Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts

2019 ◽  
Vol 17 (4) ◽  
pp. 342-351 ◽  
Author(s):  
De Cai Xiang ◽  
Bao Yu Jia ◽  
Guo Bo Quan ◽  
Bin Zhang ◽  
Qing Yong Shao ◽  
...  
Keyword(s):  
Serum Replacement ◽  
Recovery Culture ◽  
Knockout Serum Replacement

289 DERIVATION OF MOUSE EMBRYONIC STEM CELL FROM C57BL/6/EGFP STRAIN WITH FETAL CALF SERUM AND KNOCKOUT SERUM REPLACEMENT® SUPPLEMENTATION MEDIUM

2011 ◽  
Vol 23 (1) ◽  
pp. 242
Author(s):  
B. C. S. Campanha ◽  
C. S. Oliveira ◽  
D. M. Souza ◽  
C. P. Godoi ◽  
H. Fernandes ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Serum Replacement ◽  
Tetraploid Complementation ◽  
Murine Fibroblast ◽  
Esc Derivation ◽  
Knockout Serum Replacement

Embryonic stem cells (ESC) have been used in attempts to obtain specific tissues or even individuals. Embryonic stem cells are pluripotent, allowing the differentiation of cell types from 3 germ layers. The establishment of a stable lineage of ESC is a valuable tool; however, some strains of mice are less permissive to ESC derivation or generation of chimeric animals (e.g. C57BL/6). Supplementation of culture medium with FCS, in the ESC derivation, may influence the potentiality to derivation or use of these strains in tetraploid complementation assays (Sato et al. 2009 Tsukuba Res. Inst. 47, 414–422). Thus, its replacement was carried out using knockout serum replacement (KSR®) to minimize the deleterious action of serum (Wang et al. 2007 Inst. of Biotech. 23, 269–272). Embryos were obtained from 5 females of lineage C57BL6/EGFP, aged between 21 and 30 days and weighing ∼35 g, and superstimulated according (Mancini et al. 2008 Transg. Res. 17, 1015). The animals were placed for mating with fertile males of the same strain in a proportion of one to one (male:female). The copulation was confirmed by plug vaginal (0.5 days postcopulation). Embryo recovery was performed 3.5 to 4.0 days postcopulation to obtain expanded (EB) or hatched blastocysts (HB). Zona pellucida was removed from EB with the aid of pronase solution, and the whole embryos (n = 8) were placed on a 4-well dish pretreated with pig skin gelatin 0.1%, under murine fibroblast primary in DMEM medium supplemented with 7.5% FCS and 7.5% KSR®, 10 mM βmercaptoetanol, 1 mM sodium pyruvate, 2 mM L-glutamine, and 83.4 mg mL–1 amikacin for 24 h. After this period, the medium was replaced by DMEM supplemented with 15% KSR®. The colonies began to grow between 3 and 6 days after in vitro culture of the embryos. Once established, the colony was picked and placed into new plates containing murine fibroblast primary every 48 to 72 h. After 14 days, the derivation was confirmed with some proved pluripotency markers by immunofluorescence (Oct3/4, SSEA-1, and Nanog) and karyotyping for ploidy detection. The reaction was positive for all tested markers in addition to the detection of the endogenous fluorescence from EGFP protein itself (C57BL/6EGFP origin). It was concluded that ESC derivation with partial serum replacement and using a less permissive strain such as C57BL/6EGFP is feasible, although with a reduced success rate (12.5%; i.e. 1 lineage – named BCM04 – from 8 attempts). Fellowships and grants were received from FAPESP, Brazil: 09/15919-4 (BCSC), 09/16254-6 (DMS), 09/17605-7 (CPG), 06/06491-2 (MFGN), and 07/07705-9 (MFGN).

