scholarly journals 151A SIMPLE AND FAST METHOD FOR CONCURRENT DIFFERENTIAL STAINING AND TUNEL LABELLING OF BOVINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 197 ◽  
Author(s):  
A.A. Fouladi-Nashta ◽  
R. Alberio ◽  
B. Nicholas ◽  
K.H.S. Campbell ◽  
R. Webb
Keyword(s):  
Embryo Quality ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Molecular Probes ◽  
Rnase A ◽  
Differential Staining ◽  
Short Exposure ◽  
Fast Method ◽  
A Cell ◽  
Tunel Labelling

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.

119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep

2020 ◽  
Vol 32 (2) ◽  
pp. 186
Author(s):  
D. Anzalone ◽  
M. Czernik ◽  
L. Palazzese ◽  
Y. Ressaissi ◽  
P. Scapolo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Window ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Basic Research ◽  
Cell Number ◽  
Differential Staining ◽  
Assisted Reproductive Technique ◽  
Significant Difference

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 226 ◽  
Author(s):  
E. P. López-Damián ◽  
T. Fiordelisio ◽  
M. A. Lammoglia ◽  
M. Alarcón ◽  
M. Asprón ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Embryo Quality ◽  
Developmental Stages ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Nile Red ◽  
Blastocyst Stage ◽  
Transfer Program

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P < 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P < 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P < 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P < 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P < 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P < 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P < 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs. Research funding provided by UNAM-DGAPA-PAPIIT IN200810.

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

scholarly journals Multiscale analysis of single and double maternal-zygotic Myh9 and Myh10 mutants during mouse preimplantation development

2020 ◽  
Author(s):  
Markus Frederik Schliffka ◽  
Anna-Francesca Tortorelli ◽  
Özge Özgüç ◽  
Ludmilla de Plater ◽  
Oliver Polzer ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Preimplantation Development ◽  
Mammalian Development ◽  
Mammalian Embryo ◽  
Fate Specification ◽  
Heavy Chains ◽  
A Cell ◽  
The Impact

AbstractDuring the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavages divisions, morphogenetic movements and lineage specification. Recent studies identified the essential role of actomyosin contractility in driving the morphogenesis, fate specification and cytokinesis leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin-II heavy chains (NMHC) to characterize them using multiscale imaging. We find that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most attempts of cytokinesis. We find that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

2016 ◽  
Vol 28 (2) ◽  
pp. 208
Author(s):  
N. G. Alves ◽  
I. J. Ascari ◽  
L. S. A. Camargo ◽  
J. Jasmin ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
Embryo Quality ◽  
Linear Models ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Quality Data ◽  
Trophoblast Cells ◽  
Melatonin Concentration

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P > 0.05). The total number of cells was not affected (P > 0.05) by melatonin, but it was decreased (P < 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P > 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P < 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

scholarly journals Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Sergey Rodin ◽  
Liselotte Antonsson ◽  
Colin Niaudet ◽  
Oscar E. Simonson ◽  
Elina Salmela ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
A Cell ◽  
Hes Cells ◽  
Free Environment ◽  
Different Cell Types ◽  
E Cadherin

Abstract Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

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