39 results about "Inner cell mass" patented technology

A method for isolating an inner cell mass comprising the steps of immobilizing a blastocyst stage embryo having a zona pellucida, trophectoderm, and inner cell mass, creating an aperture in the blastocyst stage embryo by laser ablation, and removing the inner cell mass from the blastocyst stage embryo through the aperture. The aperture is through the zona pellucida and the trophectoderm. The laser ablation is acheived using a non-contact diode laser. The inner cell mass removed from the blastocyst stage embryo is used to establish human Embryonic Stem Cell lines.

An improved method of producing differentiated progenitor cells comprising obtaining inner cell mass cells from a blastocyst and inducing differentiation of the inner cell mass cells to produce differentiated progenitor cells. The differentiated progenitor cells may be transfected such that there is an addition, deletion or alteration of a desired gene. The differentiated progenitor cells are useful in cell therapy and as a I source of cells for the production of tissues and organs for transplantation. Also provided is a method of producing a lineage-defective human embryonic stem cell.

The present inventors discovered that genes could be introduced specifically into trophectodermal cells with high efficiency, by infecting blastocysts with viral vectors carrying an arbitrary polynucleotide, or by using a nucleic acid transfection reagent in blastocysts, from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from infection of viruses and the like) is removed. This method has no risk of infecting cells of the inner cell mass, which develops into a fetus in the future, with the introduced polynucleotide because the trophectoderm serves as a barrier. The present invention provides methods for introducing foreign genes into only placenta but not fetus, which enables rescue of genetically mutant animals from embryonic lethality due to placental abnormality and allows their birth. Furthermore, it is possible to analyze expression and effect of genes that regulate placental formation or placental function by using these methods.

The invention discloses a differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae. The method comprises the following steps: 1, carrying out immune combination on an anti-CDX2 monoclonal antibody which is adopted as a primary antibody and CDX2 molecules which are specifically expressed in the trophoblastic cells of the cattle blastulae through an immunostaining process; 2, the cattle blastulae which are cleaned and are combined with the primary antibody are immunostained with an antibody which is labeled by red fluorescence and can combine with the anti-CDX2 monoclonal antibody as a secondary antibody through the immunostaining process; and 3, whole nuclei of the cattle blastulae are subjected to fluorescent staining after completing the immunostaining to form the differential staining. On the basis of a case that CDX2 proteins specifically express in the trophoblastic cells and do not express in the inner cell mass cells, the method of the invention allows the differential staining to be carried out based on the red immunostaining of the CDX2 molecules and the blue staining of DAPI nuclei, so the accuracy is 100%, and obtained pictures are intelligible and beautiful.

The invention discloses a method for analyzing embryo quality based on molecular immunoassay of mammalian blastulas. The expressions of key transcription factors OCT4 (octamer-binding transcription factor 4) and CDX2 (caudal-related homeodomain transcription 2) respectively expressing in inner cell masses and trophectoderm cells are used as the basis for analysis of the mammalian blastulas, and cellular level and molecular level analyses are carried out based on the immunostaining results of OCT4 and CDX2, so that the conventional blastula quality assessment method is covered, and the lineage differentiation with higher accuracy is proposed to conduct assessment; the blastulas with a proper ratio of the number of the inner cell masses to the number of the trophectoderm cells and complete first lineage differentiation are used as quality blastulas. Compared with the conventional blastula quality assessment method, the method disclosed by the invention not only can assess the blastula quality at a cellular level, but also can accurately assess the blastula quality and subsequent development potential at a molecular level, and the method is simple and high in accuracy and can provide technical support for embryonic implantation.

The invention discloses an embryo pregnancy state intelligent prediction method and system. The method comprises the steps of collecting embryo images within the time of D1 to D6; inputting the embryoimage into a fragment proportion prediction network model, a blastocyst cavity and inner cell mass grade prediction network model and a trophoblast grade prediction network model to calculate and outputting a predicted fragment proportion, a predicted blastocyst cavity proportion, a predicted inner cell mass grade and a predicted trophoblast grade of the embryo image; and inputting into an embryopregnancy rate state prediction machine learning model to calculate and output an embryo pregnancy rate prediction result. According to the intelligent prediction method and the system, the whole embryo development process is monitored, the embryo pregnancy rate is obtained through calculation by means of the comprehensive scoring function, manual intervention is not needed in the prediction process, and doctors can be helped to quickly and accurately judge embryo scores.

