53 results about "Neurosphere" patented technology

A clonogenic neurosphere assay is described that carries out high throughput screens (HTS) to identify potent and / or selective modulators of proliferation, differentiation and / or renewal of neural precursor cells, neural progenitor cells and / or self-renewing and multipotent neural stem cells (NSCs). Compositions comprising the identified modulators and methods of using the modulators and compositions, in particular to treat neurological disorders (e.g. brain or CNS cancer) or damage are also disclosed.

The present invention provides methods of culturing, propagating, treating, and maintaining stem cells in the presence of a phosphate mimic. In particular, the invention relates to the propagation of stem cells in vitro, the formation of neurospheres in vitro, and to tissue culture of stem cells in the presence of a phosphate mimic. The invention further relates to the treatment of neurodegenerative disorders.

Methods and assay systems for analyzing effects of chemical and cellular agents on brain cell neurogenesis in vivo, comprising administering an agent to a test animal and determining responses of cells of brain marrow tissues, including irradiated brain marrow tissue depleted of neurogenic stems cells, and cells in brain marrow-derived neurospheres cultured in vitro.

The invention provides in vitro cell culture compositions consisting of neurospheres and culture medium, wherein the neurospheres consist of undifferentiated cells that are nestin+, glial fibrillary acid protein (GFAP)−, neurofilament (NF)−, and myelin basic protein (MBP)− and are not nestin−.

There is provided a treatment for patients suffering from neurodegenerative disease or neural injury including the steps of transplanting cultured bone marrow cells into the spinal cord or brain or injecting intravascularly bone marrow cells of a patient in need. Also provided is a method of activating the differentiation of neural cells in an injured brain including the steps of transplanting bone marrow cells adjacent to the injured brain cells and activating the endogenous central nervous system stem cells to differentiate into neurons. A method of treating injured brain or spinal cord cells is also provided including the steps of transplanting bone marrow cells near the injured brain cells and generating new neurons at the location of transplantation. A method of treating injured brain or spinal cord cells with a composite of MSCs and neurospheres.

The invention discloses a neural stem cell culture amplification method, which comprises the following steps of culturing different sources of neural stem cells with a culture solution, adding a nutritional supplement solution, replacing a fresh culture solution, selecting a certain size of neurospheres for passage and the like. A formula of a relative culture solution, the nutritional supplement solution and other compositions is also provided. The method obviously improves the proliferation efficiency of neural stem cells under static culture, no antibiotics are used, and the cost is lower. The method is suitable for industrial production.

The present invention provides a therapeutic agent for a nerve injury and a method for treating a nerve injury. One aspect of the invention is the method for treating a nerve injury by administering to a patient with a nerve injury a therapeutic agent for a nerve injury containing a differentiated cell-derived pluripotent cell obtained by forced expression of reprogramming genes such as a combination of the Oct3 / 4 gene, Sox2 gene, Klf4, and c-myc gene. in a differentiated cell; or cells obtained by inducing the aforementioned differentiated cell-derived pluripotent cells to differentiate into an embryoid body or a neurosphere.

Methods of generating an innervated muscle structures are disclosed as well as bioengineered structures for tissue repair or regeneration. The methods can include the steps of obtaining populations of smooth muscle cells and neuronal progenitor cells and then seeding the cells together onto a matrix material, followed by culturing the seeded cells to form an innervated smooth muscle cell construct of directionally oriented smooth muscle cells. In one embodiment, the neuronal progenitor cells can be seeded first as neurospheres in a biocompatible solution, e.g., a collagen / laminin solution, and allowed to gel. Next, a second suspension of smooth muscle cells can be deposited as separate layer. Multiple layer structures of alternating muscle or neuron composition can also be formed in this manner. Differentiation of the neuronal progenitor cells can be induced by exposure to a differentiation medium, such as Neurobasal A medium and / or exposure to a differentiating agent, such as B-27 supplement. The innervated muscle structures can be disposed around a tubular scaffold, e.g., a chitosan-containing tube and further cultured to form tubular, bioengineered structures and two or more innervated muscle structures can be joined together to form an elongate composite structure.

