In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test (Levine, Youngs et al. 2007). In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After four weeks of either isolation or group rearing (a period that encompasses rodent childhood and early adolescence), rats were sacrificed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post burn injury. We found that: 1) gross wound healing was significantly impaired in isolation reared rats compared to group reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group reared rats than for uninjured group reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation reared rats during burn injury compared to uninjured isolation reared rats, 5) behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.
Hepatic glucose metabolism is a key player in diseases such as obesity and diabetes as well as in antihyperglycemic drugs screening. Hepatocytes culture in two-dimensional configurations is limited in vitro model for hepatocytes to function properly, while truly practical platforms to perform three-dimensional (3D) culture are unavailable. In this work, we present a practical organoid culture method of hepatocytes for elucidation of glucose metabolism under nominal and stress conditions. Employing this new method of culturing cells within a hollow fiber reactor, hepatocytes were observed to self-assemble into 3D spherical organoids with preservation of tight junctions and display increased liver-specific functions. Compared to both monolayer culture and sandwich culture, the hepatocyte organoids displayed higher intracellular glycogen content, glucose consumption, and gluconeogenesis and approached the in vivo values, as also confirmed by gene expression of key enzymes. Moreover, hepatocyte organoids demonstrated more realistic sensitivity to hormonal challenges with insulin, glucagon, and dexamethasone. Finally, the exposure to high glucose demonstrated toxicities including alteration of mitochondrial membrane potential, lipid accumulation, and reactive oxygen species formation, similar to the in vivo responses, which was not captured by monolayer cultures. Collectively, hepatocyte organoids mimicked the in vivo functions better than hepatocyte monolayer and sandwich cultures, suggesting suitability for applications such as antihyperglycemic drugs screening.
rat hepatocytes; 3D organoid culture; hollow fiber bioreactor; glucose metabolism
We report an all-in-one platform – ScanDrop – for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a “cloud” network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2–4 days for other currently available standard detection methods.
Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.
Non-viral transfection; Lentiviruses; Primary rat hepatocytes; Huh7.5.1; Transfection efficiency; Hepatic function
Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections.
Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C.
Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls.
Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values.
MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of subnormothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.
liver transplantation; reperfusion injury; sub-normothermic machine perfusion
While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.
Dendritic cell chemotaxis is an important process involved in the acquisition of adaptive immunity. Despite several studies, our understanding of this process remains limited. One of the reasons for this is the lack of experimental models that give us real-time information on dendritic cell locomotion. Here, using tools in microfluidics, we have fabricated a microdevice that allows us to monitor dendritic cell migration in a chemokine gradient in real time. We successfully observed the migration of dendritic cells derived from a myeloid leukemia cell line (MUTZ-3) in a soluble chemokine (CCL-19) gradient. Our experiments suggest the utility of microdevices in monitoring dendritic cell chemotaxis in real time and getting important information regarding migration speeds and distances previously not available from conventional chemotaxis assays. This kind of data is useful for building mechanistic mathematical models of dendritic cell chemotaxis that may give us novel insights to the process of dendritic cell chemotaxis.
Chemotaxis; dendritic cells; BioMEMs; migration; CCL-19
Mesenchymal stem cells (MSCs) are capable of modulating the immune system and have been used to successfully treat a variety of inflammatory diseases in preclinical studies. Recent evidence has implicated paracrine signaling as the predominant mechanism of MSC therapeutic activity. We have shown in models of inflammatory organ failure that the factors secreted by MSCs are capable of enhancing survival, downregulating inflammation, and promoting endogenous repair programs that lead to the reversal of these diseases. As a marker of disease resolution, we have observed an increase in serum IL-10 when MSC-conditioned medium (MSC-CM) or lysate (MSC-Ly) is administered in vivo. Here we present an in vitro model of IL-10 release from blood cells that recapitulates this in vivo phenomenon. This assay provides a powerful tool in analyzing the potency of MSC-CM and MSC-Ly, as well as characterizing the interaction between MSC-CM and target cells in the blood.
Mesenchymal stem cell; IL-10; Potency assay; Organ injury; Inflammation; Autoimmunity; Transplantation
Deep partial thickness burns are subject to delayed necrosis of initially viable tissues surrounding the primary zone of thermally induced coagulation, which results in an expansion of the burn wound, both in area and depth, within 48 hours postburn. Neutrophil sequestration and activation leading to microvascular damage is thought to mediate this secondary tissue damage. Resolvins, a class of endogenous mediators derived from omega-3 polyunsaturated fatty acids, have been shown to regulate the resolution of inflammation. We hypothesized that exogenous resolvins could mitigate the deleterious impact of the inflammatory response in burn wounds. Using two different mouse burn injury models involving significant partial thickness injuries, we found that a systemically administered single dose of resolvin D2 (RvD2) as low as 25 pg/g bw given within an interval of up to 4 hours postburn effectively prevented thrombosis of the deep dermal vascular network and subsequent dermal necrosis. By preserving the microvascular network, RvD2 enhanced neutrophil access to the dermis, but prevented neutrophil-mediated damage through other anti-inflammatory actions, including inhibition of tumor necrosis factor-α, interleukin-1β, and neutrophil platelet–endothelial cell adhesion molecule-1. In a clinical context, RvD2 may be therapeutically useful by reducing the need for surgical debridement and the area requiring skin grafting.
