CN115029302B - Culture method for maintaining mouse embryo stem cell stem property - Google Patents
Culture method for maintaining mouse embryo stem cell stem property Download PDFInfo
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Abstract
The invention provides a culture method for maintaining the stem cell dryness of a mouse embryo, and the cells can be maintained in a brand new form under the condition and can be maintained for a long time and subcultured. The culture medium comprises N2B27 culture medium, and 2 mu MIWR1, 1 mu MPD0325901 and 50 mu M Forskolin are added. The present invention uses entirely new culture conditions and the method may be applied to the culture of embryonic stem cells of other mammals including humans. According to the invention, a brand-new method for culturing the mouse embryonic stem cells is established, so that the stem cell culturing conditions can be better optimized, a new network for regulating and controlling the pluripotency of the mouse embryonic stem cells is disclosed, understanding of different pluripotency states of the stem cells by people is deepened, and a new insight is provided for optimizing the current stem cell culturing conditions to adapt to the different pluripotency states.
Description
Technical Field
The invention relates to a method for in vitro induction and establishment of pluripotent stem cells
Background
Embryonic stem cells (Embryonic stem cells, ESCs) are derived from an inner cell mass at the blastocyst stage prior to implantation, and under certain conditions ESCs are capable of immortalizing (self-renewing) in an undifferentiated state. The cell volume is small, the morphology is similar to that of early embryo cells, the cell nucleus is large, the cell arrangement is compact, the cell edge is clear, and the cell grows in a colony shape in vitro by inhibiting differentiation culture. Can be induced into a plurality of cells (pluripotency) under certain conditions. Therefore, embryonic stem cells are often used as a very useful tool for studying the basic research of developmental biology such as animal cell tissue differentiation, embryogenesis, cell signaling network regulation and the like. Embryonic stem cells can be classified into two categories depending on their origin: primitive state derived from cell mass in preimplantation blastocysts
Embryonic stem cells (EpiSCs) derived from the initial state of embryonic epiblast (Primed) following implantation. Pluripotency of early and late ectoderm of mammal can be achieved by +.>
The embryonic stem cells in the state and the epiblast stem cells in the Primed state are represented respectively, and represent two states before and after implantation of an embryo in vivo, however, these two pluripotent states may not be sufficient to reflect the full complexity and developmental potential of the ectoderm in early development of mammals. In recent years scientists have proposed to be between +.>
And Primed pluripotent state, and cells in this state should be capable of chimerism to the embryo ICM and induction of primordial germ cells (Primordial Germ Cell PGC) in vitro. This pluripotent state is then designated as a constitutive, i.e. intermediate stem cell. This finding allows a better description of a model of the continuous development of pluripotency, thus finding and building +.>
Intermediate stem cells between the multipotent state of Primed have become entirely new hot spots. In the early experiments we screened various small molecules and performed cell induction experiments. It was finally found that when MEK/ERK and Wnt/β -catenin signaling pathways were blocked and an intracellular cAMP inducer (Forskolin) was added, the mouse embryonic stem cells were able to maintain a certain morphology and were able to be serially subcultured, and their phenotypes were different from several intermediate stem cells that have been reported. We named this pluripotent stem cell as rfISC. Under these conditions, other mammals, including humans, may also be successfully induced and established. Therefore, by establishing intermediate stem cells, a model of the continuous development of pluripotency can be better described, and the capability of dynamic change of ectoderm in early and later stages of embryo development of mammals can be better summarized. Can open up a new way for researching mammal pluripotency and researching a molecular mechanism for regulating and controlling primordial germ cell differentiation and promote the clinical application of the pluripotent stem cells.
Forskolin (Coleonol) is a potent adenylate cyclase activator. Is also an inducer of intracellular cAMP formation. Forskolin can also induce autophagy.
PD0325901 is an inhibitor of MEK/ERK signaling pathway with oral activity, selectivity and non-ATP competition, and previous studies have found that PD0325901 can maintain pluripotency of embryonic stem cells by inhibiting the expression of p-ERK1/2 and has anticancer activity against various human tumor xenografts.
IWR1 is a Wnt-beta-catenin signal channel inhibitor, can inhibit the activity of tankyras, and IWR1 can degrade beta-catenin by stabilizing Axin protein.
