211 EFFECT OF AMNIOTIC FLUID DERIVED STEM CELL-CONDITIONED MEDIUM ON DEVELOPMENT AND POST-THAW SURVIVAL OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 214
Author(s):  
K. Krautkramer ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Stem Cell ◽  
Amniotic Fluid ◽  
Lipid Content ◽  
Culture Conditions ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Step Size ◽  
Conventional Freezing

In vitro-produced (IVP) embryos have altered metabolism with lower blastocyst rates and higher lipid accumulation compared with IVP embryos. Culturing embryos with amniotic fluid derived stem cell-conditioned media (AFS) may help mimic in vivo conditions and improve embryo development. We hypothesised that culturing embryos with AFS will improve in vitro culture conditions and developmental competence. The IVP embryos were produced in 5 replicates by aspirating oocytes (n = 1469) of 2 to 8 mm from abattoir ovaries. Oocytes were matured for 23 h, fertilized with semen from 1 of 2 bulls, and cultured in a novel SOF medium (SOF for conventional freezing media) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Media was supplemented with either 0 (control), 5, or 10% AFS. Stage 7 blastocysts were stained with 1 µg mL−1 of Nile Red and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) to measure lipid content and mitochondrial polarity, respectively. Ten images per stained embryo were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Remaining blastocysts were slow frozen using 2 min of dehydration in 0.6 M sucrose before equilibration for 10 min in a conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose). Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data were analysed by general linear model using a one-way ANOVA with means separated by Tukey’s HSD. Results (Table 1) indicated no difference in development, lipid content, mitochondrial polarity, or post-thaw survival (P > 0.01), suggesting that growth factors present in AFS did not improve culture conditions. Table 1. Main effect of AFS on embryo developmental competence and post-thaw survival rates

86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

2017 ◽  
Vol 29 (1) ◽  
pp. 129 ◽  
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Equilibration Time ◽  
Step Size ◽  
A Cell ◽  
Blastocyst Rate ◽  
Mitotracker Red ◽  
Main Effects ◽  
Conventional Freezing

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage

2009 ◽  
Vol 10 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Jean Gekas ◽  
Guillaume Walther ◽  
Daniel Skuk ◽  
Emmanuel Bujold ◽  
Isabelle Harvey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Amniotic Fluid ◽  
Stem Cell Differentiation ◽  
In Vivo Study ◽  
Human Amniotic Fluid ◽  
Myogenic Lineage

scholarly journals Enrichment of Melanoma Stem-Like Cells via Sphere Assays

2021 ◽  
pp. 185-199
Author(s):  
Nabanita Mukherjee ◽  
Karoline A. Lambert ◽  
David A. Norris ◽  
Yiqun G. Shellman
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Stem Cell ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Low Density ◽  
In Vitro Techniques ◽  
Sphere Culture

AbstractSphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.

scholarly journals Osteogenic Differentiation Potential of Human Bone Marrow and Amniotic Fluid-Derived Mesenchymal Stem Cells in Vitro & in Vivo

2019 ◽  
Vol 7 (4) ◽  
pp. 507-515 ◽  
Author(s):  
Eman E. A. Mohammed ◽  
Mohamed El-Zawahry ◽  
Abdel Razik H. Farrag ◽  
Nahla N. Abdel Aziz ◽  
Wessam Sharaf-ElDin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Differentiation Potential

BACKGROUND: Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering. AIM: This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies. SUBJECTS AND METHODS: Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters. RESULTS: The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potential CONCLUSIONS: AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 178
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Nile Red ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Treatment Groups ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Conventional Freezing

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S7-S8
Author(s):  
Safina Gadeock ◽  
Cambrian Liu ◽  
Brent Polk
Keyword(s):  
Stem Cell ◽  
Stem Cell Niche ◽  
Mesenchymal Cells ◽  
Epithelial Stem Cells ◽  
Epithelial Stem Cell ◽  
Long Term Treatment ◽  
Lgr5 Expression ◽  
Cell Niche

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p&lt;0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p&lt;0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p&lt;0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p&lt;0.001; N=3) and MAPK (p&lt;0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p&lt;0.0001; N=6) and Wnt2b (-63%, p&lt;0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

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