PRODUCING METHOD OF NEURAL STEM CELLS IN BODY FROM INDUCED PLURIPOTENT STEM CELLS USING CHIMERA
Chimeric the present invention refers to pluripotent stem a subsidiary vivo derived from neural stem cell production relates to method. Induction pluripotent stem cells (induced pluripotent stem cells; iPSCs) has differentiated somatic cells to provide by the process of vaporation reverse differentiation of differentiating into all early embryo capable of versatile is LED to step is cells. Induction of differentiating into all pluripotent stem cells are the patient since feature is derived cells without rejection immunosuppression for intractable diseased cells carried out at a liquid silicon is coated according plepuring for therapeutic agent. Method for reducing sludge using pluripotent stem induction under study, the treatment of neurological disorders occupies substantial portion is. Therefore, induction pluripotent stem cells and to cause differentiation of neural stem cells or neuronal cells the techniques. new oral in extracorporeal i.e. (neurosphere) by forming a single layer (monolayer) culture method in bFGF, EGF added at a water neural stem cells in the method of differentiating the. material is used. Surface of differentiating into neural stem cells, continuously neuronal cells capable of differentiating into precursor cells since the supply can be achieved, and to cause differentiation of completely differentiated neural cells than has advantage in many ways. An induction to current method pluripotent stem cell differentiation in vitro culturing method. circumstances less efficient. The, the present inventor which has undergone the a two-step are derived from pluripotent stem derived neural stem cell production techniques has been developed. Induction pluripotent stem cells to betray implanting fabrics (or aggregation method) method for producing it and mouse chimeric using, mouse chimeric is an inductive included in brain tissue of pluripotent stem cells derived from neural stem cells by extracting the method is neural stem cells. Thus establishing an induction pluripotent stem cells chimeric derived neural stem cells neural stem cells expressing marker (Nestin, Musashi) which, normally neurons (neuron), cells, (astrocyte), oligodendrocyte (oligodendrocyte) permits their differentiation to the. In addition gene expression pattern is derived pluripotent stem cells in vitro differentiated neural stem than brain tissue derived from neural stem cells generally, was shown to be more similar and. Therefore, pluripotent stem induction the present invention refers to method for reducing sludge using chimeric mouse of brain tissue in nerve stem cells are separated the body neural stem the culture of dendritic cells novel inductively pluripotent stem cell differentiation are defined.. The present purpose of the invention the chimeric derived from pluripotent stem a subsidiary vivo neural stem cell production is provided to method. It is another object of the present invention produced according to said method a subsidiary chimeric derived from pluripotent stem is provided to neural stem cells. Said end of the, the present invention refers to (1) induction pluripotent stem cells to betray fabrics introducing or, aggregation (aggregation) to obtain an embryo chimeric has a value of a range of ; (2) said chimeric embryonic uterine the chimeric mouse for producing step ; (3) said single brain tissue of chimeric MICE of isolating and, new oral step for forming (neurosphere); and (4) said new oral forming neural stem cells derived from pluripotent stem derived from separating the chimeric including vivo derived from pluripotent stem a subsidiary method provides neural stem cell production. Specifically, induction said pluripotent stem cells transformed MICE with induction ROSA26 (neo/lacZ) and OG2 (Oct4-GFP) generated through in cross fertilization with, deformed conjugates fibroblast Oct4 factor reprogramming cells, Sox2, by overexpressing c-Myc and Klf4 reverse differentiation establishment of the but may be stem cells, not limited to blood. Particularly, said (4) step derived from pluripotent stem induction of neural stem cells are separated G418 husk that added to the step and selected although it is possible, not limited to blood. Specifically, pluripotent stem induction said neural stem cells derived from neural stem cells marker expressing Sox2 Nestin and is characterised in that it has a. "Transformed MICE with induction OG2 (Oct4-GFP)" used in the present invention the Oct4 control of an which are expressed by. from the detected text MICE transformed with GFP (Oct4-GFP). The manufacturing method of said MICE Methods in Molecular Biology, vol 254, Germ Cell Protocols, Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, is publicly known and Cloning or the like. In the present invention, "induction pluripotent stem cells (induced pluripotent stem cells; iPSCs)" has differentiated in somatic of differentiating into all early embryo capable of. cells reverse it was differentiated step. In the present invention, "chimeric (chimera)" object has an in individuals genetically different derived tissue, cells, nuclear, gene or chromosome dielectric