Bovine Adipose-derived Stem Cells Research Articles

Curcumin Inhibits Oxidative Stress and Apoptosis Induced by H2O2 in Bovine Adipose-Derived Stem Cells (bADSCs)

In livestock production, oxidative stress (OS) is ubiquitous, reducing animal productivity and product quality. Hence, investigating the mechanisms of oxidative stress in livestock and inhibiting oxidative stress-induced damage is crucial. Curcumin, a plant-derived bioactive compound, exhibits antioxidant and anti-apoptotic properties. Adipose-derived stem cells (ADSCs) from animal adipose tissue are easily accessible and possess multilineage differentiation potential. Therefore, this work utilized bovine ADSCs to establish an oxidative stress model and investigated the effects of curcumin on oxidative stress and apoptosis. Firstly, bovine ADSCs were isolated and cultured from fetal calf subcutaneous adipose tissue. Their surface markers were identified by immunofluorescence, confirming the expression of CD29, CD44, CD73, CD90, CD105 and Vimentin, but not CD34, indicative of mesenchymal stem/progenitor cell characteristics. Secondly, to explore the effects of curcumin on oxidative damage and apoptosis in bovine ADSCs, an oxidative stress model was induced using H2O2. CCK-8 assays showed significantly reduced cell viability and SOD activity, along with increased malondialdehyde (MDA) and reactive oxygen species (ROS) levels, indicating successful modeling. RT-qPCR further confirmed that 500 μM of H2O2 treatment for 24 h promoted apoptosis. Herein, CCK-8 assays indicated a significant reduction in cell viability at >8 μM of curcumin. Thirdly, using 4 μM and 8 μM of curcumin for pre-protection, 8 μM maintained SOD activity, reduced MDA and ROS, inhibited apoptosis-related gene changes (Bcl-2, Bax, Caspase-3), and suppressed apoptosis according to a TUNEL assay. Fourthly, curcumin’s autophagy-inducing potential was hypothesized, which was confirmed by increased LC3-II and decreased P62 expression upon co-treatment with 3-MA. 3-MA inhibited curcumin’s antioxidant and anti-apoptotic effects, suggesting that curcumin’s antioxidant and anti-apoptotic roles may involve autophagy induction. In conclusion, bovine ADSCs are abundant, easily accessible, and multipotent, making them suitable for in vitro expansion. Curcumin alleviated H2O2-induced oxidative stress in bovine ADSCs, with curcumin also inhibiting apoptosis, likely through autophagy induction. This study validates the protective role of curcumin in bovine ADSCs, with potential applications in livestock production.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(−). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 105 cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content.

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell-cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

Mimicking Wagyu beef fat in cultured meat: Progress in edible bovine adipose tissue production with controllable fatty acid composition.

Since the current process of livestock meat production has considerable effects on the global environment, leading to high emissions of greenhouse gases, cultured meat has recently attracted attention as a suitable alternative way to acquire animal proteins. However, while most published studies on cell-cultured meat have focused on muscle tissue culture, fat production which is an important component of the process has often been neglected from this technology, even though it can enhance the meat's final taste, aroma, tenderness, texture, and palatability. In this study, we focused on bovine muscle reconstruction by monitoring and optimizing the possible expansion rate of isolated primary bovine adipose stem cells and their adipogenesis differentiation to be fully edible for cultured meat application. After approximatively 100 days of serial passages, the bovine adipose-derived stem cells, isolated from muscle tissue, underwent 57​±​5 doublings in the edible cell culture medium condition. This implies that by cultivating and amplifying them, a minimum of 2.9​×​1022​cells can be obtained from around 10​g of fat. It was discovered that these cells retain their adipogenesis differentiation ability for at least 12 passages. Moreover, the final lipid composition could be controlled by adjusting the fatty acid composition of the culture medium during the differentiation process, resulting in organoleptic features similar to those of real fat from muscle. This was especially so for the cis isomer oleic acid percentage, an important part of high-grade Japanese Wagyu meat. These characteristics of the primary bovine adipose-derived stem cell proliferation and adipogenesis differentiation provide valuable insights for the in vitro production of meat alternatives.

Life Sciences Discovery and Technology Highlights

Life Sciences Discovery and Technology Highlights

Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors.

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cellsas precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity.

Bioprocess development for scalable production of cultivated meat.

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm2 ) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm2 ) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

ABSTRACT Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation. Abbreviations: AcA: acetic acid; AsA: ascorbic acid; ASC: adipose-derived stem cells; BSL: bovine serum lipids; DAPI: 4´,6-diamidino-2-phenylindole; DLK: delta like non-canonical notch ligand; DMEM: Dulbecco’s modified Eagle’s medium; DPBS: Dulbecco’s phosphate-buffered saline; ENG: endoglin; FABP: fatty acid binding protein; FAS: fatty acid synthase; GLUT4: glucose transporter type 4; IBMX: 3-isobutyl-1-methylxanthine; LPL: lipoprotein lipase; MSC: mesenchymal stem cells; α-MEM: α minimum essential medium; NT5E: ecto-5ʹ-nucleotidase; PDGFRα: platelet derived growth factor receptor α; PPARγ: peroxisome proliferator activated receptor γ; RPS19: ribosomal protein S19; SEM: standard error of the mean; THY1: Thy-1 cell surface antigen; TRT: treatment; TRT-Con: treatment negative control; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta

Characterization Of Bovine Adipose-Derived Stem Cells

Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory, and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers (CD73 and CD90). Meanwhile, haematopoietic markers (CD45 and CD34) are absent from bovine adipose-derived stem cells. We also induced their adipogenic differentiation in vitro.

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.

