CN109722411B - Application method of micromolecules for promoting self-renewal state of embryonic stem cells - Google Patents
Application method of micromolecules for promoting self-renewal state of embryonic stem cells Download PDFInfo
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Abstract
The invention discloses an application method of a small molecule for promoting the self-renewal state of embryonic stem cells, which comprises the passage and culture of human and mouse embryonic stem cells under the small molecule state of the invention, and the observation and detection of the self-renewal state of the cells. The invention solves the problems of easy differentiation of the states of mouse and human embryonic stem cells, complex subculture operation, high culture condition cost, inconvenience for subsequent mechanism research and the like under the traditional culture condition by adding CID755673 micromolecules, has the advantages of stable effect, economy, high efficiency, simple operation, convenience for research and the like, provides clues for improving and establishing the culture conditions of other types and species of pluripotent stem cells, and has positive promotion effect on the smooth development of the future stem cell foundation and application research.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application method of a small molecule for promoting the self-renewal state of embryonic stem cells.
Background
Stem cells are a class of cells derived from a fetus, embryo, or adult and have unlimited self-renewal and proliferative differentiation capabilities under certain conditions. The embryonic stem cell can be induced and differentiated into various tissues under certain conditions, the characteristic enables the embryonic stem cell to have attractive application prospects in basic research, transplantation therapy and gene therapy, the embryonic stem cell can be infinitely proliferated under the in-vitro proper condition in an undifferentiated state, and an inexhaustible cell source is provided for the research and application of the embryonic stem cell. The two main characteristics of the embryonic stem cell become one of powerful tools for researching gene function, screening medicine and manufacturing disease animal models, and have great application prospect in the field of regenerative medicine. The Stem cell which is most widely researched at present is a mouse Embryonic Stem cell (mESCs), which is derived from an inner cell mass of a pre-implantation embryo (day 3.5 of pregnancy) and has developmental pluripotency, and is also the first Stem cell line successfully established in vitro. Then, human Embryonic Stem Cells (hESCs) are successfully constructed, and hESCs are pluripotent Cells separated from human early Embryonic inner cell masses or hPGCs through in vitro differentiation inhibition culture, and have extremely wide application prospects. Although mouse and human ESCs share some common features, they differ in their biological properties. The CID755673 small molecule maintains the self-renewal state of human and mouse embryonic stem cells, and provides a new idea for avoiding the differentiation state of human and mouse in the culture process. The method also explores the depth and the breadth of the whole stem cell field, thereby having important scientific research value and academic significance, and the related achievements are beneficial to the safe application of the stem cells in the future.
The maintenance of human and mouse embryonic stem cells needs specific nutrients and cytokines, and if the embryonic stem cells without the cytokines have differentiation phenomena, the chemical micromolecules of CID755673 discovered by the inventor can promote the self-renewal of the embryonic stem cells under the condition of not adding some cytokines, and the chemical micromolecules can be used as future culture conditions of the embryonic stem cells, enrich the types of the culture conditions of the embryonic stem cells and optimize the culture conditions of the embryonic stem cells.
Although the optimized CID755673 culture condition can maintain the self-renewal state of the embryonic stem cells, the related cytokines are too many, which is not beneficial to the follow-up deep research of the related signal path and molecular mechanism in the cells, and the culture cost is high. On the basis, the self-renewal conditions of the human and the mouse are further improved and optimized through the attempts of gradual decrease and different permutation and combination, the self-renewal state of the embryonic stem cells of the mouse can be maintained by using only two small molecules after the optimized conditions, the self-renewal of the embryos of the human can be promoted by one small molecule, the culture effect same as the original culture condition is achieved, and the method has the advantages of time saving, labor saving, money saving, benefit for subsequent research and the like.
Disclosure of Invention
The invention provides an application method of a small molecule for promoting the self-renewal state of embryonic stem cells, aiming at solving the problems of easy differentiation, inconvenient passage operation and the like in the culture process of the embryonic stem cells.
