CN111304157B - Method for obtaining bovine initial state induced pluripotent stem cells - Google Patents
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Abstract
A method for obtaining bovine initial state induced pluripotent stem cells belongs to the field of stem cell culture, wherein bovine induced pluripotent stem cells are cultured for 3-4 days under the conditions of 5% carbon dioxide and 37 ℃, an induced culture medium is discarded, PBS buffer solution is used for washing, the culture medium is incubated at 37 ℃, clone edges are observed to be separated from feeder cells, then digestive juice is sucked, the induced culture medium is added, and bovine induced pluripotent stem cells are scraped from the feeder cells; digesting with 0.05% pancreatin at 37 ℃, adding 1mL of transformation culture medium, re-suspending into a 1.5mL centrifuge tube, centrifuging at 1000rpm for 5min, inoculating onto a fresh feeder layer, and replacing transformation culture medium for culturing until a dome shape appears, wherein cloning similar to ESCs of the mouse embryo stem cells is bovine initial state induced pluripotent stem cells. The invention solves the problems of incomplete pluripotency of the existing bovine induced pluripotent stem cells and the like.
Description
Technical Field
The invention belongs to the field of stem cell culture, and particularly relates to a method for obtaining bovine initial state induced pluripotent stem cells.
Background
Embryonic stem cells (Embryonic Stem Cells, ESCs) are a type of pluripotent stem cells isolated from an Inner Cell Mass (ICM) that can differentiate into various types of cells of the three germ layers. Mouse ESCs can be directly obtained from embryos, and the obtained ESCs are reinjected into the embryos and transplanted into parents to generate chimeric offspring. For large livestock cattle, only a class of stem cells similar in morphology to mouse epidermal stem cells (Epiblast stem cells, epiSCs) can be obtained using the same medium, but such cells are not capable of forming chimeric embryos. In recent years, researchers try to combine and screen different small molecule inhibitors in animals such as humans, pigs, monkeys and the like, and clone similar to mouse ESCs in morphological and biological characteristics is obtained. But are all transformed from morphology resembling EpiSCs and are not obtained directly from embryos. It can be seen that the dependence on small molecules is different from species to species, in other words, the pathway regulation varies widely from species to species. Up to now, in large domestic animals, particularly cattle, it has been reported that stem cells which can differentiate and develop into three germ layer tissues and cells in vitro and in vivo are obtained. These stem cells have a distinct boundary with feeder cells and express multipotent genes such as Oct4, nanog, etc., but still resemble EpiSCs in morphology and are unable to withstand single cell passaging.
Induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) are a class of cells that reprogram somatic cells into ESCs with morphological characteristics similar to ESCs, not only express multipotent genes at levels similar to ESCs, but also have the potential for multipotent differentiation and development. In cattle, iPSCs mostly take the form of EpiSCs, have the expression of multipotent genes, and accept a mechanical passage mode. This is also far from true ESCs, so in order to obtain bovine iPSCs with biological properties closer to those of early embryos, it is first necessary to obtain clones with morphological characteristics similar to those of mouse iPSCs/ESCs.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for obtaining bovine initial state induced pluripotent stem cells, which solves the problems of incomplete pluripotency and the like of the existing bovine induced pluripotent stem cells.
The invention aims at realizing the following technical scheme: a method for obtaining bovine-derived, initially-induced pluripotent stem cells, comprising the steps of:
step 1, culturing bovine induced pluripotent stem cells:
placing bovine induced pluripotent stem cells in a carbon dioxide incubator, culturing under the condition of containing 5% carbon dioxide and the temperature of 37 ℃, replacing an induction culture medium at regular time every day, culturing for 3-4 days, discarding the induction culture medium, washing with PBS buffer solution, incubating for 8-10 min at the temperature of 37 ℃ with 1mg/mL dispase dispersion enzyme, observing that cloning edges are separated from feeder cells, absorbing digestion liquid, adding the induction culture medium, scraping bovine induced pluripotent stem cells from the feeder cells, and taking the bovine induced pluripotent stem cells as bovine initial induced pluripotent stem cells for culturing bovine initial induced pluripotent stem cells;
step 2, culturing bovine initial state induced pluripotent stem cells;
the process of converting the bovine induced pluripotent stem cells obtained in the step 1 into bovine initial state induced pluripotent stem cells is as follows: and (3) digesting the bovine induced pluripotent stem cells with 0.05% pancreatin at 37 ℃ for 2min, adding 1mL of transformation culture medium, re-suspending into a 1.5mL centrifuge tube, centrifuging at 1000rpm for 5min, inoculating onto a fresh feeder layer after centrifuging, and replacing the transformation culture medium for culturing until the dome shape appears, and cloning similar to the ESCs of the mouse embryonic stem cells, namely the bovine initial state induced pluripotent stem cells.