The three small molecules PD0325901, IWR1 and Forskolin are added into a serum-free culture system of the mouse embryonic stem cells, and the mouse embryonic stem cells can grow normally under the conditions, and the phenotype of the mouse embryonic stem cells can maintain a certain state. The cells were serially passaged to find that the cells were stable under the conditions under which the phenotype was not changed, so we further examined to demonstrate that under the conditions, the cells were in intermediate stem cells, and different from several intermediate stem cells that have been reported. The establishment of intermediate stem cells can better describe a model of multipotency continuous development and better summarize the capability of dynamic change of ectoderm in early and later stages of embryo development of mammals.
Disclosure of Invention
The invention aims to provide a method for in vitro induction and establishment of pluripotent stem cells by exploring new culture conditions of mouse embryonic stem cells.
The invention solves the technical problems by adopting the technical scheme that the type and the concentration of small molecules of the probe are based on the culture of N2B 27. The mouse embryo stem cells are induced to differentiate into an intermediate stem cell by adding three small molecules of PD0325901, IWR1 and Forskolin. And such intermediate stem cells were named rfiscs. This method may also be applicable to the in vitro induction and establishment of rfiscs in other mammals, including humans.
The specific operation of the invention comprises the following steps:
(1) Taking 0.5ml gelatin-coated cell culture plate with 0.1% concentration, placing at 37deg.C and 5% CO 2 A cell culture box with the concentration of the cells,wrapping the plate for 30min;
(2) Taking P20 mouse embryo stem cells with the growth density of 70-80%, discarding the culture solution, washing once with PBS buffer solution, and removing the residual culture solution;
(3) Adding 0.3ml of trypsin with the concentration of 0.05%, digesting the cells for about 2min until the edges of the cells float, blowing the cells by a pipetting gun, sucking the cell suspension, transferring the cell suspension into a 1.5ml centrifuge tube containing 1ml of DMEM serum culture solution, continuously blowing and uniformly mixing, and stopping digestion;
(4) After centrifugation at 1000rpm for 3min, the supernatant was discarded, and 1ml of DMEM medium containing 10% fbs was added to resuspend the cells, which were counted by using a cell counter plate, and thus cell density was calculated;
(5) Taking out the packaged culture plate, discarding gelatin, and adding 1ml of DMEM culture solution containing 10% FBS into each well of the culture plate;
(6) 6.0X10 were added to each culture well 3 Adding 1 mu L of Lif, horizontally shaking in a cross shape to uniformly distribute the cells, and placing the cells into an incubator for culturing for 24 hours until the cells are completely adhered to the wall;
(7) After the cells are completely adhered, the culture medium is replaced by an N2B27 culture medium, and the following small molecules are respectively added into the cell culture plates: (1) 10ng/ml of ActivinA+2 μM IWR1, (2) 10ng/ml of ActivinA+2 μM IWR1+500nM LDN193189, (3) 10ne/ml of ActivinA+2 μM IWR1+50 μM Forskolin, (4) 10ng/ml of ActivinA+2 μM IWR1+2 μM DAPT, (5)2 μM IWR1+1 μM PD0325901, (6) 10ng/ml of ActivinA+2 μM IWR1+1 μM PD0325901, (7)2 μM IWR1+1 μM PD0325901+50 μM Forskolin, (8)2 μM IWR1+1 μM PD0325901+500nM LDN 189;
(8) The cell culture dish was placed at 37℃in 5% CO 2 The cells were cultured in a concentrated cell incubator.
Cell self-renewal state detection
Morphological observation, cells under the culture conditions (1) to (8) were observed by using a Leica DMIL inverted microscope, respectively, and as a result, it was found that when the small molecules added under the culture conditions were 2. Mu.M IWR1, 1. Mu.M PD0325901, and 50. Mu.M Forskolin, the mouse embryonic stem cells were slightly differentiated, and the phenotype was maintained in a certain morphology, and serial subculture was possible and the self-renewal state was maintained. While the remaining groups of cells had died.
The invention has the following advantages:
(1) Three small molecules, PD0325901, IWR1, forskolin, were added to DMEM medium (N2B 27 medium) containing both serum substitutes of N2 and B27 to maintain the pluripotent state of mouse embryonic stem cells. Under these conditions, the mouse embryonic stem cells can grow normally and their phenotype can maintain a certain state. The cells were found to be stable under these conditions by serial passage.
(2) Different from other intermediate stem cell induction methods, the invention uses brand-new induction conditions, and the induced cells have high stability, can be continuously passed, and have unchanged phenotype.
