CN116042508B - A kit, method and application of inducing pluripotent stem cells to differentiate into hepatocytes - Google Patents
A kit, method and application of inducing pluripotent stem cells to differentiate into hepatocytesInfo
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- CN116042508B CN116042508B CN202211696116.4A CN202211696116A CN116042508B CN 116042508 B CN116042508 B CN 116042508B CN 202211696116 A CN202211696116 A CN 202211696116A CN 116042508 B CN116042508 B CN 116042508B
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Abstract
The invention discloses a kit and a method for inducing pluripotent stem cells to differentiate into hepatocytes and application thereof. The kit comprises low-concentration resveratrol, and the invention discovers that the albumin secretion of the liver cells obtained by adding the low-concentration resveratrol to induce the expression level of the genes related to liver functions such as drug metabolizing enzyme, polarized genes and the like is obviously increased, the use of high-concentration and expensive growth factors can be reduced, the hepatic differentiation of the human multifunctional stem cells is further promoted, the method for 3D suspension directional differentiation of the human multifunctional stem cells into the liver cells is established by adopting the kit, the hepatic differentiation process of the stem cells is greatly accelerated, the differentiation cost is reduced, the maturity and the drug metabolizing capability of the liver cells are improved, and the method is more efficient and simple.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a kit and a method for inducing differentiation of pluripotent stem cells into hepatocytes and application thereof.
Background
Currently, the incidence of liver disease is significantly increasing worldwide, with in situ donor liver transplantation being the primary method of treating end-stage liver disease. In the case of shortage of donor liver organs, the patients with end-stage liver diseases and acute liver failure are clinically treated by liver cell transplantation or bioartificial liver, and each time of liver cell transplantation treatment or bioartificial liver treatment needs 10 10 hundred billion of liver cells [1]. However, due to the same lack of liver sources, sufficient hepatocytes are not available to limit clinical application to hepatocyte transplantation and bioartificial livers. In addition, the liver is a major organ of drug metabolism in the human body, and a large number of functional hepatocytes are also required for drug metabolism and toxicity tests for new drug development, drug screening, and the like.
At present, a certain gap exists between the liver function of human pluripotent stem cells (hPSCs) source liver cells obtained by a 3D differentiation method and freshly isolated human primary liver cells.
Freshly isolated Primary Human Hepatocytes (PHHs) rapidly lose their properties and function in vitro culture, and thus hepatocytes with a function comparable to PHHs have become an urgent problem [2] to be solved in clinical and biopharmaceutical fields at present.
The directional differentiation of human multifunctional stem cells into liver cells mainly comprises three stages, namely, the differentiation stage of endoderm cells, namely, the differentiation of human multifunctional stem cells into endoderm cells, the differentiation stage of liver cells, namely, the differentiation of endoderm cells into hepatic progenitor cells and hepatic precursor cells, and the differentiation stage of liver cells, namely, the differentiation of hepatic progenitor cells and hepatic precursor cells into functional mature liver cells. The subject group established a differentiation method [3,4] in the early work, successfully induced differentiation of human pluripotent stem cells cultured in two-dimensional (2D) adherence into hepatocytes.
However, the 2D differentiation system cannot mimic the 3D microenvironment of the liver in humans, and secondly, there is increasing evidence that 3D culture systems can better mimic the microenvironment in humans, promoting the generation of liver lineages and hepatocyte maturation.
In summary, the prior art has the problems of time consuming differentiation route, long time span, high time cost, more processes and more cytokines, thus more expensive culture medium and cytokines are consumed, the material cost is high, and the maturity and the function of the hepatocyte-like cells obtained by differentiating the human multifunctional stem cells are to be improved. There is an urgent need for a method of efficiently culturing hepatic cells comparable to PHHs.
[1]Chan C,Berthiaume F,Nath BD,et al.Hepatic tissue engineering for adjunct and temporary liver support:critical technologies[J].Liver Transpl,2004,10(11):1331-42.
[2]Baldari,S.,Di Rocco,G.,&Toietta,G.(2020).Current Biomedical Use of Copper Chelation Therapy.International journal of molecular sciences,21(3),1069.
[3]Duan Y,Ma X,Zou W,et al.Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells[J].Stem Cells,2010,28(4):674-86.