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

scholarly journals Factors supporting long-term culture of bovine male germ cells

2016 ◽  
Vol 28 (12) ◽  
pp. 2039 ◽  
Author(s):  
Mahesh Sahare ◽  
Sung-Min Kim ◽  
Ayagi Otomo ◽  
Kana Komatsu ◽  
Naojiro Minami ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Culture System ◽  
Normal Karyotype ◽  
Selective Inhibition ◽  
Culture Conditions ◽  
Serum Free ◽  
Serum Replacement ◽  
Knockout Serum Replacement

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte : somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

Supplementation of culture medium with knockout serum replacement improves the survival of bovine secondary follicles when compared with other protein sources during in vitro culture

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 32-36
Author(s):  
D.S. Gomes ◽  
L.B. Aragão ◽  
M.F. Lima Neto ◽  
P.A.A. Barroso ◽  
L.R.F.M. Paulino ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Survival Rates ◽  
Basal Membrane ◽  
Histological Evaluation ◽  
Serum Replacement ◽  
Before And After ◽  
Chi Squared ◽  
Knockout Serum Replacement

SummaryThe present study evaluated the effect of knockout serum replacement (KSR), fetal bovine serum (FBS) and bovine serum albumin (BSA) on the viability and growth of bovine secondary follicles cultured in vitro for 12 days. To this end, secondary follicles were isolated (185–202 μm) and cultured in vitro in TCM-199+ medium supplemented with KSR (5% and 10%), FBS (5% and 10%) or BSA (3 mg/ml) at 38.5°C with 5% CO2 in air. Follicular diameters were evaluated on days 0, 4, 8 and 12. After 12 days of culture, follicular survival analysis was performing by using calcein-AM and ethidium homodimer. Before and after culture, follicles were fixed in paraformaldehyde for histological evaluation. Follicular diameter at different days of culture were compared using the Kruskal–Wallis test, while the percentages of viable follicles were analyzed by chi-squared test (P < 0.05). Results showed that follicles cultured in the presence of KSR at both concentrations presented higher follicular survival rates than those cultured in control medium alone or supplemented with FBS or BSA. Conversely, the presence of KSR, BSA or FBS did not increase follicular diameter after 12 days of culture. Histology analysis showed that, among the tested treatments, follicles cultured in the presence of KSR had preserved rounded oocytes, juxtaposed granulosa cells and intact basal membrane. In conclusion, supplementation of culture medium with KSR increases the follicular survival of bovine secondary follicles cultured in vitro.

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa

Cryobiology ◽  
2020 ◽  
Vol 92 ◽  
pp. 208-214 ◽  
Author(s):  
Seyed Mohamad Javad Taher-Mofrad ◽  
Tohid Rezaei Topraggaleh ◽  
Niloofar Ziarati ◽  
Mustafa Numan Bucak ◽  
Mohammad Nouri ◽  
...  
Keyword(s):  
Human Spermatozoa ◽  
Serum Replacement ◽  
Knockout Serum Replacement ◽  
Serum Substitute

214 Different pluripotency maintenance supplements affect the reprogramming process and pluripotency state of bovine-induced pluripotent stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 235
Author(s):  
R. Botigelli ◽  
N. Pieri ◽  
B. Bessi ◽  
R. de Castro ◽  
K. Recchia ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Cell Reprogramming ◽  
Rt Pcr ◽  
Serum Replacement ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement ◽  
Pluripotency Maintenance

