Cell Animal Models Research Articles

An easy way to make a good anti-tumor chimeric antigen receptor T cell?

The notion that the immune system has the capacity to prevent, control and eradicate cancer has been present for many years, but truly effective potent immunotherapeutic strategies have materialized only recently. One of the most successful and effective of these strategies involves adoptive transfer of genetically engineered T cells expressing chimeric antigen receptor (CAR) targeting the B-cell antigen CD19 present on B-cell lymphomas, acute lymphoblastic leukemia and chronic lymphocytic leukemia (1,2). Although the CD19 appears to represent an excellent safe target and the initial reports of efficacy are highly promising, it is clear that many aspects of the adoptive immunotherapy protocols still must be perfected. Generation of specific anti-tumor T cells in vitro requires their stimulation and significant expansion. The quality of the in vitroegenerated T-cell product appears to be as critical as the antigenic specificity of the T cells and can profoundly affect the treatment outcome. Traditionally, these highly cytotoxic, armed effectors were thought to be the most desirable by tumor immunologists because they displayed target killing capacity in vitro. Paradoxically, however, the in vivo function of these cells in animal models and in many clinical trials was disappointing (3). Gradually, it became clear that these armed terminal effectors displayed functional features of senescence and were unable to survive, expand and eradicate the tumor in vivo (4). In contrast, T cells under various conditions promoting less terminally differentiated central memory (CM) or even earlier stem cellelike phenotype display absent or low in vitro cytotoxic effector activity but much higher in vivo anti-tumor functionality that was

Human Amnion-Derived Cells: Prospects for the Treatment of Lung Diseases

Lung diseases represent a significant burden of morbidity and mortality worldwide. Current therapies have not proven adequate in the long term and are often associated with significant side effects. There has been recent interest in the regenerative/reparative potential of cell-based therapies, including cells derived from the placental tissues. Amnion-derived cells are fetal-derived and characterized by expression profile and differentiative capacity of pluripotent cells. Moreover, because placenta is discarded after delivery, they represent an ethical source for the purposes of regenerative medicine. Amnion-derived cells are endowed with immunomodulatory, anti-inflammatory, anti-scarring and antibacterial properties, which may explain many of the beneficial effects observed with administration of the cells in animal models for a large number of inflammatory diseases. Both human amniotic epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC) have been shown to acquire in vitro and in vivo some characteristics of epithelial cells, i.e. CFTR (cystic fibrosis transmembrane conductance regulator) and surfactant proteins. Administration of hAEC or hAMSC in vivo in the bleomycin-induced lung injury model has proven their therapeutic effects in term of reduction of pulmonary fibrosis and inflammation, as well as recovery of lung mechanical function. Many biological and clinical information have to be gathered before proposing amnion-derived cells in the clinic for the treatment of acute and chronic lung diseases.

Flagellin pretreatment enhances the immunosuppressive capacity of mesenchymal stem cells in animal model of proctitis

Flagellin pretreatment enhances the immunosuppressive capacity of mesenchymal stem cells in animal model of proctitis

Concise Review: The Immune Status of Mesenchymal Stem Cells and Its Relevance for Therapeutic Application

Multipotentiality and anti-inflammatory activity, the two main properties of mesenchymal stem cells (MSCs), underlie their therapeutic prospective. During the past decade, numerous studies in animal models and clinical trials explored the potential of MSCs in the treatment of diseases associated with tissue regeneration and inflammatory control. Other qualities of MSCs: ready accessibility in bone marrow and fat tissue and rapid expansion in culture make the therapeutic use of patients' own cells feasible. The prevailing belief that MSCs are nonimmunogenic encouraged the use of unrelated donor cells in immune-competent recipients. The data emerging from studies performed with immune-incompatible cells in animal models for a wide-range of human diseases show, however, conflicting results and cast doubt on the immune privileged status of MSCs. Our analysis of the preclinical literature in this review is aimed to gain a better understanding of the therapeutic potential of immune-incompatible MSCs. Emphasis was laid on applications for enhancement of tissue repair in the absence of immune-suppressive therapy.

A Type VI Secretion System Is Involved in Pseudomonas fluorescens Bacterial Competition

Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS) is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeat protein G) into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2), suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models), but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium), and our results suggest that there may be a connection between the T6SS and the PGPR properties of this bacterium.

