233 results about "Trophoblast" patented technology

The present invention provides methods and compositions for specifically identifying a fetal cell. An initial screening of approximately 400 candidate genes by digital PCR in different fetal and adult tissues identified a subset of 24 gene markers specific for fetal nucleated RBC and trophoblasts. The specific expression of those genes was further evaluated and verified in more defined tissues and isolated cells through quantitative RT-PCR using custom Taqman probes specific for each gene. A subset of fetal cell specific markers (FCM) was tested and validated by RNA fluorescent in situ hybridization (FISH) in blood samples from non-pregnant women, and pre-termination and post-termination pregnant women. Applications of these gene markers include, but are not limited to, distinguishing a fetal cell from a maternal cell for fetal cell identification and genetic diagnosis, identifying circulating fetal cell types in maternal blood, purifying or enriching one or more fetal cells, and enumerating one or more fetal cells during fetal cell enrichment.

The present invention relates to a non-invasive method of retrieving and identifying cells particularly fetal cells and trophoblastic cells. The invention includes methods for use of the cells for identifying chromosomal abnormalities and mutations particularly for prenatal diagnosis by performing genetic diagnosis for chromosomal and single gene disorders. The invention also includes methods of confirming cells of fetal origin.

The present invention relates to a composition comprising at least one mRNA encoding a combination of antigens capable of eliciting an (adaptive) immune response in a mammal, wherein the antigens are selected from the group consisting of 5T4 (Trophoblast glycoprotein, TPBG), Survivin (Baculoviral TAP repeat-containing protein 5; BIRC5), NY-ESO-1 (New York esophageal squamous cell carcinoma 1, CTAG1B), MAGE-C1 (Melanoma antigen family C1), MAGE-C2 (Melanoma antigen family C2), and MUC1 (Mucin 1). The invention furthermore relates to a vaccine comprising at least one mRNA encoding such a combination of antigens, and to the use of said composition (for the preparation of a vaccine) and / or of the vaccine for eliciting an (adaptive) immune response for the treatment of lung cancer, preferably of non-small cell lung cancer (NSCLC), and diseases or disorders related thereto. Finally, the invention relates to kits, particularly to kits of parts, containing the composition and / or the vaccine.

A non-invasive, risk-free method of prenatal diagnosis is provided. According to the method of the present invention transcervical specimens are subjected to trophoblast-specific immuno-staining followed by FISH, PRINS, Q-FISH and / or MCB analyses and / or other DNA-based genetic analysis in order to determine fetal gender and / or identify chromosomal and / or DNA abnormalities in a fetus.

Existence of human trophoblast stem (hTS) cells has been suspected but unproved. The isolation of hTS cells is reported in the early stage of chorionic villi by expressions of FGF4, FGFR-2, Oct4, Thy-1, and stage-specific embryonic antigens distributed in different compartments of the cell. hTS cells are able to derive into specific cell phenotypes of the three primitive embryonic layers, produce chimeric reactions in mice, and retain a normal karyotype and telomere length. In hTS cells, Oct4 and fgfr-2 expressions can be knockdown by bFGF. These facts suggest that differentiation of the hTS cells play an important role in implantation and placentation. hTS cells could be apply to human cell differentiation and for gene and cell-based therapies.

A non-invasive, risk-free method of prenatal diagnosis is provided. According to the method of the present invention transcervical specimens are subjected to trophoblast-specific immuno-staining followed by FISH, PRINS, Q-FISH and / or MCB analyses and / or other DNA-based genetic analysis in order to determine fetal gender and / or identify chromosomal and / or DNA abnormalities in a fetus. Also provided is a method of in situ chromosomal, DNA and / or RNA analysis of a prestained specimen by incubating the prestained specimen in ammonium hydroxide. Also provided is a method of identifying embryonic cells according to a nucleus / cytoplasm ratio of at least 1:1 and the presence of at least variably condensed chromatin.

