To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Astrocytes/*cytology ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; Neurons/*cytology ; Stem Cells/*cytology ; Tretinoin/pharmacology

CRISPR-Cas Systems ; Embryo, Mammalian ; cytology ; metabolism ; Germ Cells ; cytology ; metabolism ; Humans ; RNA Editing ; genetics

BACKGROUNDThe efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.

METHODSHuman ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.

RESULTSEighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.

CONCLUSIONSVitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

Cell Differentiation ; Cell Survival ; Cryopreservation ; methods ; Embryo, Mammalian ; cytology ; Humans ; Osmotic Pressure ; Stem Cells ; cytology

OBJECTIVETo establish a coculture and a sequential system for human early embryo culture.

METHODSThe endometrial tissue was digested enzymatically and cultured to achieve generated and cryo-thawed endometrial monolayer cells. The generated and cryo-thawed monolayer cells were cocultured with human 2PN embryos and transferred to sequential medium every 48 hours.

RESULTSHuman endometrial cells had viability in vitro culture. The autologously generated and cryo-thawed monolayer cells were successfully obtained, and 74.04% of the cryo-thawed cells were successfully used in coculturing human early embryos. The embryos developed well, with the clinical pregnancy rate of 68.83% and the implantation rate of 44.23%.

CONCLUSIONThe autologous endometrial cell coculture and sequential culture system for human early embryo development provides a feasible method for studying human embryo development and implantation so as to improve embryo quality.

Cells, Cultured ; Coculture Techniques ; Culture Media ; Embryo, Mammalian ; cytology ; Endometrium ; cytology ; Female ; Humans

Human embryonic stem cells can be induced to differentiate into all kinds of cells in vitro to be applied to clinical medicine and scientific research. Because they have to be isolated from human embryos, any attempt to establish human stem cell line is prohibited by religion and ethics in some areas and countries. Parthogenetic embryonic stem cells have similar capacity of totipotency and proliferation, and can be established from parthenogenetic activation of discarded oocytes. This paper reviews the progress in the studies of the establishment of parthenogenetic embryonic stem cell line and its differentiation capacities.
Animals ; Cell Differentiation ; Embryo, Mammalian ; cytology ; Humans ; Parthenogenesis ; Stem Cell Transplantation ; Stem Cells ; cytology

OBJECTIVETo evaluate the accuracy of a scoring system combining zygote and embryo morphology in predicting the outcome of in vitro fertilization (IVF) treatment.

METHODSIn a study group, 117 consecutive IVF or intracytoplasmic sperm injection (ICSI) cycles with embryo transfer were carried out and 312 embryos were scored using a combined scoring system (CSS) of zygote and embryo morphology before transplantation. In a control group, a total of 420 IVF or ICSI cycles were carried out and 1176 embryos were scored using a cumulative embryo score (CES). The effects of the combined scoring system on the embryo implantation rate and pregnancy rate per cycle were analyzed.

RESULTSUsing the combined scoring system, the embryo implantation rate (27.6%) and the clinical pregnancy rate (48.7%) were significantly higher than those in the control group (20.8% and 38.6%, respectively). Also, the implantation rate of embryos scoring>or=70 (38.5%: 82 sacs/213 embryos) was significantly higher (P<0.001) than that of embryos scoring<70 (4%: 4 sacs/99 embryos). The pregnancy rate of patients with embryos scoring>or=70 using the combined scoring system (66.7%) was significantly higher (P<0.001) than that of patients with embryos scoring>or=20 using the cumulative embryo score (59.0%).

CONCLUSIONThe results suggest that selecting embryos with a high score (>or=70) using the combined scoring system could increase the implantation rate and pregnancy rate, and that using a scoring system combining assessments of human zygotes and pre-implantation embryos might predict IVF outcomes more accurately than using a cumulative embryo score.

Embryo, Mammalian ; cytology ; Female ; Fertilization in Vitro ; methods ; Humans ; Pregnancy ; Treatment Outcome ; Zygote ; cytology

OBJECTIVETo induce nonparenchymal mesenchymal stem cells (NPMSCs) differentiating into functional hepatocyte-like cells in vitro, and to identify the molecular biology and functional characteristics of those hepatocyte-like cells.

