Cultured meat is an attractive concept to address environmental and ethical issues of livestock farming. Replication of conventional meat requires fat tissue, for flavour, texture, and mouthfeel. However, cultured meat production is limited by the lack of bovine adipogenic cells that are both proliferative and differentiable. Here, we demonstrate the isolation and characterisation of spontaneously immortalised bovine preadipocytes proliferating over 100 days and 21 passages, showing adipogenic differentiation. Upon induction, the cells accumulate lipids and express key adipogenic markers. Comparative RNA sequencing and proteomic analysis between induced and non-induced cells confirmed bovine origin, cell identity, and low tumour suppressor expression. Proteomic profiling identified over 2000 proteins, with enrichment in adipogenesis-associated pathways, including PPAR signalling and the ERK1/2 cascade. Additionally, the cells demonstrate viability in suspension and strong scaffold attachment. These results establish a reproducible bovine cell source for cultured fat supporting scale-up and bioprocess research in cellular agriculture.

Cellular Agriculture (CellAg) is an attractive concept for innovative technology with the intent to provide food and nutrition complementary to existing supply streams. The past decade has seen considerable progress in the field with advancement of cellular technology that delivers the initial building blocks for meaningful implementation. The availability of natural cell-based material that can serve as nutrient-filled source for human consumption at low cost is a critical challenge for the emerging cellular agriculture industry. Therefore, here the isolation of bovine myofibroblasts of the Black Angus breed has been pursued and accomplished together with its characterisation by using RNA sequencing and proteomics through western blotting.To transition CellAg from a concept to a game changing technology for the industry, multiple challenges need to be overcome. The field requires powerful initial material, i.e., dedicated cells that can proliferate and differentiate robustly at scale. The methodology described allows for the production of healthy cells, which have been unequivocally characterized as clonal representatives of a stable myofibroblast cell line using transcriptomics and proteomics validation. Stringent and rigorous live cell monitoring of a nascent cell line derived from healthy muscle tissue allowed for stable cell growth.In this research article, a simple and precise methodology is presented for creating a bovine myofibroblast cell line (Bov.mia). Our work puts forward a low-tech use of materials and expertise that is devoid of transgenic approaches, thus creating a reliable approach for lab-scale research.

The origin of vitamin D2 in herbivorous animals was investigated in vivo in sheep and in bovine as well as mouse gastrointestinal tracts. A high concentration of 25-hydroxyvitamin D2 in blood plasma of sheep both in summer and winter appeared to be incompatible with the undetectable level of vitamin D2 in the pasture on which the sheep were grazing. Studies with bovine rumen contents from a cow grazing the same pasture as the sheep, demonstrated an increased concentration of vitamin D2 on anaerobic incubation in a ‘Rusitec’ artificial rumen, which was further enhanced when cellulose powder was added as a fermentation substrate. The colon contents of mice that were fed from weaning on a vitamin D-free diet were found to contain vitamin D2. The results of these comparative studies in 3 animal species indicated that vitamin D2 was being generated by microbial anaerobic metabolism in the gastrointestinal tract.

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200–400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage.

ABSTRACTBio-scaffolds for the cellular agriculture field require to be simple, with low associated costs. Here, a method is described to generate decellularized leaf scaffolds utilizing a simple bacterial process, yielding scaffolds with the capacity to sustain myoblast attachment, growth, and differentiation.To best develop a minimal cost decellularization process, we aimed to design the key steps of the method to be as “low-tech” as possible and not use chemical or thermal processing to produce the cellular scaffold. Decellularized leaves (DCL) from the black walnut tree (Juglans nigra) were successfully produced employing a domestic fish tank with a biological filtration system that supports an active aquatic nitrogen-fixing bacterial population, typically within 3-5 days. Following decellularization, the DCL were devoid of any pulp material as confirmed by scanning electron microscopy (SEM). DCL produced in this way are an effective cellular scaffold, and the C2C12 myoblast cell line was shown to attach, proliferate and differentiate on DCL and maintain viability up to 3 weeks post-seeding. Differentiated cellular material grew extensively over the DCL veins and larger differentiated cellular structures extended between individual DCL veins.The data presented provide a proof of concept for an inexpensive, simple, and chemical-free method for leaf decellularization, which supports myoblast attachment, growth, and differentiation. The technology provides clear applications for the cellular agriculture field where cost reduction, scalability, and simplification of established laboratory processes, such as bio scaffold production, is a key factor.