The invention provides for the production of several humanized murine antibodies specific for the antigen LK26, which is recognized by the murine antibody LK26. This antigen is expressed in all choriocarcinoma, teratocarcinoma and renal cancer cell lines whereas it is not expressed on cell lines of leukaemias, lymphomas, neuroectodermally-derived and epithelial tumour cell lines (excepting a small subset of epithelial cell lines). Furthermore, whereas renal cancer cell lines express the LK26 antigen, normal renal epithelial cells do not. Similarly, with the exception of the trophoblast, all normal adult and fetal tissues tested are negative for the LK26 phenotype. The invention also provides for numerous polynucleotide encoding humanized LK26 specific antibodies, expression vectors for producing humanized LK26 specific antibodies, and host cells for the recombinant production of the humanized antibodies. The invention also provides methods for detecting cancerous cells (in vitro and in vivo) using humanized LK26 specific antibodies. Additionally, the invention provides methods of treating cancer using LK26 specific antibodies.

Existence of human trophoblast stem (hTS) cells has been suspected but unproved. The isolation of hTS cells is reported in the early stage of chorionic villi by expressions of FGF4, fgfr-2, Oct4, Thy-1, and stage-specific embryonic antigens distributed in different compartments of the cell. hTS cells are able to derive into specific cell phenotypes of the three primitive embryonic layers, produce chimeric reactions in mice, and retain a normal karyotype and telomere length. In hTS cells, Oct4 and fgfr-2 expressions can be knockdown by bFGF. These facts suggest that differentiation of the hTS cells play an important role in implantation and placentation. hTS cells could be apply to human cell differentiation and for gene and cell-based therapies.

The invention relates to an in-vitro pure culture method for natural killer cells. The method includes the following steps that peripheral blood is collected, single karyocyte is separated, magnetic beads combined with specific antibodies are adopted to remove the NK cells, and NK cell populations are obtained; the NK cell populations are put into a culture bottle coated with multiple antibodies,and are subjected to activation culture with a complete medium containing IL-2, IL-7, IL-15, anti-CD16, OK432 and inactivated autologous serum, and inactivated NK cell populations are obtained; the inactivated NK cell populations are put into a culture bottle free of antibody coating and continue to carry out multiplication culture, and the natural killer cells are obtained. Any heterologous serumingredients and trophoblast cells are not used in the in-vitro pure culture method, and high amplification efficiency can be wholly obtained; meanwhile, the purity of the amplified NK cells reaches 90% or above, in-vitro efficient pure culture is achieved, and the clinical application requirement is conveniently met.

The invention provides for the production of several humanized murine antibodies specific for the antigen LK26, which is recognized by the murine antibody LK26. This antigen is expressed on all choriocarcinoma, teratocarcinoma and renal cancer cell lines whereas it is not expressed on cell lines of leukaemias, lymphomas, neuroectodermally-derived and epithelial tumor cell lines (excepting a small subset of epithelial cell lines). Furthermore, whereas renal cancer cell lines express the LK26 antigen, normal renal epithelial cells do not. Similarly, with the exception of the trophoblast, all normal adult and fetal tissues tested are negative for the LK26 phenotype. The invention also provides for numerous polynucleotide encoding humanized LK26 specific antibodies, expression vectors for producing humanized LK26 specific antibodies, and host cells for the recombinant production of the humanized antibodies. The invention also provides methods for detecting cancerous cells (in vitro and in vivo) using humanized LK26 specific antibodies. Additionally, the invention provides methods of treating cancer using LK26 specific antibodies.

The invention discloses a serum-free culture medium used for culturing and amplifying skin keratinocytes in vitro. It adds amino acid, vitamin, hormone, growth gene, trace elements, and antioxidant, thus able to accelerate cell breeding and delay consenescence of the keratinocytes and makes them amplified in vitro by more times. Using the culture medium to make primary culture and subculture needs no trophoblast cells and no extracellular substrates like collagen, etc prepaved at the bottom of the culture bottle to help the keratinocytes adhere to the wall and grow.