[4]Ma X,Duan Y,Tschudy-Seney B,et al.Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells[J].Stem Cells Transl Med,2013,2(6):409-19.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a kit for inducing differentiation of stem cells into hepatocytes.
The object of the second aspect of the present invention is to provide the use of resveratrol and/or a kit according to the first aspect of the present invention.
The object of the third aspect of the present invention is to provide a method for inducing stem cells into endoderm cells.
The object of the fourth aspect of the present invention is to provide a method for inducing stem cells to hepatic progenitors.
The object of the fifth aspect of the present invention is to provide a method for inducing stem cells to be hepatocytes.
The technical scheme adopted by the invention is as follows:
In a first aspect of the present invention, there is provided a kit comprising at least one of (a 1) to (a 3);
a1, a first culture medium, a second culture medium and a third culture medium;
a2, a fourth culture medium;
a3, a fifth culture medium;
The first culture medium, the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium comprise 10 nM-10 mu M Resveratrol;
The first culture medium, the second culture medium and the third culture medium also comprise Activin A;
The fourth culture medium also comprises BMP2 and BMP4;
The fifth culture medium also comprises Oncoinhibin M.
Preferably, the first medium, the second medium, the third medium, the fourth medium and the fifth medium comprise 100 nM-1 μ M Resveratrol;
More preferably, the first medium, the second medium, the third medium, the fourth medium, and the fifth medium comprise 1 μ M Resveratrol.
Preferably, the kit comprises a first medium, a second medium, a third medium, a fourth medium, and a substrate medium.
Preferably, the concentration of the Activin A in the first culture medium, the second culture medium and the third culture medium is 80-120 ng/mL.
Preferably, the concentration of BMP2 in the fourth culture medium is 5-15 ng/mL.
Preferably, the concentration of BMP4 in the fourth culture medium is 5-15 ng/mL.
Preferably, the concentration of the Oncoinhibin M in the fifth culture medium is 40-60 ng/mL.
Preferably, the first medium further comprises a gsk3β inhibitor, a basal medium;
preferably, the gsk3β inhibitor is CHIR99021.
Preferably, the concentration of the GSK3 beta inhibitor is 2-4 mu M.
Preferably, the second medium further comprises KSR, a basal medium.
Preferably, the KSR concentration in the second medium is 0.6-1 w/w%.
Preferably, the third medium further comprises KSR, basal medium.
Preferably, the KSR concentration in the third medium is 6-10 w/w%.
Preferably, the fourth medium further comprises FBS, L-glutamine, insulin, 1-thioglycerol, FGF-4, HGF, dexamethasone, dimethyl sulfoxide, basal medium.
Preferably, the concentration of FBS in the fourth culture medium is 10-30 w/w%.
Preferably, the concentration of L-glutamine in the fourth medium is 1-3 mM.
Preferably, the concentration of insulin in the fourth culture medium is 0.1-0.2U/mL.
Preferably, the concentration of the 1-thioglycerol in the fourth culture medium is 0.2-0.4 mM.
Preferably, the concentration of FGF-4 in the fourth medium is 10-30 ng/mL.
Preferably, the concentration of HGF in the fourth culture medium is 10-30 ng/mL.
Preferably, the concentration of dexamethasone in the fourth culture medium is 80-120 nM.
Preferably, the concentration of dimethyl sulfoxide in the fourth culture medium is 0.4-0.6 w/w%.
Preferably, the fifth medium further comprises HGF, dimethyl sulfoxide, dexamethasone, FGF-4, a first additive, and a basal medium.
Preferably, the concentration of HGF in the fifth culture medium is 10-30 ng/mL.
Preferably, the concentration of dimethyl sulfoxide in the fifth culture medium is 0.4-0.6 w/w%.
Preferably, the concentration of dexamethasone in the fifth culture medium is 80-120 nM.
Preferably, the concentration of FGF-4 in the fifth culture medium is 10-30 ng/mL.
Preferably, the first additive comprises ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor and GA-1000, and further preferably, the first additive consists of seven components within SingleQuots kit, 0.5mL ascorbic acid, 10.5mL BSA-FAF, 0.5mL hydrocortisone, 0.5mL transferrin, 0.5mL insulin, 0.5mL recombinant human epidermal growth factor and 0.5mL GA-1000,Single Quots kit available from Lonza under the accession number CC-4182.
