Chimeric Animals Research Articles

Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion.

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

Stem cell topography splits growth and homeostatic functions in the fish gill.

While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive un-exhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Here, we use lineage analysis in the medaka fish gill to address aSCs and report separate stem cell populations for homeostasis and growth. These aSCs are fate-restricted during the entire post-embryonic life and even during re-generation paradigms. We use chimeric animals to demonstrate that p53 mediates growth coordination among fate-restricted aSCs, suggesting a hierarchical organisation among lineages in composite organs like the fish gill. Homeostatic and growth aSCs are clonal but differ in their topology; modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesise that physical niches are main players to restrict aSCs to a homeostatic function in animals with fixed adult size.

Nephrogenic Systemic Fibrosis Is Mediated by Myeloid C-C Chemokine Receptor 2

Gadolinium-based contrast agents are implicated in several pathologic abnormalities (long-term retention in vital organs such as the skin and the brain) and are the cause of a sometimes fatal condition in patients, nephrogenic systemic fibrosis. Bone marrow-derived fibrocytes and the monocyte chemoattractant protein-1 inflammatory pathway have been implicated as mediators of the adverse effects induced by gadolinium-based contrast agents. Mechanistic studies are scant; therefore, a mouse model of nephrogenic systemic fibrosis was established. Dermal cellularity was increased in contrast-treated green fluorescent protein (GFP) chimeric mice. GFP in the skin and fibrosis were increased in the contrast-treated chimeric animals. Monocyte chemoattractant protein-1 and C-C chemokine receptor 2 were increased in the tissues from contrast-treated mice. C-C chemokine receptor 2-deficient recipients of GFP-expressing marrow had an abrogation of gadolinium-induced pathology and displayed less GFP-positive cells in the skin. Wild-type animals that received C-C chemokine receptor 2-deficient bone marrow had a complete abrogation of dermal pathology. That GFP levels and expression increase in the skin, in tandem with a fibrocyte marker, supports the blood-borne circulating fibrocyte hypothesis of the disease. As of now, fibrocyte trafficking has yet to be demonstrated. Importantly, our data demonstrate that the monocyte chemoattractant protein-1/C-C chemokine receptor 2 axis plays a critical role in the pathogenesis of nephrogenic systemic fibrosis.

Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J (n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure (P = 0.013) and mean pulmonary artery pressure (P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance (P = 0.024) and RV effective arterial elastance (P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.

Concise Review: Human-Animal Neurological Chimeras: Humanized Animals or Human Cells in an Animal?

Blastocyst complementation is an emerging methodology in which human stem cells are transferred into genetically engineered preimplantation animal embryos eventually giving rise to fully developed human tissues and organs within the animal host for use in regenerative medicine. The ethical issues surrounding this method have caused the National Institutes of Health to issue a moratorium on funding for blastocyst complementation citing the potential for human cells to substantially contribute to the brain of the chimeric animal. To address this concern, we performed an in-depth review of the neural transplantation literature to determine how the integration of human cells into the nonhuman neural circuitry has altered the behavior of the host. Despite reports of widespread integration of human cell transplants, our review of 150 transplantation studies found no evidence suggestive of humanization of the animal host, and we thus conclude that, at present, concerns over humanization should not prevent research on blastocyst complementation to continue. We suggest proceeding in a controlled and transparent manner, however, and include recommendations for future research with careful consideration for how human cells may contribute to the animal host nervous system. Stem Cells 2019;37:444-452.

A framework for the ethical assessment of chimeric animal research involving human neural tissue

BackgroundAnimal models of human diseases are often used in biomedical research in place of human subjects. However, results obtained by animal models may fail to hold true for humans. One way of addressing this problem is to make animal models more similar to humans by placing human tissue into animal models, rendering them chimeric. Since technical and ethical limitations make neurological disorders difficult to study in humans, chimeric models with human neural tissue could help advance our understanding of neuropathophysiology.Main bodyIn this article, we examine whether the introduction of human neural tissue and any consequent cognitive change is relevant to the way we ought to treat chimeras. We argue that changes in cognitive abilities are morally relevant to the extent that they increase the capacities that affect the moral status of any entity, including awareness, autonomy, and sociability. We posit that no being, regardless of species, should be treated in a way that is incommensurate with its moral status. Finally, we propose a framework that can be used to guide ethical assessment of research involving chimeras with advanced cognitive capacities.ConclusionWe advance this framework as a useful tool for bringing relevant considerations to the forefront for those considering the ethical merit of proposed chimeric research. In doing so, we examine concepts relevant to the question of how any entity may be treated, including moral status, dignity, and capacities.