A human corneal limbal stem cell-amnion tissue engineering complex using human corneal limbal fibroblasts as the trophoblast is characterized in that: it is close to the human corneal epithelium form in histology form, and is analogous to the human corneal epithelium in immunology phenotype, its ultrastructure shows that the connection between the cells grows mature and the connection between the epithelia and the amnion carrier is a close conglutination. The culture method includes the following steps: (1) preparing the amnion for culturing the human corneal limbal stem cell in vitro; (2) preparing the human corneal limbal fibroblast trophoblast; (3) preparing the human corneal epithelia suspension; (4) culturing the human corneal limbal stem cell-amnion complex. The human corneal limbal stem cell-amnion complex has analogous phenotype to the human corneal epithelium, and provides a safe and effective biology tissue engineering product to treat corneal limbal stem cell defective eye diseases.

This invention provides a fluid therapeutic placental product comprising placental cells and a placental dispersion comprising placental factors. The placental cells and the placental dispersion are derived from placental tissue. A placental tissue can optionally be an amnion, chorion, or a trophoblast-depleted chorion. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

The disclosure provided herein relates generally to mesenchymal-like stem cells “hES-T-MiSC” or “T-MSC” and the method of producing the stem cells. The method comprises culturing embryonic stem cells under conditions that the embryonic stem cells develop through an intermediate differentiation of trophoblasts, and culturing the differentiated trophoblasts to hES-T-MSC or T-MSC, T-MSC derived cells and cell lineages “T-MSC-DL” are also described. Disclosed also herein are solutions and pharmaceutical compositions comprising the T-MSC and / or T-MSC-DL, methods of making the T-MSC and T-MSC-DL, and methods of using the T-MSC and T-MSC-DL for treatment and prevention of diseases, specifically, T-MSC and T-MSC-DL are used as immunosuppressive agents to treat multiple sclerosis and autoimmune diseases.

The first method to cause a culture of human and other primate stem cells to directly and uniformly differentiate into a committed cell lineage is disclosed. Treatment of primate stem cells with a single protein trophoblast induction factor causes the cells to transform into human trophoblast cells, the precursor cells of the placenta. Several protein factors including bone morphogenic protein 4 (BMP4), BMP2, BMP7, and growth and differentiation factor 5 can serve as trophoblast-inducting factors.

The invention provides a method for culturing human induced pluripotent stem cells (iPSCs) by using human bone marrow mesenchymal stem cells as a trophoblast. The method comprises the following steps of: obtaining the human iPSCs from child foreskin fibroblasts by using third to fifth generation human bone marrow mesenchymal stem cells obtained through subculture and culture after thawing from low-temperature freezing, performing amplification culture, and inoculating into trophoblast cells to obtain the human iPSCs. In the method, the human iPSCs are cultured by using the human bone marrow mesenchymal stem cells as the trophoblast cells instead of mouse embryonic fibroblasts in the conventional method, so that the pollution of heterologous cells in a culture system is reduced, that the culture system can make the human iPSCs amplified in vitro is proved, biological characteristics and multipotency of the human iPSCs are maintained for a long time, and the possibility of clinical application of the human iPSCs is provided.

The invention discloses a human umbilical cord blood hematopoietic stem cell (HSC) high-efficiency in vitro amplification technology. According to the invention, umbilical cord mesenchymal stem cells (MSCs) and umbilical cord blood plasma are used in combination for carrying out HSC in vitro amplification. A process comprises steps that: umbilical cord MSCs are cultured as a trophoblast; a special culture solution containing umbilical cord blood plasma is adopted as an amplification medium of the HSCs; and umbilical cord blood karyotes are adopted as amplification initiator cells. According to the invention, exogenous cell factors are not required to be added, treatments such as mitomycin C or gamma-ray 21Gy irradiation are not required by the trophoblast, no 3-dimensional technology is required, and the HSC high-efficiency amplification can be carried out. The technology is advantaged in low cost, convenient amplification, and low product immunogenicity. The technology is suitable for large-scaled productions. The practical application of the technology plays an important role in clinical treatments and researches.