The invention discloses a method for quickly, directly and directionally inducing differentiation from mouse embryonic stem cells to neuroepithelial cells. The method comprises the steps of adding an induction medium containing dorsomorphin, noggin, SB431542 and CHIR99021 to a bulk clone of the mouse embryonic stem cells subjected to adherent culture, to induce the mouse embryonic stem cells to form rosette neurospheres; then adding a second stage of induction medium containing bFGF and performing suspension culture to form suspended neurospheres; and finally, digesting the neurospheres into single cells, and then performing adherent culture with a third stage of induction medium to form the neuroepithelial cells with long-term self proliferation and update. By adopting the method, the traditional EB method is abandoned, the differentiation time is shortened, and the differentiation efficiency reaches over 95%. The method can be used for quickly inducing NE cells to substitute NSC cells to treat central system injury, and has very high clinical applicability.

The invention provides a method able to realize separation, amplification and purification of embryonic cranial neural stem cells rapidly and simply. The invention uses the thermal conversion polymer N-isopropylacrylamide and polyethylene glycol to conduct three-dimensional culture on neural cells, and makes use of the characteristic that neural stem cells can proliferate in a gel environment and form neurospheres to achieve the purposes of separation and purification of the neural stem cells. The formed neurospheres are subjected to immunofluorescent labeling determination, which shows that about 93% of the cells are Nestin positive, so that the method is simple and effective for separation and purification of neural stem cells. The embryonic neural stem cells separated and purified by the method can differentiate in vitro to form neurons, oligodendrocytes and Glias.

The present invention discloses an isolation and culture method of amniotic-fluid-derived neural stem cells. The method comprises the following steps of: firstly isolating to obtain amniotic fluid cells, then adding an isolation culture medium to the amniotic fluid cells, isolating obtained neural stem cells to grow and form neurospheres, digesting and isolating the neurospheres into single cells and carrying out subculture. The isolation and culture method of the amniotic-fluid-derived neural stem cells provided by the invention opens up a novel way for the isolation and culture of the neural stem cells, and has a high success rate in the isolation and culture the neural stem cells from the amniotic fluid. The isolated amniotic-fluid-derived neural stem cells can differentiate into astrocytes, neuronal cells and other functional cells after in-vitro induction, and have feasibility, effectiveness and the potential of self-renewal, proliferation, and differentiation.

An isolated human olfactory stem cell can be prepared by culturing human tissue from olfactory neuroepithelium to form neurospheres.

The invention discloses a method for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps: 1) preparing human mesenchymal stem cells and subculturing; 2) differentiating the passaged human mesenchymal stem cells into human mesenchymal stem cells in a differentiation medium-neurosphere; 3) differentiating the human mesenchymal stem cell-neurosphere into neurons in an induction culture medium. Neurotrophic factors are added to the induction culture medium to promote the differentiation into neurons, and the results show that expressing mature nerve marker (MAP2ab) and immature nerve marker (beta-tubulin III) were significantly improved relative to the blank control. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.

The disclosure reports on the identification and isolation of adult mouse lateral ventricle subventricular zone (SVZ) neurosphere initiating cells (NICs) by flow cytometry on the basis of GlastmidEGFRhighPlexinB2highCD24− / lowO4 / PSA-NCAM− / lowTer-119 / CD45− markers (GEPCOT cells). These cells are highly mitotic and short-lived in vivo based on fate-mapping with Ascl1CreERT2 and Dlx1CreERT2. In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEP-COT cells could not form neurospheres but expressed the stem cell markers Glast-CreERT, GFAP-CreERT2, Sox2CreERT2, and Gli1C-reERT2 and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for p16Ink4a repression. These data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo.

The invention discloses a culture method for Schwann cells. The culture method comprises the following steps: S1, culturing mononuclear cells in umbilical cord blood by virtue of an MSCs culture medium to obtain P1 generation umbilical cord blood MSCs; S2, culturing the P1 generation umbilical cord blood MSCs by virtue of a neural sphere culture medium, and harvesting P3 generation neural spheres; and S3, culturing the P3 generation neural spheres by virtue of a Schwann cell culture medium, so that the Schwann cells are obtained. The culture method disclosed by the invention has the beneficial effects that the Schwann cells sourced from umbilical cord blood MSCs are cultured by virtue of a nestin+ neural sphere stage; and formation of the neural spheres is induced by adopting the P1 generation umbilical cord blood MSCs, and neural sphere formation rate reaches up to 41%. The invention also provides a specific neural sphere culture medium, the umbilical cord blood MSCs can be sufficiently induced to be formed into nestin+ neural spheres; and a specific culture scheme that nestin+ neural precursor cells are differentiated into the Schwann cells is provided, adherence and migration of the nestin+ neural precursor cells can be obviously promoted, and agglomeration and multilayer growth in a cell proliferation process are prevented.

