CN114525240A - Material and method for differentiation of pluripotent stem cells into hematopoietic stem cells - Google Patents
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Abstract
The invention discloses a material and a method for differentiating pluripotent stem cells into hematopoietic stem cells, relates to the technical field of biology, and aims at solving the problems of high difficulty, high technical cost and the like of differentiating the pluripotent stem cells into the hematopoietic stem cells. According to the invention, multi-domain induction of pluripotent stem cells to hematopoietic stem cells can be realized under the conditions of constant temperature and normal oxygen concentration in culture media generated on the basis of different culture solutions, and basement membrane extracts and vitronectin are used for providing nutrients for stem cell differentiation, so that the differentiation cost is reduced, meanwhile, the process of adding the culture solution is reduced, and further, the hematopoietic stem cells with stable state strength are formed.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a material and a method for differentiation of pluripotent stem cells into hematopoietic stem cells.
Background
Pluripotent stem cells are all types of stem cells that can develop into the body, but cannot develop into extra-embryonic tissue (or placenta), and are inner cell masses derived from blastocysts after the cleavage stage, and are one type of pluripotent stem cells.
The hematopoietic stem cell is an adult stem cell in a blood system, is a heterogeneous population, has the capacity of long-term self-renewal and the potential of differentiation into various mature blood cells, is the adult stem cell which has the longest research history and is the most deep, has important guiding significance for researching various stem cells including tumor stem cells, and the differentiation from the pluripotent stem cell to the hematopoietic stem cell aims at differentiating into the hematopoietic stem cell or repairing the hematopoietic stem cell through the expansion of the pluripotent stem cell so as to treat the hematological diseases.
Disclosure of Invention
Object of the invention
In order to solve the technical problems in the background art, the invention provides a material and a method for differentiating pluripotent stem cells into hematopoietic stem cells, which can realize multi-domain induction of differentiation of the pluripotent stem cells into the hematopoietic stem cells under the conditions of constant temperature and normal oxygen concentration in culture media generated on the basis of different culture solutions, and provide nutrients for differentiation of the stem cells by utilizing a basement membrane extract and vitronectin, so that the differentiation cost is reduced, meanwhile, the process of adding the culture solution is reduced, and then the hematopoietic stem cells with stable state strength are formed.
(II) technical scheme
The invention provides a material and a method for differentiation of pluripotent stem cells into hematopoietic stem cells, comprising a basement membrane extract, vitronectin and a cell culture medium;
the method also comprises a method for differentiating the pluripotent stem cells into the hematopoietic stem cells, and specifically comprises the following steps:
s1: preparing a basement membrane extract;
s2: preparing vitronectin;
s3: preparing culture medium, and culturing the prepared basement membrane extract and vitronectin in the culture medium
S4: and treating the induced pluripotent stem cells, and putting the treated induced pluripotent stem cells into a culture medium to culture the treated induced pluripotent stem cells together with a basement membrane extract and vitronectin to obtain hematopoietic stem cells after amplification and differentiation.
As a preferred embodiment of the present invention, the basement membrane extract in step S1 is prepared by the following specific steps:
s1: purifying stem cells, extracting a soluble basement membrane extract, performing polymerization reaction on the basement membrane extract, and freezing to obtain a gel matrix;
s2: thawing the gel matrix, diluting in culture medium, and refrigerating.
As a preferred embodiment of the present invention, the vitronectin in the step S2 is prepared by the following steps:
vitronectin was stored at-80 ℃, thawed, aliquoted, diluted with PBS reagent and incubated in medium at room temperature.
In a preferred embodiment of the present invention, the culture medium in step S3 is prepared by the following steps:
step 1: removing the refrigerated supplement, thawing, and adding to a basal medium under sterile conditions;
step 2: the medium was then heated to room temperature while avoiding light to obtain a medium.
In a preferred embodiment of the present invention, the supplement is any one of amino acids, insulin or water-soluble milk protein.
In a preferred embodiment of the present invention, the induced pluripotent stem cells are treated in step S4 as follows:
step 1: gently rotating the freezing tube in a water bath to thaw the induced pluripotent stem cells, and disinfecting the freezing tube with 70% ethanol or an equivalent disinfectant before opening;
step 2, transferring the cryoprotectant and the cell mixture from the freezing tube to a centrifugal tube by using a sterile pipette;
and step 3: slowly dropping proper culture solution into a centrifuge tube at room temperature, slightly shaking the centrifuge tube back and forth, centrifuging cells, removing and pouring out supernatant, and adding a proper amount of culture solution;
and 4, step 4: the tube was tapped to detach the cell pellet, seeded into coated 6-well plates, and the plates were gently shaken to spread the cells evenly throughout the wells for culture.
Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:
in conclusion, in the culture medium generated on the basis of different culture solutions, multi-domain induction of pluripotent stem cells to hematopoietic stem cells can be realized under the conditions of constant temperature and normal oxygen concentration, and the basement membrane extract and vitronectin are used for providing nutrients for stem cell differentiation, so that the differentiation cost is reduced, meanwhile, the process of adding the culture solution is reduced, and further, the hematopoietic stem cells with stable state strength are formed.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a schematic representation of the number of cell differentiations according to the present invention;
FIG. 2 is a graph showing the differentiation rate of cells according to the present invention.
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. In the present invention, the term "pharmaceutically acceptable" means that the indicated material is not of a nature that would cause reasonable caution in a physician avoiding administration of the material to a patient, taking into account the disease or condition to be treated and the corresponding route of administration. For example, it is generally desirable that such materials be substantially sterile, and in the present invention, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting and/or stopping one or more clinical symptoms of a disease or disorder before, during and/or after injury or intervention, and in the present invention, the term "patient" or "subject" refers to an animal, including a mammal, e.g., a mouse, dog, horse, cow, ape or human, and particularly a human, being treated with a pharmaceutical composition or according to the methods described herein.
While the various publications, patents and published patent specifications cited herein have been incorporated by reference in their entirety, for the purpose of describing a preferred embodiment of the invention, the disclosure is to be clearly and completely described below in connection with the examples, it is to be understood that the examples are illustrative of only some of the examples, and not restrictive of the scope of the invention, and that all other examples, which would be within the purview of one of ordinary skill in the art based on the examples and are intended to be protected by the present invention without the exercise of inventive faculty.
Detailed Description
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. It should be understood that throughout the drawings, identical or similar reference numerals indicate identical or similar parts and features. The drawings are only schematic representations of the concepts and principles of the embodiments of the disclosure, and do not necessarily show specific dimensions or proportions of the various embodiments of the disclosure. Certain features that are part of a particular figure may be exaggerated in order to illustrate relevant details or structures of embodiments of the present disclosure.
The first embodiment is as follows: a material and method for differentiation of pluripotent stem cells into hematopoietic stem cells:
purifying a soluble basement membrane extract from tumor cells by using a gel matrix in the stem cells, polymerizing the matrix at the temperature of 20-40 ℃ to form a reconstructed basement membrane, thawing the gel matrix, diluting the thawed gel matrix in a culture medium, refrigerating, thawing, putting the basement membrane extract into the culture medium by using a liquid transfer device, diluting the basement membrane extract by using a DMEM culture medium at a dilution ratio of 1:80, and obtaining the ECM gel matrix;
adding a 1:80 diluted ECM gel matrix into each well of a 6-well plate, rotating the culture plate to uniformly spread the ECM gel matrix on the surface of the plate, and storing the ECM gel matrix in an environment of 2-8 ℃ for cold storage overnight for at least 2 hours, or coating the ECM gel matrix with a preservative film and storing the ECM gel matrix in an environment of 2-8 ℃;
thawing vitronectin at room temperature, preparing an aliquot in a sterile polypropylene tube, freezing and storing the aliquot, gently mixing PBS buffer solution and vitronectin after thawing, diluting the vitronectin according to a ratio of 1:100, adding the diluted vitronectin into each hole of a 6-hole plate, and coating the vitronectin and incubating for 1 hour at room temperature;
taking out the basic culture medium in a sterile environment, adding an insulin supplement into the basic culture medium, equally dividing the culture medium into the volumes required by 1 week of culture, heating the basic culture medium to room temperature before use, and then avoiding illumination to place the culture medium at room temperature;
placing a cryovial filled with induced pluripotent stem cells in a water bath at 37 ℃, thawing the cells, gently rotating the cryovial in the water bath, disinfecting the cryovial with 70% ethanol or an equivalent disinfectant before opening, transferring a cryoprotectant and a cell mixture from the cryovial to a centrifuge tube with a 5 mL sterile pipette, slowly adding a proper culture solution dropwise into the centrifuge tube at room temperature, gently shaking the centrifuge tube while adding a few drops of the culture solution, centrifuging the cells for 2 minutes, and removing and pouring off a supernatant;
before inoculating cells, the plate containing the ECM gel matrix is placed back to room temperature for 10-15 minutes, coating solution is removed, induced pluripotent stem cells are added into each hole, after the cells are cultured for 5-10 minutes, the cells on the plate are taken out and placed into a culture medium containing amino acid supplement, vitronectin is added at the same time, after the cells are cultured for a period of time, the cultured cells are extracted and placed into a centrifuge tube, the centrifuge tube is tapped to separate cell precipitates, the cells are uniformly spread in a culture dish, culture solution is added, and differentiated hematopoietic stem cells are obtained after the cells are cultured for a period of time.