Novel cultured inner cell mass (CICM) cells, and cell lines, derived from ungulates, in particular, pigs and cows, and methods for their preparation are provided. The subject CICMs possess similar morphology and express cell markers identically or substantially similarly to ICMs of undifferentiated developing embryos for prolonged culturing periods. Heterologous DNA is inserted into the subject CICM cells and cell lines so as produce transgenic CICM cell which are introduced into non-human fertilized embryos to produce transgenic chimeric embryos. The transgenic chimeric embryos are transferred into recipient females where they are permitted to develop into transgenic chimeric fetuses. Recipient females give birth to transgenic chimeric animals which are capable of transmitting the heterologous DNA to their progeny. Transgenic CICM cells are also used to produce cloned transgenic embryos, fetuses and offspring.

The present invention provides a method for parthenogenetic activation by injection and subsequent removal of a sperm into a mammalian cell, for example, an oocyte, an embryo, a blastomere, an inner cell mass cell, or a morulae cell.

The invention relates to a method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing said aggregate to obtain an artificial blastocyst. This artificial blastocyst has a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. The invention further relates to a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a fetus or a live animal. The invention further pertains to an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

The invention provides a kit for detecting mammal blastula inner cell mass (ICM)/trophectoderm (TE) ratio and cell apoptosis. The kit comprises a PBS solution containing 0.5% of BSA, a PBS solution containing 2% of paraformaldehyde, a PBS solution containing 0.5% of Triton X-100 and 0.05% of Tween20, a PBS solution containing 10% of goat serum and 0.05% of Tween20, a mouse anti-CDX2 primary antibody, a rabbit anti-caspase-3 primary antibody, an isothiocyanic acid green fluorescein-labeled goat anti-mouse IgG secondary antibody, an Alexa Fluor 594 red fluorescein-labeled goat anti-rabbit IgG secondary antibody, and a PBS solution containing 20 micromoles per liter of H33342. The kit is designed based on an immunofluorescence technology, respectively utilizes a trophoblast cell surface antigen CDX2 and important caspase-3 produced in cell apoptosis as target points, respectively utilizes the primary antibodies respectively aiming at CDX2 and caspase-3 and the secondary antibodies respectively carrying the fluorescein labels as reactants for an antigen-antibody reaction, utilizes the DNA dye to dye the DNAs in cells and realizes synchronous detection of blastula ICM/TE ratio and cell apoptosis in the same embryo. The kit has good repeatability and high efficiency.

A method for obtaining a stable xeno-free hBS cell line, xeno-free hBS cell lines obtained according to said method and use thereof. The method comprises the steps of:

• • i) removing the zona pellucida from a blastocyst to obtain trophectoderm-enclosed inner cell mass by a xeno-free procedure,
  • ii) at least partly removing the trophectoderm to obtain isolated inner cell mass cells by a xeno-free procedure,
  • iii) placing the inner cell mass cells on a layer of human feeder cells in a xeno-free medium,
  • iv) co-culturing of the inner cell mass cells with human feeder cells for a time period of from about 5 days to about 50 days in a xeno-free medium,
  • v) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by a xeno-free procedure,
  • vi) selectively, transferring the inner cell mass cells or cells derived thereof to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells,
  • vii) propagating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line.

The xeno-free hBS cell line is suitable for use in medicine and in in vitro testing.

The invention provides a culture medium and a method for establishing and maintaining early embryonic-like cells of mammals. The culture medium is used for culturing pluripotent stem cells (PSC) of mammals, has definite chemical components, contains a basic culture medium for culturing stem cells, and is supplemented with an S-adenosylhomocysteine hydrolase (SAH)/polycomb inhibition complex (PRC)/EZH2 inhibitor and an HDAC inhibitor. By means of the culture medium, primate (human and non-human) animal PSC can be converted into pre-implantation internal cell mass-like cells (ICLC) or 8-cell embryonic-like cells (8CLC).

The invention discloses a simple and rapid pig blastaea differential dyeing method and belongs to the technical field of developmental biology. The method comprises the following steps: performing surfactant TritonX100 permeable membrane treatment on the pig blastaea, and performing differential dyeing on trophoderm cells on the outer layer of the blastaea and inner cell mass cells by utilizing the difference of two nucleus dyes in molecular size. According to the method, the experimental steps of performing differential dyeing on the pig blastaea are simplified, the experimental time is greatly shortened, and the experimental cost is reduced.

The invention discloses a tissue stem cell separation and function evaluation system. The tissue stem cell separation and function evaluation system comprises the following steps: placing a male mouse and a female mouse together for mating, resting the fertilized female mouse for several days to obtain oocytes in the fertilized mouse body, and reprogramming, activating and culturing the obtained oocytes in vitro to form embryonic vesicles; after embryonic vesicles are cultured, removing zona pellucida or parts of the zona pellucida from the embryonic vesicles, then removing trophoblasts from the obtained embryonic vesicles, and separating inner cell clusters. The gene sequence of the mouse is similar to that of human beings, the similarity between the whole genome of the mouse and the whole genome of the human beings is extremely high, similar characters of many diseases which are difficult to cure by the human beings can be found on the mouse body, disease treatment genes are found through experiments, and the function of each component after tissue stem cell separation is easier to obtain.