The invention is realized by the following technical scheme:
a method for applying a small molecule for promoting the self-renewal state of embryonic stem cells includes adding a CID755673 small molecule compound into a DMEM medium and a N2B27+ KSR medium to maintain the self-renewal state of mouse and human embryonic stem cells, including the passage and culture of human embryonic stem cells under the condition of CID755673 and the passage and culture of mouse embryonic stem cells under the condition of CID 755673.
The passage and culture of the human embryonic stem cells under the condition of CID755673 comprises the following steps:
(1) taking a 6-well cell culture plate, wrapping each well with 2 ml of DMEM +10 mu l of Matrigel, placing at 37 ℃ and 5% CO2Coating for 4 hours in a cell culture box with concentration;
(2) taking human embryonic stem cells growing to 70-80% density, discarding culture solution, washing the cells for 1 time by PBS, and removing residual culture solution on the cell surface;
(3) adding 1 ml of CTK to digest the embryonic stem cells, floating the cell edges after 6-8min, removing the CTK, adding 2 ml of conventional cell culture solution containing 10% FBS, blowing the cells from the culture plate by using a pipette, and transferring the cells to a 15ml sterile centrifuge tube;
(4) centrifuging at 1200 rpm for 4 min, and sucking out the supernatant;
(5) 2 ml of N2B27 containing 10% KSR was added to resuspend the cells;
(6) counting the cells in the suspension by using a blood counting chamber under a microscope, and calculating the density of the cells according to the cell count;
(7) taking the coated culture dish, removing the plate coating solution, washing with PBS once, and adding 2 ml of N2B27+ KSR culture medium into each hole of the culture plate;
(8) adding 5X 10 to the culture medium4The individual cells are horizontally shaken in a cross shape, so that the cells are uniformly distributed;
(9) adding 10 mu mol of CID755673 small molecules, and horizontally shaking in a cross shape to uniformly mix the small molecules;
(10) placing the cell culture dish at 37 ℃ and 5% CO2Culturing in a cell culture box with the concentration.
The CTK in the step 3 contains 1% Collagenase IV, 0.25% Trypsin, 20% KSR and 1mM CaCl2The PBS (1).
The N2B27 in step 5 contains 1.875 ml of DMEM/F12, 0.25 ml of N2, 1.875 ml of Neurobasal medium, 0.5 ml of B27, 2 mM L-gl [ mu ] tamine and 0.1 mM beta-mercaptoethanol in every 5 ml.
The addition of a small molecule CRT0066101 in the application can maintain the long-term self-renewal state of the embryonic stem cells of human.
The passage and culture of the mouse embryonic stem cells under the CID755673 condition comprises the following steps:
(1) taking a 6-hole cell culturePlates were plated with 2 ml of Gelatin per well at 37 ℃ in 5% CO2Coating for 1 h in a cell culture box with concentration;
(2) taking a stable cell line with the growth density of 55-65%, discarding the culture solution, carefully washing for 1 time by using a PBS (phosphate buffer solution), and discarding the residual washing solution;
(3) immediately adding 1 ml of pancreatin digestive juice to digest cells, after several minutes, floating the edges of cell colonies, carefully blowing and transferring the cells by using a micropipettor into a 15ml centrifuge tube containing 2 ml of DMEM serum culture solution, continuously and carefully blowing and uniformly mixing the cells, and stopping digestion;
(4) centrifuging the mixed suspension in the centrifugal tube at 1000 rpm for about 3min, discarding the supernatant, adding 1 ml of PBS buffer solution for washing, and centrifuging again;
(5) removing the supernatant, adding a proper amount of DMEM serum culture solution to resuspend the cells, and counting the cells;
(6) taking a proper amount of cell suspension into a 15ml centrifuge tube, adding a proper amount of DMEM serum culture medium to the total volume of 2 ml, adding 2 mu mol CID755673 micromolecules, and carefully blowing, beating and uniformly mixing;
(7) taking a well-coated cell culture dish, removing gelatin, transferring the 2 ml suspension to the culture dish, and sliding by a horizontal cross method to ensure that cells and small molecules are uniformly distributed;
(8) placing the cell culture dish at 37 ℃ and 5% CO2Culturing in cell culture box, observing cell shape and growth condition every day, and changing culture solution every other day.