(3) The invention can better describe a model of the continuous development of multipotency by establishing a brand-new intermediate stem cell, and better summarize the capability of dynamic change of ectoderm in early and later stages of embryo development of mammals.
(4) The novel pluripotent stem cells established by the invention can open up a new way for researching the pluripotency of mammals and researching a molecular mechanism for regulating and controlling the differentiation of primordial germ cells.
(5) The method may be applicable to the induction of embryonic stem cells in other mammals, including humans.
Drawings
FIG. 1 shows the cell morphology observation when the induction differentiation is performed on the P20-generation embryonic stem cells of the mice under different conditions,
each cell was cultured under different conditions, which were, in order, (1) 10ng/ml ActivinA+2μM IWR1, (2) 10ng/ml ActivinA+2μM IWR1+500nM LDN193189, (3) 10ng/ml ActivinA+2μM IWR1+50 μM Forskolin, (4) 10ng/ml ActivinA+2μM IWR1+2μM DAPT, (5)2 μM IWR1+1μM PD0325901, (6) 10ng/ml ActivinA+2μM IWR1+1μM PD 032595301, (7)2 μM IWR1+1μM PD0325901+50 μM Forskolin, (8)2 μM IWR1+1 nM PD0325901+500nM LDN 193189); it was found that when the culture conditions were 2. Mu.M IWR1+1. Mu.M PD 0325901+50. Mu.M Forskolin, the cell phenotype was slightly differentiated and maintained in its self-renewing state, while under the remaining conditions, the cells all died, so we chose to conduct serial subculture under this condition.
FIG. 2 shows the phenotypic observation of cells serially subcultured under 2. Mu.M IWR1+ 1. Mu.M PD 0325901+50. Mu.M Forskolin culture conditions, wherein cells can be serially subcultured and their phenotype does not change when they are cultured under such conditions. Experiments have shown that cells can be passaged more than 20 times in succession under these conditions and maintain their self-renewing capacity.
Several intermediate stem cells such as RSC and FSC reported in the present stage are induced and successfully established according to the culture conditions. And culturing
Cells in a pluripotent state with Primed (i.e., ESC and EpiSC). FIG. 3 is a graph showing the results of AP staining of RSC, FSC, ESC, epiSC and cells cultured under these conditions, showing that cells have a self-renewal capacity between RSC and FSC under these conditions.
Detailed Description
Examples
Mouse embryonic stem cells used in the experiments were supplied by university of south california in the united states.
Exploration of intermediate stem cell-induced culture conditions and methods, and serial subculturing of cells under the conditions explored, and multipotent comparison with several known intermediate states:
(1) Taking 0.5ml gelatin-coated cell culture plate with 0.1% concentration, placing at 37deg.C and 5% CO 2 A cell incubator with concentration, a plate for 30min;
(2) Taking P20 mouse embryo stem cells with the growth density of 70-80%, discarding the culture solution, washing once with PBS buffer solution, and removing the residual culture solution;
(3) Adding 0.3ml of trypsin with the concentration of 0.05%, digesting the cells for about 2min until the edges of the cells float, blowing the cells by a pipetting gun, sucking the cell suspension, transferring the cell suspension into a 1.5ml centrifuge tube containing 1ml of DMEM serum culture solution, continuously blowing and uniformly mixing, and stopping digestion;
(4) After centrifugation at 1000rpm for 3min, the supernatant was discarded, and 1ml of DMEM medium containing 10% fbs was added to resuspend the cells, which were counted by using a cell counter plate, and thus cell density was calculated;
(5) Taking out the packaged culture plate, discarding gelatin, and adding 1ml of DMEM culture solution containing 10% FBS into each well of the culture plate;
(6) 6.0X10 were added to each culture well 3 Adding 1 mu L of Lif, horizontally shaking in a cross shape to uniformly distribute the cells, and placing the cells into an incubator for culturing for 24 hours until the cells are completely adhered to the wall;
(7) After the cells are completely adhered, the culture medium is replaced by an N2B27 culture medium, and the following small molecules are respectively added into the cell culture plates: (1) 10ng/ml of ActivinA+2 μM IWR1, (2) 10ng/ml of ActivinA+2 μM IWR1+500nM LDN193189, (3) 10ng/ml of ActivinA+2 μM IWR1+50 μM Forskolin, (4) 10ng/ml of ActivinA+2 μM IWR1+2 μM DAPT, (5)2 μM IWR1+1 μM PD0325901, (6) 10ng/ml of ActivinA+2 μM IWR1+1 μM PD0325901, (7)2 μM IWR1+1 μM PD0325901+50 μM Forskolin, (8)2 μM IWR1+1 μM PD0325901+500nM LDN 189;
(8) The cell culture dish was placed at 37℃in 5% CO 2 The cells were cultured in a concentrated cell incubator.