Stem cell therapy in animal models of central nervous system (CNS) diseases: therapeutic role, challenges and perspectives

Many human diseases relating to central nervous system (CNS) are mimicked in animal models to evaluate the efficacy of stem cell therapy. The therapeutic role of stem cells in animal models of CNS diseases include replacement of diseased or degenerated neuron, oligodendrocytes and astrocytes with healthy ones, secretion of neurotrophic factors and delivery of therapeutics/genes. Scaffolds can be utilized for delivering stem cells in brain. Sustained delivery of stem cells, lineage specific differentiation, and enhanced neuronal network integration are the hallmarks of scaffold mediated stem cell delivery in CNS diseases. This review discusses the therapeutic role, challenges and future perspectives of stem cell therapy in animal models of CNS diseases.

Preclinical efficacy and mechanisms of mesenchymal stem cells in animal models of autoimmune diseases.

Mesenchymal stem cells (MSCs) are present in diverse tissues and organs, including bone marrow, umbilical cord, adipose tissue, and placenta. MSCs can expand easily in vitro and have regenerative stem cell properties and potent immunoregulatory activity. They inhibit the functions of dendritic cells, B cells, and T cells, but enhance those of regulatory T cells by producing immunoregulatory molecules such as transforming growth factor-β, hepatic growth factors, prostaglandin E2, interleukin-10, indolamine 2,3-dioxygenase, nitric oxide, heme oxygenase-1, and human leukocyte antigen-G. These properties make MSCs promising therapeutic candidates for the treatment of autoimmune diseases. Here, we review the preclinical studies of MSCs in animal models for systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, and experimental autoimmune encephalomyelitis, and summarize the underlying immunoregulatory mechanisms.

Animal models to test hiPS-derived hepatocytes in the context of inherited metabolic liver diseases.

Human induced pluripotent stem (hiPS) cells are established following reprogramming of somatic cells from a wide variety of tissues. Given the scarcity of adult human hepatocytes, hiPS-derived hepatocytes would be a valuable source of cells to study differentiation programs, model patient-specific diseases, test drug toxicities, and cell transplantation therapies. Although hiPS-derived hepatocytes are extensively characterized in cell culture assays, testing these cells in animal models is necessary to fully evaluate their differentiation profile and their lack of tumorigenicity. Immunodeficient mouse models harboring liver damage are effective hosts in which xenogeneic hepatocytes can engraft, proliferate, and participate in liver regeneration, thus constituting a stringent test of hepatocyte functionality. The in vivo evaluation of disease-specific hiPS-derived hepatocytes should broaden our understanding of the cellular and molecular processes involved in inherited metabolic liver disease phenotypes. Herein, we detail our methods to test the functions of hiPS-derived hepatocytes in the context of the immunodeficient Rag2(-/-)IL2Rγc(-/-)Alb-uPAtg mouse model.

HYPOXIC PRECONDITIONING OF STEM CELLS AS A NEW APPROACH TO INCREASE THE EFFICACY OF CELL THERAPY FOR MYOCARDIAL INFARCTION

During the last decade, stem cell research has developed at an accelerated pace. Various types of stem cells have been tested for myocardial infarction therapy. Despite the preclinical benefits of cell therapy success in clinical trials remains modest. The main obstacles to regeneration of the infarcted heart using stem cells are: 1) not every stem cell type can differentiate into cardiomyocytes; and 2) low survival rates of transplanted cells, due to the harsh environment of the infarcted myocardium. Hypoxic preconditioning (HP) has been shown to improve transplantation efficacy of mesenchymal stem cells and cardiac progenitor cells in animal models of myocardial infarction. It has also been shown that transplantation of preconditioned cells decreases infarct size, prevents postinfarction remodeling of the heart, and positively modulates development of ischemic cardiomyopathy. Hypoxic preconditioning also prevents extensive death of transplanted cells due to necrosis and apoptosis during long-term hypoxia or oxidative stress. The protective effect of HP is based on three main processes: (1) modification of cell phenotypes to help survival during hypoxia (enhancement of HIF-1alpha expression, ERK1/2 and Akt activation, enhancement of erythropoietin receptor expression and erythropoietin production, and an elevation in levels of antiapoptotic proteins Bcl-2 and Bcl-xL); (2) upregulation of various secretable factors including the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), and expression of VEGF-2 and HGF-receptors; (3) enhancement in the formation of CXCR4 and CXCR7 receptors, which play an important role in mobilization and homing of stem cells in the ischemic region.

Interferon‐induced programmed death‐ligand 1 (PD‐L1/B7‐H1) expression increases on human acute myeloid leukemia blast cells during treatment

While current treatment for acute myeloid leukemia is characterized by high response rates, patients' long-term outcome is still disappointing, due to frequent relapse and ineligibility of the often elderly patients for stem cell transplantation approaches. Considerable efforts have, thus, been made to incorporate immunotherapeutic approaches in the acute myeloid leukemia (AML) consolidation, with so far disappointing clinical benefit. The B7 family ligand programmed-death receptor-ligand 1 (PD-L1, B7-H1, CD274) has been recently described (with conflicting results) to be expressed on AML blast cells, and interaction with its receptor on T cells, programmed death receptor-1 (PD-1, CD279), has been shown to suppress T-cell functions and to allow survival of dormant AML cells in animal models. In this work, we analyzed freshly isolated myeloid precursor cells from healthy donors and from AML patients for PD-L1 expression with or without interferon-γ exposure at different time points during their treatment. While without IFN exposure, only minor differences were observed, we found IFN-γ-induced PD-L1 expression most prominent after initial treatment and independent of treatment outcome. Our observations support the recently suggested PD-L1-mediated adaptive immune resistance and argue for a targeting of the PD-L1/PD-1 pathway during the consolidation phase of AML treatment.

Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1).

Human mesenchymal stem/precursor cells (MSC) are similar to some other stem/progenitor cells in that they compact into spheres when cultured in hanging drops or on nonadherent surfaces. Assembly of MSC into spheres alters many of their properties, including enhanced secretion of factors that mediate inflammatory and immune responses. Here we demonstrated that MSC spontaneously aggregated into sphere-like structures after injection into a subcutaneous air pouch or the peritoneum of mice. The structures were similar to MSC spheres formed in cultures demonstrated by the increased expression of genes for inflammation-modulating factors TSG6, STC1, and COX2, a key enzyme in production of PGE2. To identify the signaling pathways involved, hanging drop cultures were used to follow the time-dependent changes in the cells as they compacted into spheres. Among the genes upregulated were genes for the stress-activated signaling pathway for IL1α/β, and the contact-dependent signaling pathway for Notch. An inhibitor of caspases reduced the upregulation of IL1A/B expression, and inhibitors of IL1 signaling decreased production of PGE2, TSG6, and STC1. Also, inhibition of IL1A/B expression and secretion of PGE2 negated the anti-inflammatory effects of MSC spheres on stimulated macrophages. Experiments with γ-secretase inhibitors suggested that Notch signaling was also required for production of PGE2 but not TSG6 or STC1. The results indicated that assembly of MSC into spheres triggers caspase-dependent IL1 signaling and the secretion of modulators of inflammation and immunity. Similar aggregation in vivo may account for some of the effects observed with administration of the cells in animal models.

Modification of Hematopoietic Stem/Progenitor Cells with CD19-Specific Chimeric Antigen Receptors as a Novel Approach for Cancer Immunotherapy

Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity.

Removing the Immune Response From Muscular Dystrophy Research

Immune responses against exogenous cells, vectors, and proteins have emerged as a significant barrier limiting development of therapies for inherited disorders. This is especially true when testing human cells and genes in animal models of disease. In this issue of Molecular Therapy, Vallese et al.1 describe an improved immunodeficient mouse model for Duchenne muscular dystrophy (DMD) that should facilitate the development of therapies. The authors show that these mice enable extensive testing of human stem cells and methods of delivery without immune-mediated rejection of donor cells. The utility of this model is further enhanced by use of a specific mutant allele that eliminates background dystrophin expression in muscle tissues, providing a sensitive platform for monitoring cell engraftment and exogenous gene expression. As such, the model could accelerate development of interventions based on both stem cell transplantation and genetic manipulation.

Radiation-Induced Bystander Signaling from Somatic Cells to Germ Cells in Caenorhabditis elegans

Recently, radiation-induced bystander effects (RIBE) have been studied in mouse models in vivo, which clearly demonstrated bystander effects among somatic cells. However, there is currently no evidence for RIBE between somatic cells and germ cells in animal models in vivo. In the current study, the model animal Caenorhabditis elegans was used to investigate the bystander signaling from somatic cells to germ cells, as well as underlying mechanisms. C. elegans body size allows for precise microbeam irradiation and the abundant mutant strains for genetic dissection relative to currently adopted mouse models make it ideal for such analysis. Our results showed that irradiation of posterior pharynx bulbs and tails of C. elegans enhanced the level of germ cell apoptosis in bystander gonads. The irradiation of posterior pharynx bulbs also increased the level of DNA damage in bystander germ cells and genomic instability in the F1 progeny of irradiated worms, suggesting a potential carcinogenic risk in progeny even only somatic cells of parents are exposed to ionizing radiation (IR). It was also shown that DNA damage-induced germ cell death machinery and MAPK signaling pathways were both involved in the induction of germ cell apoptosis by microbeam induced bystander signaling, indicating a complex cooperation among multiple signaling pathways for bystander effects from somatic cells to germ cells.

Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

It has been shown that coculture of bone marrow–derived stromal cells (BMSCs) with intervertebral disc (IVD) nucleus pulposus (NP) cells significantly activates the biological characteristics of NP cells in animal models and in humans. We therefore predicted that activated NP cells would be a useful graft source for cellular transplantation therapy in the treatment of degenerative IVDs. However, the activation protocol is based on fresh isolation and activation of NP cells, which limits the timing of clinical application. Cell transplantation therapy could be offered to more patients than is now possible if activated NP cells could be transplanted as and when required by the condition of the patient. No study has investigated the effect of cryopreservation on NP cells after enzymatic isolation. We investigated the effects of cryopreservation of canine and human NP cells in both cell and tissue form before coculture with autologous BMSCs. Cell viability, proliferation, glycosaminoglycan production, aggrecan transcriptional activity, colony generation, and gene expression profile of the cells after cryopreservation and subsequent coculture were analyzed. The influence of cryopreservation on cell chromosomal abnormalities and tumorigenesis was also studied. The results showed that there were no clear differences between the noncryopreserved and cryopreserved cells in terms of cell viability, proliferation capacity, and capacity to synthesize extracellular matrix. Furthermore, the cells showed no apparent chromosomal abnormalities or tumorigenic ability and exhibited similar patterns of gene expression. These findings suggest that by using cryopreservation, it may be possible to transplant activated NP cells upon request for patients' needs.

Circulation Editors’ Picks

<i>Circulation</i> Editors’ Picks

Lymphodepletional Strategies in Transplantation

Because lymphocytes were shown to mediate transplant rejection, their depletion has been studied as a mechanism of preventing rejection and perhaps inducing immunologic tolerance. Agents that profoundly deplete lymphocytes have included monoclonal antibodies, cytotoxic drugs, and radiation. We have studied several such agents but focused on antibodies that deplete not only peripheral blood lymphocytes, but also lymph node lymphocytes. Depletion of lymph node T lymphocytes appears to permit peripheral tolerance at least for T cells in animal models. Nevertheless, B-cell responses may be resistant to such approaches, and T memory cells are likewise relatively resistant to depleting antibodies. We review the experimental and clinical approaches to depletion strategies and outline some of the pitfalls of depletion, such as limitations of currently available agents, duration of tolerance, infection, and malignancy. It is notable that most tolerogenic strategies that have been attempted experimentally and clinically include depleting agents even when they are not named as the underlying strategy. Thus, there is an implicitly acknowledged role for reducing the precursor frequency of donor antigen-specific lymphocytes when approaching the daunting goal of transplant tolerance.

Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system

There is a need for improved therapy for acquired brain injury, which has proven resistant to treatment by numerous drugs in clinical trials and continues to represent one of the leading causes of disability worldwide. Research into cell-based therapies for the treatment of brain injury is growing rapidly, but the ideal cell source has yet to be determined. Subpopulations of cells found in amniotic fluid, which is readily obtained during routine amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity, are non-tumourigenic, and avoid the ethical complications associated with embryonic stem cells, making them a promising cell source for therapeutic purposes. Beneficial effects of amniotic fluid cell transplantation have been reported in various models of nervous system injury. However, evidence that amniotic fluid cells can differentiate into mature, functional neurons in vivo and incorporate into the existing circuitry to replace lost or damaged neurons is lacking. The mechanisms by which amniotic fluid cells improve outcomes after experimental nervous system injury remain unclear. However, studies reporting the expression and release of neurotrophic, angiogenic, and immunomodulatory factors by amniotic fluid cells suggest they may provide neuroprotection and (or) stimulate endogenous repair and remodelling processes in the injured nervous system. In this paper, we address recent research related to the neuronal differentiation of amniotic fluid-derived cells, the therapeutic efficacy of these cells in animal models of nervous system injury, and the possible mechanisms mediating the positive outcomes achieved by amniotic fluid cell transplantation.

Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region

Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here, we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3.

Strategies of Human Corneal Endothelial Tissue Regeneration

Penetrating keratoplasty has previously been the only surgical treatment for patients with corneal endothelial disorders. Recently, posterior lamellar keratoplasty has become a viable and less aggressive alternative technique. However, both transplantation techniques have disadvantages, such as non-immunologic graft failure, allograft endothelial rejection or a global shortage of donor corneas. Over the past few years, several groups have established methods for the isolation, preservation, in vitro cultivation, transplantation, and in vivo stimulation of human corneal endothelial cells in animal models. It is hoped that these new strategies will allow the treatment of more than one patient with one donor cornea, performing autologous corneal endothelium transplantation from a surgical biopsy sample, or stimulating the growth of corneal endothelial cells in vivo. However, several aspects need to be addressed before commencing clinical trials.