Development of blastocyst complementation technology without contributions to gametes and the brain.

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.

Basal Polarity Complex Scribble Is Required for Leukemic Initiation and Propagation through Negative Regulation of Apical Polarity Complex Activator Cdc42 and Hypoxia Inducing Factor-1α

Despite the introduction of tyrosine kinase inhibitor and CAR-T cell therapies, the prognosis for Ph+ and Ph-like acute lymphoblastic leukemia remains poor. In the present study, we show the role of the Scribble protein in both lymphoid and myeloid leukemogenesis. The polarity protein Scribble is a member of the basal polarity complex, which is down-regulated in many cancers, suggesting a possible tumor suppressor role, especially in so-called cancer initiating cells. Its effect and mechanisms of activity in leukemic cell fate along with its potential activity on leukemic initiating cells have only been recently started to elucidated. Using interferon-responsive inducible (Mx1-Cre) Scribble-deficient mice, we have characterized the role of Scribble in both retroviral transduction, transplantation animal models and binary, inducible stem cell initiated (Scl-tTA/TRE-BCR-ABL) serial propagation models of BCR-ABL induced leukemia. We found that Scribble expression is upregulated at both transcriptional and translational levels in p210- or p190-BCR-ABL induced leukemic progenitors. In vitro, leukemic colony formation was impaired in Scribble deficient leukemic progenitors (~48% reduction; p≤ 0.05, compared to Wt leukemic progenitors) demonstrating that Scribble is important for leukemogenesis. In vivo, the deletion of Scribble abrogates the development of myeloproliferative disease induced by p210-BCR-ABL (median survival: 70 vs 47 days in Scribble deficient and Wt chimeric mice, respectively; p≤0.05); and significantly impairs B-cell lymphoid leukemogenesis induced by p190-BCR-ABL (median survival: 80 vs 60 days for Scribble deficient and Wt chimeric animals, respectively). Mechanistically, BCR-ABL activates the apical polarity regulator Cdc42 in leukemic progenitors and this activation is inhibited by the deficiency of Scribble. The deficiency of Cdc42 does not impair leukemogenesis but the combined deficiency of Cdc42 and Scribble restores the in vivo survival (median survival: 47 days, p≤0.01 compared to Scribble deficient mice) in chimeric p190-BCR-ABL+ leukemic mice to levels similar to wild-type leukemic cells. These data indicate that Scribble-deficient leukemogenesis is dependent on oncogene induced Cdc42 activity in lymphoid progenitors. Furthermore, Scribble deficiency in leukemic progenitors increases the activation of the AMPK/mTORC1 signaling pathway and the protein expression and transcriptional activity of its downstream effector hypoxia-inducing factor-1α (HIF-1α). HIF-1α silencing by constitutive shRNA expression or inducible deletion in Scribble deleted B-lymphoid leukemic cells restored leukemic progenitor clonogenic efficiency (CFU average: 52 vs 110 per 1,000 B220+/EGFP+ BM cells, in Scribble and double Scribble/HIF-1α deficient, respectively; p≤0.01) and B-lymphoid leukemogenesis in vivo (median survival of 62 days; p≤0.05 compared with Scribble deficient chimeric animals). In addition, double deficiency of Scribble and HIF-1α restored AMPK/mTORC1 signaling to Wt leukemic levels. This data indicates that Scribble is a negative regulator of HIF-1α expression and activity, and the restoration of HIF-1α expression and activity to normal leukemic levels is necessary to restore leukemogenesis. Altogether, our data indicates that Scribble is a positive regulator of oncogenesis in leukemic progenitors, in vitro and in vivo, through Cdc42 and HIF-1α activities. DisclosuresNo relevant conflicts of interest to declare.

Specific and Synergistic Targeting of JAK2V617F Cells By Interferon Alpha and Arsenic