Further, the induction culture medium comprises the following components in percentage by weight: basal medium 87%Knockout DMEM,10%KSR,Lif (10000X), 1% streptomycin penicillin, 1% glutamine, 1% non-essential amino acids, mercaptoethanol (1000X), 3. Mu.M CHIR99021, 1. Mu.M PD0325901,8ng/mL basic fibroblast growth factor.
Further, the method for scraping the bovine induced pluripotent stem cells in the step 1 is as follows: cattle induced pluripotent stem cells are scraped from feeder cells with a gun head or cell scraper in a Z-shape from left to right and top to bottom.
The step 1 further includes: and (3) inoculating the scraped bovine induced pluripotent stem cells onto the fresh feeder cells, wherein the bovine induced pluripotent stem cells inoculated onto the fresh feeder cells account for 1/6 of the total amount of the scraped bovine induced pluripotent stem cells.
Further, the transformation medium in step 2 was 12.5mg/mL insulin, (10 x) N2supplement,1ng/mL TGF-b1,5mM SB202190,10mM SP600125,10mM SB203580, 50mg/mL Vitamin C added on the induction medium.
Through the design scheme, compared with the prior art, the invention has the following beneficial effects:
firstly, the invention fully utilizes the characteristics of an originating state and an initial state to distinguish different multipotent stages of the bovine induced multipotent stem cells (iPSCs);
second, the present invention results in bovine induced pluripotent stem cells that are closer to early embryo.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a undue limitation of the invention, in which:
FIG. 1 is a morphology of bovine induced pluripotent stem cells, induced pluripotent stem cells during culture;
FIG. 2 is a morphology of bovine initial iPSCs, forming dome-shaped clones with distinct boundaries with surrounding feeder cells 4 days after induction.
Detailed Description
The invention is further illustrated below in connection with preferred embodiments. The present examples are provided for illustration of the invention and are not intended to limit the scope of the invention. The experimental procedure of this example, in which specific conditions are not noted, is generally followed by conventional conditions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are merely exemplary.
The method for obtaining the bovine initial state induced pluripotent stem cells comprises two stages of culturing bovine induced pluripotent stem cells and culturing bovine initial state induced pluripotent stem cells:
1. culturing bovine induced pluripotent stem cells:
the culture of the bovine induced pluripotent stem cells is specifically performed as follows: placing bovine induced pluripotent stem cells in a carbon dioxide incubator, culturing under the conditions of 5% carbon dioxide and 37 ℃ of temperature, and replacing an induction culture medium every day at regular time, wherein the induction culture medium comprises the following components in percentage by weight: basic culture medium 87%Knockout DMEM,10%KSR,Lif (10000 x), 1% streptomycin penicillin, 1% glutamine, 1% nonessential amino acid, mercaptoethanol (1000 x), 3 mu M CHIR99021,1 mu M PD0325901,8ng/mL basic fibroblast growth factor, culturing for 3-4 days, and carrying out passage according to clone growth vigor and state; discarding the induction culture medium, washing with PBS buffer solution to prevent the components in the culture medium from affecting digestion effect, incubating for 9min at 37 ℃ with 1mg/mL dispase dispersion enzyme, observing separation of clone edge and feeder cells, sucking digestion solution, adding the induction culture medium, scraping cells from left to right with gun head or cell scraper in Z shape from top to bottom, thereby ensuring sufficient digestion of cells; inoculating the scraped bovine induced pluripotent stem cells onto fresh feeder cells, wherein the bovine induced pluripotent stem cells inoculated onto the fresh feeder cells account for 1/6 of the total amount of the scraped bovine induced pluripotent stem cells, so that stable propagation of the bovine induced pluripotent stem cells is ensured, and a morphological diagram of the bovine induced pluripotent stem cells in the culture process is shown in FIG. 1, wherein the morphological diagram of the bovine induced pluripotent stem cells is shown in FIG. 1;
