CN112553145A - Induced differentiation method of high-efficiency stereotyped endoderm cells - Google Patents

Induced differentiation method of high-efficiency stereotyped endoderm cells Download PDF

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CN112553145A
CN112553145A CN202011561906.2A CN202011561906A CN112553145A CN 112553145 A CN112553145 A CN 112553145A CN 202011561906 A CN202011561906 A CN 202011561906A CN 112553145 A CN112553145 A CN 112553145A
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differentiation
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definitive endoderm
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孙平楠
周小玲
周奇
钟志乾
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Shantou University Medical College
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Abstract

The invention belongs to the technical field of biology, and particularly relates to an induced differentiation method of high-efficiency definitive endoderm cells, which comprises the following steps: A. adding a TGF-beta activator, a GSK-3 inhibitor and an mTOR inhibitor into stem cells for induced differentiation, and culturing in a first-stage culture medium; B. differentiation is induced by addition of an activator of TGF- β and an inhibitor of BMP type I receptor, and the definitive endoderm cell stage is completed in the first stage medium. The invention solves the problems of high differentiation cost, low differentiation quality, incomplete differentiation, low differentiation efficiency, insufficient purity of the definitive endoderm cells obtained after differentiation and the like in the existing differentiation method.

Description

Induced differentiation method of high-efficiency stereotyped endoderm cells
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an induced differentiation method of high-efficiency definitive endoderm cells.
Background
Transplantation of human primary hepatocytes (PHH) is one of the important ways to effectively treat end-stage liver diseases and inherited metabolic liver diseases. Meanwhile, because the liver is an important place of drug metabolism, the human primary hepatocytes are also the gold standard for detecting drug metabolism and toxicity. PHH is in great global demand but its use in clinical and scientific research is greatly restricted due to limited donor sources. In addition, a hepatocyte is a highly differentiated cell that readily dedifferentiates and loses its hepatocyte function under in vitro culture conditions. Therefore, it is of great practical significance to find a PHH substitute that is effective and abundant in source. There are many researchers currently using several liver cancer cell lines (e.g., HepG2 and Huh 7). However, liver cancer cells are very different from primary human hepatocytes. For example, the liver cancer cell line has the problems of gene expression disorder, defective signal pathways, and insufficient sensitivity of natural immune response compared with normal liver cells. The mature liver-like cell chromosome is normal, is more similar to the human primary liver cell in physiological and biochemical characteristics, has complete cell signal path, can reflect the actual condition of natural cells more truly, and has greater application potential.
Human embryonic stem cells (hESCs) have a strong self-renewal capacity and can differentiate into different types of somatic cells. Liver cells (hESC-Heps) obtained by in vitro directed differentiation of human embryonic stem cells have characteristics closer to PHH and can be stably obtained in large quantities, so that the liver cells are considered as candidate cells with application potential and can replace PHH. Therefore, the development of an efficient hepatic differentiation method of stem cells has important significance for obtaining hESC-Heps which has performance closer to PHH and can be practically applied.
Typical differentiation methods mimic the patterns and stages observed during embryonic development, and reconstitute the necessary molecular and cellular cues in vitro. The differentiation of cells to specific lineages is controlled by external signals, and stem cells make specific selections in fates of various cell differentiation directions at key node moments of cell development. The existing in vitro directional differentiation method generally comprises the steps of adding various growth factors and small molecular compounds in vitro and combining corresponding different culture media to perform directional differentiation. The hESC-Heps directed differentiation method mainly comprises three steps, namely definitive endoderm differentiation, hepatic induction and hepatocyte maturation.
Definitive endoderm differentiation is the first step in the directed differentiation of stem cells into hESC-Heps, and is also an important step. In vivo animal models (e.g., mice), the pluripotent ectoderm (mouse embryonic development day 5.5, E5.5) differentiates into preprimitive stripes (about embryonic development day 6.5,. about.E 6.5) and further generates Definitive Endoderm (DE) (about embryonic development day 7.0-7.5,. about.E 7.0-E7.5). Definitive endoderm then differentiates along the anterior-posterior axis to develop into distinct foregut, midgut and hindgut regions (approximately 8.5 days of embryonic development, -E8.5), followed by development into distinct endodermal organs. The in vitro directional induced differentiation of stem cells is to firstly differentiate stem cells into precursor cells and then further induce and differentiate the precursor cells into definitive endoderm cells by simulating the mode and stage of in vivo development.
To date, different schemes of differentiation from stem cells into the endodermal lineage have been reported. Transforming growth factor beta (TGF β) family members are essential for the formation of nodes in the endoderm. High concentrations of activin a (activin a) are widely used for endodermal induction.
The existing differentiation method mainly comprises an A/C/AW/AT differentiation method.
A, i.e. three consecutive days, was differentiated by addition of activin A (100ng/ml) (final concentration of added substance in parentheses). The technology comes out: non-patent document 1: hepatology,2010.51(1): p.297-305.
C, i.e. differentiation with addition of CHIR99021 (3. mu.M) for three consecutive days (final concentration of added substance in parentheses). The technology comes out: non-patent document 2: stem Cell Reports,2015.4(5): p.939-52.