The invention provides a method for constructing a porcine naive embryonic stem cell line, and belongs to the technical field of cell biology. The method combines iPSCs technology with traditional embryonic stem cell technology to first establish a transgenic positive porcine fetal fibroblast cell line that simultaneously expresses mOCT4, mSOX2, mKLF4 and mc-MYC; obtaining a transgenic positive porcine blastocyst; and inoculating an inner cell mass of the transgenic positive porcine blastocyst to a cell feeder layer, starting DOX-induced exogenous transcription factor expression to obtain a porcine embryonic stem cell line; after the porcine embryonic stem cell line is stable in passage, stopping the expression of the exogenous mOCT4, mSOX2, mKLF4 and mc-MYC, and maintaining the stemness of stem cells by activated endogenous pOCT4, pSOX2, pKLF4 and pc-MYC to obtain the porcine naive embryonic stem cell line. The stem cells for genetic modification and long-term culture are provided forstudy of transgenic cloned pigs.

The invention discloses an extraction and separation method of embryonic stem cells. The extraction and separation method comprises the following steps: S1, the ovary of a mammal is excised, and exogenous hormone is given to make an embryo continue to develop but implantation is delayed; S2, after the embryo develops for 4-6 days, blastocysts are taken by flushing from a uterus for culture, such that trophoblastic cells grow and feeder layer cells are pushed away, and are spread on the bottom wall of a culture dish; and S3, inner cell mass (ICM) cells proliferate, and vertically grow upward toform egg cylindrical structures, the cylindrical structures are picked out by using a fine glass needle under a microscope, and digestive passaging is performed to obtain embryonic stem cell-like colonies. According to the extraction and separation method, a tissue culture method is adopted to extract and separate the embryonic stem cell; by inoculating the blastocysts on a feeder layer, the implantation of blastocysts in vivo is simulated, which is closer to the natural development process; inner cell populations proliferate vigorously, and thus, embryonic stem cell-like colonies are relatively easy to obtain; and the problems that an existing separation method of the embryonic stem cells is difficult, the cells are easy to damage, and the separation of the embryonic stem cells is influenced are solved.

The invention discloses a double staining method for a bovine in-vitro fertilization blastocyst. The method comprises the steps that the bovine in-vitro fertilization blastocyst which is cultured for 7-9 days is processed with 0.5% pronase for 55 seconds, washed in a PBS for 2-3 times, stained in PI (1% TritonX-100/PBS) with the concentration of 100 mug/ml for 20 seconds, washed in the PBS for 2-3 times, transferred into Hoechst33342 with the concentration of 45 mug/ml to be stained for 50 seconds, washed in the PBS for 2-3 times and then processed through preforming; the bovine in-vitro fertilization blastocyst is placed under an inverted fluorescence microscope after preforming is finished, observing and photographing are performed under ultraviolet light, cell nucleuses of an inner cell mass (ICM) are blue, and cell nucleuses of trophoderm cells are red. According to the double staining method, staining can be completed in 3 minutes, but at least 47 minutes are needed in previous reports; accordingly, the time needed by double staining of the blastocyst is greatly shortened, the efficiency is improved, and the bovine blastocyst quality can be quickly evaluated.

This disclosure provides ungulate embryonic stem cells (ESCs) derived from the inner cell mass of pre-implantation blastocysts or pluripotent cells from embryos. From an agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genetic engineering, and providing an experimental tool for studying human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors.

The invention discloses a blastocele fluid collecting method. The method includes steps: microneedle preparation, to be more specific, adopting a needle pulling instrument, a needle breaking instrument and a needle grinding instrument to make an injection needle in an inner diameter of 10micron and a fixing needle with a 40micron needle opening, wherein the tip of the injection needle is sharpenedby pulling; blastocele fluid collection, to be more specific, under a micromanipulator, fixing one side of an inner cell mass with the fixing needle, sucking fluid in a blastocele by the injection needle piercing into the blastocele, adding into 1MuLDPBS liquid drops, and after collection is completed, using the injection needle for transferring fluid from the blastocele to a 200microliter centrifugal tube; blastocele fluid DNA content detection. The method has advantages that by the self-made thin injection needle, the blastocele can be pierced easily, subsequent blastosphere development isslightly affected, high accuracy is realized, fluid in the single blastocele can be collected, and the novel method is provided for genetic screening before embryo implantation is provided.