METHODSHuman NPMSCs were isolated and cultured with cell culture technique. NPMSCs were induced (on 1% Matrigel as a matrix and then submitted to 2.5 mmol/L AZA pretreatment for 10-12 h), by adding HGF 10 microg/L + FGF4 10microg/L + HGM into the culture medium. The characteristics of proliferation and growth of human NPMSCs were studied with methyl thiazolyl tetrazolium (MTT). The phenotypes of NPMSCs were identified by flow cytometry, immunohistochemistry and RT-PCR. Albumin (Alb) levels in culture supernatants were determined with ELISA. Staining for glycogen of undifferentiated NPMSCs and NPMSCs derivated hepatocyte-like cells was conducted with periodic acid-Schiff (PAS) test.

RESULTSGrowth and division of adherent cells obtained from fetal livers were good and the amount of NPMSCs resourced from each fetus could be amplified to 109 cells after 10 serial subcultivations. The phenotype of NPMSCs was CD166 positive and CD34 negative. The shape of NPMSCs plated on Matrigel with FGF4 and HGF changed from long fusiform to polygonal or round on days 21-28. The rate of cell rounding was 40% and the ratio of dikaryocytes was 5%. Immunohistochemical and RT-PCR detection showed that undifferentiated NPMSCs expressed few alpha fetoprotein (AFP) and AFP mRNA, and did not express any of the liver-specific transcription factors or cytoplasmic markers. Many cells in early induction expressed GATA4, AFP and CK18 proteins and their mRNAs, and their expressions were reduced at the late induction, but the expressions of Alb, CK18, GST-and hepatocyte transcription factor HNF1increased gradually. The ratio of Alb and CK18 positive cells was 60%. Undifferentiated NPMSCs did not produce Alb. Alb production by induced NPMSCs increased in a time-dependent manner. Glycogen storage was first seen on day 14, and maximum levels were seen after day 28.

CONCLUSIONSThere are MSCs among nonparenchymal cells of fetal livers. A high ratio of hepatocyte-like cells was obtained under our induction condition. NPMSCs differentiate firstly into hepatocyte precursors, and then differentiate into mature hepatocytes and hepatocyte-like cells with positive hepatocyte markers. The induced NPMSCs have hepatocyte specific functional features.

Cell Differentiation ; Cell Separation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fetus ; cytology ; Hepatocytes ; cytology ; Humans ; Liver ; embryology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs).

METHODSSSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR.

RESULTSSSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes.

CONCLUSIONThe feeder layer of hEFs supports the growth of human spermatogonial stem cells.

Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; Humans ; Male ; Spermatogonia ; cytology ; Stem Cells ; cytology

UNLABELLEDEmbryonic germ (EG) cells are pluripotent cells derived from primordial germ cells (PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells in vitro can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The aim of this study is to investigate the potentiality of human EG cells differentiation into cardiomyocytes. Inducing human EG cells with the method of murine ES cells differentiation into cardiomyocytes, supplemented with 0.75%-1% DMSO, 20% NBS, 10(-7) mM RA and 20% cardiomyocytes conditioned medium.

RESULTS20 heart-like (rhythmic beating cell masses were observed in vitro culture and delayed human EG cells, which beat spontaneously from 20-120 times per minute and maintained beating for 2-15 days, periodic acid's staining (PAS), Myoglobin and a-actin immunological histology positive were all positive and reacted with K+, Ca2+ and adrenalin. Relatively unorganized myofibrillar bundles or more organized sarcomeres, z-bands or a gap junction, the presence of desmosomes in a few cells of the cell masses was observed with transmision electron microscope, which initially demonstrated that these cells were cardiomyocytes. We could not get rhythmly beating cardiomyocytes with 0.75%-1% DMSO, 10-7 mM RA and 20% cardiomyocytes conditioned medium,but in which the percentage of cardiac alpha-actin immunostaining positive cells were increased. The results first demonstrated that human EG cells can differentiate into rhythmic beating cardiomyocytes in vitro and suggests that human EG cells may represent a new potent resource for cardiomyocytes transplantation therapy for myocardium infarction.

Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; cytology ; Germ Cells ; cytology ; Humans ; Myocytes, Cardiac ; cytology ; Pluripotent Stem Cells ; cytology