After the emergence of induced cell reprogramming, achieved through the addition of Yamanaka transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) to somatic cells, the number of studies regarding induction and maintenance of pluripotency has increased greatly. The success of bovine iPSCs (biPSCs) was first described by Summer et al. (2011 J. Anim. Sci. 89, 2708-2716; https://doi.org/10.2527/jas.2010-3666); however, investigations on the pluripotent state of biPSCs are still needed because different protocols and characterisation profiles have since been used. The aim of this study was to produce biPSC lines supplemented with different pluripotency maintenance agents to improve self-renewal and pluripotency maintenance. For that, bovine fetal (50 days) fibroblasts (3×104) were transduced with lentivirus harbouring mouse OSKM transcription factors. The cells were further cultured in reprogramming medium (Dulbecco's modified Eagle's medium/F12 KO and 20% KSR (knockout serum replacement)) supplemented with basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), bFGF+2i or LIF+2i (where 2i inhibitors are PD0325901 and CHIR99021). The capacity for cell reprogramming was analysed by colony formation and maintenance after manually and enzymatic passaging and alkaline phosphatase (AP) activity detection; additionally, pluripotency state was assessed by reverse transcription (RT)-PCR (pluripotency biomarkers: OCT4, NANOG, and SOX2; naïve state: STELLA, LIFr, and ESRRb; primed state: OTX2 and FGF5; and mouse (m)OSKM and invitro differentiation assay (embryoid body formation). Statistical analysis was performed using the JMP software (SAS Institute Inc.). All treatments were successful at generating colonies after 28 days of mOSKM transduction, with 32 colonies in bFGF (0.53% efficiency), 21 colonies in bFGF+2i (0.35% efficiency), 5 colonies in LIF (0.08% efficiency), and 3 colonies in LIF+2i (0.05% efficiency) treatments/groups. As an initial pluripotency test, all colonies were positive for AP activity at passage 3. The colonies were cultured for at least 25 passages (±200 days) except for those from the LIF+2i treatment, which were not able to remain viable after 15 passages. Gene expression analysis of the pluripotency (naïve and primed) biomarkers in biPSCs by RT-PCR revealed that colonies from the bFGF treatment were upregulated in NANOG, OCT4, (pluripotency biomarkers), and STELLA (naïve biomarker) (P&lt;0.05) compared with bFGF+2i and LIF groups. There were no differences in expression of SOX2 (pluripotency biomarker gene) and naïve/primed biomarkers (OXT2, LIFr, and ESRRb) (P&gt;0.05). Additionally, the relative abundance of mOSKM was not different between groups (P&gt;0.05). For further pluripotency analysis, biPS colonies were tested for the invitro differentiation assay, and all colonies tested were able to form embryoid bodies. In conclusion, bovine fetal fibroblasts were successfully reprogrammed when using OSKM in all medium tested; however, LIF+2i treatment did not grow beyond 25 passages. Further tests should be performed to determine the pluripotency status of these biPSCs. We acknowledge FAPESP for funding (grant nos. 2012/50533-2, 2015/26816-5, and 2016/16841-2).

scholarly journals Establishment of Bovine-Induced Pluripotent Stem Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10489
Author(s):  
Yue Su ◽  
Ling Wang ◽  
Zhiqiang Fan ◽  
Ying Liu ◽  
Jiaqi Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Colony Morphology ◽  
Significant Advance ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

scholarly journals Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency

2015 ◽  
Author(s):  
Asaf Zviran ◽  
Yoach Rais ◽  
Nofar Mor ◽  
Noa Novershtern ◽  
Jacob H Hanna
Keyword(s):  
Pluripotent Stem Cell ◽  
Primordial Germ Cells ◽  
Induced Pluripotent Stem Cell ◽  
Major Pathway ◽  
Serum Replacement ◽  
Epiblast Stem Cells ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement ◽  
Formation Efficiency

Our group has published a study on induced pluripotent stem cell (iPSC) reprogramming (Rais et al. Nature 20131) that reached the following conclusions: a) Mbd3/NuRD is a repressor of inducing naïve pluripotency from mouse Epiblast stem cells (EpiSCs), primordial germ cells (PGCs), murine somatic cells and human secondary fibroblasts; b) Up to 100% iPSC formation efficiency can be achieved via optimized Mbd3/NuRD depletion, in concert with optimized OKSM delivery and naïve pluripotency conditions (2i supplement applied only after 48 hours, human LIF, hypoxia and Vitamin C containing Knockout serum replacement)1. This represented the first proof for deterministic/near-deterministic iPSC reprogramming, and highlighted a previously unappreciated role for Mbd3/NuRD in hampering the re-establishment of pluripotency. Recent reports have seemingly provided contradictory results and attempted to dispute our iPSC efficiency quantifications and/or the role of Mbd3/NuRD in blocking reprogramming2,3. Here we provide a detailed response to these reports based on extended discussions and providing new data. The synthesis presented herein disagrees with claims made by Silva, Hendrich, Bertone and colleagues2,3, and reconfirms that Mbd3/NuRD is a major pathway that inhibits the maintenance and induction of pluripotency1. Further, we foresee that its controlled manipulation is likely to become an integral pathway for inducing and maintaining naïve pluripotency in a variety of species.

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