2. culturing bovine initial state induced pluripotent stem cells:
the process of converting the bovine induced pluripotent stem cells into bovine initial induced pluripotent stem cells is as follows: bovine induced pluripotent stem cells were digested with 0.05% pancreatin at 37 ℃ for 2min, resuspended in 1.5mL centrifuge tube with 1mL transformation medium added 12.5mg/mL insulin based on induction medium, (10 x) N2supplement,1ng/mL TGF-b1,5mM SB202190,10mM SP600125,10mM SB203580, 50mg/mL Vitamin C, centrifuged at 1000rpm for 5min, centrifuged, inoculated onto fresh feeder layer, and the transformation medium was replaced for culture until dome-like mouse ESCs-like clones appeared, i.e. bovine-initiated pluripotent stem cells. Turning in detail to FIG. 2, FIG. 2 shows a morphology of bovine initial iPSCs, forming dome-shaped clones that are well defined with surrounding feeder cells 4 days after induction.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions that would be within the spirit and scope of the invention are also within the scope of the invention, which is defined by the following claims.
Claims (3)
1. A method for obtaining bovine-derived, initially-induced pluripotent stem cells, comprising the steps of:
step 1, culturing bovine induced pluripotent stem cells:
placing bovine induced pluripotent stem cells in a carbon dioxide incubator, culturing under the condition of containing 5% carbon dioxide and the temperature of 37 ℃, replacing an induction culture medium at regular time every day, culturing for 3-4 days, discarding the induction culture medium, washing with PBS buffer solution, incubating for 8-10 min at the temperature of 37 ℃ with 1mg/mL dispase dispersion enzyme, observing that cloning edges are separated from feeder cells, absorbing digestion liquid, adding the induction culture medium, scraping bovine induced pluripotent stem cells from the feeder cells, and taking the bovine induced pluripotent stem cells as bovine initial induced pluripotent stem cells for culturing bovine initial induced pluripotent stem cells;
step 2, culturing bovine initial state induced pluripotent stem cells;
the process of converting the bovine induced pluripotent stem cells obtained in the step 1 into bovine initial state induced pluripotent stem cells is as follows: digesting bovine induced pluripotent stem cells with 0.05% pancreatin at 37 ℃ for 2min, adding 1mL of transformation culture medium, re-suspending into a 1.5mL centrifuge tube, centrifuging at 1000rpm for 5min, inoculating onto a fresh feeder layer after centrifuging, and replacing transformation culture medium for culturing until dome shape appears, and cloning similar to ESCs of mouse embryonic stem cells, namely bovine initial induced pluripotent stem cells;
the induction culture medium comprises the following components in percentage by weight: basic medium 87% knockoutdmem,10% ksr, lif (10000 x), 1% streptomycin penicillin, 1% glutamine, 1% non-essential amino acids, mercaptoethanol (1000 x), 3 μ MCHIR99021,1 μ MPD0325901,8ng/mL basic fibroblast growth factor;
wherein, the transformation medium in the step 2 is added with 12.5mg/mL insulin, (10 x) N2 supply, 1ng/mLTGF-b1,5mM SB202190,10mM SP600125,10mM SB203580, 50mg/mLVitamin C on the basis of the induction medium.
2. The method for obtaining bovine-naive induced pluripotent stem cells according to claim 1, wherein: the method for scraping the bovine induced pluripotent stem cells in the step 1 is as follows: cattle induced pluripotent stem cells are scraped from feeder cells with a gun head or cell scraper in a Z-shape from left to right and top to bottom.
3. The method for obtaining bovine-naive induced pluripotent stem cells according to claim 1, wherein: step 1 further comprises inoculating the scraped bovine induced pluripotent stem cells to fresh feeder cells, wherein the bovine induced pluripotent stem cells inoculated to the fresh feeder cells account for 1/6 of the total amount of the scraped bovine induced pluripotent stem cells.
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