N.M. and T.D. co-first authors, J.L.V. and Hd.T. co-last authorsIntroduction:We have shown in clinical trials and preclinical studies on Myeloproliferative Neoplasms (MPN) that interferon alpha (IFN) is able to specifically target JAK2V617F cells. However, achievement of complete molecular responses is rare and often requires long-term exposures to IFN, increasing the risk of toxicity. The mechanism implicated in the specific targeting of JAK2V617F cells remains elusive and its elucidation is crucial to improve efficiency of IFN therapy. PML is an IFN transcriptional target that drives the assembly of PML-nuclear bodies (NB), activating P53 or RB checkpoints and ultimately leading to cell cycle arrest or senescence. We explored the hypothesis that PML may contribute to IFN efficacy. This would then predict enhancement of IFN activity by arsenic (AS), a potent inducer of NB formation.Methods:To analyze IFN and/or AS effect on JAK2V617F-driven MPN, we developed chimeric animals transplanted with a mixture of bone marrow (BM) from wild type (WT) mice and conditional JAK2V617F KI/vav-Cre/Ubi-GFP mice monitoring allele burden by GFP expression. Animals, suffering from a polycythemia vera (PV)-like disease, were treated by IFN, AS or IFN/AS combination for 8 to 14 weeks. Blood was analyzed every 2 weeks. After treatment, animals were either (1) sacrificed to analyze BM, spleen and disease-initiating cells in secondary recipients or (2) kept alive to study disease recurrence. We also developed an UT-7 human megakaryoblastic cell line isogenic model by transducing JAK2WT or JAK2V617F. MPN patient samples were tested in clonogenic assays. Finally, the role of PML was assessed in the JAK2V617F-positive HEL cell line transduced by different shRNA for PML knock-down.Results:In vivo experiments demonstrated that IFN vs non-treated (NT) reduced leukocytosis (p<0.001), the platelet number (p<0.001), erythrocytosis (p=0.005) and the JAK2V617F allele burden in granulocytes (p<0.01), platelets (p=0.03) and RBC (p=0.03). More importantly, addition of AS improved the efficacy of IFN for reducing leukocytosis (p<0.03) and erythrocytosis (p=0.02) at 4-6 weeks, but not the platelet number. The effect of the IFN/AS combination was particularly striking in improving the reduction of allele burden compared to IFN alone in granulocytes (p=0.005), platelets (p=0.001) and RBC (p=0.001) (see figure). Improvement of IFN efficacy by addition of AS was also observed on splenomegaly and SLAM progenitor allele burden. The IFN/As combination efficiently decreased relapse rates (48%, p=0.007 vs NT) compared to IFN alone (73%, p=0.2 vs NT), as assessed on blood cells, allele burden and splenomegaly several weeks after treatment discontinuation. Moreover, none of the secondary mice transplanted with BM from IFN/AS-treated mice developed the disease, while 68% of the mice transplanted with BM from IFN alone-treated mice did. These data demonstrate the dramatic effect of the IFN/AS combination on disease initiating cells.We then look for underlying molecular mechanism. The number and size of PML-NB were increased in JAK2V617F vs. WT UT-7 cells at baseline (4.0 vs 6.6 dots/cell, p= 0.0003). As expected, IFN/AS significantly increased PML-NB in both cell types, but this increase was more important in JAK2V617F cells. Cell cycle arrest induced by IFN/AS was more prominent in UT-7 JAK2V617F cells compared to isogenic control, showing the specificity of the combination for mutant cells. IFN/AS had synergistic effects on JAK2V617F cells in terms of inhibition of proliferation and induction of senescence in contrast to single therapies. Critically, PML knock-down in the JAK2V617F-positive HEL cell line reduced the anti-proliferative effect and pro-senescent activity of the IFN/AS combination showing the key role of PML in these processes.Finally, comparison of JAK2V617F-positive CD34+ primary cells from PV patients to normal CD34+ cells demonstrated that IFN/AS specifically decreased proliferation and enhanced senescence as well as NB formation in JAK2V617F cells. Moreover, IFN/AS had a drastic effect on the clonogenic potential of BFU-E progenitors derived from 10 PV patients with a specific targeting of JAK2V617F colonies, which were reduced from 67% to 32%.Conclusion:Altogether, our results strongly suggest involvement of PML in IFN therapy of MPN and the potential utility of AS to greatly improve its efficacy. DisclosuresKiladjian:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies.

ABSTRACTXenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted in vivo visualization of migratory events. Here, we have developed a xenotransplantation technique that transiently generates hundreds of hematopoietic tissue chimeric embryos by transplanting murine bone marrow cells into zebrafish blastulae. In contrast to previous methods, this procedure allows mammalian cell integration into the fish developmental hematopoietic program, which results in chimeric animals containing distinct phenotypes of murine blood cells in both circulation and the hematopoietic niche. Murine cells in chimeric animals express antigens related to (i) hematopoietic stem and progenitor cells, (ii) active cell proliferation and (iii) myeloid cell lineages. We verified the utility of this method by monitoring zebrafish chimeras during development using in vivo non-invasive imaging to show novel murine cell behaviors, such as homing to primitive and definitive hematopoietic tissues, dynamic hematopoietic cell and hematopoietic niche interactions, and response to bacterial infection. Overall, transplantation into the zebrafish blastula provides a useful method that simplifies the generation of numerous chimeric animals and expands the range of murine cell behaviors that can be studied in zebrafish chimeras. In addition, integration of murine cells into the host hematopoietic system during development suggests highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals.This article has an associated First Person interview with the first author of the paper.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