AW, i.e.three consecutive days, was differentiated by addition of activin A (100ng/ml) + Wnt3a (100ng/ml) (final concentration of added material in parentheses). The technology comes out: non-patent document 3: proc Natl Acad Sci USA 2008; 105:12301-12306.
AT, i.e., for three consecutive days, was differentiated by the addition of activin A (100ng/ml) + Torin 2(15nM) (final concentration of added material in parentheses). The technology comes out: non-patent document 4: nat Commun,2015.6: p.7212.
The main problems of the current differentiation method include two aspects: on one hand, the differentiation quality is a problem, and the existing differentiation method also has the problems of incomplete differentiation, low differentiation efficiency, insufficient purity of the obtained definitive endoderm cells after differentiation (insufficient homogeneity of the obtained definitive endoderm cells) and the like. On the other hand, the differentiation cost is a problem, and most of the inducers used in the existing differentiation methods are expensive growth factors, which results in high production cost. These problems have resulted in inefficient and expensive existing differentiation, which has greatly limited the large-scale production of stem cell-derived definitive endoderm. Therefore, there is an urgent need for an efficient and cost-effective differentiation method. We have established a new differentiation method in the present invention to obtain more efficient and pure definitive endoderm and hepatocyte cells for future research and application.
Disclosure of Invention
The invention aims to solve the problems of high differentiation cost, low differentiation quality, incomplete differentiation, low differentiation efficiency, insufficient purity of definitive endoderm cells obtained after differentiation and the like of the existing differentiation method, and the invention optimizes and combines the proportion of different small molecules and cytokines by analyzing a key signal path in the DE differentiation process to ensure that hESCs are efficiently differentiated towards DE and simultaneously block differentiation towards other lineages, thereby establishing an efficient definitive endoderm cell induction method and further obtaining more mature hepatic-like cells with the function of hepatocytes.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for inducing differentiation of high-efficiency definitive endoderm cells comprises the following steps:
A. adding a TGF-beta activator, a GSK-3 inhibitor and an mTOR inhibitor into stem cells for induced differentiation, and culturing in a first-stage culture medium;
B. differentiation is induced by addition of an activator of TGF- β and an inhibitor of BMP type I receptor, and the definitive endoderm cell stage is completed in the first stage medium.
In order to obtain more efficient and pure definitive endoderm cells and further more mature liver-like cells in the stage of definitive endoderm induction formation, a novel method for obtaining definitive endoderm and liver-like cells by efficiently and directionally inducing hESCs is established by optimizing the combination of induced differentiation factors. The definitive endoderm differentiation method of ACT + AD is obtained by optimizing the proportion and the concentration of various growth factors and small molecular compounds. Specifically, differentiation is induced by the addition of TGF- β activator (A), GSK-3 inhibitor (C), mTOR inhibitor (T) and BMP type I receptor inhibitor (D) at the induction of definitive endoderm stage.
In the stage of inducing definitive endoderm, activation of TGF-beta and Wnt signal paths and simultaneous inhibition of mToR signal paths are used for efficient induced differentiation, namely A, C, T is added on the first day of differentiation for induced differentiation, activation of TGF-beta and inhibition of BMP signal paths are used for blocking differentiation of stem cells to mesoderm (mesoderm) while efficiently inducing differentiation of definitive endoderm on the second and third days of differentiation, A and D are added for induced differentiation on the second and third days of differentiation, and the stem cell differentiation medium is used as a stem cell differentiation medium in the first stage of stem cell differentiation to the stage of definitive endoderm.
Preferably, after completion of the definitive endoderm cell stage, differentiation is further induced into cells of a tissue comprising one of liver, pancreas, lung, intestine and stomach.
Since stem cells can be further differentiated into tissues such as liver, pancreas, lung, intestine, and stomach after being differentiated into definitive endoderm cells, the above cells can be differentiated by this method, but the subsequent differentiation is different.
Preferably, the TGF- β activator comprises Activin a; the GSK-3 inhibitor comprises one or more of LY2090314, CHIR-98014, BIO-acetoxyime, AZD2858, SAR502250, CHIR99021 and CHIR-99021 trihydrochloride; the mTOR inhibitor comprises one or more of Rapamycin, mTOR inhibitor-3, Torin 2; the BMP type I receptor inhibitor comprises one or more of K02288 and LDN193189/DM 3189.
It is known to those skilled in the art that similar effects can be achieved with any particular class of TGF- β activators (A), GSK-3 inhibitors (C), mTOR inhibitors (T) and BMP type I receptor inhibitors (D).
Preferably, the TGF-beta activator is used at a concentration of 5-20 ng/ml, the GSK-3 inhibitor is used at a concentration of 1-10 mu M, the mTOR inhibitor is used at a concentration of 10-1000 nM, and the BMP type I receptor inhibitor is used at a concentration of 10-1000 nM.
Preferably, the concentration of Activin A used is 20 ng/ml.
Preferably, CHIR99021 is used at a concentration of 3. mu.M.
Preferably, the Torin2 is used at a concentration of 15 nM.
Preferably, the DM3189 is used at a concentration of 250 nM.
