Establishment of a Transplantation Model of PDAC-Derived Liver Metastases.

BACKGROUND: The highly metastatic nature of pancreatic ductal adenocarcinoma (PDAC) and the difficulty to achieve favorable patient outcomes emphasize the need for novel therapeutic solutions. For preclinical evaluations, genetically engineered mouse models are often used to mimic human PDAC but frequently fail to replicate synchronous development and metastatic spread. This study aimed to develop a transplantation model to achieve synchronous and homogenous PDAC growth with controlled metastatic patterns in the liver. METHODS: To generate an orthotopic PDAC model, the DT6606 cell line was injected into the pancreas head of C57BL/6 mice, and their survival was monitored over time. To generate a heterotopic transplantation model, growing doses of three PDAC cell lines (DT6606, DT6606lm, and K8484) were injected into the portal vein of mice. Magnetic resonance imaging (MRI) was used to monitor metastatic progression, and histologic analysis was performed. RESULTS: Orthotopically injected mice succumbed to the tumor within an 11-week period (average survival time, 78.2 ± 4.45 days). Post-mortem examinations failed to identify liver metastasis. In the intraportal model, 2 × 105 DT6606 cells resulted in an absence of liver metastases by day 21, whereas 5 × 104 DT6606lm cells and 7 × 104 K8484 cells resulted in steady metastatic growth. Higher doses caused significant metastatic liver involvement. The use of K8484 cells ensured the growth of tumors closely resembling the histopathologic characteristics of human PDAC. CONCLUSIONS: This report details the authors' efforts to establish an "optimal" murine model for inducing metastatic PDAC, which is critical for advancing our understanding of the disease and developing more effective treatments.

Skeletal Muscle Proteomic Profile Revealed Gender-Related Metabolic Responses in a Diet-Induced Obesity Animal Model.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC-MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxidative to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comorbidities.

A New Model of Chronic Mycobacterium abscessus Lung Infection in Immunocompetent Mice.

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades, affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge, we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection for up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of lymphocytes, aggregates of vacuolated histiocytes and a few neutrophils, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

Four-class tumor staging for early diagnosis and monitoring of murine pancreatic cancer using magnetic resonance and ultrasound.

Background: The widely used genetically engineered mouse LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre, termed KPC, spontaneously develops pancreatic cancer mirroring all phases of the carcinogenesis but in asynchronous manner. Preclinical studies need defined criteria for the enrollment of the KPC sharing the same stage of carcinogenesis. Aim: To define a tumor-staging criteria using magnetic resonance (MR) and ultrasound (US) and then to correlate the imaging stage with overall survival of KPC mice. Methods: Forty KPC (2- to 5-month-old mice) were imaged by axial fat-saturated T2-weighted sequences at MR and by brightness mode US to establish criteria for tumor staging. Immunohistopathology was used to validate imaging. A second cohort of 25 KPC was used to correlate imaging stage with survival by Kaplan-Meier analysis. Results: We defined a four-class tumor staging system ranking from stages 1 to 4. Stage 1 was described as radiologically healthy pancreas; precursor lesions were detectable in histology only. Cystic papillary neoplasms, besides other premalignant alterations, marked stage 2 in the absence of cancer nodules. Stages 3 and 4 identified mice affected by overt pancreatic cancer with size <5 or ≥5 mm, respectively. Regarding the prognosis, this staging system correlated with disease-related mortality whatever may be the KPC age when they staged. Conclusion: This imaging-based four-class tumor staging is an effective and safe method to stage pancreatic cancer development in KPC. As a result, regardless of their age, KPC mice can be synchronized based on prognosis or on a specific phase of tumorigenesis, such as the early but already radiologically detectable one (stage 2).

Utrophin up-regulation by artificial transcription factors induces muscle rescue and impacts the neuromuscular junction in mdx mice.

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy (DMD). In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZF-ATFs) that target the utrophin 'A' promoter. We have previously shown that the ZF-ATF "Jazz", either by transgenic manipulation or by systemic adeno-associated viral delivery, induces significant rescue of muscle function in dystrophic "mdx" mice. We present the full characterization of an upgraded version of Jazz gene named "JZif1" designed to minimize any possible host immune response. JZif1 was engineered on the Zif268 gene-backbone using selective amino acid substitutions to address JZif1 to the utrophin 'A' promoter. Here, we show that JZif1 induces remarkable amelioration of the pathological phenotype in mdx mice. To investigate the molecular mechanisms underlying Jazz and JZif1 induced muscle functional rescue, we focused on utrophin related pathways. Coherently with utrophin subcellular localization and role in neuromuscular junction (NMJ) plasticity, we found that our ZF-ATFs positively impact the NMJ. We report on ZF-ATF effects on post-synaptic membranes in myogenic cell line, as well as in wild type and mdx mice. These results candidate our ZF-ATFs as novel therapeutic molecules for DMD treatment.