The calcium-sensing receptor (CaSR) is an important regulator of epidermal function. We previously reported that knockdown of the CaSR or treatment with its negative allosteric modulator, NPS-2143, significantly reduced UV-induced DNA damage, a key factor in skin cancer development. We subsequently wanted to test whether topical NPS-2143 could also reduce UV-DNA damage, immune suppression, or skin tumour development in mice. In this study, topical application of NPS-2143 (228 or 2280 pmol/cm2) to Skh:hr1 female mice reduced UV-induced cyclobutane pyrimidine dimers (CPD) (p < 0.05) and oxidative DNA damage (8-OHdG) (p < 0.05) to a similar extent as the known photoprotective agent 1,25(OH)2 vitamin D3 (calcitriol, 1,25D). Topical NPS-2143 failed to rescue UV-induced immunosuppression in a contact hypersensitivity study. In a chronic UV photocarcinogenesis protocol, topical NPS-2143 reduced squamous cell carcinomas for only up to 24 weeks (p < 0.02) but had no other effect on skin tumour development. In human keratinocytes, 1,25D, which protected mice from UV-induced skin tumours, significantly reduced UV-upregulated p-CREB expression (p < 0.01), a potential early anti-tumour marker, while NPS-2143 had no effect. This result, together with the failure to reduce UV-induced immunosuppression, may explain why the reduction in UV-DNA damage in mice with NPS-2143 was not sufficient to inhibit skin tumour formation.

Ultraviolet radiation (UVR) induces immunosuppression and DNA damage, both of which contribute to the rising global incidence of skin cancer including melanoma. Nucleotide excision repair, which is activated upon UVR-induced DNA damage, is linked to expression of interleukin-12 (IL-12) which serves to limit immunosuppression and augment the DNA repair process. Herein, we report an immunomodulating peptide, designated IK14800, that not only elicits secretion of IL-12, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) but also reduces DNA damage in the skin following exposure to UVR. Combined with re-invigoration of exhausted CD4+ T cells, inhibition of UVR-induced MMP-1 release and suppression of B16F10 melanoma metastases, IK14800 offers an opportunity to gain further insight into mechanisms underlying the development and progression of skin cancers.

This chapter provides an overview of current cultured meat technology. It starts by first discussing the limitations of conventional food systems, focusing on the environmental, social and economic impacts these systems can have. The chapter moves on to provide a rationale for a transition to cellular agriculture, specifically addressing the health, sustainability and economic benefits cellular agriculture could have. A section on advances in cellular agriculture is also included, which is followed by an overview of current challenges for the cellular agriculture field.

Patients with type 2 diabetes mellitus (T2DM) experience a higher risk of fractures despite paradoxically exhibiting normal to high bone mineral density (BMD). This has drawn into question the applicability to T2DM of conventional fracture reduction treatments that aim to retain BMD. In a primary human osteoblast culture system, high glucose levels (25 mM) impaired cell proliferation and matrix mineralization compared to physiological glucose levels (5 mM). Treatment with parathyroid hormone (PTH, 10 nM), a bone anabolic agent, and cinacalcet (CN, 1 µM), a calcimimetic able to target the Ca 2+ -sensing receptor (CaSR), were tested for their effects on proliferation and differentiation. Strikingly, CN+PTH co-treatment was shown to promote cell growth and matrix mineralization under both physiological and high glucose conditions. CN+PTH reduced apoptosis by 0.9-fold/0.4-fold as measured by Caspase-3 activity assay, increased alkaline phosphatase (ALP) expression by 1.5-fold/twofold, increased the ratio of nuclear factor κ-B ligand (RANKL) to osteoprotegerin (OPG) by 2.1-fold/1.6-fold, and increased CaSR expression by 1.7-fold/4.6-fold (physiological glucose/high glucose). Collectively, these findings indicate a potential for CN+PTH combination therapy as a method to ameliorate the negative impact of chronic high blood glucose on bone remodeling.

The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce "over-irradiation products" such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.

ABSTRACTThe epidermis maintains a cellular calcium gradient that supports keratinocyte differentiation from its basal layers (low) to outer layers (high) leading to the development of the stratum corneum, which resists penetration of UV radiation. The calcium‐sensing receptor (CaSR) expressed in keratinocytes responds to the calcium gradient with signals that promote differentiation. In this study, we investigated whether the CaSR is involved more directly in protection from UV damage in studies of human keratinocytes in primary culture and in mouse skin studied in vivo. siRNA‐directed reductions in CaSR protein levels in human keratinocytes significantly reduced UV‐induced direct cyclobutane pyrimidine dimers (CPD) by ~80% and oxidative DNA damage (8‐OHdG) by ~65% compared with control transfected cells. Similarly, in untransfected cells, the CaSR negative modulator, NPS‐2143 (500 nm), reduced UV‐induced CPD and 8‐OHdG by ~70%. NPS‐2143 also enhanced DNA repair and reduced reactive oxygen species (ROS) by ~35% in UV‐exposed keratinocytes, consistent with reduced DNA damage after UV exposure. Topical application of NPS‐2143 also protected hairless Skh:hr1 mice from UV‐induced CPD, oxidative DNA damage and inflammation, similar to the reductions observed in response to the well‐known photoprotection agent 1,25(OH)2D3 (calcitriol). Thus, negative modulators of the CaSR offer a new approach to reducing UV‐induced skin damage.

Melanoma is the most dangerous form of skin cancer, with poor prognosis in advanced stages. Vitamin D, also produced by ultraviolet radiation, is known for its anti-proliferative properties in some cancers including mel-anoma. While vitamin D deficiency has been associated with advanced melanoma stage and higher levels of vitamin D have been associated with better outcomes, the role for vitamin D in melanoma remains unclear. Vitamin D synthesis is initiated upon UVB exposure of skin cells and results in formation of the active metabolite 1,25-dihydroxyvitamin D3 (1,25D). We have previously demonstrated that 1,25D plays a role in protection against ultraviolet radiation-induced DNA damage, immune suppression, and skin carcinogenesis. In this study 1,25D significantly reduced cell viability and increased caspase levels in human melanoma cell lines. This effect was not present in cells that lacked both phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well-known tumour suppressor, and the vitamin D receptor (VDR). PTEN is frequently lost or mutated in mel-anoma. Incubation of selected melanoma cell lines with 1,25D resulted in significant increases in PTEN levels and downregulation of the AKT pathway and its downstream effectors. This suggests that 1,25D may act to reduce melanoma cell viability by targeting PTEN.

Increased risks of skeletal fractures are common in patients with impaired glucose handling and type 2 diabetes mellitus (T2DM). The pathogenesis of skeletal fragility in these patients remains ill-defined as patients present with normal to high bone mineral density. With increasing cases of glucose intolerance and T2DM it is imperative that we develop an accurate rodent model for further investigation. We hypothesized that a high fat diet (60%) administered to developing male C57BL/6J mice that had not reached skeletal maturity would over represent bone microarchitectural implications, and that skeletally mature mice would better represent adult-onset glucose intolerance and the pre-diabetes phenotype. Two groups of developing (8 week) and mature (12 week) male C57BL/6J mice were placed onto either a normal chow (NC) or high fat diet (HFD) for 10 weeks. Oral glucose tolerance tests were performed throughout the study period. Long bones were excised and analysed for ex vivo biomechanical testing, micro-computed tomography, 2D histomorphometry and gene/protein expression analyses. The HFD increased fasting blood glucose and significantly reduced glucose tolerance in both age groups by week 7 of the diets. The HFD reduced biomechanical strength, both cortical and trabecular indices in the developing mice, but only affected cortical outcomes in the mature mice. Similar results were reflected in the 2D histomorphometry. Tibial gene expression revealed decreased bone formation in the HFD mice of both age groups, i.e., decreased osteocalcin expression and increased sclerostin RNA expression. In the mature mice only, while the HFD led to a non-significant reduction in runt-related transcription factor 2 (Runx2) RNA expression, this decrease became significant at the protein level in the femora. Our mature HFD mouse model more accurately represents late-onset impaired glucose tolerance/pre-T2DM cases in humans and can be used to uncover potential insights into reduced bone formation as a mechanism of skeletal fragility in these patients.