Preferably, the basal medium of the first medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium.
Preferably, the basal medium of the second medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium.
Preferably, the basal medium of the third medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium.
Preferably, the basal medium of the fourth medium is at least one of RPMI1640 and IMDM medium.
Preferably, the basal medium of the fifth medium is a hepatocyte basal medium.
In a second aspect of the invention, there is provided the use of resveratrol and/or a kit according to the first aspect of the invention in at least one of b1 to b 6;
b1, preparing endoderm cells;
b2, preparing hepatic progenitors;
b3, preparing liver cells;
b4, preparing a product of endoderm cells;
b5 preparing a hepatic progenitor product;
b6, preparing a hepatocyte product.
According to a third aspect of the invention, there is provided a method for preparing endoderm cells, culturing stem cells in a first culture medium according to the first aspect of the invention for 12-36 hours, in a second culture medium according to the first aspect of the invention for 12-36 hours, and in a third culture medium according to the first aspect of the invention for 12-36 hours.
According to a fourth aspect of the present invention, there is provided a method for preparing hepatic progenitors, comprising culturing endoderm cells in the fourth medium according to the first aspect of the present invention for 120 to 168 hours to obtain hepatic progenitors.
Preferably, the fourth culture medium is replaced every 16-32 hours in the culture process.
Preferably, the endoderm cells are prepared by the method of the third aspect of the invention.
In a fifth aspect of the present invention, there is provided a method for preparing hepatocytes, comprising culturing hepatic progenitors in the fifth medium according to the first aspect of the present invention for 120-168 h to obtain hepatocytes.
Preferably, the fifth culture medium is replaced every 16-32 hours in the culture process.
Preferably, the hepatic progenitors are prepared by the method described in the fourth aspect of the invention.
Preferably, the stem cells are human stem cells having a multipotent differentiation potential.
Preferably, the human stem cells having multipotential differentiation potential are human pluripotent stem cells, human parthenogenesis stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells or cord blood stem cells.
Preferably, the stem cells are stem cell pellets.
Preferably, the stem cell pellet is prepared by mixing stem cells with a digestive juice, incubating, discarding the digestive juice, adding a mTESR1 culture medium containing a Rock inhibitor, re-suspending, inoculating the mixture into the mTESR1 culture medium containing the Rock inhibitor at a density of 25-100 ten thousand/mL, and culturing for 12-36 h.
Preferably, the stem cells are stem cells which have a cell coverage rate of 70% -80% and are regular in cloning edge and free of differentiated cells 4-5 days after passage.
Preferably, the Rock inhibitor is Y-27632.
Preferably, the Rock inhibitor has a final concentration of 8-12 mu M in mTESR1 medium.
The beneficial effects of the invention are as follows:
On the basis of the laboratory early-stage hepatocyte differentiation method, the invention prepares a novel kit for inducing the differentiation of pluripotent stem cells into hepatocytes by changing the culture mode (from 2D culture to 3D culture), replacing part of cytokines and adding low-concentration resveratrol. The invention discovers for the first time that the hepatic cell obtained after resveratrol is added has obviously increased hepatic function related gene expression levels such as the secretion of histone, the gene expression level of drug metabolizing enzyme, polarized gene and the like. Compared with the prior original differentiation scheme in the laboratory, the invention replaces the high-concentration and expensive growth factors by the combined use of the small molecular compound resveratrol and the low-concentration growth factors, reduces the cost, can further promote the hepatic differentiation of the human multifunctional stem cells, improves the maturity of the liver cells and the metabolism capability of medicines, differentiates the human multifunctional stem cells into more mature liver cells under the 3D suspension culture differentiation condition, shortens the differentiation time and improves the liver function of the liver cells.
