Hypothermic Oxygenated Perfusion Improves Vascular and Contractile Function by Preserving Endothelial Nitric Oxide Production in Cardiac Grafts Obtained With Donation After Circulatory Death.

BACKGROUND Cardiac donation after circulatory death is a promising option to increase graft availability. Graft preservation with 30 minutes of hypothermic oxygenated perfusion (HOPE) before normothermic machine perfusion may improve cardiac recovery as compared with cold static storage, the current clinical standard. We investigated the role of preserved nitric oxide synthase activity during HOPE on its beneficial effects. METHODS AND RESULTS Using a rat model of donation after circulatory death, hearts underwent in situ ischemia (21 minutes), were explanted for a cold storage period (30 minutes), and then reperfused under normothermic conditions (60 minutes) with left ventricular loading. Three cold storage conditions were compared: cold static storage, HOPE, and HOPE with Nω-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor). To evaluate potential confounding effects of high coronary flow during early reperfusion in HOPE hearts, bradykinin was administered to normalize coronary flow to HOPE levels in 2 additional groups (cold static storage and HOPE with Nω-nitro-L-arginine methyl ester). Cardiac recovery was significantly improved in HOPE versus cold static storage hearts, as determined by cardiac output, left ventricular work, contraction and relaxation rates, and coronary flow (P<0.05). Furthermore, HOPE attenuated postreperfusion calcium overload. Strikingly, the addition of Nω-nitro-L-arginine methyl ester during HOPE largely abolished its beneficial effects, even when early reperfusion coronary flow was normalized to HOPE levels. CONCLUSIONS HOPE provides superior preservation of ventricular and vascular function compared with the current clinical standard. Importantly, HOPE's beneficial effects require preservation of nitric oxide synthase activity during the cold storage. Therefore, the application of HOPE before normothermic machine perfusion is a promising approach to optimize graft recovery in donation after circulatory death cardiac grafts

Mimicking the Intervertebral Disc Microenvironment for Expansion of Nucleus Pulposus Progenitor Cells in a Context of Cell Therapy

INTRODUCTION: Low back pain (LBP) is a global health concern that affects as many as 75–80% of people during their lifetime. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, the progenitor cell population was described in the nucleus pulposus (NP) of the IVD. These cells, positive for the Tie2 marker, have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Here, we study the influence of the culture method and of the microenvironment on the human NP progenitors and their differentiation potential in vitro. METHODS:Cells were obtained from human NP tissue from trauma patients undergoing spinal surgery. Briefly, after mild overnight digestion, the NP tissue cells were cultured in 2D (monolayer) or 3D (alginate beads) conditions with medium supplemented in ascorbic acid. After 2 weeks, cells from 2D or 3D culture were expanded on fibronectin-coating flasks with medium supplemented in FGF-2 to mimic the native microenvironment of NP cells. Subsequently, expanded NP cells were then characterized by cytometry (CD105, CD90, CD73, CD45, CD34, and Tie2) and tri-lineage differentiation, which was analyzed by qPCR and histology. RESULTS: Cytometry analysis, after 2D- or 3D-expansion showed the presence of 0.1 % and 78.2 % of Tie2+ NP progenitors, respectively. Concerning the chondrogenic differentiation assay, the detection of glycosaminoglycans in the culture medium was drastically increased for 3D-expanded cells (11-fold) vs 2D-expanded cells. Moreover, the relative gene expression of collagen type 2 and aggrecan was also increased (600-fold and 2-fold, respectively). Regarding osteogenic differentiation assay, relative gene expression for osteopontin increased for 3D- (150-fold) vs 2D-expanded cells. However, no difference was observed between 2D and 3D expansion for the adipogenic differentiation assay. DISCUSSION & CONCLUSIONS: The present study shows that 3D expansion of NP cells better preserves the progenitor's cells population and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. This project not only has a scientific impact by evaluating the role of native physiological niches on the functionality of NP progenitors but could also lead to an innovative clinical approach with cell therapy for IVD regeneration and repair. Acknowledgments: Financial support was received from iPSpine H2020 project #825925

Pre-ischemic Lactate Levels Affect the Post-ischemic Recovery in an Isolated Rat Heart Model of Donation After Circulatory Death (DCD)

