CN113388573A

CN113388573A - Method for obtaining liver organoid composed of liver double-phenotype cells derived from hPSC

Info

Publication number: CN113388573A
Application number: CN202110940522.XA
Authority: CN
Inventors: 葛啸虎; 吴迪; 徐迎; 曹宁; 田应洲
Original assignee: Tianjiu Regenerative Medicine Tianjin Technology Co ltd
Current assignee: Qijia Technology Suzhou Co ltd
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-09-14
Anticipated expiration: 2041-08-17
Also published as: CN113388573B

Abstract

The invention provides a method for obtaining a liver organoid composed of liver double-phenotype cells derived from hPSC, which comprises the steps of firstly inducing human pluripotent stem cells to differentiate to form 2D liver precursor cells and 3D liver epithelial-like spherical structures, then separating the 3D liver epithelial-like spherical structures, carrying out liver double-phenotype activation, and continuously culturing to form the liver organoid composed of the liver double-phenotype cells on the basis. The method induces hPSC into liver double-phenotype cells for the first time, further forms liver organoid consisting of the liver double-phenotype cells, realizes the differentiation of human pluripotent stem cells into a liver organoid system with liver-series double-phenotype liver cells, and can be applied to the repair of chronic liver injury.

Description

Method for obtaining liver organoid composed of liver double-phenotype cells derived from hPSC

Technical Field

The invention relates to the field of stem cell biology and regenerative medicine, in particular to a method for obtaining a liver organoid consisting of liver double-phenotype cells derived from hPSC.

Background

The liver is one of the most regenerative organs in the human body. For mechanical injury, the aim of tissue repair is mainly achieved by the liver cells through self replication; for chronic Liver injury such as alcoholic Liver injury and drug-induced Liver injury, Liver cells cannot contribute to repair, and Liver regeneration is achieved by intrahepatic bile duct epithelial cells (Liver regeneration-mechanisms and modules to clinical application [ J ]. Nature reviews Gastroenterology & hepatology, 2016, 13(8): 473-. A series of evidences from recent studies show that in the state of chronic liver damage, intrahepatic bile duct epithelial cells can be differentiated into an intermediate state of cells, and the key characteristics are as follows: expressing both markers for hepatic progenitors and markers for mature hepatocytes, are called hepatic bi-phenotypic cells (In vitro expansion of primary human hepatocytes with efficacy probability [ J ]. Cell stem Cell, 2018, 23(6): 806-. Such cells will further proliferate and differentiate into hepatocytes, thereby achieving lesion repair (viral liver infection conversion of bipolar epithelial cells into liver cells, Cell stem cells, 2018, 23(1): 114-122. e 3). Therefore, the liver double-phenotype cell has important application value in the aspect of chronic injury repair.

However, such intermediate cells are not present in healthy liver and are induced only after signaling chronic liver damage. At present, there is no data indicating that the liver bi-phenotype cell can be obtained in vitro by inducing differentiation of human pluripotent stem cells (hpscs).

Disclosure of Invention

In view of the above, the present invention provides a method for obtaining a liver organoid composed of hPSC-derived liver bi-phenotypic cells, which uses hPSC as seed cells to generate a 3D liver organoid composed of liver bi-phenotypic cells by in vitro differentiation induction.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a method for obtaining a liver organoid composed of hepatic bi-phenotypic cells derived from hpscs, the method comprising the steps of:

s1, inducing the differentiation of the human pluripotent stem cells to form 2D hepatic precursor cells and 3D hepatic epithelial-like spherical structures;

s2, separating the 3D liver epithelial-like spherical structure, activating the liver double phenotype, and continuously culturing to form the liver organoid consisting of the liver double phenotype cells on the basis.

Further, the S1 includes the following steps:

s11, inducing the differentiation of the human pluripotent stem cells to form 2D definitive endoderm-like cells;

s12, inducing 2D definitive endoderm-like cells to differentiate to form 2D hepatic progenitor-like cells;

and S13, inducing the 2D liver progenitor-like cells to differentiate to form 2D liver precursor cells and 3D liver epithelial-like spherical structures.

Further, the S11 includes the following steps:

s111, culturing the human pluripotent stem cells in a first induction culture medium for 24h, wherein the first induction culture medium contains RPMI-1640 culture medium as a basal medium, and is added with 50-300ng/ml of rhGDF8, 1-5 mu M of CHIR99021, 1-3% of B27 (-insulin), 0.2-1% of GlutaMAX and 0.5-2% of NEAA;

and S112, culturing the cells obtained in the step S111 in a second induction culture medium for 24 hours, wherein the second induction culture medium contains RPMI-1640 culture medium as a basic culture medium, and 50-300ng/ml of rhGDF8, 1-3% of B27 (-insulin), 0.2-1% of GlutaMAX and 0.5-2% of NEAA are added.

Preferably, the concentration of rhGDF8 (recombinant human growth differentiation factor 8) in S111 and S112 is 100 ng/ml.

Preferably, the concentration of CHIR99021 (GSK-3 inhibitor) in S111 is 2. mu.M.