The invention provides a preparation method of a 3D (Three Dimensional) brain organ. The preparation method comprises the following steps: digesting neurospheres obtained by an RONA method into singlecells through accutase; after counting, inoculating a cell culture plate with the single cells and culturing in a culture medium A; after culturing to the 7th day, continually culturing in a culturemedium B; when culturing to the 25th to 35th day, covering the neurospheres with Matrigel; then continually culturing to the 55th to 65th day; after covering the neurospheres with the Matrigel for thesecond time, continually culturing. The invention further provides a culture medium for culturing the 3D brain organ. According to the preparation method provided by the invention, the highly-purified neurospheres obtained by the RONA method can be controlled and cultured by utilizing a relatively simple method to obtain the real 3D brain organ with uniform size and structure; the 3D brain organhas a six-layer cortical structure of a brain and each subtype of inhibitory medial nerve cells; the 3D brain organ is applicable to in-vitro disease researches, medicine screening and the like and has great industrialization significance.

Methods of generating an innervated muscle structures are disclosed as well as bioengineered structures for tissue repair or regeneration. The methods can include the steps of obtaining populations of smooth muscle cells and neuronal progenitor cells and then seeding the cells together onto a matrix material, followed by culturing the seeded cells to form an innervated smooth muscle cell construct of directionally oriented smooth muscle cells. In one embodiment, the neuronal progenitor cells can be seeded first as neurospheres in a biocompatiable solution, e.g., a collagen / laminin solution, and allowed to gel. Next, a second suspension of smooth muscle cells can be deposited as separate layer. Multiple layer structures of alternating muscle or neuron composition can also be formed in this manner. Differentiation of the neuronal progenitor cells can be induced by exposure to a differentiation medium, such as Neurobasal A medium and / or exposure to a differentiating agent, such as B-27 supplement. The innervated muscle structures can be disposed around a tubular scaffold, e.g., a chitosan-containing tube and further cultured to form tubular, bioengineered structures and two or more innervated muscle structures can be joined together to form an elongate composite structure.

The invention provides methods for inducing or enhancing neovascularization following ischemia by transplanting an effective amount of human central nervous system stem cells. The human central nervous system stem cells can be grown as neurospheres or in adherent culture. Also provided are methods for inducing the repair of ischemic tissue in a patient and methods for treating stroke in a patient suffering therefrom.

The invention relates to a method for inducing neural stem cells in vitro to directionally differentiate into neurons. The method includes: using fetal rat brain tissue of an ICR (institute of cancerresearch) pregnant rat as a raw material to carry out primary culture on neural stem cells so as to obtain neurospheres, centrifuging to remove supernate, washing precipitate with PBS (phosphate buffered solution), allowing digestion to occur, adding a culture solution to terminate the digestion, centrifuging to remove supernate, adding a growth culture solution into the precipitate, carrying outsecondary culture on the neural stem cells, digesting the fourth-generation or fifth-generation neurospheres to prepare a single-cell suspension, spreading the single-cell suspension to a 12-orifice cell culture plate coated with poly-l-ornithine and laminin under the cell density of 5*104 to 5*106 cells per mL, culturing overnight, adding a differentiation culture solution on the second day, andcarrying out differentiation culture to obtain mature neurons. The method is simple and well repeatable, allows the differentiation ratio of neural stem cells to neurons to be evidently increased, canpromote purity differentiation of neurons and helps attain the neurons with good growth state and high purity.

A medium containing hepatocyte growth factor (HGF) was shown to induce neurosphere formation. Furthermore, the addition of HGF to a culture medium containing FGF-2, EGF, or both increased both the size and number of newly formed neurospheres. Thus, the present invention relates to a growth medium comprising HGF for culturing neural stem cells and methods for culturing the cells using the culture medium.

