Cultured beef: from small biopsy to substantial quantity.

Cultured meat is an emerging technology with the potential to solve huge challenges related to the environmental, ethical, and health implications of conventional meat production. Establishing the basic science of cultured meat has been the primary focus of the last decade but it is now feasible that cultured meat products will enter the market within the next 3 to 4 years. This proximity to market introduction demands an evaluation of aspects of the cultured meat production process that have not yet been outlined or discussed in significant detail. For example, one technological approach for the production of cultured meat uses adult muscle stem cells, the limited proliferative capacity of which necessitates repeated collection of tissue samples via biopsies of living donor animals. The selection of donor animals and the details of biopsy processes must be optimized, as this is a key bottleneck in the cultured meat production process. The number of stem cells harvested from a biopsy, together with their proliferative capacity, determines a 'multiplicity factor' achieved by a cultured meat production process, thus dictating the reduction in number of animals required to produce a given quantity of meat. This article considers potential scenarios for these critical upstream steps, focusing on the production of cultured beef as an example. Considerations related to donor selection and details of the biopsy process are discussed in detail. The practicalities of various scenarios for cultured beef production, the health of donor animals, and regulatory issues associated with the safety of cultured meat for consumers are also considered. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tick Saliva Protein Evasin-3 Allows for Visualization of Inflammation in Arteries through Interactions with CXC-Type Chemokines Deposited on Activated Endothelium.

Atherosclerosis is one of the leading causes of mortality in developed and developing countries. The onset of atherosclerosis development is accompanied by overexpression of several inflammatory chemokines. Neutralization of these chemokines by chemokine-binding agents attenuates atherosclerosis progression. Here, we studied structural binding features of the tick protein Evasin-3 to chemokine (C-X-C motif) ligand 1 (CXCL1). We showed that Evasin-3-bound CXCL1 is unable to activate the CXCR2 receptor, but retains affinity to glycosaminoglycans. This observation was exploited to detect inflammation by visualizing a group of closely related CXC-type chemokines deposited on cell walls in human endothelial cells and murine carotid arteries by a fluorescent Evasin-3 conjugate. This work highlights the applicability of tick-derived chemokine-binding conjugates as a platform for the development of new agents for inflammation imaging.

Shear Stress and VE-Cadherin.

Objective- Vascular fusion represents an important mechanism of vessel enlargement during development; however, its significance in postnatal vessel enlargement is still unknown. During fusion, 2 adjoining vessels merge to share 1 larger lumen. The aim of this research was to identify the molecular mechanism responsible for vascular fusion. Approach and Results- We previously showed that both low shear stress and DAPT ( N-[ N-(3,5-difluorophenacetyl)-L-alanyl]- S-phenylglycine t-butyl ester) treatment in the embryo result in a hyperfused vascular plexus and that increasing shear stress levels could prevent DAPT-induced fusion. We, therefore, investigated vascular endothelial-cadherin (VEC) phosphorylation because this is a common downstream target of low shear stress and DAPT treatment. VEC phosphorylation increases after DAPT treatment and decreased shear stress. The increased phosphorylation occurred independent of the cleavage of the Notch intracellular domain. Increasing shear stress rescues hyperfusion by DAPT treatment by causing the association of the phosphatase vascular endothelial-protein tyrosine phosphatase with VEC, counteracting VEC phosphorylation. Finally, Src (proto-oncogene tyrosine-protein kinase Src) inhibition prevents VEC phosphorylation in endothelial cells and can rescue hyperfusion induced by low shear stress and DAPT treatment. Moesin, a VEC target that was previously reported to mediate endothelial cell rearrangement during lumenization, relocalizes to cell membranes in vascular beds undergoing hyperfusion. Conclusions- This study provides the first evidence that VEC phosphorylation, induced by DAPT treatment and low shear stress, is involved in the process of fusion during vascular remodeling.

Cultured Meat in Islamic Perspective.