The invention discloses an in-vitro expansion method, a kit and an application of umbilical cord blood NK (nature killer) cells. The in-vitro expansion method of the umbilical cord blood NK cells comprises the processes of activated culture and enrichment culture of the umbilical cord blood NK cells from isolated cord blood mononuclear cells. Compared with existing methods for isolating and expanding the umbilical cord blood NK cells, the in-vitro expansion method has the advantages that cell sorting is needless, trophoblast cells are needless, operation is simple, universality is good, cost is low, adopted reagents do not contain components of animal origin, safety is good, the yield of the NK cells is high, purity of the obtained cord blood NK cells is up to 90% or higher, the total number of the cells can be up to 10<10-11> / part of cord blood, and the killing capacity for tumor cells is high, thereby playing a role in preparation of tumor immunotherapy drugs and tumor immunotherapy.

The present invention discloses a dedicated multi-index protein chip for female infertility detection and a kit thereof. The protein chip consists of a substrate, protein detection indexes arranged on the substrate in an array mode and a contrast coating for the detection indexes. The protein detection indexes cover ovary antigen (OAg), endometrium antigen (EAg), sperm antigen (SAg), zona pellucida antigen (ZAg), human chorionic gonadotropin antigen (HCG), trophoblast antigen (TAg) and cardiolipin antigen (CAg). The present invention overcomes the defects of the existing autoimmunological detection products on market, such as single detection function, lagged detection indexes and no integration. Besides the indexes of the product (patent application No: 200510102466.3), the protein chip also has the ACA index. The density of all indexes can be quantitatively detected, and the cross reaction of all indexes is eliminated. The present invention not only has high specificity and high sensitivity, but also has high accuracy and precision.

The invention discloses a conditional Cas9 expression induced swine trophoblastic cell line and an establishment method and application thereof. The method includes: subjecting a lentiviral vector, for controlling Cas9 gene expression, of a Tet-on system to virus packaging, concentrating and purifying; incubating lentivirus with a typical swine trophoblastic cell line, changing fluid, adding puromycin for drug screening, diluting cells to single ones, and culturing to obtain the single-cell-source swine trophoblastic cell line with Cas9 expression under Tet-on regulation. The swine trophoblastic cell line is simple in operation and convenient to use and has a potential utilization value in researching of key functional genes of some trophoblast cells and screening of swine gene target spots. The cell line is expected to be a significant cell material for researching swine placenta development and applicable to correlation researches on swine early-stage placenta development and uterine pregnancy mechanisms and also has a high reference value in research of human early embryonic development.

A non-invasive, risk-free method of prenatal diagnosis is provided. According to the method of the present invention cell specimens are subjected to molecular and morphological methods which allow trophoblast identification. Trophoblasts identified according to the teachings of the present invention can be further examined to thereby prenatally diagnosing a fetus. Also provided is a method of in situ chromosomal, DNA and / or RNA analysis of a prestained specimen by incubating the prestained specimen in ammonium hydroxide. Also provided is a method of identifying embryonic cells according to a nucleus / cytoplasm ratio of at least 0.3 and the presence of at least variably condensed chromatin.

The invention relates to a stem cell culture medium which contains certain chemical components as follows: water-soluble non-polyelectrolyte polymers, inorganic salts, vitamins, amino acids, glucose, transferrin, insulin or a substitute with the same functions as the insulin (the IGF-1 / 2), and a fibroblast growth factor. The stem cell culture medium containing the certain chemical components is free of additives from an animal source, namely various animal products, trophoblast cells and conditioned mediums, and has the advantages that cell differentiation resulted from non-absolute purification of the traditional culture medium blood products can be avoided; a totipotential stem cell culture medium can be provided to effectively increase human totipotential stem cells (including the hESC and the hiPSC) in vitro, achieve a remarkable effect, and substitute for the traditional culture mediums from an animal source; due to the adoption of the stem cell culture medium stem cell culture medium, the quality stability of each product batch can be ensured effectively to facilitate the product standardization.

The invention relates to an integrated detection reaction orifice plate and protein chip reagent box for detecting six sterility indexes. And the reaction orifice plate comprises a substrate and reaction orifices on the substrate and the reaction orifices comprise 3-384 sample orifices, 1-300 negative contrast orifices, and 1-300 positive quality control orifices, where at the bottom of each reaction hole is solid carrier, coated with micro lattice of Sperm antigen (SAg), Zona pellucida antigen (ZPAg), Endometrium antigen (EMAg), Ovary antigen (OAg), Trophoblast antigen (TAg), and Human Chorionic Gonadotropin antigen (HCGAg) antigens or more. And the reaction orifice plate and reagent can simply and conveniently, rapidly and accurately implement simultaneously detection of six sterility indexes for many persons.