From the Editor’s desk....: November 2018

From the Editor’s desk....: November 2018

Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease that is expressed constitutively by many haematopoietic and non-haematopoietic tissues. It exists as a membrane-associated protein, as well as in an active, soluble form (herein called sDPP4), present at high concentrations in bodily fluids. Despite the proposed use of sDPP4 as a biomarker for multiple diseases, its cellular sources are not well defined. Here, we report that individuals with congenital lymphocyte immunodeficiency had markedly lower serum concentrations of sDPP4, which were restored upon successful treatment and restoration of lymphocyte haematopoiesis. Using irradiated lymphopenic mice and wild-type to Dpp4-/- reciprocal bone marrow chimeric animals, we found that haematopoietic cells were a major source of circulating sDPP4. Furthermore, activation of human and mouse T lymphocytes resulted in increased sDPP4, providing a mechanistic link between immune system activation and sDPP4 concentration. Finally, we observed that acute viral infection induced a transient increase in sDPP4, which correlated with the expansion of antigen-specific CD8+ T cell responses. Our study demonstrates that sDPP4 concentrations are determined by the frequency and activation state of lymphocyte populations. Insights from these studies will support the use of sDPP4 concentration as a biomarker for inflammatory and infectious diseases.

SMA CLINICAL DATA, OUTCOME MEASURES AND REGISTRIES: P.86Cognitive assessment in spinal muscular atrophy type 1-2 using eye tracking system

Spinal muscular atrophy (SMA) is a devastating inherited disorder caused by ubiquitous deficiency in the SMN protein. SMA main feature is the progressive loss of motor activity caused by motor neurons degeneration leading to muscle atrophy. Recent evidence suggests that SMA is a systemic disease. In the brain SMN is expressed in many cell types in addition to motoneurons. Brain morphology changes have been described, and circadian dysregulation, abnormal fatty acid metabolism have been observed. Symptoms of signs of a systemic disease may likely become increasingly apparent in treated children treated, where neuromuscular symptoms are alleviated and life extended. Literature on cognitive aspects of SMA type 1-2 is poor and divergent. Indeed, physical impairments make usual IQ tests often unusable in many SMA children. Therefore, we developed an eye tracking system (Tobii®) for the assessment of cognitive functions in SMA patients belonging to the severe end of the spectrum. The set of tests contains the following subtests: Matrix subtest of Weschler non-verbal scale of ability for the study of fluid reasoning and perceptual reasoning. Recognition subtest of Weschler non-verbal scale of ability for the study of visuo-spatial memory. Peabody picture vocabulary test for the study of receptive vocabulary (French). The chimeric animal stroop test to study inhibition. Face recognition subtest of NEPSY-II to study face encoding, discrimination and recognition. Picture complement test to study the spatial reasoning. Matching pair test. Fourty children from 2 to 12 years old were enrolled: 10 SMA type 1, 10 SMA type 2, and 20 age and sex matched control patients. The methodology and first results will be presented. At the era of new treatments, in particular for SMA type 1, many questions are opened and could be partially answered by this approach: What is the natural history of cognition in SMA type 1 (and 2)? What is the impact of new treatment? How to organize the environment of SMA type 1 children in order to achieve optimal cognitive development (language, cognition, participation)? The developed protocol aims at validating eye tracking assessment tool for cognitive functions in SMA type 1 and 2. This study will help to adapt the overall management of patients and to evaluate the impact on cognition of future SMA treatments.

Chimeric liver transplantation reveals interspecific graft remodelling

A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested. We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45 ± 3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic). Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients). Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed. Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.

Ethical rejections of xenotransplantation? The potential and challenges of using human-pig chimeras to create organs for transplantation.