The amount of cytokine, small molecule compound or drug required for different cells is different, but the concentration added is constant. For example, a 12-well plate will typically employ 1ml of daily medium, at which time 20ng of TGF- β activator (typically a formulated stock solution, e.g. 20. mu.g/ml) will be added. However, if a 6-well plate or other differentiation is used, the medium to be added will vary accordingly, but the concentration will be constant.
Preferably, said step a is for 1 day; step B was for 2 days.
Preferably, the tissue is liver; after the definitive endoderm cell stage is completed, further inducing differentiation comprises the following steps:
C. after finishing the stage of definitive endoderm, entering a stage of inducing hepatic progenitor cells, adding dimethyl sulfoxide (DMSO) for inducing differentiation, and finishing the stage of inducing hepatic progenitor cells in a second-stage culture medium;
D. after finishing the stage of inducing hepatic progenitor cells, entering a stage of inducing hepatic-like cells, adding hepatocyte growth factor, tumor suppressor and glucocorticoid dexamethasone (Dex) for inducing differentiation, and finishing the stage of inducing hepatic-like cells in a third-stage culture medium;
E. and finishing the stage of inducing the liver-like cells to obtain the liver-like cells.
Inducing differentiation; adding dimethyl sulfoxide (DMSO) to induce differentiation at the stage of inducing hepatic progenitor cells; in the stage of inducing hepatocyte-like cells, Hepatocyte Growth Factor (HGF), oncostatin M (OSM) and glucocorticoid dexamethasone (Dex) are added to induce differentiation. DMSO and Dex are inducers for inducing differentiation of definitive endoderm cells into liver-like cells, are inducers for subsequent differentiation, and are essential for differentiation.
The liver-like cells obtained by the differentiation method can be used for researches such as drug screening, natural immunity research and HBV infection, and can also be applied to cell therapy such as treatment of hepatic fibrosis.
Preferably, the hepatocyte growth factor is HGF, and the oncostatin is oncostatin M (OSM).
Preferably, the concentration of the dimethyl sulfoxide is 1%, the concentration of the hepatocyte growth factor is 10ng/ml, the concentration of the oncostatin is 20ng/ml, and the concentration of the glucocorticoid dexamethasone is 100 nM.
Preferably, the first stage culture medium stage is RPMI1640 culture medium consisting of penicillin-streptomycin double antibody and B-27.
Preferably, the second-stage culture medium is a Knockout DMEM culture medium consisting of penicillin-streptomycin double antibody, Glutamx, DMSO and Knockout-SR.
Preferably, the third-stage medium is an L-15 medium consisting of penicillin-streptomycin double antibody, Glutamx, Insulin-Transferrin-Selenium, ascortisone acid, hydrocortisone-21-hemisuccinate, Tryptose Phosphonate Broth, and FBS.
The penicillin-streptomycin double-antibody refers to a double antibiotic system consisting of penicillin-streptomycin, and B-27 refers to B-27Supplement, which is a serum-free additive (Gibco, cat. No. 17504044). Glutamax refers to glutamine, DMSO refers to dimethyl sulfoxide, ascorbyl acid refers to ascorbic acid, hydrocortisone-21-hemisuccinate refers to hydrocortisone, Insulin-Transferrin-Selenium refers to Insulin-Transferrin-Selenium factor, and Tryptose Phosphonate Broth refers to a culture medium. Different culture mediums are selected at different differentiation stages, which is beneficial to better differentiation and growth of cells at specific stages.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
compared with the prior art, the method optimizes the induced differentiation method for differentiating the stem cells into the definitive endoderm, and can induce and differentiate the stem cells into the more efficient and pure definitive endoderm (DE cells) so as to be more beneficial to the later differentiation process. Specifically, the expression level of the stem gene of the definitive endoderm cells obtained by differentiation is lower, the expression level of the specific gene of the definitive endoderm is higher, and the expression level of the specific gene of the mesoderm is lower. Meanwhile, the flow-type result shows that the proportion of DE marker gene positive cells is higher (95.8%). Meanwhile, the DE differentiation is carried out by using the method, only one growth factor ActivinA is needed, and the use concentration of the growth factor ActivinA can be reduced to 20ng/ml (which is 1/5 of the common use amount), so that the cost of differentiation is greatly reduced.
