Animal Models Of Transplantation Research Articles

Surgical Protocol for Partial Heart Transplantation in Growing Piglets.

Partial heart transplantation is a new approach to deliver growing heart valve implants. Partial heart transplants differ from heart transplants because only the part of the heart containing the necessary heart valve is transplanted. This allows partial heart transplants to grow, similar to the valves in heart transplants. However, the transplant biology of partial heart transplantation remains unexplored. This is a critical barrier to progress of the field. Without knowledge about the specific transplant biology of partial heart transplantation, children with partial heart transplants are empirically treated like children with heart transplants because the valves in heart transplants are known to grow. In order to progress the field, an animal model for partial heart transplantation is necessary. Here, we contribute our surgical protocol for partial heart transplantation in growing piglets. All aspects of partial heart transplantation, including the donor procedure, the recipient procedure, and recipient perioperative care are described in detail. There are important nuances in the conduct of virtually all aspects of open heart surgery that differs in piglets from humans. Our surgical protocol, which is based on our experience with 34 piglets, will allow other investigators to leverage our experience to seek fundamental knowledge about the nature of partial heart transplants. This is significant because the partial heart transplant model in piglets is complex and very resource intensive.

Animal models for partial heart transplantation.

Partial heart transplantation delivers growing heart valve implants by transplanting the part of the heart containing the necessary heart valve only. In contrast to heart transplantation, partial heart transplantation spares the native ventricles. This has important implications for partial heart transplant biology, including the allowable ischemia time, optimal graft preservation, primary graft dysfunction, immune rejection, and optimal immunosuppression. Exploration of partial heart transplant biology will depend on suitable animal models. Here we review our experience with partial heart transplantation in rodents, piglets, and non-human primates. This review is based on our experience with partial heart transplantation using over 100 rodents, over 50 piglets and one baboon. Suitable animal models for partial heart transplantation include rodent heterotopic partial heart transplantation, piglet orthotopic partial heart transplantation, and non-human primate partial heart xenotransplantation. Rodent models are relatively cheap and offer extensive availability of research tools. However, rodent open-heart surgery is technically not feasible. This limits rodents to heterotopic partial heart transplant models. Piglets are comparable in size to children. This allows for open-heart surgery using clinical grade equipment for orthoptic partial heart transplantation. Piglets also grow rapidly, which is useful for studying partial heart transplant growth. Finally, nonhuman primates are immunologically most closely related to humans. Therefore, nonhuman primates are most suitable for studying partial heart transplant immunobiology and xenotransplantation. Animal research is a privilege that is contingent on utilitarian ethics and the 3R principles of replacement, reduction and refinement. This privilege allows the research community to seek fundamental knowledge about partial heart transplantation, and to apply this knowledge to enhance the health of children who require partial heart transplants.

Development of a Large Animal Model of Ischemia-free Liver Transplantation in Pigs.

In organ transplantation, ischemia, and reperfusion injury (IRI) is considered as an inevitable event and the major contributor to graft failure. Ischemia-free liver transplantation (IFLT) is a novel transplant procedure that can prevent IRI and provide better transplant outcomes. However, a large animal model of IFLT has not been reported. Therefore, we develop a new, reproducible, and stable model of IFLT in pigs for investigating mechanisms of IFLT in IRI. Ten pigs were subjected to IFLT or conventional liver transplantation (CLT). Donor livers in IFLT underwent 6-h continuous normothermic machine perfusion (NMP) throughout graft procurement, preservation, and implantation, whereas livers in CLT were subjected to 6-h cold storage before implantation. The early reperfusion injury was compared between the 2 groups. Continuous bile production, low lactate, and liver enzyme levels were observed during NMP in IFLT. All animals survived after liver transplantation. The posttransplant graft function was improved with IFLT when compared with CLT. Minimal histologic changes, fewer apoptotic hepatocytes, less sinusoidal endothelial cell injury, and proinflammatory cytokine (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) release after graft revascularization were documented in the IFLT group versus the CLT group. We report that the concept of IFLT is achievable in pigs. This innovation provides a potential strategy to investigate the mechanisms of IRI and provide better transplant outcomes for clinical practice.

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies.