characteristics such as a composite animal circulation promoted.. Furthermore, produced according to method of the present invention refers to said chimeric derived from pluripotent stem a subsidiary provides neural stem cells from regions where. Chimeric the present invention refers to pluripotent stem a subsidiary vivo derived from neural stem cell production relates to method, induction pluripotent stem cells to betray implanting fabrics (or aggregation method) method for producing it and mouse chimeric using, mouse chimeric is an inductive included in brain tissue of pluripotent stem cells derived from neural stem cells by extracting the method is neural stem cells. Pluripotent stem induction the present invention refers to method for reducing sludge using chimeric mouse of brain tissue in nerve stem cells are separated the body neural stem the culture of dendritic cells novel inductively pluripotent stem cell differentiation can be period of time and thus utilized as techniques. Call is held at the extracorporeal Figure 1 reverse differentiation vivo from stem cells differentiated neural line thereof occurring chimeric device, in order to establish an gun week blades, presenting a experimental process forming. reverse differentiation Figure 2 form a chimeric stem cells in an. of the results of an. (A) reverse differentiation stem cells embryonic and generally dish of aggregation in in culturing colonies obtained has a value of a range of (aggregation) exhibits chimeric embryo. (B) chimeric embryo the uterine mouse scene being, , the mobile phone confirms the embryos uterine to 13.5 dpc is result. (C) reverse differentiation stem cells are embryo contribute to form a cementous the click signal except for the head for the body portion. of the results of an demonstrated that despite dyeing X-gal. Differentiated in the body from embryonic chimeric Figure 3 reverse differentiation derived stem cells to obtain neural stem cells from regions where, forming in a culture new oralnew oral exhibits and extended from neural stem cells. Vivo from stem cells reverse differentiation Figure 4 traits in stem cells out with an adaptive neuronal differentiation marker neural stem cells Nestin, verify Sox2 gene is expressed of the results of an.. Hereinafter, embodiment a to the present invention more rapidly and to reduce a memory.. Just, and/or at least two different such embodiment the present invention is limited not. < 실시예 1>Chimeric (chimera) forming Call is held at the extracorporeal reverse differentiation vivo from stem cells differentiated neural line thereof occurring forming chimeric device, in order to establish an gun week the ground terminal of experiment (also 1). Used in the present invention stem cells reverse differentiation ROSA26 (neo/lacZ) and OG2 (Oct4-GFP) generated through in cross fertilization with transformed MICE with induction, deformed conjugates fibroblast Oct4 factor reprogramming cells, Sox2, Klf4, using Retrovirus a c-Myc by overexpressing is cell line establishment of the. Thus reverse differentiation Oct4-GFP surface is retained universal stem cells which is expressed using experiment (staining) dyeing X-gal positive reaction capable of verifying which even to medium drug G418. maintain the potential difference between the gate. Such reverse differentiation stem cells in the body, using the characteristics derived stem cells differentiated reverse differentiation can establish a neural stem cell lines. reverse differentiation first chimeric stem cells in an embryonic and in a generally dish of aggregation in in culturing colonies reverse differentiation stem cells (aggregation) has a value of a range of chimeric embryo can be obtained (also 2A). Universal, and having reverse differentiation stem cells has a receiving unit storing cell mass (ICM) the n bit parallel data inputted to confirm the, such chimeric embryo was mouse uterine implantation in being, scene. Identifying embryos uterine to 13.5 dpc can be (also 2B). reverse differentiation stem cells are embryo contribute to form a cementous the click signal except for the head for the barrel portion are result on experiment dyeing X-gal X-gal it is a cultivation voice portion and of the basic area which is filled with a mixture of the make sure that a chimeric formed stem cells reverse differentiation it has been confirmed (also 2C). < 실시예 2>Chimeric embryonic from reverse differentiation stem cells differentiated in the body derived neural stem cells isolating and culturing the pancreatic progenitor cells Chimeric embryonic from reverse differentiation derived stem cells differentiated in the body to obtain neural stem cells Trypsin/EDTA the head portion is separated into single cell using, new oral to form made an attempt to culture in a culture new oral. new oral DMEM/F12 (Gibco) 100x N2 (Gibco) to the culture, 50x B27 (Gibco), 1x penicillin/Streptomyces/glutamine (Gibco), 0.6% glucose (sigma), 20ng/ml EGF, bFGF (Gibco) adding a, the third to eo. 10-15 in a culture new oral is installed at culture days, new oral with which capable of confirming the, 0.1% gelatin (gelatin) for new oral formed coated with diamond-like culture dish in nerve stem cell expansion in a culture culture the movable member is driven by which extends out from the implant body new oral. neural stem cells from regions where (also 3A). 