Two small molecules of CID755673 and PD0325901 are added into the passage and culture of the mouse embryonic stem cells under the condition of CID755673, so that the mouse embryonic stem cells can be maintained in a long-term self-renewal state.
The invention has the advantages that:
(1) the invention adopts a method of adding CID755673 micromolecules into DMEM culture medium with 10% FBS content and N2B27+ KSR culture medium to promote the self-renewal of mouse and human embryonic stem cells, under the culture condition, the cell growth condition is good, and the self-renewal of human and mouse embryonic stem cells can be maintained for a short time;
(2) two small molecules, namely CID755673 and PD0325901, are added in the culture of mouse embryonic stem cells, and a CRT0066101 small molecule is added in the culture of human embryonic stem cells, so that the cells can be maintained in the optimal growth state, the self-renewal of the embryonic stem cells is promoted for a long time, the operation steps and the flow are simplified, and the development of basic research is facilitated;
(3) compared with the existing culture conditions of human and mouse embryonic stem cells, the invention has the advantages of more economy and high efficiency, improves the differentiation phenomenon in the culture process of human and mouse embryonic stem cells, and can obviously save the culture cost and time; meanwhile, the addition types of the cell factors are simplified, a culture system is optimized, and the subsequent deep exploration on related signal paths and molecular regulation and control mechanisms in cells is facilitated;
(4) the culture conditions discovered by the invention can provide clues for improving the culture conditions of the prior human and mouse embryonic stem cells and other kinds of pluripotent stem cells.
Drawings
FIG. 1 shows the morphology of P5 mouse embryonic stem cells cultured under three conditions under a phase contrast microscope and the result of AP staining of the embryonic stem cells.
FIG. 2 shows the morphology of P0 embryonic stem cells cultured under three conditions under a phase contrast microscope and the result of AP staining of the embryonic stem cells.
FIG. 3 shows the immunofluorescence staining assay for self-renewing marker proteins of P3 mouse embryonic stem cells cultured under three conditions.
Detailed Description
The technical scheme of the invention is further explained by combining the specific examples as follows:
human embryonic stem cell line HES2 and mouse embryonic stem cell line 46C were used in the experiments and were provided by professor Longus at southern California university, USA.
Example 1
Passage and culture of human embryonic stem cells under CID755673 conditions
(1) Taking a 6-well cell culture plate, wrapping each well with 2 ml of DMEM +10 mu l of Matrigel, placing at 37 ℃ and 5% CO2Coating for 4 hours in a cell culture box with concentration;
(2) taking human embryonic stem cells growing to 70-80% density, discarding the culture solution, and washing the cells for 1 time by PBS to remove the residual culture solution on the cell surface;
(3) adding 1 ml CTK (1% Collagenase IV +0.25% Trypsin +20% KSR +1mM CaCl)2PBS) for about 7 min, floating the cell edge, discarding CTK, adding 2 ml of conventional cell culture solution containing 10% FBS, blowing the cell from the culture plate by using a pipette, and transferring the cell to a 15ml sterile centrifuge tube;
(4) centrifuging at 1200 rpm for 4 min, and sucking out the supernatant;
(5) adding 2 ml of N2B27 containing 10% KSR (5 ml of N2B27 containing 1.875 ml of DMEM/F12, 0.25 ml of N2, 1.875 ml of Ne mu robasal medium, 0.5 ml of B27, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol) to resuspend the cells;
(6) counting the cells in the suspension by using a blood counting chamber under a microscope, and calculating the density of the cells according to the cell count;
(7) taking the coated culture dish, removing the plate coating solution, washing with PBS once, and adding 2 ml of N2B27+ KSR culture medium into each hole of the culture plate;
(8) adding 5X 10 to the culture medium4The individual cells are horizontally shaken in a cross shape, so that the cells are uniformly distributed;
(9) adding 10 mu mol of CID755673, horizontally shaking the cross shape to uniformly mix the two groups of culture conditions, and setting two groups of culture conditions as a control, wherein one group is not added with factors, and the other group is added with 10 mu mol of CID 755673;
(10) the cell culture dish was placed in a cell culture chamber at 37 ℃ and 5% CO2 concentration for culture.