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.

The three-dimensional formation of bio-engineered tissue for applications such as cell-based meat requires critical interaction between the bioscaffold and cellular biomass. To explore the features underlying this interaction, we have assessed the commercially available bacterial nanocellulose (BNC) product from Cass Materials for its suitability to serve as a bioscaffold for murine myoblast attachment, proliferation, and differentiation. Rigorous application of both scanning electron microscopy and transmission electron microscopy reveals cellular details of this interaction. While the retention rate of myoblast cells appears low, BNC is able to provide effective surface parameters for the formation of anchor points to form mature myotubes. Understanding the principles that govern this interaction is important for the successful scaling of these materials into edible, commercially viable, and nutritious biomass.

ABSTRACTThe vitamin D hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2D3), and related compounds derived from vitamin D3 or lumisterol as a result of metabolism via the enzyme CYP11A1, have been shown, when applied 24 hours before or immediately after UV irradiation, to protect human skin cells and skin from DNA damage due to UV exposure, by reducing both cyclobutane pyrimidine dimers (CPD) and oxidative damage in the form of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdG). We now report that knockdown of either the vitamin D receptor or the endoplasmic reticulum protein ERp57 by small, interfering RNA (siRNA) abolished the reductions in UV‐induced DNA damage with 20‐hydroxyvitamin D3 or 24‐hydroxylumisterol3, as previously shown for 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced oxygen consumption rates in UV‐exposed and sham‐exposed human keratinocytes and reduced phosphorylation of cyclic AMP response binding element protein (CREB). Both these actions have been shown to inhibit skin carcinogenesis after chronic UV exposure, consistent with the anticarcinogenic activity of 1,25(OH)2D3. The requirement for a vitamin D receptor for the photoprotective actions of 1,25(OH)2D3 and of naturally occurring CYP11A1‐derived vitamin D–related compounds may explain why mice lacking the vitamin D receptor in skin are more susceptible to UV‐induced skin cancers, whereas mice lacking the 1α‐hydroxylase and thus unable to make 1,25(OH)2D3 are not more susceptible. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-β−/−) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-β−/−/−− versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-β−/− mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-β−/− mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-β−/−, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-β.

Vitamin D, unlike the micronutrients, vitamins A, E, and K, is largely obtained not from food, but by the action of solar ultraviolet (UV) light on its precursor, 7-dehydrocholesterol, in skin. With the decline in UV light intensity in winter, most skin production of vitamin D occurs in summer. Since no defined storage organ or tissue has been found for vitamin D, it has been assumed that an adequate vitamin D status in winter can only be maintained by oral supplementation. Skeletal muscle cells have now been shown to incorporate the vitamin D-binding protein (DBP) from blood into the cell cytoplasm where it binds to cytoplasmic actin. This intracellular DBP provides an array of specific binding sites for 25-hydroxyvitamin D (25(OH)D), which diffuses into the cell from the extracellular fluid. When intracellular DBP undergoes proteolytic breakdown, the bound 25(OH)D is then released and diffuses back into the blood. This uptake and release of 25(OH)D by muscle accounts for the very long half-life of this metabolite in the circulation. Since 25(OH)D concentration in the blood declines in winter, its cycling in and out of muscle cells appears to be upregulated. Parathyroid hormone is the most likely factor enhancing the repeated cycling of 25(OH)D between skeletal muscle and blood. This mechanism appears to have evolved to maintain an adequate vitamin D status in winter.

Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds.

Severe vitamin D deficiency—25-hydroxyvitamin D (25OHD) concentrations below around 25–30 nmol/L—may lead to growth plate disorganization and mineralization abnormalities in children (rickets) and mineralization defects throughout the skeleton (osteomalacia) and proximal muscle weakness. Both problems are reversed with vitamin D treatment. Apart from this musculoskeletal dysfunction at very low vitamin D levels, there is apparent inconsistency in the available data about whether concentrations of 25OHD below around 50 nmol/L cause muscle function impairment and increase the risk of fracture. This narrative review provides evidence to support the contention that improving vitamin D status, up to around 50 nmol/L, plays a small causal role in optimizing bone and muscle function as well as reducing overall mortality.