2-Deoxy-d-Glucose Ameliorates PKD Progression.

Autosomal dominant polycystic kidney disease (ADPKD) is an important cause of ESRD for which there exists no approved therapy in the United States. Defective glucose metabolism has been identified as a feature of ADPKD, and inhibition of glycolysis using glucose analogs ameliorates aggressive PKD in preclinical models. Here, we investigated the effects of chronic treatment with low doses of the glucose analog 2-deoxy-d-glucose (2DG) on ADPKD progression in orthologous and slowly progressive murine models created by inducible inactivation of the Pkd1 gene postnatally. As previously reported, early inactivation (postnatal days 11 and 12) of Pkd1 resulted in PKD developing within weeks, whereas late inactivation (postnatal days 25-28) resulted in PKD developing in months. Irrespective of the timing of Pkd1 gene inactivation, cystic kidneys showed enhanced uptake of (13)C-glucose and conversion to (13)C-lactate. Administration of 2DG restored normal renal levels of the phosphorylated forms of AMP-activated protein kinase and its target acetyl-CoA carboxylase. Furthermore, 2DG greatly retarded disease progression in both model systems, reducing the increase in total kidney volume and cystic index and markedly reducing CD45-positive cell infiltration. Notably, chronic administration of low doses (100 mg/kg 5 days per week) of 2DG did not result in any obvious sign of toxicity as assessed by analysis of brain and heart histology as well as behavioral tests. Our data provide proof of principle support for the use of 2DG as a therapeutic strategy in ADPKD.

Leukocyte HMGB1 is required for vessel remodeling in regenerating muscles.

Signals of tissue necrosis, damage-associated molecular patterns (DAMPs), cause inflammation. Leukocytes migrating into injured tissues tonically release DAMPs, including the high mobility group box 1 protein (HMGB1). In the absence of suitable models, the relative role of DAMPs released because of necrosis or leukocyte activation has not, so far, been dissected. We have generated a mouse model lacking Hmgb1 in the hematopoietic system and studied the response to acute sterile injury of the skeletal muscle. Regenerating fibers are significantly less numerous at earlier time points and smaller at the end of the process. Leukocyte Hmgb1 licenses the skeletal muscle to react to hypoxia, to express angiopoietin-2, and to initiate angiogenesis in response to injury. Vascularization of the regenerating tissue is selectively jeopardized in the absence of leukocyte Hmgb1, revealing that it controls the nutrient and oxygen supply to the regenerating tissue. Altogether, our results reveal a novel nonredundant role for leukocyte Hmgb1 in the repair of injured skeletal muscle.

Lipid dysmetabolism in ceruloplasmin-deficient mice revealed both in vivo and ex vivo by MRI, MRS and NMR analyses.

Ceruloplasmin (Cp) is a ferroxidase that plays a role in cellular iron homeostasis and is mainly expressed in the liver and secreted into the blood. Cp is also produced by adipose tissue, which releases it as an adipokine. Although a dysfunctional interaction of iron with the metabolism of lipids has been associated with several metabolic diseases, the role of Cp in adipose tissue metabolism and in the interplay between hepatocytes and adipocytes has been poorly investigated. We previously found that Cp-deficient (CpKO) mice become overweight and demonstrate adipose tissue accumulation together with liver steatosis during aging, suggestive of lipid dysmetabolism. In the present study, we investigated the lipid alterations which occur during aging in adipose tissue and liver of CpKO and wild-type mice both in vivo and ex vivo. During aging of CpKO mice, we observed adipose tissue accumulation and liver lipid deposition, both of which are associated with macrophage infiltration. Liver lipid deposition was characterized by accumulation of triglycerides, fatty acids and ω-3 fatty acids, as well as by a switch from unsaturated to saturated fatty acids, which is characteristic of lipid storage. Liver steatosis was preceded by iron deposition and macrophage infiltration, and this was observed to be already occurring in younger CpKO mice. The accumulation of ω-3 fatty acids, which can only be acquired through diet, was associated with body weight increase in CpKO mice despite food intake being equal to that of wild-type mice, thus underlining the alterations in lipid metabolism/catabolism in Cp-deficient animals.

Inhibition of asparagine synthetase effectively retards polycystic kidney disease progression.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.

Gold nanorods assisted photothermal therapy of bladder cancer in mice: A computational study on the effects of gold nanorods distribution at the centre, periphery, and surface of bladder cancer.