Introduction: Donation after circulatory death (DCD) could substantially improve donor heart availability. In DCD, the heart is not only exposed to a period of warm ischemia, but also to a damaging pre-ischemic phase. We hypothesized that the DCD-relevant pre-ischemic lactate levels negatively affect the post-ischemic functional and mitochondrial recovery in an isolated rat heart model of DCD. Methods: Isolated, working rat hearts underwent 28.5′ of global ischemia and 60′ of reperfusion. Prior to ischemia, hearts were perfused with one of three pre-ischemic lactate levels: no lactate (0 Lac), physiologic lactate (0.5 mM; 0.5 Lac), or DCD-relevant lactate (1 mM; 1 Lac). In a fourth group, an inhibitor of the mitochondrial calcium uniporter was added in reperfusion to 1 Lac hearts (1 Lac + Ru360). Results: During reperfusion, left ventricular work (heart rate-developed pressure product) was significantly greater in 0.5 Lac hearts compared to 0 Lac or 1 Lac. In 1 vs. 0.5 Lac hearts, in parallel with a decreased function, cellular and mitochondrial damage was greater, tissue calcium content tended to increase, while oxidative stress damage tended to decrease. The addition of Ru360 to 1 Lac hearts partially abrogated the negative effects of the DCD-relevant pre-ischemic lactate levels (greater post-ischemic left ventricular work and less cytochrome c release in 1 Lac+Ru360 vs. 1 Lac). Conclusion: DCD-relevant levels of pre-ischemic lactate (1 mM) reduce contractile, cellular, and mitochondrial recovery during reperfusion compared to physiologic lactate levels. Inhibition of mitochondrial calcium uptake during early reperfusion improves the post-ischemic recovery of 1 Lac hearts, indicating calcium overload as a potential therapeutic reperfusion target for DCD hearts

Spheroid-like Cultures for Cell Expansion of Angiopoietin Receptor-1 (aka. Tie2) positive Cells from the human Intervertebral Disc

INTRODUCTION:Low back pain is the leading cause of disability worldwide (1). Nevertheless, the mechanism of the intervertebral disc (IVD) degeneration is still not clear. In this context, the nucleus pulposus (NP) and more precisely NP progenitor cells (NPPCs) present in the IVD, positive for angiopoietin-1 receptor (aka. Tie2) display multipotent and stem capacity (2,3). In this study, the first aim was to determine whether spheroid formation in suspension-culture will increase the amount/percentage of NPPCs during the expansion compared to traditional monolayer culture. The second aim of this study was to investigate if the percentage of NPPCs will be enriched even further by the resuspension of the spheroid-like cultured cells (=1st generation) and reformation of those spheroids one more time (= 2nd generation). METHODS:Human NP tissues from trauma patients (N=3) were obtained with written ethical consent and isolated by a two-step digestion protocol (3). The NP cells were resuspended and frozen at -150°C after reaching confluence of passage 0. At passage 1, NP cells were seeded in standard or ultra-low attachment tissue culture flasks with 2.5 ng/ml FGF-2 in low glucose - DMEM (supplemented with 10 % FBS). Flow cytometry was used to analyze and quantify the percentage of NPPCs using Tie2 antibody. We defined the spheroids formed after passage 1 NPCs as 1st generation spheroid. We obtained the 2nd generation spheroids by resuspending the 1st-generation-spheroid and reassembly. The NPCs from 1st and 2nd spheroid were quantified by CFU-assay. RESULTS:As a result, the percentage of NPPCs in monolayer culture condition was reaching 7 ± 2 % (Mean±SEM), however, in the 1st and 2nd generation spheroids culture condition, we were observing 20 ± 10 % and 28 ± 6% of Tie2+ cells, respectively. Concerning the CFU-assay, the NPCs from the 2nd generation spheroid formed 30 CFU-S per 1,000 cells, which were twice more CFU-S compared to the 1st generation spheroid. DISCUSSION & CONCLUSIONS:From these data we conclude than the spheroid-like formation of NPCs would be a more efficient method for expansion and enrichment of NPPCs than monolayer expansion in a context of future cell therapy. Acknowledgements:Financial support was received from iPSpine H2020 project under grant agreement #825925 and China Scholarship Council to X.Z

Open- vs. closed-chest pig models of donation after circulatory death.