Drawings
FIG. 1 is a schematic diagram of the differentiation process of stem cells and inducers, cell morphology and specific marker proteins at various stages according to the present invention;
FIG. 2 is a HE staining visualization of cells of the invention;
FIG. 3 is a PAS staining view of the liver-like cells of the present invention;
FIG. 4 is an experimental observation picture of the uptake of indocyanine green by the liver-like cells of the invention;
FIG. 5 is an experimental overview of HBcAg immunofluorescence after infection of liver-like cells with HBV in accordance with the present invention;
FIG. 6 is a flow-type staining result chart of DE specific marker genes of cells obtained by different differentiation methods of stem cells;
FIG. 7 is a graph showing comparison of expression levels of sternness, DE and mesoderm-specific marker genes of cells obtained by different differentiation methods of the stem cells of the present invention;
FIG. 8 is a graph showing the comparison of the expression levels of specific gene mRNA in the stem cells induced to differentiate for 8 days and 18 days by different differentiation methods;
FIG. 9 is a comparison graph of the enzyme activity test of the P450 cell obtained by different differentiation methods of the stem cell of the present invention;
FIG. 10 is a graph comparing the survival rate of the liver-like cells of the present invention and the conventional liver cancer cell lines after 24h treatment with different concentrations of the drug;
FIG. 11 is a graph comparing the innate immune response assay elicited by the hepatocyte-like cells of the invention with a commonly used hepatoma cell line transfected with polyI: C;
FIG. 12 is a graph showing comparison between stem cell desiccation and DE-specific marker gene expression levels of the stem cells of the present invention obtained by AW and ACT + AD (ActivinA at different concentrations) differentiation methods.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Taking induced differentiation of liver-like cells as an example, the induced differentiation method of the high-efficiency definitive endoderm cells comprises the following steps:
A. day0, adding A (activin A), C (CHIR99021) and T (Torin 2) into stem cells to induce differentiation, and culturing in the first stage culture medium, wherein the concentration of A is 20ng/ml, C is 3 μ M, and T is 15 nM;
B. day1, differentiation was induced by addition of A and D (LDN193189/DM3189), A was used at a concentration of 20ng/ml and D was 250nM, and the definitive endoderm cell stage was completed in the first stage medium for 2 days.
C. Day3, entering into liver progenitor cell induction stage after finishing definitive endoderm cell stage, adding dimethyl sulfoxide (DMSO) to induce differentiation, finishing liver progenitor cell induction stage in second stage culture medium, wherein DMSO concentration is 1%;
D. day8, entering into the stage of inducing liver-like cells after finishing the stage of inducing liver progenitor cells, adding Hepatocyte Growth Factor (HGF), tumor suppressor M and glucocorticoid dexamethasone (Dex) to induce differentiation, finishing the stage of inducing liver-like cells in a third stage culture medium, wherein the HGF use concentration is 10ng/ml, the tumor suppressor M use concentration is 20ng/ml, and the Dex use concentration is 100 nM;
E. day18, completing the stage of inducing liver-like cells, obtaining liver-like cells.
The first stage culture medium is preferably RPMI1640 medium consisting of diabody and B-27, the second stage culture medium is preferably Knockout DMEM medium consisting of diabody, Glutamx, DMSO and Knockout-SR, and the third stage culture medium is preferably L-15 medium consisting of diabody, Glutamx, Insulin-Transferrin-Selenium, ascortisone acid, hydrocortisone-21-hemisuccinate, Tryptose Phosphonate Broth and FBS).
In order to obtain more efficient and pure qualitative endoderm cells and further obtain more mature liver-like cells in the stage of definitive endoderm induction formation, a novel method for obtaining definitive endoderm and liver-like cells by efficiently and directionally inducing hESCs is established. The definitive endoderm differentiation method of ACT + AD is obtained by optimizing the proportion and the concentration of various growth factors and small molecular compounds. Specifically, differentiation is induced by the addition of A (TGF- β activator), C (GSK-3 inhibitor), T (mTOR inhibitor) and D (BMP type I receptor inhibitor) at the induction of the definitive endoderm stage.
In the stage of inducing definitive endoderm, activation of TGF-beta and Wnt signal paths and simultaneous inhibition of mToR signal paths are used for efficient induced differentiation, namely A, C, T is added on the first day of differentiation for induced differentiation, activation of TGF-beta and inhibition of BMP signal paths are used for blocking differentiation of stem cells to mesoderm (mesoderm) while efficiently inducing differentiation of definitive endoderm on the second and third days of differentiation, A and D are added for induced differentiation on the second and third days of differentiation, and the stem cell differentiation medium is used as a stem cell differentiation medium in the first stage of stem cell differentiation to the stage of definitive endoderm.
The differentiation process of stem cells and inducers, cell morphology and specific marker proteins at various stages are shown in FIG. 1. As can be seen from FIG. 1, the cells before induced differentiation were originally in the typical stem cell morphology, and were small in size, large in nucleus, high in nuclear-to-mass ratio, and densely arranged. In this case, the cells generally grow in colony form, and the formed cell clones have distinct boundaries and various forms (mostly, island-like, cluster-like, or oval-like). After induced differentiation, the cells gradually become typical hepatocyte shapes, the volume is increased, the cells are in polygonal shapes and are uniformly distributed, obvious boundaries exist among the cells, and the cells with double cores or multiple cores can be seen under a microscope. From the cell morphology and specific marker proteins, it can be seen that this differentiation method can obtain liver-like cells with typical hepatocyte characteristics.
The liver-like cells obtained by this differentiation method can be used for studies such as drug screening, natural immunity studies and HBV infection studies.
Example 2
Taking induced differentiation of liver-like cells as an example, the induced differentiation method of the high-efficiency definitive endoderm cells comprises the following steps:
A. day0, adding A (activin A), C (LY2090314) and T (rapamycin) into stem cells to induce differentiation, and culturing in the first stage culture medium, wherein A is used at a concentration of 100ng/ml, C is 10 μ M, and T is 1000 nM;
B. day1, differentiation was induced by addition of A and D (K02288), A was used at a concentration of 10ng/ml and D was 1000nM, and definitive endoderm cell stage was completed in the first stage medium for 2 days.