Example two: a material and method for differentiation of pluripotent stem cells into hematopoietic stem cells, which comprises replacing insulin supplementation in a culture medium with amino acid supplementation, on the basis of example one;
purifying a soluble basement membrane extract from tumor cells by using a gel matrix in the stem cells, polymerizing the matrix at the temperature of 20-40 ℃ to form a reconstructed basement membrane, thawing the gel matrix, diluting the thawed gel matrix in a culture medium, refrigerating, thawing, putting the basement membrane extract into the culture medium by using a liquid transfer device, diluting the basement membrane extract by using a DMEM culture medium at a dilution ratio of 1:80, and obtaining the ECM gel matrix;
adding a 1:80 diluted ECM gel matrix into each well of a 6-well plate, rotating the culture plate to uniformly spread the ECM gel matrix on the surface of the plate, and storing the ECM gel matrix in an environment of 2-8 ℃ for cold storage overnight for at least 2 hours, or coating the ECM gel matrix with a preservative film and storing the ECM gel matrix in an environment of 2-8 ℃;
thawing vitronectin at room temperature, preparing an aliquot in a sterile polypropylene tube, freezing and storing the aliquot, gently mixing PBS buffer solution and vitronectin after thawing, diluting the vitronectin according to a ratio of 1:100, adding the diluted vitronectin into each hole of a 6-hole plate, and coating the vitronectin and incubating for 1 hour at room temperature;
taking out the basic culture medium in a sterile environment, adding an amino acid supplement into the basic culture medium, equally dividing the culture medium into the volumes required by 1 week of culture, heating the basic culture medium to room temperature before use, and standing the culture medium at room temperature without illumination;
placing a cryovial filled with induced pluripotent stem cells in a water bath at 37 ℃, thawing the cells, gently rotating the cryovial in the water bath, disinfecting the cryovial with 70% ethanol or an equivalent disinfectant before opening, transferring a cryoprotectant and a cell mixture from the cryovial to a centrifuge tube with a 5 mL sterile pipette, slowly adding a proper culture solution dropwise into the centrifuge tube at room temperature, gently shaking the centrifuge tube while adding a few drops of the culture solution, centrifuging the cells for 2 minutes, and removing and pouring off a supernatant;
before inoculating cells, the plate containing the ECM gel matrix is placed back to room temperature for 10-15 minutes, coating solution is removed, induced pluripotent stem cells are added into each hole, after the cells are cultured for 5-10 minutes, the cells on the plate are taken out and placed into a culture medium containing amino acid supplement, vitronectin is added at the same time, after the cells are cultured for a period of time, the cultured cells are extracted and placed into a centrifuge tube, the centrifuge tube is tapped to separate cell precipitates, the cells are uniformly spread in a culture dish, culture solution is added, and differentiated hematopoietic stem cells are obtained after the cells are cultured for a period of time.