Example 2
Mouse embryonic stem cell subculture and culture under CID755673 condition
(1) A6-well cell culture plate was prepared, each well was coated with 2 ml of Gelatin, and the plate was incubated at 37 ℃ with 5% CO2Coating for 1 h in a cell culture box with concentration;
(2) taking a stable cell line with the growth density of about 60%, discarding the culture solution, carefully washing for 1 time by using a PBS (phosphate buffer solution), and discarding the residual washing solution;
(3) immediately adding 1 ml of pancreatin digestive juice (Trypsin-EDTA) to digest cells, after several minutes, floating the edges of cell colonies, carefully blowing and transferring the cells into a 15ml centrifuge tube containing 2 ml of DMEM serum culture solution by using a micropipettor, continuously and carefully blowing and uniformly mixing the cells, and stopping digestion;
(4) centrifuging the mixed suspension in the centrifugal tube at 1000 rpm for about 3min, discarding the supernatant, adding 1 ml of PBS buffer solution for washing, and centrifuging again;
(5) removing the supernatant, adding a proper amount of DMEM serum culture solution to resuspend the cells, and counting the cells;
(6) taking a proper amount of cell suspension in a 15ml centrifuge tube, adding a proper amount of DMEM serum culture medium to the total volume of 2 ml, adding 10 mu mol CID755673 according to the proportion, and carefully blowing, beating and uniformly mixing;
(7) taking a well-coated cell culture dish, removing gelatin, transferring the 2 ml suspension to the culture dish, and sliding by a horizontal cross method to ensure that cells and small molecules are uniformly distributed;
(8) the cell culture dish was placed at 37 ℃ in 5% CO2Culturing in cell culture box, observing cell shape and growth condition every day, and changing culture solution every other day.
Example 3
Self-renewal indicator detection after CID755673 small molecule
(1) Morphological observation
When the embryonic stem cells of mice which are not added with any small molecules (used as a control group), are added with only CID755673 small molecules and are cultured for different days are observed by using a Leica DMi8 inverted microscope, the cells show a tightly-packed monolayer form compared with the control group, the colony boundary is clear, and the morphological characteristics of self renewal are met, but only adding CID755673 can only maintain the embryonic stem cells of the mice to be self-renewed for a short time, so that the addition of two small molecules of CID755673 and PD0325901 can also maintain the embryonic stem cells of the mice to be self-renewed for a long time.
The human embryonic stem cells which are not added with any small molecules (used as a control group), are only added with CID755673 small molecules and are cultured for different days are observed by using a Leica DMi8 inverted microscope, the cells are found to present a tightly-packed monolayer form compared with the control group, the colony boundary is clear and accords with the form characteristics of self-renewal, the transient self-renewal state of the human embryonic stem cells can be maintained only by adding CID755673, and the long-term self-renewal state of the human embryonic stem cells can be maintained by screening out the small molecules CRT 0061 through deeper research.
After the cells are passaged for many times, the culture forms of different generations of cells are observed, and the self-renewal characteristics are kept.
(2) AP staining (alkaline phosphate staining) for detecting whether cells have differentiated or not
After subculturing for 5 passages in different culture conditions, AP staining was performed. Firstly, preparing a staining agent, dissolving an 8523R capsule in 48ml of water, storing at 4 ℃ in dark place, taking a proper amount of the staining agent, mixing the staining agent with SLBJ6060V, and performing 25: 1, are mixed well to form the coloring agent. Removing the culture solution from the cultured stem cells, and adding PBS for washing once; cells were fixed for 2 min by adding 1 ml of 4% Paraformaldehyde (PFA); discarding PFA, adding PBS to wash twice, and washing out PFA residue; 2 ml of stain is added into each culture well; standing for 40 minutes in dark; discarding the staining solution, and washing twice with PBS; after adding an appropriate amount of PBS, the mixture was placed under a Leica DMi8 inverted microscope and photographed. Observing the coloration state to judge the self-renewal state of the cells.