The shortage of donor livers has led to an increased use of organs from expanded criteria donors. Included are livers with steatosis, a metabolic abnormality that increases the likelihood of graft complications post-transplantation. After a brief introduction on the etiology, pathophysiology, categories and experimental models of hepatic steatosis, we herein review the methods to rescue steatotic donor livers before transplantation applied in clinical and experimental studies. The methods span the spectrum of encouraging donor weight loss, employing drug therapy, heat shock preconditioning, ischemia preconditioning and selective anesthesia on donors, and the treatment on isolated grafts during preservation. These methods work at different stages of transplantation process, although share similar molecular mechanisms including lipid metabolism stimulation through enzymes or nuclear receptor e.g., peroxisomal proliferator-activated receptor, or anti-inflammation through suppressing cytokines e.g., tumor necrosis factor-α, or antioxidant therapies to alleviate oxidative stress. This similarity of molecular mechanisms implies possible future attempts to reinforce each approach by repeating the same treatment approach at several stages of procurement and preservation, as well as utilizing these alternative approaches in tandem.
Liver transplantation; Steatosis; Donor liver; Clinical; Experimental
Initially hailed as the ultimate solution to organ failure, engineering of vascularized tissues such as the liver has stalled because of the need for a well-structured circulatory system that can maintain the cells seeded inside the construct. A new approach has evolved to overcome this obstacle. Whole-organ decellularization is a method that retains most of the native vascular structures of the organ, providing microcirculatory support and structure, which can be anastomosed with the recipient circulation. The technique was first applied to the heart and then adapted for the liver. Several studies have shown that cells can be eliminated, the extracellular matrix and vasculature are reasonably preserved and, after repopulation with hepatocytes, these grafts can perform hepatic functions in vitro and in vivo. Progress is rapidly being made as researchers are addressing several key challenges to whole-organ tissue engineering, such as ensuring correct cell distribution, nonparenchymal cell seeding, blood compatibility, immunological concerns, and the source of cells and matrices.
Purpose of review
The capacity of the liver to regenerate and maintain a constant size despite injury is unique. However, the exact mechanisms are not completely clear. Cell transplantation has been proposed as an alternative treatment of liver diseases. Recent progress has been reported on the generation of stem/progenitor cells that may differentiate towards the hepatic lineage. However, it is currently difficult to determine which of the stem/progenitor cell populations are the best for therapy of a given disease.
The limited access to donor human hepatocytes has opened a great interest on the generation of hepatocyte-like cells. Several potential cell sources have been identified. However, general standardization of the methods to evaluate these cells is particularly important for the promise of stem/progenitor-derived hepatocyte-based therapies. Moreover, innovations aimed at improving hepatocyte delivery, survival and engraftment have recently opened the field of organ engineering that may improve the perspective of liver repopulation.
Here we review current evidence reported from the perspective of potential clinical applications of different hepatic cell sources with repopulation capacities and the future perspectives and tools that can facilitate the translation of laboratory work into clinical success.
Hepatocyte transplantation; xenogenic hepatocytes; stem cell-derived hepatocytes; liver tissue engineering
Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.
The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system.
Livers from 12 Yorkshire pigs (20–30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly.
Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts.
Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.
donor after cardiac death (DCD); normothermic machine perfusion (NMP); warm ischemia; transplantation; ATP
Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.
Cytokine; Immune response; Immunomodulation; Macrophage; Microencapsulation; Vaccine
Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and its regulatory mechanisms remain poorly understood. In the present study, we evaluated the effect of a blockage in terminal keratinocytic lineage differentiation in the Foxn1−/− nude phenotype on the epithelial progeny. Most notably we found a constitutive upregulation of LIM homeobox protein 2 (Lhx2), a marker gene of epithelial stem cellness indispensible for hair cycle progression. However, histological evidence along with an erratic, acyclic rise of otherwise suppressed CyclinD1 levels along with several key markers of keratinocyte lineage differentiation indicate a frustrated expansion of epithelial stem cell niches in skin. In addition, CD49f/CD34/CD200–based profiling demonstrated highly significant shifts in subpopulations of epithelial progeny. Intriguingly this appeared to include the expansion of Oct4+ stem cells in dermal fractions of skin isolates in the Foxn1 knock-out opposed to wild type. Overall our findings indicate that the Foxn1−/− phenotype has a strong impact on epithelial progeny and thus offers a promising model to study maintenance and regulation of stem cell niches within skin not feasible in other in vitro or in vivo models.
The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fiber bioreactor devices in which the cells (separated by a semipermeable polyethersulfone (PES) membrane) can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibers. The cells also preserved their immunophenotype under physiologic fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow fiber bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.
Mesenchymal Stem Cell; Dialysis; Acute Kidney Failure
Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid P. vanderplanki  to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese Hamster Ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4 M trehalose for 4 h at 37 °C, a seven-fold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14 % relative humidity. After desiccation to 2.60 g of water per gram dry weight, a 170 % increase in viability and a 400 % increase in growth (after 7 days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.
Drug-induced liver injury (DILI) limits the development and utilization of numerous therapeutic compounds, and consequently presents major challenges to the pharmaceutical industry and clinical medicine1, 2. Acetaminophen (APAP) containing compounds are among the most frequently prescribed drugs, and also the most common cause of DILI3. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and APAP-induced hepatotoxicity. We report that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation, and death. We identified a small molecule inhibitor of Cx32 as a novel hepatoprotectant that achieves the same result in wildtype mice when coadministered with known hepatotoxic drugs. These findings demonstrate that gap junction inhibition is an effective therapy for limiting DILI, and suggest a novel pharmaceutical strategy to improve drug safety.
Biomaterials; cell-material interaction; drug delivery; multifunctional coatings; nanoporous materials
Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.
Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in-vitro and in-vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In-vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 hours post- injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post SCI indicated that as few as 5×104 encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in-vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.
Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET.
Mitoneet; Autodock; Docking; Fluorescence; Iron–sulfur; Thiazolidinedione