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat liver transplantation (LTx) protocols typically use an orthotopic model where the recipients' native liver is removed and replaced with a donor liver. This technically demanding surgical procedure requires advanced micro-surgical skills and is further complicated by lengthy anhepatic and lower body ischemia times. This prompted the development of a less complicated heterotopic method that can be performed faster with no anhepatic or lower body ischemia time, reducing post-surgery stress for the recipient animal. This heterotopic LTx protocol includes two main steps: excising the liver from the donor rat and transplanting the whole liver into the recipient rat. During the excision of the donor liver, the surgeon ligates the supra-hepatic vena cava (SHVC) and hepatic artery (HA). On the recipient side, the surgeon removes the left kidney and positions the donor liver with the portal vein (PV), infra-hepatic vena cava (IHVC), and bile duct facing the renal vessels. Further, the surgeon anastomoses the recipient's renal vein end to end with the IHVC of the liver and arterializes the PV with the renal artery using a stent. A hepaticoureterostomy is utilized for biliary drainage by anastomosing the bile duct to the recipient's ureter, permitting the discharge of bile via the bladder. The average duration of the transplantation was 130 min, cold ischemia duration was around 35 min, and warm ischemia duration was less than 25 min. Hematoxylin and eosin histology of the auxiliary liver from syngeneic transplants showed normal hepatocyte structure with no significant parenchymal alterations 30 days post-transplant. In contrast, 8-day post-transplant allogeneic graft specimens demonstrated extensive lymphocytic infiltration with a Banff Schema rejection activity index score of 9. Therefore, this LTx method facilitates a low morbidity rejection model alternative to orthotopic LTx.

Mitochondria as secretory organelles and therapeutic cargos.

Mitochondria have been primarily considered intracellular organelles that are responsible for generating energy for cell survival. However, accumulating evidence suggests that mitochondria are secreted into the extracellular space under physiological and pathological conditions, and these secreted mitochondria play diverse roles by regulating metabolism, the immune response, or the differentiation/maturation in target cells. Furthermore, increasing amount of research shows the therapeutic effects of local or systemic administration of mitochondria in various disease models. These findings have led to growing interest in exploring mitochondria as potential therapeutic agents. Here, we discuss the emerging roles of mitochondria as extracellularly secreted organelles to shed light on their functions beyond energy production. Additionally, we provide information on therapeutic outcomes of mitochondrial transplantation in animal models of diseases and an update on ongoing clinical trials, underscoring the potential of using mitochondria as a novel therapeutic intervention.

Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models.

Mesenchymal stem cells (MSCs) can alleviate graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). MSCs-derived exosomes (MEXs) can mirror the biological function of their parent cells. Whether MEXs can alleviate GVHD like their parent cells or not is unclear. In this study, we investigate the effects of MEXs on GVHD and graft-versus-leukemia (GVL) effect in vitro and in HSCT animal models. MSCs were produced using bone marrow mononuclear cells (MNCs), and MEXs were separated from the supernatants of MSCs. Electron microscopy, western blot, and nanoparticle tracking analysis (NTA) were used to determine the characteristics of MEXs. The immunomodulatory function of MEXs and their effects on GVHD and GVL were examined in vitro and in vivo. Like other cell-type derived exosomes, our data revealed that MEXs were also disc-shaped vesicles with a diameter of 100-200 nm under electron microscopy and were positive for the exosomal hallmark proteins. MEXs can notably inhibit the expression of costimulatory molecules and functional cytokine secretion of dendritic cells (DCs). Meanwhile, MEXs can exert suppressive effects on T lymphocyte proliferation and activation. Moreover, MEXs can also encourage the polarization of macrophages toward the M2 type. In animal HSCT models, MEXs can promote the differentiation of Treg cells in spleens, decrease the GVHD score, increase the survival rate of mice, and preserve the cytotoxic antileukemia effects of CD8+ T lymphocytes from recipient mice. These findings showed that MEXs exert their effects by inhibiting the immunomodulatory function of DCs, macrophages, and T lymphocytes. In the animal model, MEXs ameliorate the clinical symptoms of GVHD, while maintaining the antitumor effects of CD8+ T lymphocytes. Therefore, it can be inferred that MEXs can separate GVHD from GVL in HSCT. Our study suggests that MEXs have broad clinical application potential in the prevention and treatment of GVHD in HSCT in the near future.

Optimizing Immature Testicular Tissue and Cell Transplantation Results: Comparing Transplantation Sites and Scaffolds.