The invention also provides a method for inducing stem cells to differentiate into stem cells, which establishes a more efficient and cost-saving method for differentiating human multifunctional stem cells into liver cells by 3D suspension orientation through adopting the kit provided by the invention, wherein the first differentiation stage only needs 3 days, the second differentiation stage only needs 6 days, and the third differentiation stage only needs 8 days. Compared with the traditional 2D differentiation method (32 days are required for differentiation), the method greatly accelerates the liver differentiation process of stem cells, reduces the differentiation cost, detects the differentiation efficiency at each differentiation stage, and proves that the finally differentiated liver cells have albumin secretion capacity and drug metabolism function. And it is verified that the resveratrol with low concentration can promote liver function level of liver cells under the 3D suspension culture condition, and improve maturity of the liver cells.
Drawings
Fig. 1 is a route diagram of directional induced differentiation of human multifunctional stem cells into hepatocytes, fig. 1A is a route diagram of directional induced differentiation of 2D adherent cultured human multifunctional stem cells into hepatocytes, with a scale of=100 μm, and fig. 1B is a route diagram of directional induced differentiation of 3D suspension cultured human multifunctional stem cells into hepatocytes, with a scale of=100 μm.
FIG. 2 is a graph showing the directional induced differentiation of human pluripotent stem cells into qualitative endoderm. Fig. 2A is a route map of directional induced differentiation of human multifunctional stem cells into endodermal cell pellets, fig. 2B is a cell flow detection map of SOX17 and FOXA2 of endodermal cell pellets, and fig. 2C is a cell morphology change image of directional induced differentiation of human multifunctional stem cells into endodermal cell pellets, with a scale bar=100 μm. FIG. 2D is an immunofluorescent staining of DAPI, FOXA2 of endodermal cell spheres. FIG. 2E is a graph showing the directed induction of qualitative endodermal gene expression by human pluripotent stem cells under three different 3D conditions.
FIG. 3 shows the directed induced differentiation of 3D suspension cultured hepatocyte spheroids into hepatocyte spheroids with the addition of 1. Mu.M resveratrol. Fig. 3A is a roadmap and a hepatocyte pellet image of the directed induced differentiation of human multifunctional stem cells into the hepatocyte pellet, the scale=100 μm, fig. 3B is the expression of ALB, AFP, CYP A4 of the hepatocyte pellet, fig. 3C is the expression of CPS1, FOXO1, BSEP, NTCP of the hepatocyte pellet, fig. 3D is the expression of the drug metabolizing enzyme CYP3A4 of the hepatocyte pellet induced by rifampin at 25 μm, and fig. 3E is the secretion of the hepatocyte pellet albumin.
FIG. 4 is a graph showing the results of screening resveratrol for concentrations used by using a cytotoxicity assay.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1A kit for inducing differentiation of Stem cells into endodermal cells
The first medium (DE Medium 1) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +3. Mu.M CHIR99021 (Selleck, CT 99021) +1. Mu. M Resveratrol (Selleck, S3934);
A second medium (DE medium 2) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +0.8% KSR (Thermo, 10828028) +1 μ MResveratrol (Selleck, S3934);
the third medium (DE medium 3) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +8% KSR (Thermo, 10828028) +1 μ M Resveratrol (Selleck, S3934).
Example 2A kit for inducing differentiation of Stem cells into hepatocytes
The first medium (DE Medium 1) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +3. Mu.M CHIR99021 (Selleck, CT 99021) +1. Mu. M Resveratrol (Selleck, S3934);
A second medium (DE medium 2) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +0.8% KSR (Thermo, 10828028) +1 μ MResveratrol (Selleck, S3934);
the third medium (DE Medium 3) RPMI1640 (Gibco, 61870036) +100ng/ML ACTIVIN A (recombinant human activin-A, peprotech, 120-14) +8% KSR (Thermo, 10828028) +1 μ M Resveratrol (Selleck, S3934);
Fourth Medium (HDM Medium) IMDM MEDIA (Gibco, 31980030) +20% FBS (fetal bovine serum, VISTECH, SE 100-B7953) +2mM L-glutamine (L-glutamine, gibco, 25030081) +0.126U/mL human insulin (recombinant human insulin, sigma,91077C-11 MG) +0.3mM1-thioglycerol (1-thioglycerol, sigma, M6145) +20ng/mL FGF-4 (fibroblast growth factor-4, peprotech, 100-18B-50) +20ng/mL HGF (hepatocyte growth factor, peprotech, 100-39) +10ng/mL BMP2 (bone morphogenetic protein 2, peprotech, 120-02) +10ng/mL BMP4 (bone morphogenetic protein 4, peprotech, 120-05) +0.5% DMSO (dimethyl sulfoxide, MP media) +100, 100-18B-50) +20ng/mL BMP2 (Sigma, 100-18B-50) +20S 3934;
Fifth medium (HCM medium) Hepatocyte basal medium (Lonza) (hepatocyte basal medium, lonza, CC-3911) + Single Quots kit (Lonza) (Lonza, CC-4182, specifically including seven additional components Ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, rhEGF, GA-1000) +20ng/mL HGF (hepatocyte growth factor, peprotech, 100-39) +50ng/mL oncostatin M (Oncoinhibin M, peprotech, 300-10) +0.5% DMSO (dimethyl sulfoxide, MP biomedica, 196055) +100nM dexamethasone (dexamethasone, sigma, D4902) +20ng/mL FGF4 (fibroblast growth factor-4, peprotech, 100-18B-50) +1 μ M Resveratrol (Selleck, S3934).