BACKGROUND AND OBJECTIVES: Gold nanorod-assisted photothermal therapy (GNR-PTT) is a cancer treatment whereby GNRs incorporated into the tumour act as photo-absorbers to elevate the thermal destruction effect. In the case of bladder, there are few possible routes to target the tumour with GNRs, namely peri/intra-tumoural injection and intravesical instillation of GNRs. These two approaches lead to different GNR distribution inside the tumour and can affect the treatment outcome. METHODOLOGY: The present study investigates the effects of heterogeneous GNR distribution in a typical setup of GNR-PTT. Three cases were considered. Case 1 considered the GNRs at the tumour centre, while Case 2 represents a hypothetical scenario where GNRs are distributed at the tumour periphery; these two cases represent intratumoural accumulation with different degree of GNR spread inside the tumour. Case 3 is achieved when GNRs target the exposed tumoural surface that is invading the bladder wall, when they are delivered by intravesical instillation. RESULTS: Results indicate that for a laser power of 0.6 W and GNR volume fraction of 0.01%, Case 2 and 3 were successful in achieving complete tumour eradication after 330 and 470 s of laser irradiation, respectively. Case 1 failed to form complete tumour damage when the GNRs are concentrated at the tumour centre but managed to produce complete tumour damage if the spread of GNRs is wider. Results from Case 2 also demonstrated a different heating profile from Case 1, suggesting that thermal ablation during GNR-PTT is dependant on the GNRs distribution inside the tumour. Case 3 shows similar results to Case 2 whereby gradual but uniform heating is observed. Cases 2 and 3 show that uniformly heating the tumour can reduce damage to the surrounding tissues. CONCLUSIONS: Different GNR distribution associated with the different methods of introducing GNRs to the bladder during GNR-PTT affect the treatment outcome of bladder cancer in mice. Insufficient spreading during intratumoural injection of GNRs can render the treatment ineffective, while administered via intravesical instillation. GNR distribution achieved through intravesical instillation present some advantages over intratumoural injection and is worthy of further exploration.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Targeted inducible delivery of immunoactivating cytokines reprograms glioblastoma microenvironment and inhibits growth in mouse models.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector-based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

Single-shot morpho-functional and structural characterization of the left-ventricle in a mouse model of acute ischemia-reperfusion injury with an optimized 3D IntraGate cine FLASH sequence at 7T MR.

Preclinical cardiac MR is challenging and time-consuming. A fast and comprehensive acquisition protocol and standardized image post-processing may improve preclinical research, reducing acquisition time, costs and variability of results. In the present study, we evaluated the feasibility of a contrast-enhanced 3D IntraGate steady-state cine sequence (ce-3D-IG-cine) with short acquisition time (11 min) for a single-shot combined characterization of left ventricle (LV) remodeling and infarct size (IS) in a mouse model of acute ischemia-reperfusion injury. Sixteen male C57BL/6N mice underwent 7T cardiac MR (Bruker, BioSpec 70/30) including optimized ce-3D-IG-cine (total scan time 11 min) at day 1, 5 and 28 after surgery. LV end-diastolic volume (EDVMR) and ejection fraction (EFMR) extracted from MR were compared to ones from short-axis (SA-EDVecho, SA-EFecho) and parasternal long-axis (LA-EDVecho, LA-EFecho) echocardiography. IS was manually and semiautomatically segmented from ce-3D-IG-cine using different standard deviation (SD +2, +3, +4, +5, +6 in respect to a reference tissue). Mice were sacrificed at day 28, immediately after imaging. IS at day 28 was compared to injury burden at histology. MR and echocardiographic morpho-functional parameters were compared, as IS from MR and histology. Bland-Altman plots were used to assess the agreement in ischemic burden segmentation. Volumetric and functional parameters measured on ce-3D-IG-cine correlated to the correspondent echocardiographic parameter (EDVMR vs SA-EDVecho: ρ = 0.813; EDVMR vs LA-EDVecho: ρ = 0.845; EFMR vs SA-EFecho ρ = 0.612; EFMR vs LA-EFecho ρ = 0.791; p < 0.001 in all cases). Manually segmented IS strongly correlated with the scar at histology (ρ = 0.904, p < 0.001). A threshold of +3SD showed the highest performance for semiautomatic assessment of IS compared to manual segmentation (ρ = 0.965, p < 0.001), with an overall reproducibility of 73%, and a peak reproducibility of 80% at day 1. The ce-3D-IG-cine sequence, manually or semiautomatically segmented using 3SD threshold, allows fast and comprehensive LV morpho-functional and structural characterization in myocardial ischemia-reperfusion injury model.

Increased mediastinal fat and impaired left ventricular energy metabolism in young men with newly found fatty liver.