Cell self-renewal state detection:
(1) Morphological observation, cells under the culture conditions (1) to (8) were observed by using a Leica DMIL inverted microscope, respectively, and as a result, it was found that when the small molecules added under the culture conditions were 2. Mu.M IWR1, 1. Mu.M PD0325901, and 50. Mu.M Forskolin, the mouse embryonic stem cells were slightly differentiated, and the phenotype was maintained in a certain morphology, and serial subculture was possible and the self-renewal state was maintained. While the remaining groups of cells were dead, as shown in figures 1 and 2.
(2) Alkaline phosphatase (Alkaline Phosphatase, AP) staining detects self-renewing conditions as follows:
a. inoculating RSC, FSC, ESC, epiSC and proper amount of cells under the condition, and culturing for a certain time to perform AP staining;
b. preparing BCIP/NBT dyeing working solution according to the description of the AP dyeing kit;
c. removing culture solution in the cell culture plate, washing with PBS for 3-5 times each for 3-5min, adding 500 μl 4% paraformaldehyde to fix cells for 1-2min, removing the fixing solution, and washing with PBS for 3-5 times each for 3-5min;
d. after the last washing is finished, removing the washing liquid, and adding a proper amount of BCIP/NBT dyeing working liquid to ensure that cells can be fully covered;
e. incubation for 30min or longer (up to 24 hours) at room temperature in dark until the color develops to the expected depth;
f. removing BCIP/NBT dyeing working solution, and washing with PBS for 1-2 times to terminate the color reaction;
g. finally, adding a proper amount of PBS, placing the culture plate on a Leica DMIL inverted microscope to observe the cell staining condition, and judging the self-renewing state of the cells. As shown in FIG. 3, it can be seen that the number of AP staining positive clones was between RSC and FSC under this condition. So its versatility is between RSC and FSC.
Claims (3)
1. A culture method for maintaining the stem property of mouse embryo stem cells is characterized in that three small molecules of 2 mu M IWR1, 1 mu M PD0325901 and 50 mu M Forskolin are added into an N2B27 culture medium to maintain the pluripotent state of the mouse embryo stem cells.
2. The method of claim 1, wherein the mouse embryonic stem cells are passaged and cultured under three small molecule conditions of PD0325901, IWR1, forskolin:
(1) Taking 0.5ml gelatin-coated cell culture plate with 0.1% concentration, placing at 37deg.C and 5% CO 2 A cell incubator with concentration, a plate for 30min;
(2) Taking P20 mouse embryo stem cells with the growth density of 70-80%, discarding the culture solution, washing once with PBS buffer solution, and removing the residual culture solution;
(3) Adding 0.3ml of trypsin with the concentration of 0.05%, digesting the cells for about 2min until the edges of the cells float, blowing the cells by a pipetting gun, sucking the cell suspension, transferring the cell suspension into a 1.5ml centrifuge tube containing 1ml of DMEM serum culture solution, continuously blowing and uniformly mixing, and stopping digestion;
(4) After centrifugation at 1000rpm for 3min, the supernatant was discarded, and 1ml of DMEM medium containing 10% fbs was added to resuspend the cells, which were counted by using a cell counter plate, and thus cell density was calculated;
(5) Taking out the packaged culture plate, discarding gelatin, and adding 1ml of DMEM culture solution containing 10% FBS into each well of the culture plate;
(6) 6.0X10 were added to each culture well 3 Adding 1 mu L of LIF, horizontally shaking in a cross shape to uniformly distribute the cells, and culturing in an incubator for 24 hours until the cells are completely adhered to the wall;
(7) After the cells are completely adhered, the culture medium is replaced by an N2B27 culture medium, and the following small molecules are added into a cell culture plate: 2. Mu.M IWR1, 1. Mu.M PD0325901 and 50. Mu.M Forskolin:
(8) The cell culture dish was placed at 37℃in 5% CO 2 The cells were cultured in a concentrated cell incubator.
3. The method of claim 2, wherein the cells are maintained in a morphology and can be serially subcultured when 2 μm IWR1, 1 μm PD0325901, 50 μm Forskolin are added.
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