the interactions between the negatively charged groups of GAGNPs and the edges of LA. The shear-thinning behavior of the hydrogel protected encapsulated cells from aggressive shear stresses during bioprinting. The bioinks could be printed straightforwardly into shape-persistent and free-standing structures with high aspect ratios. Rheological studies demonstrated fast recovery of GLgels over multiple strain cycles. In vitro studies confirmed the ability of GLgels to support cell growth, proliferation, and spreading. In vitro osteogenic differentiation of pre-osteoblasts murine bone marrow stromal cells encapsulated inside the GLgels was also demonstrated through evaluation of ALP activity and calcium deposition. The subcutaneous implantation of the GLgel in rats confirmed its in vivo biocompatibility and biodegradability. The engineered shear-thinning hydrogel with osteoinductive characteristics can be used as a new bioink for 3D printing of constructs for bone tissue engineering applications.

Commentary: A Cost-Effective Method to Enhance Adenoviral Transduction of Primary Murine Osteoblasts and Bone Marrow Stromal Cells.

A Commentary on A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells by Buo, A. M., Williams, M. S., Kerr, J. P., and Stains, J. P. (2016). Bone Res. 4:16021. doi: 10.1038/boneres.2016.21

β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2.

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

Metabolic programming determines the lineage-differentiation fate of murine bone marrow stromal progenitor cells

Enhanced bone marrow adipogenesis and impaired osteoblastogenesis have been observed in obesity, suggesting that the metabolic microenvironment regulates bone marrow adipocyte and osteoblast progenitor differentiation fate. To determine the molecular mechanisms, we studied two immortalized murine cell lines of adipocyte or osteoblast progenitors (BMSCsadipo and BMSCsosteo, respectively) under basal and adipogenic culture conditions. At baseline, BMSCsadipo, and BMSCsosteo exhibit a distinct metabolic program evidenced by the presence of specific global gene expression, cellular bioenergetics, and metabolomic signatures that are dependent on insulin signaling and glycolysis in BMSCsosteo versus oxidative phosphorylation in BMSCsadipo. To test the flexibility of the metabolic program, we treated BMSCsadipo with parathyroid hormone, S961 (an inhibitor of insulin signaling) and oligomycin (an inhibitor of oxidative phosphorylation). The treatment induced significant changes in cellular bioenergetics that were associated with decreased adipocytic differentiation. Similarly, 12 weeks of a high-fat diet in mice led to the expansion of adipocyte progenitors, enhanced adipocyte differentiation and insulin signaling in cultured BMSCs. Our data demonstrate that BMSC progenitors possess a distinct metabolic program and are poised to respond to exogenous metabolic cues that regulate their differentiation fate.

Thrombin: A Driver or Attenuator of Bone Disease on Hemophilia?

Introduction: Hemophilic bone disease is a pathology inherent to factor VIII (FVIII) and factor IX (FIX) deficiency present in both adult and pediatric persons with hemophilia (PWH). This pathology is also present in mouse models. We have previously demonstrated that FVIII replacement restores lost mineralization activity in osteoblast-like cells from FVIII-deficient mice. We hypothesized that bone disease in PWH is secondary to decreased thrombin generation in PWH. Methods: Murine bone marrow stromal cells isolated from FVIII knockout (FVIII-/-) and wild-type (FVIII+/+) C57BL mice and human osteosarcoma (HOS) TE85 cells were cultured in α-MEM with 0-10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL Amphotericin B. Ten millimolar ß-glycerophosphate and 50 µg/mL ascorbic acid were added to induce differentiation and mineralization. Mineralized deposits were stained with alizarin red. Alkaline phosphatase (ALP) activity and culture protein content were measured using colorimetric assays. Results: In , the addition of thrombin in serum-free conditions increases cell proliferation ~25% (p=5x10-4, ANCOVA) and decreases mineralization up to 84% (p=1x10-6) (Figure 1). Thrombin's effect on ALP activity in HOS TE85 cells is negative at low cell numbers and positive at high cell numbers (Figure 1). Osteoblast-like cells from FVIII-/- mice mineralize 70% less than those from FVIII+/+ cells (p=3x10-6). Administering FVIII to cells from FVIII-/- mice ameliorates the mineralization deficiency (Figure 1), while mineralization in cells from FVIII+/+ mice does not change (p=0.66). FVIII-/- cells express 30% lower levels of ALP activity than FVIII+/+ (p=3x10-4). FVIII+/+ cells proliferate 26% more rapidly than FVIII-/- cells (p=4x10-7, ANCOVA), but this is not affected by the addition of FVIII in vitro (p=0.45). The addition of FVIII causes a ~33% decrease in ALP activity in osteoblast-like cells from both FVIII-/- (p=2x10-4) and FVIII+/+ (p=5x10-5) mice. Although thrombin impacts osteoblast differentiation and function, it has paradoxical effects on their mineralization, suggesting thrombin deficiency is not the sole driver of reduced bone health in PWH. Deficient mineralization in cells from FVIII-/- mice compared to those from FVIII+/+ mice is corrected with FVIII replacement in vivo or in vitro, suggesting that circulating FVIII has a positive role in these cells' mineralization. More rapid proliferation in cells from FVIII+/+ mice likely contributes to the increase in mineralization, yet, as proliferation is unchanged by FVIII replacement, it appears unlikely that this effect occurs via the same mechanism. Thrombin causes increased proliferation of HOS TE85 cells, yet also causes a significant decline in mineralization activity (Figure 1). Whether this is a paradoxical effect present only in this transformed cell line is yet undetermined. ALP activity increases when HOS TE85 cells are given thrombin in vivo during the log phase of growth (Figure 1), indicating a positive effect on the osteoblast-like function of the cells. However, this notion is undermined by the decrease in mineralization. Identifying the role of FVIII, FIX, thrombin, and active protein C in bone mineralization in PWH is proving to be a challenging undertaking, yet it remains critical, especially as new drugs with the potential to supplant FVIII prophylaxis hit the market. Future experiments will include thrombin and a thrombin inhibitor, a PAR1 agonist and antagonist, a PAR2 antagonist, and an APC neutralizing antibody. Upcoming findings will be discussed. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership.