liver-cold-storage-transplantation-cold-adaptive-daurian-ground

Liver Cold Storage and Transplantation in the Cold-Adaptive Daurian Ground Squirrels

In liver transplantation, the functional recovery of donor grafts following extended cold storage is a major challenge. The same obstacle applies to ...

post-liver-transplantation-management-specimen-collection-daurian

Post-Liver Transplantation Management and Specimen Collection in Daurian Ground Squirrels for Single-Cell RNA Sequencing

Graft functional recovery and graft-host immunocompatibility represent major prognostic concerns following liver transplantation. Critically, conventional ...

Sun Yat-sen University

Organ Transplantation Research Center of Guangdong Province

The Third Affiliated Hospital of Sun Yat-sen University

Guangdong Province Engineering Laboratory for Transplantation Medicine

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1β) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.

NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration.

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities.

Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Comparative physiologists are often interested in adaptive physiological phenomena found in unconventional model organisms; however, research on these species is frequently constrained by the limited availability of investigative tools. Here, we propose that induced pluripotent stem cells (iPSCs) from unconventional model organisms may retain certain species-specific features that can consequently be investigated in depth ; we use hibernating mammals as an example. Many species (including ground squirrels, bats and bears) can enter a prolonged state of physiological dormancy known as hibernation to survive unfavorable seasonal conditions. Our understanding of the mechanisms underpinning the rapid transition and adaptation to a hypothermic, metabolically suppressed winter torpor state remains limited partially because of the lack of an easily accessible model. To address the fascinating unanswered questions underlying hibernation biology, we have developed a powerful model system: iPSCs from a hibernating species, the thirteen-lined ground squirrel (). These stem cells can potentially be differentiated into any cell type, and can be used for the analysis of cell-autonomous mechanisms that facilitate adaptation to hibernation and for comparisons with non-hibernators. Furthermore, we can manipulate candidate molecular and cellular pathways underlying relevant physiological phenomena by pharmacological or RNAi-based methods, and CRISPR/Cas9 gene editing. Moreover, iPSC strategies can be applied to other species (e.g. seals, naked mole rats, humming birds) for  studies on adaptation to extreme physiological conditions. In this Commentary, we discuss factors to consider when attempting to generate iPSCs from unconventional model organisms, based on our experience with the thirteen-lined ground squirrel.

Induced pluripotent stem cells as a tool for comparative physiology: lessons from the thirteen-lined ground squirrel.

Therapeutic hypothermia (TH) is potentially an important therapy for central nervous system (CNS) trauma. However, its clinical application remains controversial, hampered by two major factors: (1) Many of the CNS injury sites, such as the optic nerve (ON), are deeply buried, preventing access for local TH. The alternative is to apply TH systemically, which significantly limits the applicable temperature range. (2) Even with possible access for 'local refrigeration', cold-induced cellular damage offsets the benefit of TH. Here we present a clinically translatable model of traumatic optic neuropathy (TON) by applying clinical trans-nasal endoscopic surgery to goats and non-human primates. This model faithfully recapitulates clinical features of TON such as the injury site (pre-chiasmatic ON), the spatiotemporal pattern of neural degeneration, and the accessibility of local treatments with large operating space. We also developed a computer program to simplify the endoscopic procedure and expand this model to other large animal species. Moreover, applying a cold-protective treatment, inspired by our previous hibernation research, enables us to deliver deep hypothermia (4 °C) locally to mitigate inflammation and metabolic stress (indicated by the transcriptomic changes after injury) without cold-induced cellular damage, and confers prominent neuroprotection both structurally and functionally. Intriguingly, neither treatment alone was effective, demonstrating that in situ deep hypothermia combined with cold protection constitutes a breakthrough for TH as a therapy for TON and other CNS traumas.

Cold protection allows local cryotherapy in a clinical-relevant model of traumatic optic neuropathy.

 Hibernating thirteen-lined ground squirrels (GS; ) are naturally adapted to prolonged periods of ultraprofound hypothermia (body temperature < 5 ºC) during torpor, and drastic oscillations of body temperature and ischemia/reperfusion-like stress during their short euthermic interbout arousals. Thus, their superior adaptability may hold tremendous promise for the advancement of donor organ cold preservation and subsequent organ transplantation. However, bridging hibernation research and translational medicine has been impeded by a dearth of  research tools, till the recent establishment of the GS induced pluripotent stem cells (iPSCs). In this study, we reported the generation of functional hepatocyte-like cells (HLCs) from GS iPSCs. As temperature and oxygen supply affect cellular metabolism, we hypothesized that the GS HLCs can metabolically counter drastic temperature and oxygen supply changes. Differentially regulated metabolites can be evaluated and included into the preservation solution to mitigate temperature and ischemia/reperfusion-associated damage to donor livers.  A protocol has been developed to produce GS iPSCs-derived HLCs. Comparative metabolomic analysis on GS HLCs and human donor liver samples revealed changes in metabolites caused by cold storage and rewarming. Human embryonic stem cell (ESC)-derived HLCs and  cold preservation and reperfusion of isolated rat livers were used to assess candidate metabolites that may have protective effects against preservation-related injuries.  GS iPSCs were efficiently differentiated into expandable, cryopreservation-compatible and functional HLCs. Metabolomic analysis unveiled distinct changes of mitochondrial metabolites between GS and human cells following cold storage and rewarming. GS and human HLC-based experiments indicated that the metabolism of 5-aminolevulinate (5-ALA) is key to restricting free radical production during rewarming. Survival of human HLCs was significantly increased following cold exposure and rewarming, as supplemented 5-ALA enhanced Complex III activity and improved mitochondrial respiration. Further, 5-ALA mitigated damage in rat livers following 48-h cold preservation and  reperfusion. Metabolomic and transcriptomic analyses revealed that supplemented 5-ALA promoted both anabolic and catabolic activities while alleviating cell death, inflammation, hypoxia and other stress responses in isolated perfused rat livers.  In the liver, rewarming from ultraprofound hypothermia imposes complex metabolic challenges and stresses on the mitochondria. Metabolites such as 5-ALA can help alleviate mitochondrial stress. Supplementing 5-ALA to the liver preservation solution can substantially improve the functional recovery of rat livers following prolonged cold preservation, rewarming and reperfusion.

5-Aminolevulinate improves metabolic recovery and cell survival of the liver following cold preservation.

Inadequate remnant volume and regenerative ability of the liver pose life-threatening risks to patients after partial liver transplantation (PLT) or partial hepatectomy (PHx), while few clinical treatments focus on safely accelerating regeneration. Recently, we discovered that supplementing 5-aminolevulinate (5-ALA) improves liver cold adaptation and functional recovery, leading us to uncover a correlation between 5-ALA metabolic activities and post-PLT recovery. In a mouse 2/3 PHx model, 5-ALA supplements enhanced liver regeneration, promoting infiltration and polarization of anti-inflammatory macrophages via P53 signaling. Intriguingly, chemokine receptor CX3CR1 functions to counterbalance these effects. Genetic ablation or pharmacological inhibition of CX3CR1 (AZD8797; phase II trial candidate) augmented the macrophagic production of insulin-like growth factor 1 (IGF-1) and subsequent hepatocyte growth factor (HGF) production by hepatic stellate cells. Thus, short-term treatments with both 5-ALA and AZD8797 demonstrated pro-regeneration outcomes superior to 5-ALA-only treatments in mice after PHx. Overall, our findings may inspire safe and effective strategies to better treat PLT and PHx patients.

Synergy of 5-aminolevulinate supplement and CX3CR1 suppression promotes liver regeneration via elevated IGF-1 signaling.