C. Day3, entering into liver progenitor cell induction stage after finishing definitive endoderm cell stage, adding dimethyl sulfoxide (DMSO) to induce differentiation, finishing liver progenitor cell induction stage in second stage culture medium, wherein DMSO concentration is 1%;
D. day8, entering into the stage of inducing liver-like cells after finishing the stage of inducing liver progenitor cells, adding Hepatocyte Growth Factor (HGF), tumor suppressor M and glucocorticoid dexamethasone (Dex) to induce differentiation, finishing the stage of inducing liver-like cells in a third stage culture medium, wherein the HGF use concentration is 10ng/ml, the tumor suppressor M use concentration is 20ng/ml, and the Dex use concentration is 100 nM;
E. day18, completing the stage of inducing liver-like cells, obtaining liver-like cells.
The first stage culture medium is preferably RPMI1640 medium consisting of diabody and B-27, the second stage culture medium is preferably Knockout DMEM medium consisting of diabody, Glutamx, DMSO and Knockout-SR, and the third stage culture medium is preferably L-15 medium consisting of diabody, Glutamx, Insulin-Transferrin-Selenium, ascortisone acid, hydrocortisone-21-hemisuccinate, Tryptose Phosphonate Broth and FBS).
The cells before induced differentiation are originally in a typical stem cell shape, and have the advantages of small volume, large cell nucleus, high nuclear-to-mass ratio and compact cell arrangement. In this case, the cells generally grow in colony form, and the formed cell clones have distinct boundaries and various forms (mostly, island-like, cluster-like, or oval-like). After induced differentiation, the cells gradually become typical hepatocyte shapes, the volume is increased, the cells are in polygonal shapes and are uniformly distributed, obvious boundaries exist among the cells, and the cells with double cores or multiple cores can be seen under a microscope. From the cell morphology and specific marker proteins, it can be seen that this differentiation method can obtain liver-like cells with typical hepatocyte characteristics.
The liver-like cells obtained by this differentiation method can be used for studies such as drug screening, natural immunity studies and HBV infection studies.
Example 3
Taking induced differentiation of liver-like cells as an example, the induced differentiation method of the high-efficiency definitive endoderm cells comprises the following steps:
A. day0, adding A (activin A), C (SAR502250) and T (mTOR inhibitor-3) into stem cells for induced differentiation, and culturing in a first-stage culture medium, wherein the use concentration of A is 5ng/ml, C is 1 μ M, and T is 10 nM;
B. day1, differentiation was induced by addition of A and D (LDN193189), A was used at a concentration of 5ng/ml and D was 10nM, and definitive endoderm cell stage was completed in the first stage medium for 2 days.
C. Day3, entering into liver progenitor cell induction stage after finishing definitive endoderm cell stage, adding dimethyl sulfoxide (DMSO) to induce differentiation, finishing liver progenitor cell induction stage in second stage culture medium, wherein DMSO concentration is 1%;
D. day8, entering into the stage of inducing liver-like cells after finishing the stage of inducing liver progenitor cells, adding Hepatocyte Growth Factor (HGF), tumor suppressor M and glucocorticoid dexamethasone (Dex) to induce differentiation, finishing the stage of inducing liver-like cells in a third stage culture medium, wherein the HGF use concentration is 10ng/ml, the tumor suppressor M use concentration is 20ng/ml, and the Dex use concentration is 100 nM;
E. day18, completing the stage of inducing liver-like cells, obtaining liver-like cells.
The first stage culture medium is preferably RPMI1640 medium consisting of diabody and B-27, the second stage culture medium is preferably Knockout DMEM medium consisting of diabody, Glutamx, DMSO and Knockout-SR, and the third stage culture medium is preferably L-15 medium consisting of diabody, Glutamx, Insulin-Transferrin-Selenium, ascortisone acid, hydrocortisone-21-hemisuccinate, Tryptose Phosphonate Broth and FBS).
The cells before induced differentiation are originally in a typical stem cell shape, and have the advantages of small volume, large cell nucleus, high nuclear-to-mass ratio and compact cell arrangement. In this case, the cells generally grow in colony form, and the formed cell clones have distinct boundaries and various forms (mostly, island-like, cluster-like, or oval-like). After induced differentiation, the cells gradually become typical hepatocyte shapes, the volume is increased, the cells are in polygonal shapes and are uniformly distributed, obvious boundaries exist among the cells, and the cells with double cores or multiple cores can be seen under a microscope. From the cell morphology and specific marker proteins, it can be seen that this differentiation method can obtain liver-like cells with typical hepatocyte characteristics.
The liver-like cells obtained by this differentiation method can be used for studies such as drug screening, natural immunity studies and HBV infection studies.
Comparative example 1
The same procedure and control of conditions as in example 1 were used, with the difference that the differentiation method A was used, i.e. step A and step B were replaced by: differentiation was carried out in first stage medium with addition of Activin A (100ng/ml) for three consecutive days.
Comparative example 2
The same procedure and control of conditions as in example 1 were used, with the difference that the differentiation method C was used, i.e., step A and step B were replaced with: differentiation was carried out in first stage medium with addition of Activin A (100ng/ml) for three consecutive days.