The invention discloses a method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells. The method includes: processing a placenta sample to obtain processed placenta tissues; digesting the tissues to obtain cell suspension; performing discontinuous Percoll density gradient separation to obtain trophoblast and placental mesenchymal stem cells; purifying the trophoblast; further separating and culturing the placental mesenchymal stem cells. The method has the advantages that HyQTase and DNAse I are used to jointly digest the tissues, and a large amount of trophoblast and placental mesenchymal stem cells with ideal cell purity and activity is obtained through the density gradient separation; digestion fragments, fibroblast and red blood cells are removed, the trophoblast and the placental mesenchymal stem cells are distinguished from each other, the purity of the trophoblast can reach 90%, the non-adherence upper layers of the placental mesenchymal stem cells are removed through a differential adhesion method, serum-free medium is added into a culture flask to continue the culture of the placental mesenchymal stem cells, and high purity of the placental mesenchymal stem cells is achieved.

Methods for isolating and purifying fetal trophoblasts from a mucus sample obtained from the uterine cavity of a pregnant female. The mucus sample is transported from a clinical collection facility to a laboratory in a transportation medium so the cells remain viable. The mucus sample is then subjected to precise processing steps, including treatment with mucolytic agents or mucinases, sugar hydrolysis enzymes, nucleases, and proteases to provide fetal cells, the outer surfaces of which are so essentially completely devoid of attached mucosal biological material that they are then isolated in greater numbers than previously had been possible. The isolated cells are in appropriate condition to immediately be effectively subjected to FISH or to other molecular diagnostics.

The invention discloses an umbilical cord blood Treg cell in-vitro amplification method based on trophoblastic cells and application. The specific technical method comprises the steps that firstly, umbilical cord Wharton's jelly mesenchymal stem cells are adopted as the trophoblastic cells to induce preliminary proliferation of Treg cells in umbilical cord blood mononuclear cells; then, pure Tregcells are obtained through magnetic bead sorting; and finally, the Treg cells are stimulated to be rapidly amplified by using optimized amplification factors. According to the amplification method, human AB plasma, IL-2, rapamycin, an RARA agonist and a DNA methyltransferase inhibitor are used as the optimized amplification factors, and a large number of umbilical cord blood Treg cells with high purity and high activity can be prepared within two weeks. Umbilical cord blood is used as a raw material for Treg cell amplification, batch preparation can be achieved, and Treg cell quality fluctuation caused by individual differences of samples can be reduced. The umbilical cord blood Treg cells have low immunogenicity and can be used as universal cells for clinical research, such as autoimmunediseases, graft-versus-host diseases and the like.

The invention relates to the field of gene engineering and cytobiology, in particular to a preparation method for efficiently amplifying NK cells by utilizing trophoblasts. The construction method comprises the following steps: (1) constructing a pHR-mbIL-21 plasmid vector, a pHR-4-1BBL plasmid vector and a pHR-MICA plasmid vector; (2) preparing recombinant lentiviruses by using pHR-mbIL-21, pHR-4-1BBL and pHR-MICA plasmid vectors respectively; (3) preparing K562-mbIL-21-4-1BBL-MICA trophoblasts; (4) extracting PBMC cells; and (5) carrying out in-vitro amplification on NK cells. The inventionprovides the preparation method of NK cells. The method employs the step of independently constructing plasmid vectors for expressing mbIL-21, 4-1BBL and MICA molecules, K562 is infected with recombinant lentivirus, the NK cells can be prepared, the preparation method overcomes the defects in the prior art, the K562 cells simultaneously expressed by IL-21, 4-1BBL and MICA molecules are used as trophoblasts for amplification culture of the NK cells, the amplification multiple of the NK cells reaches up to 890 times, the purity of the prepared NK cells reaches 92.2%, and the repeatability of theamplification multiple between different PBMC cells is good.