 Liver resection and transplantation surgeries are accompanied by hepatic ischemia-reperfusion (HIR) injury that hampers the subsequent liver recovery. Given that the liver is the main organ for metabolism and detoxification, ischemia-reperfusion in essence bestows metabolic stress upon the liver and disrupts local metabolic and immune homeostasis. Most of the recent and current research works concerning HIR have been focusing on addressing HIR-induced hepatic injury and inflammation, instead of dealing with the metabolic reprogramming and restoration of redox homeostasis. As our previous work uncovers the importance of 5-aminolevulinate (5-ALA) synthesis during stress adaptation, here we evaluate the effects of supplementing 5-ALA to mitigate HIR injury.  5-ALA was supplemented into the mice or cultured cells during the ischemic or oxygen-glucose deprivation (OGD) phase. Following reperfusion or reoxygenation, cellular metabolism and energy homeostasis, mitochondrial production of reactive oxygen species (ROS) and transcriptomic changes were evaluated in HIR mouse models or cultured hepatocytes and macrophages. Liver injury, hepatocytic functional tests, and macrophagic M1/M2 polarization were assessed.  Dynamic changes in the expression of key enzymes in 5-ALA metabolism were first confirmed in donor and mouse liver samples following HIR. Supplemented 5-ALA modulated mouse hepatic lipid metabolism and reduced ATP production in macrophages following HIR, resulting in elevation of anti-inflammatory M2 polarization. Mechanistically, 5-ALA down-regulates macrophagic chemokine receptor CX3CR1 via the repression of RelA following OGD and reoxygenation (OGD/R).  KO mice demonstrated milder liver injuries and more macrophage M2 polarization after HIR. M2 macrophage-secreted chitinase-like protein 3 (CHIL3; CHI3L1 in human) is an important HIR-induced effector downstream of CX3CR1 deficiency. Addition of CHIL3/CHI3L1 alone improved hepatocellular metabolism and reduced OGD/R-inflicted injuries in cultured mouse and human hepatocytes. Combined treatment with 5-ALA and CHIL3 during the ischemic phase facilitated lipid metabolism and ATP production in the mouse liver following HIR.  Our results reveal that supplementing 5-ALA promotes macrophagic M2 polarization via downregulation of RelA and CX3CR1 in mice following HIR, while M2 macrophage-produced CHIL3/CHI3L1 also manifests beneficial effects to the recovery of hepatic metabolism. 5-ALA and CHIL3/CHI3L1 together mitigate HIR-induced mitochondrial dysfunction and hepatocellular injuries, which may be developed into safe and effective clinical treatments to attenuate HIR injuries.

5-aminolevulinate and CHIL3/CHI3L1 treatment amid ischemia aids liver metabolism and reduces ischemia-reperfusion injury.

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage.

Cold and ischemia/reperfusion (IR)-associated injuries are seemingly inevitable during liver transplantation and hepatectomy. Because Syrian hamsters demonstrate intrinsic tolerance to transplantation-like stimuli, cross-species comparative metabolomic analyses were conducted with hamster, rat, and donor liver samples to seek hepatic cold and IR-adaptive mechanisms. Lower hepatic phosphocholine contents were found in recipients with early graft-dysfunction and with virus-caused cirrhosis or high model for end-stage liver disease scores (≥30). Choline/phosphocholine deficiency in cultured human THLE-2 hepatocytes and animal models weakened hepatocellular cold tolerance and recovery of glutathione and ATP production, which was rescued by phosphocholine supplements. Among the biological processes impacted by choline/phosphocholine deficiency, 3 lipid-related metabolic processes were downregulated, whereas phosphocholine elevated the expression of genes in methylation processes. Consistently, in THLE-2, phosphocholine enhanced the overall RNA mA methylation, among which the transcript stability of fatty acid desaturase 6 (FADS6) was improved. FADS6 functioned as a key phosphocholine effector in the production of polyunsaturated fatty acids, which may facilitate the hepatocellular recovery of energy and redox homeostasis. Thus, our study reveals the choline-phosphocholine metabolism and its downstream FADS6 functions in hepatic adaptation to cold and IR, which may inspire new strategies to monitor donor liver quality and improve recipient recovery from the liver transplantation process.