Example three: a material and method for differentiation of pluripotent stem cells into hematopoietic stem cells, wherein the material and method are based on the first embodiment, wherein insulin supplement is replaced with water milk protein;
purifying a soluble basement membrane extract from tumor cells by using a gel matrix in the stem cells, polymerizing the matrix at the temperature of 20-40 ℃ to form a reconstructed basement membrane, thawing the gel matrix, diluting the thawed gel matrix in a culture medium, refrigerating, thawing, putting the basement membrane extract into the culture medium by using a liquid transfer device, diluting the basement membrane extract by using a DMEM culture medium at a dilution ratio of 1:80, and obtaining the ECM gel matrix;
adding a 1:80 diluted ECM gel matrix into each well of a 6-well plate, rotating the culture plate to uniformly spread the ECM gel matrix on the surface of the plate, and storing the ECM gel matrix in an environment of 2-8 ℃ for cold storage overnight for at least 2 hours, or coating the ECM gel matrix with a preservative film and storing the ECM gel matrix in an environment of 2-8 ℃;
thawing vitronectin at room temperature, preparing an aliquot in a sterile polypropylene tube, freezing and storing the aliquot, gently mixing PBS buffer solution and vitronectin after thawing, diluting the vitronectin according to a ratio of 1:100, adding the diluted vitronectin into each hole of a 6-hole plate, and coating the vitronectin and incubating for 1 hour at room temperature;
taking out the basic culture medium in a sterile environment, adding a water-lactoprotein supplement into the basic culture medium, equally dividing the culture medium into the required volumes for culturing for 1 week, and before use, heating the basic culture medium to room temperature, and then avoiding illumination to place the culture medium at room temperature;
placing a cryovial filled with induced pluripotent stem cells in a water bath at 37 ℃, thawing the cells, gently rotating the cryovial in the water bath, disinfecting the cryovial with 70% ethanol or an equivalent disinfectant before opening, transferring a cryoprotectant and a cell mixture from the cryovial to a centrifuge tube with a 5 mL sterile pipette, slowly adding a proper culture solution dropwise into the centrifuge tube at room temperature, gently shaking the centrifuge tube while adding a few drops of the culture solution, centrifuging the cells for 2 minutes, and removing and pouring off a supernatant;
before inoculating cells, the plate containing the ECM gel matrix is placed back to room temperature for 10-15 minutes, coating solution is removed, induced pluripotent stem cells are added into each hole, after the cells are cultured for 5-10 minutes, the cells on the plate are taken out and placed into a culture medium containing amino acid supplement, vitronectin is added at the same time, after the cells are cultured for a period of time, the cultured cells are extracted and placed into a centrifuge tube, the centrifuge tube is tapped to separate cell precipitates, the cells are uniformly spread in a culture dish, culture solution is added, and differentiated hematopoietic stem cells are obtained after the cells are cultured for a period of time.
Experimental example: the number of cells differentiated and the rate of differentiation of the pluripotent stem cells into hematopoietic stem cells were recorded in the same manner as in examples 1 to 3.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.
Claims (6)
1. A material for differentiation of pluripotent stem cells into hematopoietic stem cells, comprising a basement membrane extract, vitronectin and a cell culture medium;
the method also comprises a method for differentiating the pluripotent stem cells into the hematopoietic stem cells, and specifically comprises the following steps:
s1: preparing a basement membrane extract;
s2: preparing vitronectin;
s3: preparing culture medium, and culturing the prepared basement membrane extract and vitronectin in the culture medium
S4: and treating the induced pluripotent stem cells, and putting the treated induced pluripotent stem cells into a culture medium to culture the treated induced pluripotent stem cells together with a basement membrane extract and vitronectin to obtain hematopoietic stem cells after amplification and differentiation.
2. The material and method for the differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 1, wherein the basement membrane extract of step S1 is prepared by the following steps:
s1: purifying stem cells, extracting a soluble basement membrane extract, performing polymerization reaction on the basement membrane extract, and freezing to obtain a gel matrix;
s2: thawing the gel matrix, diluting in culture medium, and refrigerating.
3. The material and method for the differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 1, wherein the vitronectin in the step S2 is prepared by the following steps:
vitronectin is stored in an environment with the temperature of-80 ℃, an aliquot sample is thawed, diluted by a PBS reagent and placed in a culture medium to be incubated at room temperature.
4. The material and method for differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 1, wherein the medium in step S3 is prepared by the following steps:
step 1: removing the refrigerated supplement, thawing, and adding to a basal medium under sterile conditions;
step 2: the medium was then heated to room temperature while avoiding light to obtain a medium.
5. The material and method for differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 4, wherein the supplement is any one of amino acids, insulin or lactalbumin.
6. The material and method for differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 1, wherein the induced pluripotent stem cells are treated in step S4 as follows:
step 1: gently rotating the freezing tube in a water bath to thaw the induced pluripotent stem cells, and disinfecting the freezing tube with 70% ethanol or an equivalent disinfectant before opening;
step 2, transferring the cryoprotectant and the cell mixture from the freezing tube to a centrifugal tube by using a sterile pipette;
and step 3: slowly dropping proper culture solution into a centrifuge tube at room temperature, slightly shaking the centrifuge tube back and forth, centrifuging cells, removing and pouring out supernatant, and adding a proper amount of culture solution;
and 4, step 4: the tubes were tapped to detach the cell pellet and seeded into coated 6-well plates, and the plates were gently shaken to spread the cells evenly throughout the wells for culture.
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