The embodiment also provides a directional induced differentiation method of the 3D suspension cultured human multifunctional stem cells to the liver cells, and the route diagram is shown in figure 1.
Example 3 resveratrol concentration selection
A method of inducing a cytotoxicity test comprising the steps of:
S1. hepavg resuscitation:
1) Selecting a tube of HepaRG (supplied by Huang Lizhen teacher laboratory of the university of North China university of medical science) with good freezing state, taking out the freezing tube from liquid nitrogen, and rapidly putting into a 37 ℃ water bath for melting;
2) The caps were carefully opened, the cells were transferred to a centrifuge tube containing 5mL of DMEM high sugar (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE 100-B7953), centrifuged at 1500rpm for 5 minutes, after centrifugation was completed, the supernatant was aspirated off, resuspended in 10mL of DMEM high sugar (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE 100-B7953), and the cell suspension was inoculated into 10 cm dishes and shaken cross-wise.
S2. hepavg passage:
1) After sucking and discarding the culture medium, adding 1mL of calcium-magnesium-free PBS (phosphate buffer solution) to clean the cells, adding 3mL of 0.05% pancreatin (Procell, PB 180222) after sucking and discarding, placing the cells back into a CO2 incubator to incubate for 2-4 minutes, adding 3mL of culture medium, and repeatedly blowing and cloning by using a pipetting gun to finally obtain single cell suspension;
2) 1500rpm, centrifugation for 5min, after centrifugation, the supernatant was aspirated, resuspended in 10mL of DMEM high-sugar (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE 100-B7953), and the cell suspension was inoculated into a 10 cm dish and homogenized by shaking.
S3 cytotoxicity test Cell Counting Kit-8 (CCK-8 kit, (Dongren, CK 04):
1) Preparing cell suspension, adding 1mL of calcium-magnesium-free PBS to clean cells after the culture medium is sucked and removed by the HepaRG cells, adding 3mL of 0.05% pancreatin (Procell, PB 180222) after the suction and removal, placing the cells into a CO2 incubator for incubation for 2-4 minutes, adding 3mL of culture medium, and repeatedly blowing and cloning by using a pipetting gun to finally obtain single cell suspension;
2) 1500rpm, centrifugation for 5min, after centrifugation, the supernatant was aspirated, resuspended in 10mL of DMEM high-glucose (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE 100-B7953), and counted.
3) 5000 Cells were seeded in 96-well plates at approximately 100ul of 3 replicates per well.
4) The cells were placed in an incubator for 24 hours. The resveratrol concentration per well was 0nM,10nM,100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M, 1mM, respectively.
4) 10U of CCK-8 solution was added to each well, and the plates were placed in an incubator for incubation for 1h.
5) The absorbance (OD) at 450nm was measured by a microplate reader.
Based on comparison of cytotoxicity test results (fig. 4), 1 μm resveratrol was finally selected as the final additive concentration. Comparative example 1A kit for inducing differentiation of stem cells into hepatocytes
The kit of comparative example 1 was different from that of example 2 in that Resveratrol was not added, and the other was the same as in example 2.