The invention discloses a color sorter which comprises a feeding unit, a supplying unit and a color sorter main body; the feeding unit comprises a feeding bracket, a supporting seat, a vibrator, a first base, a feeding groove and a groove body angle regulation device; the outlet of the feeding groove is connected with the supplying unit; the supplying unit comprises a supplying funnel and a stirring device arranged in the supplying funnel; the stirring device comprises an eccentric wheel, a driving rod, a driven rod and a stirring rod; and the supplying unit is arranged above the color sortermain body. Compared with the prior art, the color sorter has the beneficial effects that through the arrangement of the groove body angle regulation device, the color sorter can be suitable for sorting various materials, and thus the production cost is lowered; and the stirring device of the supplying unit can realize stirring of the materials in the transportation process, can effectively avoid the phenomenon that the materials pile together and is beneficial to being beneficial to promoting the formation of neurospheres improving of the color sorting efficiency.

The invention discloses an immunoglobulin staining method for a neurosphere. The method comprises the steps of (1) neurosphere cleaning: taking neurospheres in suspension culture, centrifuging, discarding supernatant, resuspending the neurospheres, centrifuging again, and discarding supernatant, (2) fixing, (3) permeability and blocking, (4) primary antibody incubation, (5) rinsing, (6) secondaryantibody incubation, (7) rinsing, and (8) mounting: adding a PBS buffer solution, resuspending the neurospheres, dropwise dropping the neurospheres on an object slide, dropwise adding an anti-fluorescence quencher, covering with cover glass, flattening and packaging the neurospheres, and observing with a fluorescence microscopy. According to the method, cells are collected at a tube bottom by directly using low-speed centrifugation by using the characteristics of large diameter and visibility for a naked eye of the neurospheres, the loss of the neurospheres in a staining process is avoided, the original cell morphology and internal structure of the neurospheres can be maintained, the change of antigenic properties caused in a cell climbing process is effectively avoided, the neurospheres are directly pressed by using a physical pressing method, the neurospheres are pressed into a film shape, the cutting is not needed, and the requirements for equipment are low.

Method for generating monoclonal antibodies that recognize progenitor cells. Said method comprises immunization of an Armenian hamster with neurospheres obtained from olfactory bulb cells from a 13.5-day mouse embryo and subsequent selection of the antibodies by means of neurosphere flow cytometry in the presence of propidium iodide. The antibodies thus obtained may be of use in the enrichment of cell cultures in progenitor cells, primarily neural progenitor cells.

Method for generating monoclonal antibodies that recognize progenitor cells. Said method comprises immunization of an Armenian hamster with neurospheres obtained from olfactory bulb cells from a 13.5-day mouse embryo and subsequent selection of the antibodies by means of neurosphere flow cytometry in the presence of propidium iodide. The antibodies thus obtained may be of use in the enrichment of cell cultures in progenitor cells, primarily neural progenitor cells.

The present invention relates to a method for preparing a GM1 gangliosidosis human cell model based on induced pluripotent stem cells (iPSCs) and iPSCs originated neural progenitor cells, and a use of the GM1 model above for the development of a GM1 gangliosidosis treating agent. The iPSCs originated from GM1 patient fibroblasts can be differentiated into neural progenitor cells (NPCs) and neurosphere cells that can emulate the characteristics shown in GM1 patient, so that the said cells can be efficiently used for the investigation of intracellular GM1 symptoms such as the GM1 gangliosidosis and lysosome accumulation and the gene expression pattern change. So, the GM1 cell model of the present invention can be efficiently used for the study of GM1 development mechanism and the study for the development of a therapeutic agent for the disease. The present inventors also established the inflammasome inhibitor rhIL1RA or Z-YVAD-FMK by using the above GM1 cell model and further confirmed that it can be efficiently used as a relieving / treating agent of GM1 gangliosidosis.In addition, the molecular symptoms of GM1 patient could be reproduced in the transformed cells having the E186A mutation which is newly identified as the GM1 gangliosidosis causing protein mutation. Therefore, the mutant cells containing the induced E186A mutation can be efficiently used as the GM1 gangliosidosis cell model.