Preferably, in S111 and S112, the mass concentration of B27 (-insulin) (B27 serum-free nutritional supplement (insulin-free type)) is 2%.

Preferably, the mass concentration of GlutaMAX (a substitute for L-glutamine) in S111, S112 is 1%.

Preferably, the mass concentration of NEAA (non-essential amino acids) in S111 and S112 is 1%.

Further, the S12 includes the following steps:

culturing the 2D definitive endoderm-like cells obtained in the step S11 in a third induction medium containing IMDM medium (Iscove modified DMEM basal medium) as a basal medium, and adding 10-20% KSR, 0.5-1% DMSO, 0.1-0.5mM b-ME, 0.2-1% GlutaMAX, and 0.5-2% NEAA, for 4-6 days.

Preferably, in S12, KSR (Knockout serum replacement) is present at a mass concentration of 20%.

Preferably, the mass concentration of DMSO (dimethyl sulfoxide) in S12 is 1%.

Preferably, in S12, the concentration of b-ME (2-mercaptoethanol) is 0.1 mM.

Preferably, the mass concentration of GlutaMAX in S12 is 1%.

Preferably, in S12, the mass concentration of NEAA is 1%.

Further, the S13 includes the following steps:

culturing the 2D liver progenitor-like cells obtained in the step S12 in a fourth medium containing Advanced DMEM/F12 medium (modified DMEM/F12 base medium) as a basal medium, and adding 0.5-5. mu. M A83-01, 5-40. mu.M FH1, 5-20. mu.M FPH1, 10-20. mu.M HH, 0.1-1. mu.M DEX, 0.5-1% B27 (-insulin), 0.5-1% KSR, 0.2-1% GlutaMAX and 1-2% NEAA, for 8-10 days.

Preferably, the concentration of A83-01 (TGF-. beta.type I receptors ALK5, ALK4 and ALK7 inhibitors) in S13 is 0.5. mu.M.

Preferably, in S13, FH1 (hepatocyte function enhancer) concentration is 20 μ M.

Preferably, in S13, FPH1 (hepatocyte functional proliferation enhancer) is at a concentration of 20 μ M.

Preferably, in S13, the concentration of HH (hydrocortisone) is 10 μ M.

Preferably, in S13, DEX (dexamethasone) is 0.5 μ M.

Preferably, in S13, the mass concentration of B27 (-insulin) is 1%.

Preferably, in S13, the mass concentration of KSR is 1%.

Preferably, the mass concentration of GlutaMAX in S13 is 1%.

Preferably, in S13, the mass concentration of NEAA is 1%.

Further, the culture conditions of the S11, S12 and S13 steps are all 37 ℃ and 5% CO₂And the medium was replaced with new medium every 24 h.

Further, the S2 includes the following steps:

s21, picking a 3D liver epithelium sample spherical structure and mechanically scattering the spherical structure;

s22, coating by 3D matrigel, and performing in vitro activation culture by using a liver 3D activation culture medium, wherein the liver 3D activation culture medium contains Advanced DMEM/F12 culture medium as a basic culture medium, and 5-20mM HEPES, 5-20nM Gastrin I, 1-3mM NAC, 2-10mM NIC, 0.5-15 μ M FSK, 0.5-15 μ M A83-01, 100-3000 ng/ml rhRSPO1, 50-400ng/ml rhFGF10, 10-200ng/ml rhEGF, 10-200ng/ml rhHGF, 0.5-1% B27 (-insulin) and 0.5-1% N2 are added.

Preferably, the concentration of HEPES (N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid) in S22 is 15 mM.

Preferably, in S22, Gastrin I is present at a concentration of 5 nM.

Preferably, the concentration of NAC (acetylcysteine) in S22 is 1.25 mM.

Preferably, the concentration of NIC (nicotinamide) in S22 is 10 mM.

Preferably, in S22, the concentration of FSK (forskolin) is 10 μ M.

Preferably, in S22, the concentration of A83-01 is 5. mu.M.

Preferably, the concentration of rhRSPO1 (recombinant human R-spondin-1) in S22 is 1. mu.g/ml.

Preferably, the concentration of rhFGF10 (recombinant human fibroblast growth factor 10) in S22 is 100 ng/ml.

Preferably, the concentration of rhEGF (recombinant human epithelial growth factor) in S22 is 50 ng/ml.

Preferably, rhHGF (recombinant human hepatocyte growth factor) concentration in S22 is 25 ng/ml.

Preferably, in S22, the mass concentration of B27 (-insulin) is 1%.

Preferably, the mass concentration of N2 (N2 serum-free cell nutrition supplement) in S22 is 1%.

Further, the S22 includes the following steps:

s221, 3D culture

Resuspending the minced cell structures using cold matrigel (gfr), dropping onto a 37 ℃ pre-heated cell culture plate until they solidify to form a 3D water-drop-like structure;

s222, hepatic bi-phenotypic activation

Sufficient liver 3D activation medium was added to completely cover the 3D water droplet structure formed by solidification and incubation was continued for 9-12 days.