Application example 1
A method for efficiently inducing qualitative endoderm by 3D suspension orientation of human multifunctional stem cells (hPSCs), which comprises the following steps (endoderm cell differentiation stage):
S1, single-cell passaging hPSC, namely selecting hPSC with good state, wherein the cell coverage rate reaches about 70% -80% 4-5 days after hPSC passaging, cloning hPSC with regular edges and no differentiated cells, adding 1mL of calcium-magnesium-free PBS to clean cells after absorbing and discarding the culture medium, adding 1mL GCDR after absorbing and discarding, putting the culture medium into a CO 2 incubator for incubation for 2-4 minutes, adding GCDR in the absorbing and discarding holes, adding 1mL of mTESR1 culture medium containing 10 mu M of Y-27632 (Rocki), and repeatedly blowing and cloning by using a pipette to finally obtain single-cell suspension;
S2, cell inoculation, namely sucking 20 mu l of single cell suspension, staining with trypan blue, counting by using a blood cell counting plate, sucking 100 ten thousand single cell suspensions by using a corning low-adhesion six-hole culture plate, inoculating the culture plate into a hole, supplementing mTESR1 culture medium containing 10 mu M Y-27632 until 2mL of liquid is contained in the hole, and uniformly suspending cells in the culture solution by shaking the hole plate in a crisscross manner, wherein the culture plate is marked as a day 0, and human pluripotent stem cell spheres with the diameter of about 100 mu M are formed after 3-5 days;
S3, cell replacement, namely cleaning the human pluripotent stem cell spheres by using 1mL of calcium-magnesium-free PBS (phosphate buffer solution) per well after 3-5 days (day 1 of differentiation), adding 2mL of DE culture medium 1 after suction, replacing DE culture medium 2 after 48 hours (day 2 of differentiation), and replacing DE culture medium 3 after 72 hours (day 3 of differentiation);
S4, harvesting cells, namely photographing endoderm cell pellets when culturing is carried out on the 3 rd day, collecting the endoderm cell pellets, naturally settling for 1-2 minutes, adding 1mL PBS to clean the endoderm cell pellets after removing the supernatant, naturally settling for 1-2 minutes, and carrying out qPCR (quantitative polymerase chain reaction) after extracting RNA (ribonucleic acid) after removing the supernatant to detect the expression quantity of SOX17 and FOXA2 genes.
The experimental results are shown in FIG. 2, the directional induced differentiation of the human pluripotent stem cell H9 into the endoderm cell pellet in the 3D suspension culture, wherein FIG. 2A is a roadmap of the directional induced differentiation of the human pluripotent stem cell into the endoderm cell pellet, FIG. 2B is a flow detection of SOX17 and FOXA2 of the endoderm cell pellet, FIG. 2C is a morphological change image of the directional induced differentiation of the human pluripotent stem cell into the endoderm cell pellet, FIG. 2D is an immunofluorescent staining of DAPI and FOXA2 of the endoderm cell pellet, and the preparation of the endoderm marker gene expression amount of the cell pellet by using the kit in the embodiment 1 is higher (FIG. 2E). Therefore, the kit of this example 1 can allow successful and efficient differentiation of human pluripotent stem cells into endodermal cell pellets.
Application example 2
A method for efficiently inducing and differentiating human multifunctional stem cells (hPSCs) in 3D suspension and orientation comprises the following steps:
S1, single-cell passaging hPSC, namely selecting hPSC with good state (the state is good, when the hPSC is passaged for 4-5 days, the cell coverage rate reaches about 70% -80%, cloning edge is regular and hPSC without differentiated cells), adding 1mL of calcium-magnesium-free PBS to clean cells after absorbing and discarding the culture medium, adding 1mL GCDR after absorbing and discarding, placing the cells into a CO 2 incubator to incubate for 2-4 minutes, then absorbing and discarding GCDR in a hole, adding 1mL of mTESR1 culture medium containing 10 mu M of Y-27632 (Rocki), and repeatedly blowing and cloning by using a pipetting gun to finally obtain single-cell suspension;
s2, cell inoculation, namely sucking 20 mu l of single cell suspension, staining with trypan blue, counting by using a blood cell counting plate, sucking a low-adhesion corning six-hole culture plate, sucking a mTESR1 culture medium containing 50-200 mu M of single cell suspension and inoculating the culture plate into a hole, supplementing 2mL of liquid in the hole, and shaking the hole plate in a crisscross manner to enable the cells to be uniformly suspended in the culture solution, wherein the culture solution is marked as a day 0, and human pluripotent stem cell spheres with the diameters of 50-70 mu M are formed after 3-5 days;
S3, cell replacement, namely cleaning the human pluripotent stem cell spheres by using 1mL of calcium-magnesium-free PBS (phosphate buffer solution) per well after 3-5 days (day 1 of differentiation), adding 2mL of DE culture medium 1 after suction, replacing DE culture medium 2 after 48 hours (day 2 of differentiation), and replacing DE culture medium 3 after 72 hours (day 3 of differentiation);
S4, cell replacement at the stage of directional induction of hepatic progenitors, namely cleaning hepatic progenitors with 1mL of calcium-magnesium-free PBS per well on days 6-8 (day 4 of differentiation), adding 2mL of HDM culture medium after suction and disposal, and replacing 2mL of HDM culture medium every day on days 7-11 (day 5-9 of differentiation).