EMBO Reports (2018) 19: e46337[OpenUrl][1][FREE Full Text][2] Gene editing and stem cell biotechnologies are being applied to create chimeric animals with human organs, which could alleviate the current shortage of human organs for transplantation. While the medical benefits of xenotransplantation appear to be significant, we discuss specific ethical, medical, cultural, and financial challenges and potential solutions that need to be addressed before clinical trials using organs from human‐animal chimeras should begin. > The use of animal organs to replace defective organs in human patients dates back at least to 1838 when the first pig‐to‐human corneal transplant was performed. Organ transplantation has a long history. The use of animal organs to replace defective organs in human patients dates back at least to 1838 when the first pig‐to‐human corneal transplant was performed. The first human skin transplant was done in 1869, the first successful kidney transplant between fraternal twins took place in 1954, and the first heart transplant was performed in 1967. Aortic valve replacement with a porcine heart valve was first reported 2 years later in 1969 and continues to be used to this day. Notwithstanding, of the clinical successes, organ transplantation has been held back by a huge shortage of people willing to donate their organs. Currently, more than 115,000 people are organ transplant candidates in the USA; 75,000 of these are on the national organ waiting list (https://optn.transplant.hrsa.gov). About 25% of patients on the list die before an organ becomes available. Xenotransplantation—using organs from animals for transplantation—has great potential to alleviate this organ shortage. However, tissue rejection and infections have greatly limited the effectiveness of cross‐species transplantations. Recent advances in biotechnology, including gene editing and stem cell science, may provide the key elements needed to create animals that grow human organs for transplantation. Research is currently focusing on growing human hearts, … [1]: {openurl}?query=rft.jtitle%253DEMBO%2BReports%26rft_id%253Dinfo%253Adoi%252F10.15252%252Fembr.201846337%26rft_id%253Dinfo%253Apmid%252F29991538%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/ijlink?linkType=FULL&journalCode=embor&resid=19/8/e46337&atom=%2Fembor%2F19%2F8%2Fe46337.atom

The Ethics of Chimera Creation in Stem Cell Research

To summarize and clarify the main ethical issues surrounding the generation and use of human-animal chimeras in stem cell research. US federal funding remains restricted for certain forms of human-animal chimera research despite the widespread promulgation and acceptance of national and international stem cell-specific research guidelines. Stem cell-based human-animal chimera research can promote advances in basic human developmental biology, disease modeling, and new drug discoveries. National and international guidelines for this form of stem cell research have been developed over the past several years but ethical concerns persist around human dignity and the possible moral humanization of chimeric animals. These concerns can be defused and ought to be replaced with a more tractable focus on animal welfare.

Молекулярные подходы безопасной и контролируемой Т-клеточной терапии

Chimeric antigen receptor-modified T-cell therapy (CAR-T therapy) is one of the fastest developing areas of immuno-oncology. Over the past decade, it has revolutionized the cell therapy modality and expedited its pace of development, from optimization of the structure of chimeric antigen receptors and animal model experiments to successful clinical application. The initial designs of the CAR configuration focused on increasing T-cell activation, cytotoxicity, and persistence. However, the first attempts to treat patients with CAR T cells have demonstrated the need for increased safety and controlled activation of genetically modified T cells. Herein, we summarize the different molecular approaches to engineering chimeric antigen receptors for reducing the potential clinical risks of T-cell therapy.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection.

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs.Highlights: (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver. (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle. (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo.Research in Context To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

The role of transcription factor T-bet in B cell mediated responses to Plasmodium infection

Abstract Malaria is a global health concern which affects over 200 million individuals worldwide. Although the immune system rapidly responds to Plasmodium infection with specific antibodies, natural antibodies fail to establish long term protection, often leading to repetitive infections and chronicity. Recent studies showed that in a chronic malaria setting both B cells and T cells induce expression of T-bet transcription factor. It is not completely understood whether this is a part of the specific host defense or a result of disease pathogenesis. Here using transgenic mouse models, we show that knockout of T-bet or IFN-g similarly reduces survival of mice compared to WT upon infection with Plasmodium chabaudi, therefore suggesting a protective role for T-bet. To determine the specific role T-bet plays in B cell responses to Plasmodium, we first identified that T-bet expression in mouse B cells are triggered by dual stimulation of IFN-g and B cell receptor. Similarly using Tbet-zsgreen transgenic mice we showed that conditions specific to Plasmodium chabaudi infection drive T-bet expression in multiple B cell subsets in a time dependent fashion. However, no such induction is shown when mice are immunized with T-dependent protein antigens adjuvanted with Alum. On the other hand, bone marrow chimeric animals reconstituted with equal amounts of congenically labeled WT and T-bet KO bone marrow cells showed that for most B cell subsets the lack of T-bet does not change the ratio of WT and KO cells upon Plasmodium infection indicating that T-bet expression may not be equally critical for all B cell responses to infection. These findings suggest a complex role of T-bet in the development of humoral responses.