For patients who had testicular tissue cryopreserved before receiving gonadotoxic therapies, transplantation of testicular tissues and cells has been recommended as a potential therapeutic option. There are no studies that indicate the generation of sperm after human immature testicular tissue (ITT) or spermatogonial stem cells (SSCs) transplantation. The use of releasing scaffolds and localized drug delivery systems as well as the optimizing transplantation site can play an effective role in increasing the efficiency and improving the quality of testicular tissue and cell transplantation in animal models. Current research is focused on optimizing ITT and cell transplantation, the use of releasing scaffolds, and the selection of the right transplantation site that might restore sperm production or male infertility treatment. By searching the PubMed and Google Scholar databases, original and review papers were collected. Search terms were relevant for SSCs and tissue transplantation. In this review, we'll focus on the potential advantages of using scaffolds and choosing the right transplantation site to improve transplantation outcomes.

Calcineurin Inhibition in Deceased Organ Donors: A Systematic Review and Meta-analysis of Preclinical Studies.

Preconditioning deceased organ donors with calcineurin inhibitors (CNIs) may reduce ischemia-reperfusion injury to improve transplant outcomes. We searched MEDLINE, EMBASE, Cochrane Library, and conference proceedings for animal models of organ donation and transplantation, comparing donor treatment with CNIs with either placebo or no intervention, and evaluating outcomes for organ transplantation. Reviewers independently screened and selected studies, abstracted data, and assessed the risk of bias and clinical relevance of included studies. Where possible, we pooled results using meta-analysis; otherwise, we summarized findings descriptively. Eighteen studies used various animals and a range of CNI agents and doses and evaluated their effects on a variety of transplant outcomes. The risk of bias and clinical applicability were poorly reported. Pooled analyses suggested benefit of CNI treatment on early graft function in renal transplants (3 studies; serum creatinine: ratio of means [RoM] 0.54; 95% confidence interval [CI], 0.34-0.86) but not for liver transplants (2 studies; serum alanine transaminase: RoM 0.61; 95% CI, 0.30-1.26; and serum aspartate aminotransferase: RoM 0.58; 95% CI, 0.26-1.31). We found no reduction in graft loss at 7 d (2 studies; risk ratio 0.54; 95% CI, 0.08-3.42). CNI treatment was associated with reduced transplant recipient levels of interleukin-6 (4 studies; RoM 0.36; 95% CI, 0.19-0.70), tumor necrosis factor-alpha (5 studies; RoM 0.36; 95% CI, 0.12-1.03), and cellular apoptosis (4 studies; RoM 0.30; 95% CI, 0.19-0.47). Although this compendium of animal experiments suggests that donor preconditioning with CNIs may improve early kidney graft function, the limited ability to reproduce a true clinical environment in animal experiments and to assess for risk of bias in these experiments is a serious weakness that precludes current clinical application.

Swine Partial Liver Transplantation Model for Practicing Living Donor Liver Transplantation Based on a New Liver Segmentation Method.

Living donor liver transplantation (LDLT) is one of the most technically demanding and complicated procedures. However, unlike deceased donor liver transplantation, there is no suitable animal model for practicing LDLT. Herein, we propose a new liver segmentation method and a feasible pig LDLT model for practicing for LDLT in humans. Four Landrace pigs weighing 25, 25, 27, and 28 kg were used as donors and recipients to establish a partial liver transplantation model. Partial liver transplantation was performed using a right liver and a left liver, respectively, based on a new segmentation system compatible with that of humans. We established a new segmentation system for porcine liver transplantation and a partial liver transplantation model. For right liver transplantation, 91 and 142 min were required to operate on the donor and recipient, respectively; for left liver transplantation, 57 and 104 min were required to operate on the donor and recipient, respectively. All pigs that underwent partial liver transplantation remained alive until the operation was completed. It is expected that this new pig model based on the new segmentation system will be suitable as an educational tool for LDLT training and will replace the existing animal models for partial liver transplantation.

Sheep as a Model for Liver Transplantation.