Comparative example 3
The same procedure and condition control as in example 1 were used, except that the AW differentiation method was used, i.e., step A and step B were replaced with: differentiation was carried out in first stage medium for three consecutive days with addition of Activin A (100ng/ml) + Wnt3a (100 ng/ml).
Comparative example 4
The same procedure and condition control as in example 1 were used, except that the AT differentiation method was used, that is, step A and step B were replaced with: differentiation was carried out in the first phase medium, i.e.three consecutive days, with addition of Activin A (100ng/ml) + Torin 2(15 nM).
Effect example 1
Hematoxylin-eosin staining (HE staining) is one of the commonly used methods for cell staining. The hematoxylin stains the nucleus blue and the eosin stain the cytoplasm purple. The cells obtained in example 1 were observed for HE staining at a scale of 50 μm, and the results are shown in FIG. 2.
As can be seen in fig. 2, HE staining revealed the formation of binuclear cells (the portion of the rectangular box selected was visible), while binuclear or polynuclear cells are typical morphology of mature hepatocytes. The formation of binuclear nuclei was observed in HE staining results of liver-like cells obtained after differentiation, demonstrating that the stem cells have been morphologically successfully differentiated into hepatocytes.
Effect example 2
The liver-like cells obtained in example 1 were observed by Periodic Acid-Schiff reaction staining (PAS staining) at a scale of 100. mu.m, and the results are shown in FIG. 3.
Periodic acid can oxidize hydroxyl groups on two adjacent carbons of the saccharide into aldehyde groups, and the oxidized aldehyde groups can react with Schiff reagent to show purple red. The cells to be detected (liver-like cells differentiated according to the method) are sucked away from the culture medium, washed for 1-2 times by adding PBS, and fixed by adding 4% paraformaldehyde fixing solution or ice methanol. After fixing for 1h, adding double-distilled water to wash for 4 times, and each time for 2 min. Adding oxidant to cover (300 μ l in 24-well plate), and standing at room temperature (25-30 deg.C) for 8min, preferably not more than 10 min. The double-distilled water is used for immersion washing for 3 times, each time for 1 min. Add Schiff Reagent to cover, and place in the shade at room temperature (25-30 ℃) for dip dyeing for 20 min. Washing with double distilled water for 3 times, each for 3-4 min. Covering with sujilin staining solution, and staining cell nucleus for 2 min. Adding double distilled water, washing for 3-4min, and turning blue. And adding double distilled water to perform under-mirror shooting. As can be seen from the results in FIG. 3, the differentiated hepatocyte-like cells were almost all stained purple red by PAS staining, which proves that they had excellent glycogen accumulating effect.
Effect example 3
The liver-like cells obtained in example 1 were subjected to experimental observation of indocyanine green uptake at a scale bar of 100 μm, and the results are shown in fig. 4.
Indocyanine green is a dye drug used to examine liver function and effective blood flow of the liver, and can be efficiently and selectively taken up by hepatocytes and released from the hepatocytes. In this experiment, a solution of indocyanine green was prepared, i.e. a certain amount of indocyanine green powder was weighed and dissolved in DMSO, typically in a 1g/ml stock solution. The cells (liver-like cells differentiated according to the method) were added to the well of the cell at a final concentration of 1mg/ml and diluted with the medium. After incubation for 1-2h in an incubator, wash 3 times with PBS and take pictures. As can be seen from fig. 4, most of the liver-like cells are green, which proves that most of the liver-like cells take up indocyanine green, i.e., the pigment uptake rate of the liver-like cells is high, which proves that the liver-like cells obtained by differentiation in example 1 have high degree of differentiation and maturation.
Effect example 4
The results of the observation of HBcAg immunofluorescence after HBV infection of the liver-like cells obtained in example 1 are shown in FIG. 5, with a scale bar of 100. mu.m. Cells differentiated to about 14 days by this differentiation method were infected. The concentrated hepg2.2.15 supernatant was added to the infection medium, and 2% DMSO and 10% 40% PEG8000 were added, mixed well and added to the wells to be infected. The uninfected control (Negative control) was prepared without the concentrated supernatant, and the remaining conditions were the same as those in the infected wells. The HBV infection-inhibiting drug CsA (cyclosporin A), which is treated with this drug at the same time, was used to reduce the amount of HBV infection. Infection time is 16-24 hours, virus supernatant is discarded after infection, the virus supernatant is slowly washed twice by PBS, then fresh complete culture medium is replaced, supernatant is collected every two days until the 9 th day, and fixed cells are subjected to HBcAg immunofluorescence experiment. In the figure, DAPI is the result of DAPI staining, i.e., the result of cell nucleus staining, HBcAg is the result of target protein HBc protein, and MERGE is the synthetic graph of the result of DAPI staining and HBcAg staining.
As can be seen from FIG. 5, HBcAg was detected in HBV-infected cells (the bright spot in HBcAg in the figure is HBcAg detected after infection) compared to the negative control, and the amount of HBcAg detected in cells to which CsA, a drug for suppressing HBV infection, was added was significantly reduced, demonstrating that the differentiated hepatocyte-like cells of example 1 were successfully infected with HBV.