S5, directionally inducing the cell replacement liquid in the mature stage of the liver cells, namely cleaning the liver progenitor cell balls by using 1mL of calcium-magnesium-free PBS (phosphate buffer solution) in each hole on 12-14 days (10 days of differentiation), adding 2mL of HCM (hydrogen chloride) culture medium after suction and rejection, and replacing 2mL of HCM culture medium every day on 13-17 days (11-15 days of differentiation).
S6, harvesting the cells, namely photographing the hepatocyte pellets every day, collecting the hepatocyte pellets after the hepatocytes are mature, naturally settling for 1-2 minutes, collecting culture medium supernatant, detecting the secretion amount of human Albumin (ALB) by using an ELISA kit, then adding 1mL of PBS to clean the hepatocyte pellets, naturally settling for 1-2 minutes, discarding the supernatant, and then carrying out qPCR to detect the expression amounts of human Alpha Fetoprotein (AFP), ALB and CYP3A4 genes.
The experimental results are shown in FIG. 3, the 3D suspension culture of the hepatic progenitors pellets to the hepatic pellets induced differentiation under the condition of adding 1uM resveratrol, FIG. 3A is a roadmap of the hepatic progenitors pellets to the hepatic pellets and a morphological image of the hepatic pellets maturation, and FIG. 3B is qPCR detection of functional genes ALB, AFP and CYP3A4 of the hepatic pellets, and it can be obtained from the graph that compared with the control group (comparative example 1), the resveratrol group alpha fetoprotein gene is increased, and the albumin gene and the drug metabolism gene are also obviously increased. Thus, the successful transition of hepatic progenitors to hepatocyte spheroid maturation FIG. 3C shows qPCR detection of CPS1, FOXO1, BSEP, NTCP of hepatocyte spheroids, these four genes representing ammonia metabolism, autophagy, basal and apical membrane polarization, respectively. Compared with the control group, the CPS1, the BSEP and the NTCP of the resveratrol group have obviously increased expression, and the FOXO1 has obviously decreased expression. Ammonia metabolism and polarization of representative resveratrol group hepatocytes were enhanced and autophagy was reduced, FIG. 3D is the expression of the drug metabolizing enzyme CYP3A4, rifampicin induced hepatocyte spheroid at 25. Mu.M. After induction of liver cell pellets for 48 hours with 25 μm rifampicin, 1mL PBS was subsequently added to wash the liver cell pellets, and after 1-2 minutes of natural sedimentation, qPCR detection was performed after discarding the supernatant, and drug metabolizing enzyme induction experiments were performed with differentiated liver cell pellets, which was an important marker of mature liver cells for induction of response. Compared with a control group, the addition of 1 mu MResveratrol can obviously up-regulate the expression level of the metabolic enzyme gene, which shows that the differentiated hepatocyte balls of the embodiment can react to induction and stimulation of drugs, have good drug metabolism function and have potential to become a drug screening cell model, and FIG. 3E shows that the ALB content in the supernatant of the detection culture medium of the Elisa in the maturation stage of the hepatocyte balls is 102.8430ug/mL and 138.4630ug/mL respectively, which shows that the hepatocyte balls after resveratrol treatment have stronger albumin secretion capacity.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
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