Fatty liver is characterized by metabolic abnormalities at the liver, but also at skeletal muscle and adipose tissue sites. It is hypothesized that the heart may be suffering metabolic alterations, and this study was undertaken to ascertain whether individuals with fatty liver have left ventricular (LV) alterations of energy metabolism, structure, and function and abnormal amounts of epicardial fat as a specific marker of visceral fat accumulation. To this end we studied young, nondiabetic men matched for anthropometric features with (n = 21) or without (n = 21) fatty liver by means of (1) cardiac magnetic resonance imaging (MRI); (2) cardiac (31)P-MR spectroscopy (MRS); and (3) hepatic (1)H-MRS to assess quantitatively the intrahepatic fat (IHF) content. Insulin sensitivity was determined by the updated HOMA-2 computer model. Individuals with fatty liver showed reduced insulin sensitivity, increased serum free fatty acid (FFA), and E-selectin, abnormal adipokine concentrations, and higher blood pressure. LV morphology and systolic and diastolic functions were not different; however, in the scanned intrathoracic region, the intrapericardial (7.8 +/- 3.1 versus 5.9 +/- 2.5 cm(2); P < 0.05) and extrapericardial (11.7 +/- 6.1 versus 7.8 +/- 3.2 cm(2); P < 0.03) fat was increased in men with fatty liver compared with those without fatty liver. The phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio, a recognized in vivo marker of myocardial energy metabolism, was reduced in men with fatty liver in comparison with normals (1.85 +/- 0.35 versus 2.11 +/- 0.31; P < 0.016). In conclusion, in newly found individuals with fatty liver, fat was accumulated in the epicardial area and despite normal LV morphological features and systolic and diastolic functions, they had abnormal LV energy metabolism.

Glucose metabolism during tumorigenesis in the genetic mouse model of pancreatic cancer.

AIM: More than 40% of pancreatic ductal adenocarcinoma (PDAC) patients have glucose intolerance or diabetes. The association has led to two hypotheses: PDAC causes diabetes or diabetes shares risk factors for the development of PDAC. In order to elucidate the relationship between diabetes and PDAC, we investigated the glucose metabolism during tumorigenesis in the LSL-KrasG12D/+; LSL-Trp53R172H/+; and Pdx-1-Cre (KPC) mouse, a genetically engineered model of PDAC. METHODS: Male and female KPCs have been fed with standard diet (SD) or high-fat diet (HFD). The imaging-based 4-class tumor staging was used to follow pancreatic cancer development. Not fasting glycemia, 4-h fasting glycemia, insulin, C-peptide, glucose tolerance after OGTT and abdominal fat volume were measured during tumorigenesis. RESULTS: PDAC development did not lead to an overt diabetic phenotype or to any alterations in glucose tolerance in KPC fed with SD. Consumption of HFD induced higher body weight/abdominal fat volume and worsened glucose homeostasis both in control CRE mice and only in early tumorigenesis stages of the KPC mice, excluding that the cancer development itself acts as a trigger for the onset of dysmetabolic features. CONCLUSION: Our data demonstrate that carcinogenesis in KPC mice is not associated with paraneoplastic diabetes.

Short-term evaluation of cardiac morphology, function, metabolism and structure following diagnosis of adult-onset growth hormone deficiency.

OBJECTIVE: The impact of growth hormone (GH) deficiency of the adult on cardiovascular function remains only partially elucidated. Purpose of this study was to test cardiac function in adult GH deficient patients using cardiac magnetic resonance (CMR). DESIGN: Cardiac magnetic resonance (CMR) techniques, including cardiac 31P MR spectroscopy and evaluation of gadolinium late-enhancement, were applied to assess simultaneously, in a cross-sectional fashion, morphological, functional, metabolic, and structural parameters of the left (LV) and right ventricle (RV) in 15 patients with adult onset GH deficiency. Fifteen healthy individuals served as controls. RESULTS: In GH deficient patients LV systolic function (EF%: 61 ±â€¯1.7 vs 62.1 ±â€¯0.8; p = .44) was not different in spite of a lower LV mass (83.2 ±â€¯5.3 vs 145.3 ±â€¯11.9 g; p = .001), a subclinical impairment of diastolic function (E/A peak ratio: 1.6 ±â€¯0.2 vs 2.1 ±â€¯0.2 p = .05), and a trend for lower PCr/ATP ratio (2.1 ±â€¯0.8 vs 2.3 ±â€¯0.1 p = .07). The RV showed reduced chamber size (end diastolic volume 123.8 ±â€¯9 vs 147.9 ±â€¯7.6 mL; p = .021) with preserved mass. No structural alterations of the LV and RV at late-enhancement were detected in these patients. CONCLUSIONS: GH deficient patients represent a unique model of reduced LV myocardial mass in which major structural and metabolic alterations are lacking. Mal-adaptive mechanisms developing in the long term in response to GH deficiency and more severely affecting the LV remain to be elucidated.

The Suv420h histone methyltransferases regulate PPAR-γ and energy expenditure in response to environmental stimuli.

Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.