Experimental animal liver transplantation is the initial step, before the application of the procedure on humans. Canine and swinetransplantation were used to perfect the technical aspects of the procedure. Small animals such as rats were mainly utilized to study the metabolic and immunological aspects of liver transplantation. In this paper, we describe our experience with attempting liver transplantation in a sheep animal model. The animal model used for both donor and recipientwas outbred male weanling sheep of Naimi strain (Ovis aries, Awassi). They weigh between 25 and 35 kg. They were put under general anesthesia. Harvested livers were kept in cold storage. Recipients underwent hepatectomy, after construction of an active portal systemic bypass using a Medtronic pump. The implantation was done with caval replacement and direct portal anastomosis. The hepatic artery with its attachments to the aortal was anastomosed directly to the recipient aorta. Twelve pairs (24 sheep) were utilized for donor and recipient surgery. Donor surgery was completed successfully in all 12 cases. Recipient surgery was not completed in three cases, whenanimals were lost in the implantation phase, before reperfusion mainly due to uncontrolled bleeding, resulting in hemodynamic instability.We also lost five recipients immediately after reperfusion, mainly due to post-perfusion bleeding and hemodynamic instability. Four recipients stayed alive after the implantation. We demonstrated the feasibility of using sheep as an animal model for liver transplantation. We described the similarities of sheep liver to that ofhumans, as well as the technical difficulties. This model is suitable in situations where other well-established models are not available for cultural or religious reasons. Further refinement in the technical aspects will be needed, as well as investigation of the biochemical outcome and long-term survival.

CRISPR/Cas Systems for Therapeutic Purposes: Applications in Disease Prevention, Mechanism Study, and Treatment

CRISPR/Cas systems were novel multifunctional genome editing techniques, and the modification of Cas proteins endowed them with more efficient and precise properties. Herein, we systematically outlined the principles and applications of CRISPR/Cas systems in disease prevention, mechanism research, and gene treatment of diseases. Specifically, we detailed the systems widely utilized, containing the Cas9, Cas12a, and Cas13a systems, and summarized the current status of the medical applications of these systems in terms of vaccine design, trait improvement, animal models of human diseases, organ transplantation, gene therapy, and gene detection. We concluded that CRISPR/Cas systems had great potential to advance medical research and practice but also should be carefully regulated and supervised to ensure their safety and efficacy.

Human brain organoid transplantation: ethical implications of enhancing specific cerebral functions in small-animal models

Brain organoids are self-organizing, three-dimensional tissues derived from pluripotent stem cells that recapitulate many aspects of the cellular diversity and architectural features of the developing brain. Recently, there has been growing interest in using human brain organoid transplantation in animal models as a means of addressing the limitations of in vitro culture, such as the lack of vascularization, and to explore the potential of organoids for neural repair. While there has been substantial debate on the ethical implications of brain organoid research, particularly the potential for organoids to exhibit higher-order brain functions such as consciousness, the impact of human organoid grafts on animal hosts has been less extensively discussed. Enhancement of host animal brain function may not be technically feasible at this time, but it is imperative to carefully consider the moral significance of these potential outcomes. Here, we discuss the ethical implications of enhancing somatosensation, motor processes, memory, and basic socialization in small-animal models. We consider the moral implications of such outcomes and if safeguards are needed to accommodate any increased moral status of animals transplanted with human brain organoids.

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML. MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells. ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells. ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.

Wnt3a Loaded Hydroxyapatite Nanowire@Mesoporous Silica Core-Shell Nanocomposite Promotes the Regeneration of Dentin-Pulp Complex via Angiogenesis, Oxidative Stress Resistance and Odontogenic Induction of Stem Cells.

Pulp exposure often leads to pulp necrosis, root fractures and ultimate tooth loss. The repair of exposure site with pulp capping treatment is of great significance to preserve pulp vitality, but its efficacy is impaired by the low bioactivity of capping materials and cell injuries from the local accumulation of oxidative stress. This study develops a Wnt3a loaded hydroxyapatite nanowire@mesoporous silica (Wnt3a-HANW@MpSi) core-shell nanocomposite for the pulp capping treatments. The ultralong and highly flexible hydroxyapatite nanowires provide the framework for the composites, and the mesoporous silica shell endows the composite with the capacity of efficiently loading/releasing Wnt3a and Si ions. Under the in vitro investigation, Wnt3a-HANW@MpSi not only promotes the oxidative stress resistance of dental pulp stem cells (DPSCs), enhances their migration and odontogenic differentiation, but also exhibits superior property of angiogenesis in vitro. Revealed by the transcriptome analysis, the underlying mechanisms of odontogenic enhancement by Wnt3a-HANW@MpSi are closely related to multiple biological processes and signaling pathways toward pulp/dentin regeneration. Besides, an animal model of subcutaneous transplantation demonstrates the significant reinforcement of the formation of dentin-pulp complex-like tissues and blood vessels by Wnt3a-HANW@MpSi in vivo. These results indicate promising potential of Wnt3a-HANW@MpSi in treatments of dental pulp exposure. This article is protected by copyright. All rights reserved.