Cultured meat is a promising product that is derived through biotechnology that partially circumvents animal physiology, thereby being potentially more sustainable, environmentally friendly and animal friendly than traditional livestock meat. Such a novel technology that can impact many consumers evokes ethical, philosophical and religious discussions. For the Islamic community, the crucial question is whether cultured meat is halal, meaning compliant with Islamic laws. Since the culturing of meat is a new discovery, invention and innovation by scientists that has never been discussed by classical jurists (fuqaha'), an ijtihad by contemporary jurists must look for and provide answers for every technology introduced, whether it comply the requirements of Islamic law or not. So, this article will discuss an Islamic perspective on cultured meat based on the original scripture in the Qur'an and interpretations by authoritative Islamic jurists. The halal status of cultured meat can be resolve through identifying the source cell and culture medium used in culturing the meat. The halal cultured meat can be obtained if the stem cell is extracted from a (Halal) slaughtered animal, and no blood or serum is used in the process. The impact of this innovation will give positive results in the environmental and sustain the livestock industry.

Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats.

BACKGROUND: Disturbances in coronary microcirculatory function, such as the endothelial glycocalyx, are early hallmarks in the development of obesity and insulin resistance. Accordingly, in the present study myocardial microcirculatory perfusion during rest and stress was assessed following metformin or sulodexide therapy in a rat model of diet-induced obesity. Additionally, the effect of degradation of the glycocalyx on myocardial perfusion was assessed in chow-fed rats. METHODS: Rats were fed a high fat diet (HFD) for 8 weeks and were divided into a group without therapy, and groups that received the anti-diabetic drug metformin or the glycocalyx-stabilizing drug sulodexide in their drinking water during the last 4 weeks of the feeding period. Myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. In an acute experimental setting, hyaluronidase was administered to chow-fed control rats to determine the effect of enzymatical degradation of the glycocalyx on myocardial perfusion. RESULTS: HFD-rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. We confirmed our earlier findings that the robust increase in myocardial perfusion in chow-fed rats after an adenosine challenge (+56%, p = 0.002) is blunted in HFD rats (+8%, p = 0.68). In contrast, 4-weeks treatment with metformin or sulodexide partly restored the increase in myocardial perfusion during adenosine infusion in HFD rats (+81%, p = 0.002 and +37%, p = 0.02, respectively). Treating chow-fed rats acutely with hyaluronidase, to enzymatically degrade the glyocalyx, completely blunted the increase in myocardial perfusion during stress. CONCLUSIONS: In early stages of HFD-induced insulin resistance myocardial perfusion becomes compromised, a process that can be countered by treatment with both metformin and sulodexide. The adverse effect of acute glycocalyx degradation and protective effect of long-term sulodexide administration on myocardial perfusion provides indirect evidence, suggesting a role for the glycocalyx in preserving coronary microvascular function in pre-diabetic animals.

SPECT and PET imaging of angiogenesis and arteriogenesis in pre-clinical models of myocardial ischemia and peripheral vascular disease.

PURPOSE: The extent of neovascularization determines the clinical outcome of coronary artery disease and other occlusive cardiovascular disorders. Monitoring of neovascularization is therefore highly important. This review article will elaborately discuss preclinical studies aimed at validating new nuclear angiogenesis and arteriogenesis tracers. Additionally, we will briefly address possible obstacles that should be considered when designing an arteriogenesis radiotracer. METHODS: A structured medline search was the base of this review, which gives an overview on different radiopharmaceuticals that have been evaluated in preclinical models. RESULTS: Neovascularization is a collective term used to indicate different processes such as angiogenesis and arteriogenesis. However, while it is assumed that sensitive detection through nuclear imaging will facilitate translation of successful therapeutic interventions in preclinical models to the bedside, we still lack specific tracers for neovascularization imaging. Most nuclear imaging research to date has focused on angiogenesis, leaving nuclear arteriogenesis imaging largely overlooked. CONCLUSION: Although angiogenesis is the process which is best understood, there is no scarcity in theoretical targets for arteriogenesis imaging.

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

AIMS: The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. METHODS AND RESULTS: After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CONCLUSION: CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Early impairment of coronary microvascular perfusion capacity in rats on a high fat diet.

BACKGROUND: It remains to be established if, and to what extent, the coronary microcirculation becomes compromised during the development of obesity and insulin resistance. Recent studies suggest that changes in endothelial glycocalyx properties contribute to microvascular dysfunction under (pre-)diabetic conditions. Accordingly, early effects of diet-induced obesity on myocardial perfusion and function were studied in rats under baseline and hyperaemic conditions. METHODS: Rats were fed a high fat diet (HFD) for 6 weeks and myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. RESULTS: HFD-fed rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. Early diet-induced obesity did not lead to hypertension or cardiac hypertrophic remodeling. In chow-fed, control rats a robust increase in cardiac microvascular perfusion was observed upon adenosine infusion (+40%; p < 0.05). In contrast, the adenosine response was abrogated in rats on a HFD (+8%; N.S.). HFD neither resulted in rarefaction or loss of glycocalyx integrity in skeletal muscle, nor reduced staining intensity of the glycocalyx of cardiac capillaries. CONCLUSIONS: Alterations in coronary microcirculatory function as assessed by first-pass perfusion MRI represent one of the earliest obesity-related cardiac adaptations that can be assessed non-invasively. In this early stage of insulin resistance, disturbances in glycocalyx barrier properties appeared not to contribute to the observed changes in coronary microvascular function.

Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo.

OBJECTIVE: Macrophages show extreme heterogeneity and different subsets have been characterized by their activation route and their function. For instance, macrophage subsets are distinct by acting differently under pathophysiological conditions such as inflammation and cancer. Macrophages also contribute to angiogenesis, but the role of various specific subsets in angiogenesis has not been thoroughly investigated. METHODS AND RESULTS: Matrigel supplemented with macrophage subsets [induced by IFNγ (M1), IL-4 (M2a) or IL-10 (M2c)] was injected subcutaneously in C57BL/6 J mice and analyzed by CD31 staining after 14 days. Increased numbers of endothelial cells and tubular structures were observed in M2-enriched plugs compared to control and other subsets. Additionally, more tubular structures formed in vitro in the presence of M2 macrophages or their conditioned medium. To identify a mechanism for the pro-angiogenic effect, gene expression of angiogenic growth factors was analyzed. Induced expression of basic fibroblast growth factor (Fgf2), insulin-like growth factor-1 (Igf1), chemokine (C-C motif) ligand 2 (Ccl2) and placental growth factor (Pgf) was observed in M2 macrophages. Using a blocking antibody of PlGF to inhibit M2c induced angiogenesis resulted in mildly reduced (40 %) tube formation whereas neutralization of FGF-2 (M2a) signaling by sFGFR1-IIIc affected tube formation by nearly 75 %. CONCLUSIONS: These results indicate that macrophages polarized towards an M2 phenotype have a higher angiogenic potential compared to other subsets. Furthermore, we propose FGF signaling for M2a- and PlGF signaling for M2c-induced angiogenesis as possible working mechanisms, yet, further research should elucidate the exact mechanism for M2-induced angiogenesis.

Plaque-associated vasa vasorum in aged apolipoprotein E-deficient mice exhibit proatherogenic functional features in vivo.

OBJECTIVE: Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo. METHODS AND RESULTS: Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E-deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 µm, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries. CONCLUSIONS: Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).

The role of cardiovascular magnetic resonance imaging and computed tomography angiography in suspected non-ST-elevation myocardial infarction patients: design and rationale of the CARdiovascular Magnetic rEsoNance imaging and computed Tomography Angiography (CARMENTA) trial.

BACKGROUND: Although high-sensitivity cardiac troponin (hs-cTn) substantially improves the early detection of myocardial injury, it lacks specificity for acute myocardial infarction (MI). In suspected non-ST-elevation MI, invasive coronary angiography (ICA) remains necessary to distinguish between acute MI and noncoronary myocardial disease (eg, myocarditis), unnecessarily subjecting the latter to ICA and associated complications. This trial investigates whether implementing cardiovascular magnetic resonance (CMR) or computed tomography angiography (CTA) early in the diagnostic process may help to differentiate between coronary and noncoronary myocardial disease, thereby preventing unnecessary ICA. STUDY DESIGN: In this prospective, single-center, randomized controlled clinical trial, 321 consecutive patients with acute chest pain, elevated hs-cTnT, and nondiagnostic electrocardiogram are randomized to 1 of 3 strategies: (1) CMR, or (2) CTA early in the diagnostic process, or (3) routine clinical management. In the 2 investigational arms of the study, results of CMR or CTA will guide further clinical management. It is expected that noncoronary myocardial disease is detected more frequently after early noninvasive imaging as compared with routine clinical management, and unnecessary ICA will be prevented. The primary end point is the total number of patients undergoing ICA during initial admission. Secondary end points are 30-day and 1-year clinical outcome (major adverse cardiac events and major procedure-related complications), time to final diagnosis, quality of life, and cost-effectiveness. CONCLUSION: The CARMENTA trial investigates whether implementing CTA or CMR early in the diagnostic process in suspected non-ST-elevation MI based on elevated hs-cTnT can prevent unnecessary ICA as compared with routine clinical management, with no detrimental effect on clinical outcome.

A simplified and defined serum-free medium for cultivating fat across species.

Cultivated meat is a promising technology with the potential to mitigate the ethical and environmental issues associated with traditional meat. Fat plays a key role in the meat flavor; therefore, development of suitable adipogenic protocols for livestock is essential. The traditional adipogenic cocktail containing IBMX, dexamethasone, insulin and rosiglitazone is not food-compatible. Here, we demonstrate that of the four inducers only insulin and rosiglitazone are necessary in both serum-free (DMAD) and serum-containing media, with DMAD outperforming FBS. Two glucocorticoid receptor activators, progesterone and hydrocortisone, found in DMAD and FBS, affect differentiation homogeneity, without playing an essential role in activating adipogenic genes. Importantly, this protocol leads to mature adipocytes in 3D culture. This was demonstrated in both media types and in four species: ruminant and monogastric. We therefore propose a simplified one-step adipogenic protocol which, given the replacement of rosiglitazone by a food-compatible PPARγ agonist, is suitable for making cultivated fat.

Protocol for differentiation of bovine adipogenic progenitor cells embedded in alginate sheets.

Here, we present a cost-effective protocol to differentiate bovine fibro-adipogenic progenitors in a thin hydrogel sheet adherent to 96-well plates. We describe steps for the embedding and culturing of cells in alginate sheets, culture maintenance, and analysis. Compared to alternative three-dimensional (3D) models such as hydrogel-based microfibers, this approach simplifies automation while retaining efficient maturation of adipocytes. Embedded cells are still subjected to a 3D environment, but the sheets can be handled and analyzed like two-dimensional cultures.

Single-cell analysis of bovine muscle-derived cell types for cultured meat production.

Cultured meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cells, including muscle fibers, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterization and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterize cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favor the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with time-resolved flow cytometric analysis, we characterize dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.

Short-Stranded Zein Fibers for Muscle Tissue Engineering in Alginate-Based Composite Hydrogels.

Cultivated meat is a nascent technology that aims to create an environmentally and animal-friendly alternative to conventional meat. Producing skeletal muscle tissue in an animal-free system allowing for high levels of myofusion and maturation is important for the nutritional and sensorial value of cultivated meat. Alginate is an attractive biomaterial to support muscle formation as it is food-safe, sustainable and cheap and can be crosslinked using non-toxic methods. Although alginate can be functionalized to promote cell attachment, limitations in its mechanical properties, including form, viscosity, and stress relaxation, hinder the cellular capacity for myogenic differentiation and maturation in alginate-based hydrogels. Here, we show that the addition of electrospun short-stranded zein fibers increased hydrogel degradation, resulting in faster compaction, improved cell-gel interaction, and enhanced alignment of bovine muscle precursor cells. We conclude that fiber-hydrogel composites are a promising approach to support optimal formation of 3D constructs, by improving tissue stability and thus prolonging culture duration. Together, this improves muscle-related protein content by facilitating myogenic differentiation and priming muscle organoids for maturation.

Automated multiscale vessel analysis for the quantification of MR angiography of peripheral arteriogenesis.

PURPOSE: To automatically analyze the time course of collateralization in a rat hindlimb ischemia model based on signal intensity distribution (SID). MATERIALS AND METHODS: Time-of-flight magnetic resonance angiograms (TOF-MRA) were acquired in eight rats at 2, 7, and 21 days after unilateral femoral artery ligation. Analysis was performed on maximum intensity projections filtered with multiscale vessel enhancement filter. Differences in SID between ligated limb and a reference region were monitored over time and compared to manual collateral artery identification. RESULTS: The differences in SID correlated well with the number of collateral arteries found with manual quantification. The time courses of ultrasmall (diameter ≪0.5 mm) and small (diameter ≈0.5 mm) collateral artery development could be differentiated, revealing that maturation of the collaterals and enlargement of their feeding arteries occurred mainly after the first week postligation. CONCLUSION: SID analysis performed on axial maximum intensity projections is easy to implement, fast, and objective and provides more insight in the time course of arteriogenesis than manual identification.

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment.

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.

Soluble Jagged-1 inhibits neointima formation by attenuating Notch-Herp2 signaling.

OBJECTIVE: Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. METHODS AND RESULTS: Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 -treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. CONCLUSIONS: Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.