50x N2 (Gibco) to the neural stem cell expansion culture DMEM/F12 (Gibco), 1x penicillin/Streptomyces/glutamine (Gibco), (BSA; Gibco) 50 micro g/ml small serum albumin , 10ng/ml EGF, bFGF (Gibco) adding a, the third to eo. reverse differentiation established neural stem cell lines derived from the stem cells includes neural stem cells to supervis ion portion confirms a X-gal dyeing the embodiment. Neural stem cell lines established from chimeric head body portion and similarly X-gal it is a cultivation dyeing voice cells which is filled with a mixture of cells it has been confirmed (also 3B left). Chimeric neural stem cells from neural stem cells derived from the stem cells reverse differentiation to obtain only drug G418 neural stem cell expansion culture made an attempt to culture added to. Result neural stem cells derived from embryonic generally G418 dead in a culture containing it is confirmed that the n bit parallel data inputted, a there are any cells it is a cultivation dyeing X-gal it has been confirmed (also 3B intermediate). After selecting through the medicated neural stem cell expansion paclitaxel the movable member is driven by sebocytes are excellently reverse differentiation stem cells derived neural stem cell line for establishment of the was capable of confirming the (also 3B right). reverse differentiation vivo from stem cells out with an adaptive neuronal differentiation marker neural stem cells Nestin traits in stem cells, is found to is expressed Sox2 gene (also 4). Established hepatic stem cells after washed with PBS, for controlling piezoelectric vibration 4% 20 minutes with an aldehyde function at a normal temperature by fixing and. 0.1% Triton-X 100 was then being washed with PBS for 5 minutes after treatment stop solution through 30-60 minutes was black water slurry to room temperature. 5 1 and washing irrigating solutions minutes after the processing antibodies difference was the composition is left for during night in 4 °C. Next 10 into 3 burn irrigating solutions washing and through antibodies difference 2 2 time 30 minutes was black water slurry to room temperature for processing DAPI minutes. 3-5 into 10 again and, after washing the solution cleaning burn 3 stem cells dyeing it is found out that using the fluorescent. reverse differentiation derived traits in stem cells used in neural stem cells Nestin (Chemicon) the antibody is anti-difference 1, the first voice portion out of an anti-Sox2 (Chemicon). Each result refers to surface, produced by the present invention according to method shell did stem cell and shell did derived traits in stem cells for antibodies each all neural stem cells (Immunocytochemistry) immunocytochemical wherein sequences as signal reaction, each stem cells produced that exhibits the characteristics of a is determined to. The present invention relates to a producing method of neural stem cells in the body from induced pluripotent stem cells (iPSCs) and the method is to prepare chimeric mice using a method which injects iPSCs into blastocyst (or an aggregation method) and to produce neural stem cells by extracting neural stem cells included brain tissues of the chimeric mice from iPSCs. The present invention can be used as a new differentiation technology of iPSCs which cultures neural stem cells in the body by separating neural stem cells from brain tissues of the chimeric mice using iPSCs. COPYRIGHT KIPO 2015 (1) induction pluripotent stem cells to betray fabrics introducing or, aggregation (aggregation) to obtain an embryo chimeric has a value of a range of ; (2) said chimeric embryonic uterine the chimeric mouse for producing step ; (3) said single brain tissue of chimeric MICE of isolating and, new oral step for forming (neurosphere); and (4) said new oral forming neural stem cells derived from pluripotent stem derived from separating the chimeric including derived from pluripotent stem a subsidiary vivo neural stem cell production method. According to Claim 1, said induction pluripotent stem cells transformed MICE with induction ROSA26 (neo/lacZ) and OG2 (Oct4-GFP) generated through in cross fertilization with, deformed conjugates fibroblast Oct4 factor reprogramming cells, Sox2, by overexpressing c-Myc and Klf4 reverse differentiation establishment of the chimeric characterized by stem cells is derived from pluripotent stem a subsidiary vivo neural stem cell production method. According to Claim 1, said (4) step derived from pluripotent stem induction of neural stem cells are separated G418 husk that added to the step to screening chimeric characterized by derived from pluripotent stem a subsidiary vivo neural stem cell production method. According to Claim 1, pluripotent stem induction said neural stem cells derived from neural stem cells marker Sox2 and Nestin to expressing chimeric characterized by derived from pluripotent stem a subsidiary vivo neural stem cell production method. Number 1 anti to number 4 terms either anti chimeric produced according to method a subsidiary neural stem cells derived from pluripotent stem.