The invention discloses an immortalized human neural stem cell line and a preparation method thereof. The method comprises the following steps: (1) isolating and culturing primary human hippocampus-derived neural stem cells; (2) constructing a recombinant lentiviral vector containing L-myc gene; (3) carrying out packaging production of the above recombinant lentiviral and collecting supernatant; (4) selecting virus supernatant with appropriate concentration for transfection and screening of stem cells and constructing the immortalized human neural stem cell line. The preparation method of immortalized cell line provided by the invention is a novel stable and safe and effective immortalized coding strategy; the prepared immortalized human neural stem cell line has all the biological characteristics of primary neural stem cells, and also has the feature of normal three-line differentiation at the same time, can be successfully differentiated into neurons, astrocytes and oligodendrocytes,and the growth rate of such cell line and ability to form neurospheres are stronger than the growth rate of primary cells, and the cell line can still proliferate rapidly after 20 generations withouttumorigenicity.

The invention discloses a method for preparing neural stem cells by adopting fibroblasts. The invention provides the method for preparing the neural stem cells. The neural stem cells are obtained through suspension culture of the isolated fibroblasts. The mouse fibroblasts are grown into a three-dimensional stereoscopic spherical shape by a low-attachment physical method, and thus a neural ball form is simulated. Experimental results show that through the three-dimensional spherical culture, the mouse fibroblasts (embryonic fibroblasts and tail-tip fibroblasts) can be transdifferentiated into neural stem cell-like cells. The method for preparing the neural stem cells is simple in operation and low in cost, and has significant values in preparation and application of the neural stem cells.

In order to solve the problems of cell apoptosis, mutation, cell damage, and neural stem cell differentiation of neural stem cells, long operating time, uneven sizes of cell globes and the like which are caused in the passage process of neural stem cells, the invention provides a stem cell globe cutting and collecting device. The stem cell globe cutting and collecting device comprises a stem cell globe cutter, a stem cell globe collecting bottle, and an air pump provided with positive-pressure and negative-pressure two-way air extracting functions, wherein stem cell globe cutter is connected with the stem cell globe collecting bottle through a pipeline, the stem cell globe cutter is provided with a pipe, and two sieves arranged at an interval are fixed in the pipe. With the adoption of the device, and a stem cell globe cutting and separating passage method, the technical problem of large possibilities of differentiation and death existing in the in-vitro culture of the neural stem cells is solved, the purposes of long-term culture and amplification of the human neural stem cells are achieved, and in addition, the properties of the human neural stem cells are stable, and the quality of the human neural stem cells is controllable.

The invention discloses an immortalized human neural stem cell line and a preparation method thereof, and a recombinant virus vector and an application thereof. The preparation method includes the following steps: (1) constructing the recombinant lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MYCL-3Flag of an L-myc gene; (2) packaging and producing lentivirus, and collecting supernatant; and (3)adopting the lentivirus supernatant to perform transfection, screening and construction of the immortalized human neural stem cell line on human neural stem cells. The preparation method is a novel, stable, safe and effective immortalization coding strategy; the prepared immortalized human neural stem cell line has all the biological characteristics of primary neural stem cells and also has normalcharacteristics of three-line differentiation, so that the cell line can successfully differentiate neurons, astrocytes and oligodendrocytes; and the growth speed and the ability to become neurospheres of the cell line are stronger than primary cells, so that the cell line can still be rapidly propagated after the successful passing of 25 generations and has no tumorigenicity.

The invention belongs to the field of biotechnology, relates to a method for stem cell culture and discloses application of VX-702 to preparation of a neural stem cell culture medium for promoting proliferation of the neural stem cells or maintaining dryness of the neural stem cells. The VX-702 can effectively increase the proliferation of the neural stem cells, promote formation of neural stem cell neurospheres, and significantly increase the expression of neural stem cell markers, such as NESTIN and SOX2. The invention also provides a method for promoting the proliferation of the neural stemcells. The method includes the steps of culturing the neural stem cells with the neural stem cell culture medium containing the VX-702 under the conditions of 35 -39 DEG C and 3%-8% of CO2 in an adherent culture mode. The VX-702 is used as an additive, the neural stem cell culture medium and the method for promoting the proliferation of the neural stem cells are provided, and building of a stableand reliable in-vitro culture model of the neural stem cells is facilitated.