BACKGROUND During donation after circulatory death (DCD), cardiac grafts are exposed to potentially damaging conditions that can impact their quality and post-transplantation outcomes. In a clinical DCD setting, patients have closed chests in most cases, while many experimental models have used open-chest conditions. We therefore aimed to investigate and characterize differences in open- vs. closed-chest porcine models. METHODS Withdrawal of life-sustaining therapy (WLST) was simulated in anesthetized juvenile male pigs by stopping mechanical ventilation following the administration of a neuromuscular block. Functional warm ischemic time (fWIT) was defined to start when systolic arterial pressure was <50 mmHg. Hemodynamic changes and blood chemistry were analyzed. Two experimental groups were compared: (i) an open-chest group with sternotomy prior to WLST and (ii) a closed-chest group with sternotomy after fWIT. RESULTS Hemodynamic changes during the progression from WLST to fWIT were initiated by a rapid decline in blood oxygen saturation and a subsequent cardiovascular hyperdynamic (HD) period characterized by temporary elevations in heart rates and arterial pressures in both groups. Subsequently, heart rate and systolic arterial pressure decreased until fWIT was reached. Pigs in the open-chest group displayed a more rapid transition to the HD phase after WLST, with peak heart rate and peak rate-pressure product occurring significantly earlier. Furthermore, the HD phase duration tended to be shorter and less intense (lower peak rate-pressure product) in the open-chest group than in the closed-chest group. DISCUSSION Progression from WLST to fWIT was more rapid, and the hemodynamic changes tended to be less pronounced in the open-chest group than in the closed-chest group. Our findings support clear differences between open- and closed-chest models of DCD. Therefore, recommendations for clinical DCD protocols based on findings in open-chest models must be interpreted with care

BMP Antagonists - A Possible Cause for Spinal Non-Fusion?

INTRODUCTION:Spinal fusion is a procedure where the intervertebral disc (IVD) is removed and two adjacent vertebrae are forced to fuse by compression. This procedure is the most commonly applied procedure to achieve spinal stability and relief of back pain. However, non-successful fusion leads to pseudo-athrosis and ongoing pain. There is increasing evidence that supraphysiological doses of BMP2 and burst-release of this cytokine did not generate satisfying results in clinical studies. Current hypothesis was raised that IVD cells and/or tissue seem to inhibit the action of BMP2. In this overview we summarize the current evidence that BMPs might be inhibited by the secretome of human IVD cells, i.e., nucleus pulposus cells (NPC), annulus fibrosus cells (AFC) and cartilaginous endplate (CEPC) cells. METHODS:We stimulated low-passage (2-3) human bonemarrow-derived mesenchymal stromal cells (MSCs) and femoral hip-derived osteoblasts (OBs) and co-cultured these with allogeneic IVD cells obtained from spinal surgery. We then stimulated MSCs and the OBs in monolayer and osteogenic medium, whereas IVD cells were kept in 3D alginate bead culture and separated by high density pore culture inserts (0.4 µm pore size). We quantified relative gene expression at bone-relevant genes, alkaline phosphatase (ALP) activity and Alizarin red (ALZR) staining after 21 days. Furthermore, to test the effect of a previously investigated BMP2 analog to block the inhibitors, cells were further stimulated with 100 ng/mL BMP2 and/or L51P. RESULTS:We found significant inhibitory effects of IVD cells onto MSCs undergoing differentiation in presence of NPC, AFC and CEPC as shown in reduced osteogenic gene expression, ALZR staining and ALP activity (N = 11 donors paired on each side). In the case of allogeneic human OBs only a trend towards inhibition could be demonstrated (N = 7 donors on each side). The addition of L51P to the coculture recovered ossification. On the side of the IVD cells BMP2 and/or L51P had a strong chondrogenic effect. DISCUSSION & CONCLUSIONS:Our data suggested evidence for inhibition for MSCs. However, OBs did not show the same inhibitory effects but showed a trend in presence of IVD’s secretome. This warrants for animal models where the donor variance can be better controlled. ACKNOWLEDGEMENTS:This work was supported by a start-up grant from the Center for Applied Biotechnology and Molecular Medicine (CABMM). Further funds were received from the Swiss Society of Orthopaedics (SGOT), the clinical trials unit (CTU) of Bern University Hospital, and by a Eurospine Task Force Research grant #2019_22

Macrophage-derived extracellular vesicles alter cardiac recovery and metabolism in a rat heart model of donation after circulatory death.