Effect example 5
The liver-like cells obtained in example 1 and comparative examples 1 to 4 were subjected to a DE-specific marker gene flow staining test, and a group of blank controls Day0 (i.e., undifferentiated hESCs) without induction was set, with the results shown in FIG. 6.
As can be seen from FIG. 6, the flow-type results of the liver-like cells obtained by the method of the present invention show a higher proportion of cells positive for the definitive endoderm marker gene.
Effect example 6
The cells obtained in example 1 and comparative examples 1 to 4 were examined for the expression levels of stem cell sternness, definitive endoderm and mesoderm-specific marker genes, and a set of blank control Day0 (i.e., undifferentiated hESC) was set and plotted as Log2FC (i.e., - Δ Δ CT) against GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The results of the experiment are shown in FIG. 7.
Wherein OCT4 and SOX2 refer to specific marker genes of stem cells; SOX17, FOXA2, FZD8, GATA4, CXCR4, and FOXA1 refer to specific marker genes for Day3 definitive endoderm cells; CDX1, EVX1, MSX1, PDGFR, MESP2 and MSGN1 refer to specific marker genes for Day3 mesodermal cells. As can be seen from FIG. 7, the novel induction method is more efficient and pure in differentiation than the previous methods, i.e., the cells obtained after differentiation by the method of the present invention have higher expression levels of their endoderm-specific genes and lower expression levels of mesoderm and sternness genes.
Effect example 7
The mRNA expression levels of the stage-specific genes were compared between example 1 and comparative examples 1 to 4 after 8 days and 18 days of induced differentiation, respectively, as shown in FIG. 8.
Among them, Day8 refers to cells after induced differentiation for three days by A, C, AW, AT and ACT + AD, and 5 days by treating them with the second-stage medium under the same conditions. Day18 refers to cells that were induced to differentiate for three days by A, C, AW, AT and ACT + AD, treated with the second medium under the same conditions for 5 days, and then treated with the third medium under the same conditions for 10 days. Wherein TTR, TBX3, HNF4A, AFP, CEBPA and PROX1 refer to specific marker genes of Day8 hepatic progenitor cells; AAT, ALB, TTR, ASGR1, NTCP refer to the specific marker genes of Day18 liver-like cells. Results cells obtained by the A differentiation method were used as a control group and plotted as Log2FC (i.e., - Δ Δ CT) against GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
As can be seen from FIG. 8, the liver progenitor cells and liver-like cells obtained by the ACT + AD differentiation method exhibited higher levels of stage-specific marker genes, i.e., liver progenitor cells and liver-like cells were obtained more efficiently, as compared to other differentiation methods.
Effect example 8
The liver-like cells obtained in example 1 and comparative examples 1 to 4 were subjected to a P450 enzyme activity test, and the results are shown in FIG. 9 using a commonly used liver cancer cell line HepG2 as a control group.
Cytochrome P450 (CYP 450) is an important subject for drug metabolism research, and cytochrome P450 of liver plays an important role in drug metabolism. Cytochrome P4503a4 enzyme (CYP3a4) is an important member of the cytochrome P450 enzyme family, occurs primarily in the liver and small intestine, and is one of the most common and multifaceted in drug metabolism. As can be seen in FIG. 9, HLCs obtained by ACT + AD differentiation have higher P450 enzyme activity than HLCs and HepG2 obtained by other methods.
Effect example 9
The liver-like cells of example 1 were treated with various concentrations of the drug for 24h and then tested for cell viability, using the conventional liver cancer cell line HepG2 as a control group, and the results are shown in FIG. 10.
As can be seen in FIG. 10, for the non-hepatotoxic drug ursodeoxycholic acid (UDCA), HepG2 and HLCs showed essentially no significant change in cell survival at low, medium and high concentrations, i.e., the drug was not hepatotoxic. However, the difference is that troglitazone (troglitazone), which is a hepatotoxic drug, basically has no obvious change in the survival rate of both cells at low concentrations of 7.5. mu.M and the like, but the survival rate of HLCs is obviously reduced at medium concentrations of 15-60. mu.M and the like, namely the drug toxicity effect is obvious, and the survival rate of HepG2 cells is basically not reduced. Compared with PHH treated by troglitazone in other literature results, the response pattern of HLCs obtained after differentiation to drugs is more similar to that of the PHH compared with HepG2, which indicates that the liver cancer cell line is not sensitive enough to drug toxicity, and the model of drug screening is not as good as that of the HLCs.
Effect example 10
The hepatocyte-like cells of example 1 were tested for the initiation of innate immune response by transfection with poly I: C.
The hepatocyte-like cells of example 1 were transfected with the hepatoma cell lines HepG2 and Huh7 to polyI: C, and RNA of the cells was collected 0, 4, 16, and 24 hours after transfection for detection of various natural immune factors. The results of recording the mRNA expression levels of various natural immune factors after transfection of poly I: C with HLCs, HepG2 and Huh7 were plotted against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) with 0h transfection (i.e., untransfected) as a control, and the results are shown in FIG. 11.