Animal Models for Heart Transplantation Focusing on the Pathological Conditions.

Cardiac transplant recipients face many complications due to transplant rejection. Scientists must conduct animal experiments to study disease onset mechanisms and develop countermeasures. Therefore, many animal models have been developed for research topics including immunopathology of graft rejection, immunosuppressive therapies, anastomotic techniques, and graft preservation techniques. Small experimental animals include rodents, rabbits, and guinea pigs. They have a high metabolic rate, high reproductive rate, small size for easy handling, and low cost. Additionally, they have genetically modified strains for pathological mechanisms research; however, there is a lacuna, as these research results rarely translate directly to clinical applications. Large animals, including canines, pigs, and non-human primates, have anatomical structures and physiological states that are similar to those of humans; therefore, they are often used to validate the results obtained from small animal studies and directly speculate on the feasibility of applying these results in clinical practice. Before 2023, PubMed Central® at the United States National Institute of Health's National Library of Medicine was used for literature searches on the animal models for heart transplantation focusing on the pathological conditions. Unpublished reports and abstracts from conferences were excluded from this review article. We discussed the applications of small- and large-animal models in heart transplantation-related studies. This review article aimed to provide researchers with a complete understanding of animal models for heart transplantation by focusing on the pathological conditions created by each model.

MHC Class I Ligands of Rhesus Macaque Killer Cell Ig-like Receptors.

Definition of MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology, and transplantation. To provide a more complete foundation for studying NK cell responses, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Macaca mulatta (Mamu)-A, -B, -E, -F, -I, and -AG alleles. KIR-MHC class I interactions were identified by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. Ligands for 12 KIRs of previously unknown specificity were identified that fell into three general categories: interactions with multiple Mamu-Bw4 molecules, interactions with Mamu-A-related molecules, including allotypes of Mamu-AG and the hybrid Mamu-B*045:03 molecule, or interactions with Mamu-A1*012:01. Whereas most KIRs found to interact with Mamu-Bw4 are inhibitory, most of the KIRs that interact with Mamu-AG are activating. The KIRs that recognize Mamu-A1*012:01 belong to a phylogenetically distinct group of macaque KIRs with a 3-aa deletion in the D0 domain that is also present in human KIR3DL1/S1 and KIR3DL2. This study more than doubles the number of rhesus macaque KIRs with defined MHC class I ligands and identifies interactions with Mamu-AG, -B*045, and -A1*012. These findings support overlapping, but nonredundant, patterns of ligand recognition that reflect extensive functional diversification of these receptors.

Xenogeneic transplantation of mitochondria induces muscle regeneration in an in vivo rat model of dexamethasone-induced atrophy.

Muscle atrophy significantly impairs health and quality of life; however, there is still no cure. Recently, the possibility of regeneration in muscle atrophic cells was suggested through mitochondrial transfer. Therefore, we attempted to prove the efficacy of mitochondrial transplantation in animal models. To this end, we prepared intact mitochondria from umbilical cord-derived mesenchymal stem cells maintaining their membrane potential. To examine the efficacy of mitochondrial transplantation on muscle regeneration, we measured muscle mass, cross-sectional area of muscle fiber, and changes in muscle-specific protein. In addition, changes in the signaling mechanisms related to muscle atrophy were evaluated. As a result, in mitochondrial transplantation, the muscle mass increased by 1.5-fold and the lactate concentration decreased by 2.5-fold at 1 week in dexamethasone-induced atrophic muscles. In addition, a 2.3-fold increase in the expression of desmin protein, a muscle regeneration marker, showed a significant recovery in MT 5µg group. Importantly, the muscle-specific ubiquitin E3-ligases MAFbx and MuRF-1 were significantly decreased through AMPK-mediated Akt-FoxO signaling pathway by mitochondrial transplantation compared with the saline group, reaching a level similar to that in the control. Based on these results, mitochondrial transplantation may have therapeutic applications in the treatment of atrophic muscle disorders.