Conditions to which the cardiac graft is exposed during transplantation with donation after circulatory death (DCD) can trigger the recruitment of macrophages that are either unpolarized (M0) or pro-inflammatory (M1) as well as the release of extracellular vesicles (EV). We aimed to characterize the effects of M0 and M1 macrophage-derived EV administration on post-ischaemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. Isolated rat hearts were subjected to 20 min aerobic perfusion, followed by 27 min global, warm ischaemia or continued aerobic perfusion and 60 min reperfusion with or without intravascular administration of EV. Four experimental groups were compared: (1) no ischaemia, no EV; (2) ischaemia, no EV; (3) ischaemia with M0-macrophage-dervied EV; (4) ischaemia with M1-macrophage-derived EV. Post-ischaemic ventricular and metabolic recovery were evaluated. During reperfusion, ventricular function was decreased in untreated ischaemic and M1-EV hearts, but not in M0-EV hearts, compared to non-ischaemic hearts (p < 0.05). In parallel with the reduced functional recovery in M1-EV versus M0-EV ischaemic hearts, rates of glycolysis from exogenous glucose and oxidative metabolism tended to be lower, while rates of glycogenolysis and lactate release tended to be higher. EV from M0- and M1-macrophages differentially affect post-ischaemic cardiac recovery, potentially by altering glucose metabolism in a rat model of DCD. Targeted EV therapy may be a useful approach for modulating cardiac energy metabolism and optimizing graft quality in the setting of DCD

Trilineage Potency of Human Nucleus Pulposus Cells before and after Cryo-Preservation

INTRODUCTION:Low back pain (LBP) is a major cause of disability in many countries, affecting more than half a billion people worldwide. A promising and future-oriented approach to treat LBP is cell therapy using stem or progenitor cells. Over the last decade, cells positive for Tie2 and mesenchymal stromal cell markers have been found within the nucleus pulposus (NP) of human intervertebral discs (IVD). However, little is known about the effect of expansion and cryo-preservation on here called “heterogenic” human NP cells (hNPCs) and their stemness in a context of cell therapy for regeneration of the IVD. Therefore, the aim of our study was to expand hNPCs whilst investigating their differentiation potential before and after cryo-preservation and to find and optimal approach to cryo-preserve them. METHODS:HNPCs from three human trauma patients (32, 55 and 69 years old) undergoing spinal surgery were isolated with a mild two-step digestion protocol. After subsequent expansion until complete confluency, hNPCs were separated and then differentiated into osteogenic, adipogenic or chondrogenic lineages for 21 days or were cryo-preserved for one week at -150°C with five cryo-preservation media (90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO); 90% low glucose medium + 10% DMSO and three commercially available media) to compare their effect on the cell’s viability and differentiation potential. Cell viability was determined with trypan blue and by cytometry employing propidium iodide. The differentiation potential was assessed using histological analysis and qPCR. RESULTS:HNPCs cultured in osteogenic medium showed a significant (p<0.01) higher expression of calcium deposits (up to 11-fold) vs. controls, indicating osteogenic differentiation. Furthermore, evidence for adipogenic and chondrogenic differentiation was observed using histological analysis and determining genes typical for chondrogenic and adipogenic lineages like collagen type 2 (up to 350-fold) or adiponectin (up to 3’700-fold). In addition, most hNPCs maintained their differentiation potential, even after cryo-preservation and independent of the cryo-preservation medium used. The hNPCs’ cell viability after storing for one week at -150°C was very similar for all conditions (~85% cell viability). DISCUSSION & CONCLUSIONS:The study showed heterogenic hNPCs have trilineage potential and as such possess stem cell characteristics. Therefore, they can potentially be used for future clinical trials concerning cell therapy for IVD regeneration. Furthermore, commercially available cryo-preservation media seem to perform just as well as homemade media in terms of cell viability and maintaining hNPCs differentiation potential