FIG. 1 is a phase contrast microscopic morphology of P5 mouse embryonic stem cells cultured under three conditions and the result of AP staining (alkaline phosphate staining) of the embryonic stem cells, wherein the upper half is a phase contrast microscopic morphology, and the upper left is a mouse embryonic stem cell cultured without any factor added, and is in a differentiated state; the middle upper side is cells cultured under the condition of adding CID755673, and the characteristics of short-term self-renewal state are met; the upper right side is the cells cultured by adding CID755673 and PD0325901, which is in accordance with the characteristic of long-term self-renewal state.
The lower half shows the result of AP staining, the left lower side shows the embryonic stem cells of mice cultured without any factor, the middle lower side shows the embryonic stem cells of mice cultured with CID755673, and the right lower side shows the embryonic stem cells of mice cultured with CID755673 and PD0325901, the undifferentiated cells will be stained, and the differentiated cells will not be stained.
FIG. 2 is a phase contrast microscopic morphology of P0 embryonic stem cells cultured under three conditions and the result of AP staining (alkaline phosphate staining) of the embryonic stem cells, wherein the upper half is a phase contrast microscopic morphology, and the upper left is a human embryonic stem cell cultured without any factor added, in a differentiated state; the middle upper side is the human embryonic stem cell cultured under the condition of adding CID755673, which accords with the characteristic of short-term self-renewal state; the upper right side is the human embryonic stem cell cultured by adding CRT0066101, which accords with the characteristic of long-term self-renewal state.
The lower half is the result of AP staining, the left bottom side is the human embryonic stem cells cultured without any factor, the middle bottom side is the human embryonic stem cells cultured with CID755673, the right bottom side is the embryonic stem cells cultured with CRT0066101, the undifferentiated cells will be stained, and the differentiated cells will not be stained.
(3) Expression of self-renewal marker protein Oct4 detected by immunofluorescence staining
Subculturing human embryonic stem cells and mouse embryonic stem cells for 5 generations under the condition of adding CID755673 micromolecules, removing culture solution, rinsing the cells for 1 time by PBS, adding PFA (paraformaldehyde) with the concentration of 4%, and fixing for 20 min at room temperature;
discarding PFA, rinsing the cells 1 times with PBS, adding blocking solution (PBS containing 5% bovine serum albumin and 0.2% Triton X-100), placing the cells in a 37 ℃ incubator, and incubating for 1 h;
discarding the blocking solution, rinsing the cells 1 time with PBS, then adding a diluent (containing 5% bovine serum albumin, 0.2% Triton X-100, 1% N3Na PBS) containing an Oct4 (1: 200, Santa Cr muz, muSA) primary antibody respectively, and standing overnight at 4 ℃;
the next day, the primary antibody is discarded; rinsing the cells with PBS 3 times, washing every 5 min, then respectively adding secondary antibody (1: 1000, Biyun day) carrying 488 fluorescent group and dilution of Hoechst (1: 5000, Invitrogen) dye (PBS containing 5% bovine serum albumin), wrapping the culture plate with tinfoil paper, and incubating for 1 h at 37 ℃ in the dark;
discarding the secondary antibody diluent, rinsing the cells with PBS 3 times, washing once every 3min, and then observing and taking pictures under a Leica DMi3000B type inverted fluorescence microscope;
FIG. 3 shows the self-renewal marker protein detection of P3 mouse embryonic stem cells cultured under three conditions, using immunofluorescence staining. When observed under a fluorescent microscope, Oct4 protein exhibited green fluorescence; hoechst dye is used for displaying the position of cell nucleus, and blue fluorescence is shown under a fluorescence microscope; merge is the fusion of the map of Oct4 protein showing green fluorescence with the map of Hoechst dye showing the location of the nucleus.
Immunofluorescence results show that the mouse embryonic stem cells and human embryonic stem cells with exogenous small molecule CID755673 are high in Oct4 expression, indicating that the cells are in a self-renewal state, and the cells are differentiated.
Claims (7)
1. An application method of a small molecule for promoting the self-renewal state of embryonic stem cells is characterized in that a CID755673 small molecule compound is added into a culture medium of N2B27+ KSR to maintain the self-renewal state of human embryonic stem cells, and the human embryonic stem cells are passaged and cultured under the condition of CID 755673.