Pre-Treatment of Transplant Donors with Hydrogen Sulfide to Protect against Warm and Cold Ischemia-Reperfusion Injury in Kidney and Other Transplantable Solid Organs

Ischemia-reperfusion injury (IRI), a pathological condition resulting from prolonged cessation and subsequent restoration of blood flow to a tissue, is an inevitable consequence of solid organ transplantation. Current organ preservation strategies, such as static cold storage (SCS), are aimed at reducing IRI. However, prolonged SCS exacerbates IRI. Recent research has examined pre-treatment approaches to more effectively attenuate IRI. Hydrogen sulfide (H2S), the third established member of a family of gaseous signaling molecules, has been shown to target the pathophysiology of IRI and thus appears to be a viable candidate that can overcome the transplant surgeon's enemy. This review discusses pre-treatment of renal grafts and other transplantable organs with H2S to mitigate transplantation-induced IRI in animal models of transplantation. In addition, ethical principles of pre-treatment and potential applications of H2S pre-treatment in the prevention of other IRI-associated conditions are discussed.

Feasibility of implantable microdosing devices to perform in vivo pharmacotyping in pancreatic cancer.

742 Background: Current biomarkers offer modest predictive capacity for drug sensitivity testing in pancreatic cancer. Patient-specific pharmacotyping offers the potential for a more direct, personalized prediction model. Recently, an implantable microdevice was developed to facilitate in situ multi-agent pharmacotyping and has since entered clinical trials in multiple cancer types, but had not yet been tested in pancreatic cancer. Here we evaluated the use of a microdosing device (microdevice) in a mouse xenotransplant model of pancreatic cancer as proof of principle to inform future translation to the clinic. Methods: A microdevice consists of a cylindrical frame and 18 wells embedded with standard chemotherapy agents (5 FU, gemcitabine, SN 38, paclitaxel, oxaliplatin), a non-toxic polyethylene glycol (PEG) matrix as control, and doxorubicin, the auto-fluorescent properties of which allow us to determine the distance of drug diffusion for each tumor. Devices were implanted into murine xenotransplanted human organoids (hM1A and hM1E Table 1). Microdevices retrieved at 4h, 8h, and 24h post-implantation together with tumors were fixed in 10% formalin. The tissue was embedded in paraffin and sectioned parallel to the device. A distance-dependent drug diffusion rate and concentration profiles with doxorubicin were evaluated. Co-immunohistochemistry (IHC) for cleaved caspase-3 (CC3), gamma H2AX (apoptosis markers), and human mitochondria (human cancer cell marker) was performed in the area of drug diffusion to determine the cytotoxic effects of chemotherapy. Results: 15 animals were enrolled in the experiment with 100% postoperative survival. One device per animal was placed, and all devices were successfully retrieved. Microdevice insertion and exposure to PEG had no effect on tumor integrity. At 250-350µm from the well, we observed intensity from doxorubicin, suggesting drug diffusion. The signal on differential response to drugs was detected as early as 4 hours. Conclusions: Utilization of implantable microdevice to predictcancer drug sensitivity is feasible in organoid transplantation animal models of pancreatic cancer. Future work is needed to expand this model to other cancer drugs and biomarkers to evaluate the cancer drug sensitivity and to test the feasibility of this model to study the effects of chemotherapy on the tumor microenvironment. [Table: see text]

Research Progress of Single-cell Transcriptome Sequencing Technology in Physiological Hematopoiesis--Review

Hematopoiesis starts from the embryo and runs through the entire life of a living body, which is a multi-stage and complex dynamic process that is regulated by multiple pathways, involving a variety of cells and hematopoietic anatomical locations. During the development of the mammalian hematopoietic system, the currently known hematopoietic anatomical locations mainly include yolk sac, aorta-gonad-mesonephros, fetal liver, bone marrow, and thymus. The first three are mainly responsible for hematopoiesis during the embryonic and fetal period, while bone marrow is the main place for postnatal hematopoiesis, and thymus is mainly responsible for the differentiation of T lymphocytes. Integrating flow cytometry, in vitro cell culture and in vivo animal transplantation models, early researchers conducted an in-depth analysis of the differentiation pathways of hematopoietic cells. However, due to technical limitations, it is difficult to track the single-cell hematopoietic activity of hematopoietic organs. Transcriptome sequencing at the single-cell level provides researchers with a unique perspective, making it possible to draw the most detailed cell fate transition maps of the hematopoietic development of living organisms, and providing new ideas for the diagnosis and treatment of hematological tumors. In this article, we reviewed the research progress in the use of large-scale single-cell transcriptome sequencing in the field of physiological hematopoiesis in recent years.