Cross-species metabolomic profiling reveals phosphocholine-mediated liver protection from cold and ischemia/reperfusion.

Skeletal muscle plays a significant role in both local and systemic energy metabolism. The current investigation aims to explore the role of the Bambi gene in skeletal muscle, focusing on its implications for muscle hypertrophy and systemic metabolism. We hypothesize that skeletal muscle-specific deletion of Bambi induces muscle hypertrophy, improves metabolic performance, and activates thermogenic adipocytes via the reprogramming of progenitor of iWAT, offering potential therapeutic strategies for metabolic syndromes. Leveraging the Chromatin immunoprecipitation (ChIP)-seq and bioinformatics analysis, Bambi gene is shown to be a direct target of HIF2α, which is further confirmed by ChIP-qPCR and promoter luciferase assay. Skeletal muscle-specific Bambi deletion led to significant muscle hypertrophy and improved metabolic parameters, even under high-fat diet conditions. This deletion induced metabolic reprogramming of stromal vascular fractions (SVFs) into thermogenic adipocytes, contributing to systemic metabolic improvements, potentially through the secretory factor. Notably, mice with skeletal muscle-specific Bambi deletion demonstrate resistance to high-fat diet-induced metabolic disorders, highlighting a potential therapeutic pathway for metabolic syndrome management. Thus, skeletal muscle-specific deletion of Bambi triggers muscle growth, enhances metabolic performance, and activates thermogenic adipocytes. These findings suggest Bambi as a novel therapeutic target for metabolic syndromes, providing new insights into the interaction between muscle hypertrophy and systemic metabolic improvement. The study underscores the potential of manipulating muscle physiology to regulate whole-body metabolism, offering a novel perspective on treating metabolic disorders.

Skeletal muscle-specific Bambi deletion induces hypertrophy and oxidative switching coupling with adipocyte thermogenesis against metabolic disorders.

The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.

ArticleTotal : 2	Year
Publication title	2025
Publication title	2025

Article	Year
NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration. International journal of molecular sciences\| PubMed ID: 26760997	2016
Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas. Journal of cellular physiology\| PubMed ID: 28542832	2018
iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications. Cell\| PubMed ID: 29576452	2018
Induced pluripotent stem cells as a tool for comparative physiology: lessons from the thirteen-lined ground squirrel. The Journal of experimental biology\| PubMed ID: 31585999	2019
Cold protection allows local cryotherapy in a clinical-relevant model of traumatic optic neuropathy. eLife\| PubMed ID: 35352678	2022
5-Aminolevulinate improves metabolic recovery and cell survival of the liver following cold preservation. Theranostics\| PubMed ID: 35401816	2022
Synergy of 5-aminolevulinate supplement and CX3CR1 suppression promotes liver regeneration via elevated IGF-1 signaling. Cell reports\| PubMed ID: 37578861	2023
5-aminolevulinate and CHIL3/CHI3L1 treatment amid ischemia aids liver metabolism and reduces ischemia-reperfusion injury. Theranostics\| PubMed ID: 37771779	2023
A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance. Acta pharmaceutica Sinica. B\| PubMed ID: 38261805	2024
Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage. Nature communications\| PubMed ID: 38570500	2024
Cross-species metabolomic profiling reveals phosphocholine-mediated liver protection from cold and ischemia/reperfusion. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 38878865	2024
Skeletal muscle-specific Bambi deletion induces hypertrophy and oxidative switching coupling with adipocyte thermogenesis against metabolic disorders. Science China. Life sciences\| PubMed ID: 39821828	2025
AIDA directly connects sympathetic innervation to adaptive thermogenesis by UCP1. Nature cell biology\| PubMed ID: 33664495	2021

Jingxing Ou

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