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Derivation and characterization of human embryonic stem cell lines from poor quality embryos

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Journal of Genetics and Genomics 2009年第4期36卷 229-239页

作者： Weiqiang Liu, Yifei Yin, Xiaolin Long, Yumei Luo, Yonghua Jiang, Wenhong Zhang, Hongzi Du, Shaoying Li, Yuhong Zheng, Qing Li, Xinjie Chen, Baoping Liao, Guohong Xiao, Weihua Wang, Xiaofang Sun Guangzhou Key Laboratory of Reproductive and Genetics Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Guangzhou 510150, China Guangzhou Key Laboratory of Reproductive and Genetic Institute of Gynecology and Obstetrics The Third Affiliated Hospital of Guangzhou Medical College Guangzhou China

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

关键词： embryonic stem ceils poor quality embryos inner cell mass mechanical isolation immunosurgery

Research on Isolation and Clone of Embryonic Stem cell-Like in Bovine

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Agricultural Sciences in China 2002年第4期1卷 450-458页

作者： AN Li-long, YANG Qi, XIAO Mei, FENG Xiu-Liang, YANG Chun-rong, LEI An-min, GAO Zhi-min, DOU Zhong-ying and QIU Huai( Agriculture College of Zhanjiang Ocean University, Zhanjiang 524008 , P. R . China Key laboratory of AnimalReproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China , Northwestern Scienceand Technology University of Agriculture and Forestry, yangling 712100 , P.R. China) Agriculture College of Zhanjiang Ocean University Zhanjiang 524008 P. R. China Key laboratory of Animal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China Northwestern Science and Technology University of Agriculture and Forestry Yangling 712100 P.R. China

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

关键词： Bovine Murine Embryonic stem cell inner cell mass Isolation Clone

Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast cell Feeder Layer

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Agricultural Sciences in China 2002年第4期1卷 459-465页

作者： AN Li-long, XIAO Mei, FENG Xiu-Liang, DOU Zhong-ying, QIU Huai,YANG Qi, LEI An-min, YANG Chun-rong and GAO Zhi-min( Agriculture College of Zhanjiang Ocean University, Zhanjiang 524008 , China Key Laboratory of Animal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China , Northwestern Science and Technology University ofAgriculture and Forestry, Yangling 712100 , P.R. China) Agriculture College of Zhanjiang Ocean University Zhanjiang 524008 China Key Laboratory of Animal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China Northwestern Science and Technology University of Agriculture and Fo Key Laboratory of Animal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China Northwestern Science and Technology University of Agriculture and Forestry Yangling 712100 P. R. China

The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the

关键词： Bovine Murine inner cell mass Culture Behavior

Influence of Blastocysts Morphological Score on Pregnancy Outcomes in Frozen-thawed Blastocyst Transfers: a Retrospective Study of 741 Cycles

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2014年第5期34卷 750-754页

作者：刘琳李艳辉丁晓芳耿育红陈春艳高颖 Reproductive Medical Center Union HospitalTongji Medical CollegeHuazhong University of Science and Technology

The influence of inner cell mass （ICM） and trophectoderm （TE） score on pregnancy out- comes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastosysts transfer was performed. All cycles were divided into four groups based on the number and morphological score of blastocysts： S-ICM B/TE B group （n=91）, the single blastocyst transfer oflCM B and TE B; D-ICM B/TE B group （n=579）, double blastocysts transfer oflCM B/TE B; D-1CM B/TE C group （n=35）, double blastocysts transfer of ICM B/TE C; and D-ICM C/TE B group （n=36）, double blastocysts transfer ofTE B/ICM C. The pregnancy outcomes were compared among the four groups. As compared with D-ICM B/TE C group, the clinical pregnancy rate, implantation rate and multiple pregnancy rate were increased in D-ICM B/TE B group （74.96% vs. 57.14%, 57.43% vs. 37.14%, and .48.62% vs. 25%, respectively, P〈0.05 for all）. Clinical pregnancy rate and implantation rate in D-ICM B/TE B group were also higher than in D-ICM C/TE B group （74.96% vs. 50%, and 57.43% vs. 33.33%, both P〈0.05）. Multivariable Logistic regression analysis indicated that ICM score was a better predictive parameter for clinical pregnancy （OR=3.05, CI 1.70-5.46, P〈0.001）, while the trophectoderm score was a better one for early abortion （OR=0.074, CI 0.03-0.19, P〈0.001）. Clinical pregnancy rate and multiple pregnancy rate in S-ICM B/TE B group were significantly lower than those in D-ICM B/TE B group （46.15% vs. 74.96%, and 2.38% vs. 48.62%, both P〈0.05）, but there was no si~,,niflcant difference in the implantation rate between the two groups. It was suggested that the higher score of ICM and TE may be indicative of the better pregnancy outcomes. The ICM score is a better predictor of clinical pregnancy than TE, while TE score is a better one in predicting early abortion. Sin- gle ICM B/TE B blastocyst transfer in frozen-thawed cycles can also get satisfactory pregnancy out- comes.

关键词： thawed embryos transfer vitrification cryopreservation inner cell mass trophectoderm cell pregnancy outcome

The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores

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The derivation of two additional human embryonic stem cell l...

Murine Fertilized Ovum,Blastomere and Morula cells Lacking SP Phenotype

中华医学会第九次全国物理医学与康复学学术会议

作者： CHEN Hong Rehabilitation Department,Tongji Hospital,Tongji medical college,Huazhong University of Science and Technology,Wuhan,430030

Embryonic stem cells are derived from inner cell mass of blastocysts. They have infinite proliferative capability and can differentiate into all types of adult cells (heart cells,skin cells,neural cells and et al.) of the body under special conditions,therefore,providing unique opportunities for rehabilitation of diseases and disorders with no therapies or at best,par- tially effective ones such as cerebral ischemia,diabetes,heart attack and et ***,immune rejection can lead to the failure of human embryonic stem cell (hES cell) *** approach to address the problem is to establish hES cell line banks that have cells of various HLA *** to the limited source of human embryos and to ethical reasons,the hES cell lines are not readily *** the blastocysts that develop from those discarded D3 embryos with low morphological scores can be used as a source to establish hESC lines,the problem with availability of human embryos may be *** study was undertaken to determine whether discarded day 3 embryos with low morphological scores could develop into blastocysts and produce hES cell lines. Methods D3 embryos with low morphological scores were assessed according to Steer CV scoring system and cultured in sequential culture medium to blasto- cyst *** were evaluated by Gardner’s scale and the relation between blastocyst formation rate and D3 score was investigated. (1) Trophectoderm cells were separated from the ICMs by immunosurgery and isolated ICMs were cultured for 5-8 days on mitomycin-treated mouse embryonic fibroblasts (MEF).Colonies derived from the ICMs were passed every 4-7 days and evaluated for cell surface markers including AKP,OCT-4,SSEA-4,SSEA-1, TRA-1-60,TRA-1-81,differentiation potentials and karyotypes. Results (1) A total of 19 blastocysts were obtained from 130 embryos (quality score＜ 16)and blastulation rate was 14.62%.Most of blastocysts cavitated or ex- panded on day 6.A total of 10.17% had full or ex

关键词： embryonic stem cell embryo blastocyst inner cell mass in vitro fertilization in vivo differentiation in vitro differentiation

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Murine Fertilized Ovum,Blastomere and Morula Cells Lacking S...