209 EPIGENETIC MODIFICATION WITH ZEBULARINE AND VALPROIC ACID AND EXPRESSION OF PLURIPOTENCY GENES IN BOVINE ADIPOSE STEM CELLS

Bovine adipose-derived stem cells (ASC), a form of adult stem cells, are somatic cells that have similar characteristics of embryonic stem (ES) cells. ASC are multipotent and have been found to express genes associated with pluripotency, Oct4, Sox2 and Nanog. The unique properties of ASC make them a desirable source for reprogramming experiments. When somatic cells are reprogrammed, there are certain epigenetic changes or modifications that must occur. Epigenetic modifications will change the chromatin configuration without changing the DNA sequence. Somatic cells can be exposed to small molecules that induce some of these changes. Two epigenetic modifying factors are a DNA methyltranferase inhibitor, zebularine (Zeb) and a histone deacetylase inhibitor, valproic acid (VPA). By altering gene expression with these epigenetic modifiers, the cells may be stimulated to reprogram more efficiently than untreated cells. Three lines of bovine ASC were isolated from the stromal fraction of adipose tissue and expanded through 3 passages in DMEM (high glucose) supplemented with 10% fetal bovine serum. Cells were cultured by addition of 5 mM VPA or 100 μM Zeb to the culture medium and cultured for 5, 7, 10 or 14 days before mRNA was isolated and converted to cDNA. Control groups, consisting of untreated bovine ASC from the same cell lines, were cultured for the same time periods as the treatment groups. Quantitative RT-PCR was performed to determine transcript levels for Oct4, Sox2, Nanog and poly adenylate polymerase (PAP) as a reference gene. Transcript levels were quantified by relative quantification using the ΔΔCT method and expressed as ratios of the target genes (Oct4, Sox2 and Nanog) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine ADS cells. Normalized ratios were log-transformed before analysis by ANOVA to correct for lack of normality. A one-way ANOVA was completed to test the statistical significance among the control and treatment groups. Pairwise comparison was conducted to test expression levels between individual treatments. When cells were treated for 5 days, Oct4 and Nanog expression levels were increased (P ≤ 0.05) between the control and both treatment groups. Expression of Sox2 was not different (P = 0.06) across treatment groups. Based on pair wise comparisons gene expression in VPA and Zeb treated groups were not different. Treatment for 7, 10, or 14 days did not result in any difference in transcript levels between the treatment groups and control for any of the genes analysed. VPA and Zeb treatment for 5 days may have produced a partial reprogramming. This partial reprogramming could aid in the bovine ASC reaching complete pluripotency when combined with other reprogramming techniques. This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.

The Effect of Serial Passaging on the Proliferation and Differentiation of Bovine Adipose-Derived Stem Cells

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.

288 INDUCING PLURIPOTENCY IN BOVINE SOMATIC STEM CELLS

Embryonic stem cells are pluripotent cells that can self-replicate indefinitely. There is an interest in producing pluripotent cells by reprogramming somatic cells. Porcine, murine, and human somatic cells are able to reprogram after exposure to viral vectors expressing Sox2, Oct-4, Klf4, and c-Myc. Exposure of somatic cells to these epigenetic modifying factors has been shown to increase expression of endogenous pluripotency factors, especially Oct-4 and Nanog. There have been limited attempts to induce pluripotency in bovine somatic cells. Bovine adipose-derived stem (ADS) cells were infected with a lentiviral vector expressing murine Sox2, Oct-4, Klf4, and c-Myc. In addition, bovine ADS cells were exposed to valproic acid (VPA), a histone deacetylase inhibitor, and zebularine (Zeb), a DNA methyltransferase 1 inhibitor. When two cell lines (‘A’ and ‘B’) of bovine ADS cells (between passages 2–6) were infected with the lentiviral vector, cells were able to form embryonic-stem-cell-like colonies on BD Matrigel (BD, Franklin Lakes, NJ, USA). Bovine ADS cells were exposed to lentiviral particles for 24 h and then changed to a Matrigel-coated culture dish after 3 days. These infected cells were stained on days 10 and 15 with an alkaline phosphatase stain, which resulted in positive staining cell colonies. The RNA was isolated from these cells, and expression of endogenous Oct-4 and Nanog was analysed by semiquantitative RT-PCR. The intensity of electrophoresis bands was compared between treatments as ratios of the gene of interest to an internal control gene, Poly A. The infected cells from each cell line had higher Oct-4 and Nanog expression than did noninfected cells from the same lines. For cell lines A and B, the ratios of Oct-4 increased from 0.87 to 1.27 and 2.38 to 8.77, respectively, for each line. The ratios for Nanog were also increased for A and B from 0.78 to 1.17 and 1.71 to 14.26, respectively. Furthermore, bovine ADS cells exposed to VPA and Zeb were also analysed for expression of endogenous Oct-4 and Nanog by semiquantitative RT-PCR. Bovine ADS cells from lines A and B described above were plated and allowed to culture overnight. After 24 h, the treatment group was changed to a culture medium containing 100 μM Zeb. Valproic acid (5 mM) was added to the treatment group 24 h later. The medium was changed for treatment and controls every 3 days (with the treatment group receiving Zeb and VPA). On day 14, RNA was isolated from cells of the control and treatment groups, and expression of endogenous Oct-4 and Nanog was analysed as described above. In cell line B, the expression of Oct-4 and Nanog was four times that of the control (with ratios of Oct-4 increasing from 0.20 to 0.82). However, in cell line A, the levels were slightly lower than those of the control (Oct-4 ratios decreased from 0.80 to 0.56). We hypothesise that increasing levels of Oct-4 and Nanog with epigenetic modifiers such as VPA or Zeb in conjunction with lentiviral expression vectors will result in higher induced pluripotency rates. This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.