As can be seen from FIG. 11, the expression of each gene of the innate immunity increased with time, and sharply increased within 16h after transfection, with a markedly reduced increase from 16h to 24 h. Comparing the three cells, the natural immune response of the liver-like cells obtained by differentiation (with more perfect cell path compared with liver cancer cells) after transfection of polyI: C can be found to be more sensitive than liver cancer cell lines HepG2 and Huh 7. Therefore, the ACT + AD differentiated HLCs are more suitable for the research of natural immunity.
Effect example 11
The same operations and condition controls as in example 1 were used except that the concentrations of A used were changed to 0, 40, 60, 80 and 100ng/ml, respectively, and the other operations and condition controls were the same as in example 1.
The cells obtained were compared with those obtained in example 1 and comparative example 3 for the determination of the sternness of the cells and the expression level of the DE-specific marker gene, and a set of blank control Day0 (i.e., undifferentiated hESC) was set and plotted as Log2FC (i.e., - Δ Δ Δ CT) against GAPDH (glyceraldehyde 3-phosphate dehydrogenase), the results of which are shown in FIG. 12.
Wherein OCT4 and SOX2 refer to specific marker genes of stem cells; SOX17, FOXA2, FZD8, GATA4, CXCR4, and FOXA1 refer to specific marker genes for Day3 definitive endoderm cells. As can be seen from FIG. 12, the cell results obtained by differentiating with different concentrations of ActivinA show that there is no significant difference between the expression levels of different concentrations of ActivinA (except 0ng/ml) of desiccation and definitive endoderm specific genes, so that the concentration of the growth factor ActivinA can be reduced to 20 ng/ml.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.

Claims (9)

1. A method for inducing differentiation of high-efficiency definitive endoderm cells is characterized by comprising the following steps:
A. adding a TGF-beta activator, a GSK-3 inhibitor and an mTOR inhibitor into stem cells for induced differentiation, and culturing in a first-stage culture medium;
B. differentiation is induced by addition of an activator of TGF- β and an inhibitor of BMP type I receptor, and the definitive endoderm cell stage is completed in the first stage medium.
2. The method for inducing differentiation of high-potency definitive endoderm cells according to claim 1, wherein after completion of the definitive endoderm cell stage, further differentiation into cells of a tissue comprising one of liver, pancreas, lung, intestine and stomach is induced.
3. The method of inducing differentiation of high-potency definitive endoderm cells of claim 1, wherein the TGF- β activator comprises Activin a; the GSK-3 inhibitor comprises one or more of LY2090314, CHIR-98014, BIO-acetoxyime, AZD2858, SAR502250, CHIR99021 and CHIR-99021 trihydrochloride; the mTOR inhibitor comprises one or more of Rapamycin, mTOR inhibitor-3, Torin 2; the BMP type I receptor inhibitor comprises one or more of K02288 and LDN193189/DM 3189.
4. The method of inducing differentiation of high-potency definitive endoderm cells according to claim 1, wherein the TGF- β activator is used at a concentration of 5 to 20ng/ml, the GSK-3 inhibitor is used at a concentration of 1 to 10 μ M, the mTOR inhibitor is used at a concentration of 10 to 1000nM, and the BMP type I receptor inhibitor is used at a concentration of 10 to 1000 nM.
5. The method for inducing differentiation of high-potency definitive endoderm cells according to claim 1, wherein the duration of step a is 1 day; step B was for 2 days.
6. The method of inducing differentiation of high-potency definitive endoderm cells according to claim 1, wherein the tissue is liver; after the definitive endoderm cell stage is completed, further inducing differentiation comprises the following steps:
C. after finishing the stage of definitive endoderm, entering a stage of inducing hepatic progenitor cells, adding dimethyl sulfoxide for inducing differentiation, and finishing the stage of inducing hepatic progenitor cells in a second-stage culture medium;
D. after finishing the stage of inducing hepatic progenitor cells, entering a stage of inducing hepatic-like cells, adding hepatocyte growth factor, tumor suppressor and glucocorticoid dexamethasone for inducing differentiation, and finishing the stage of inducing hepatic-like cells in a third-stage culture medium;
E. and finishing the stage of inducing the liver-like cells to obtain the liver-like cells.
7. The method of inducing differentiation of high-potency definitive endoderm cells according to claim 6, wherein the hepatocyte growth factor is HGF and the anti-tumor agent is anti-tumor agent M.
8. The method for inducing differentiation of high-potency definitive endoderm cells according to claim 6, wherein the concentration of dimethyl sulfoxide is 1%, the concentration of hepatocyte growth factor is 10ng/ml, the concentration of oncostatin is 20ng/ml, and the concentration of glucocorticoid dexamethasone is 100 nM.
9. The method for inducing differentiation of high-potency definitive endoderm cells according to claim 6, wherein the first-stage medium stage is RPMI1640 medium consisting of penicillin-streptomycin double antibody and B-27; the second-stage culture medium is a Knockout DMEM culture medium consisting of a penicillin-streptomycin double antibody, Glutamax, DMSO and Knockout-SR; the third-stage culture medium is an L-15 culture medium consisting of penicillin-streptomycin double antibody, Glutamx, Insulin-Transferrin-Selenium, ascorbic acid, hydrocortisone-21-hemisuccinate, Tryptose Phosphonate Broth and FBS.
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