2. The application method of the micromolecules for promoting the self-renewal state of the embryonic stem cells is characterized in that a CID755673 micromolecule compound is added into a DMEM medium to maintain the self-renewal state of the embryonic stem cells of the mice, and the embryonic stem cells of the mice are passaged and cultured under the condition of CID 755673.
3. The method of use according to claim 1, wherein said human embryonic stem cells are passaged and cultured under CID755673 conditions, comprising the steps of:
(1) taking a 6-well cell culture plate, wrapping each well with 2 ml of DMEM +10 mu l of Matrigel, placing at 37 ℃ and 5% CO2Coating for 4 hours in a cell culture box with concentration;
(2) taking human embryonic stem cells growing to 70-80% density, discarding culture solution, washing the cells for 1 time by PBS, and removing residual culture solution on the cell surface;
(3) adding 1 ml of CTK to digest the embryonic stem cells, floating the cell edges after 6-8min, removing the CTK, adding 2 ml of conventional cell culture solution containing 10% FBS, blowing the cells from the culture plate by using a pipette, and transferring the cells to a 15ml sterile centrifuge tube;
(4) centrifuging at 1200 rpm for 4 min, and sucking out the supernatant;
(5) 2 ml of N2B27 containing 10% KSR was added to resuspend the cells;
(6) counting the cells in the suspension by using a blood counting chamber under a microscope, and calculating the density of the cells according to the cell count;
(7) taking the coated culture dish, removing the plate coating solution, washing with PBS once, and adding 2 ml of N2B27+ KSR culture medium into each hole of the culture plate;
(8) adding 5X 10 to the culture medium4The individual cells are horizontally shaken in a cross shape, so that the cells are uniformly distributed;
(9) adding 10 mu mol of CID755673 small molecules, and horizontally shaking in a cross shape to uniformly mix the small molecules;
(10) placing the cell culture dish at 37 ℃ and 5% CO2Culturing in a cell culture box with the concentration.
4. The use of claim 3, wherein the CTK in step (3) comprises 1% Collagenase IV, 0.25% Trypsin, 20% KSR, 1mM CaCl2The PBS (1).
5. The use of claim 3, wherein said N2B27 in step (5) contains 1.875 ml of DMEM/F12, 0.25 ml of N2, 1.875 ml of Neurobasal medium, 0.5 ml of B27, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol per 5 ml.
6. The method of use according to claim 2, wherein said mouse embryonic stem cells are passaged and cultured under CID755673 conditions, comprising the steps of:
(1) a6-well cell culture plate was prepared, each well was coated with 2 ml of Gelatin, and the plate was incubated at 37 ℃ with 5% CO2Coating for 1 h in a cell culture box with concentration;
(2) taking a stable cell line with the growth density of 55-65%, discarding the culture solution, carefully washing for 1 time by using a PBS (phosphate buffer solution), and discarding the residual washing solution;
(3) immediately adding 1 ml of pancreatin digestive juice to digest cells, after several minutes, floating the edges of cell colonies, carefully blowing and transferring the cells by using a micropipettor into a 15ml centrifuge tube containing 2 ml of DMEM serum culture solution, continuously and carefully blowing and uniformly mixing the cells, and stopping digestion;
(4) centrifuging the mixed suspension in the centrifugal tube at 1000 rpm for about 3min, discarding the supernatant, adding 1 ml of PBS buffer solution for washing, and centrifuging again;
(5) removing the supernatant, adding a proper amount of DMEM serum culture solution to resuspend the cells, and counting the cells;
(6) taking a proper amount of cell suspension into a 15ml centrifuge tube, adding a proper amount of DMEM serum culture medium to the total volume of 2 ml, adding 10 mu mol CID755673 micromolecules, and carefully blowing, beating and uniformly mixing;
(7) taking a well-coated cell culture dish, removing gelatin, transferring the 2 ml suspension to the culture dish, and sliding by a horizontal cross method to ensure that cells and small molecules are uniformly distributed;
(8) placing the cell culture dish at 37 ℃ and 5% CO2Culturing in cell culture box, observing cell shape and growth condition every day, and changing culture solution every other day.
7. The use of claim 6, wherein the addition of two small molecules CID755673 and PD0325901 can maintain the embryonic stem cells of the mouse in a long-term self-renewal state.
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