Further, the culture conditions of S22 were 37 ℃ and 5% CO₂。

Further, the culture medium was changed only 1 time for the first 3 days during the S222 culture, and the culture medium was changed 1 time every 2 days thereafter.

Compared with the prior art, the method for obtaining the liver organoid consisting of the liver double-phenotype cells derived from the hPSC has the following advantages:

the method for obtaining the liver organoid composed of the liver double-phenotype cells derived from the hPSC induces the hPSC into the liver double-phenotype cells for the first time, and further forms the liver organoid composed of the liver double-phenotype cells, thereby realizing the differentiation of the human pluripotent stem cells into the liver organoid system with the liver-series double-phenotype liver cells, and being applicable to the repair of chronic liver injury.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a flow chart of the induction of hPSC differentiation into liver organoids comprised of liver bi-phenotypic cells;

FIG. 2 is a diagram showing the result of the multifunctional immunofluorescence assay for hPSC;

FIG. 3 is a graph of 2D definitive endoderm-like cells;

FIG. 4 is a graph of the 2D definitive endoderm markers SOX17 and FOXA2 fluorescent quantitative PCR results;

FIG. 5 is a 2D liver progenitor-like cell map;

FIG. 6 is a graph of the results of fluorescent quantitative PCR of the hepatic progenitor cell marker AFP and the mature hepatic cell marker ALB;

FIG. 7A is a diagram showing the structure of 2D hepatic precursor cells and 3D hepatic epithelial-like spheroids, B is a diagram showing the structure of 3D hepatic epithelial-like spheroids, and C is a diagram showing 2D hepatic precursor cells;

FIG. 8A shows the 3D culture and liver biphenotypic activation status after picking the upper 3D epithelioid spherical structure; b is a liver organoid consisting of liver double-phenotype cells; c is the state of 3D culture and liver double phenotype activation after the lower layer 2D liver precursor cells are mechanically crushed; d is dead 2D liver precursor cells;

FIG. 9 shows immunofluorescence assay of liver organoids composed of liver bi-phenotypic cells.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

The reagents used in the examples are shown in Table 1, the primer sequences are shown in Table 2, and the antibodies used are shown in Table 3.

The process of obtaining liver organoid composed of liver bi-phenotype cells derived from hPSC of the present invention is shown in figure 1, and the existing 2D hepatocyte differentiation method is firstly modified to generate 3D liver epithelial-like globular structure characterized by EPCAM + on the basis of inducing differentiation in vitro to form 2D liver precursor cell population. And then picking the structure, mechanically scattering the structure, coating the structure with 3D matrigel, and performing in-vitro activation culture by using a liver 3D activation culture medium to form the 3D liver organoid with the hepatic double-phenotype liver cells.

The method comprises the following steps:

stage 1: inducing the differentiation of human pluripotent stem cells to form 2D hepatic precursor cells and 3D hepatic epithelial-like spherical structures;

the specific scheme is as follows:

1) before differentiation, the hPSC pluripotency must be tested.

The immunofluorescence results are shown in FIG. 2, panel (a): a bright field photograph of the cells; FIG. (b): OCT4 staining results; FIG. (c): SSEA4 staining results; FIG. (d): stacking the results of OCT4 and SSEA4 staining to judge the double positive rate, wherein the double positive rate is more than 98% on the premise that the expression modes of the pluripotency markers OCT4 and SSEA4 are correct; FIG. (e): DAPI staining to characterize nuclei; FIG. f: the staining stack of OCT4+ SSEA4+ DAPI was used to determine the double positive rate of correct expression pattern, and the double positive rate was > 98%, and the effect of graph (f) is that since 2 proteins of OCT4 and SSEA4 are located in the nucleus, the determination of double positive is not only seen in the stack of both, but also in the stack of the nucleus, i.e. the three are required. It can be shown from FIG. 2 that hPSC has good differentiation potential.

After 30-60% confluence, differentiation was initiated (designated Day 0).

2) Inducing the differentiation of human pluripotent stem cells into 2D definitive endoderm-like cells (Day 1-2);

[ 1 ] and Day 1: culturing human pluripotent stem cells (hPSCs) in a first induction medium for 24h, wherein the first induction medium contains RPMI-1640 medium as a basal medium, and 100ng/ml of rhGDF8, 2. mu.M CHIR99021, 2% B27 (-insulin), 1% GlutaMAX and 1% NEAA are added;

②, Day 2: and culturing the cells obtained in the step S111 in a second induction culture medium for 24h, wherein the second induction culture medium contains RPMI-1640 culture medium as a basic culture medium, and 100ng/ml of rhGDF8, 2% of B27 (-insulin), 1% of GlutaMAX and 1% of NEAA are added.

After Day 2, the cells exhibited typical endoderm cell morphology, as shown in figure 3; the fluorescence quantitative PCR results are shown in FIG. 4, and show that definitive endoderm markers SOX17 and FOXA2 show the highest expression peak in Day 2, indicating that the differentiation of the definitive endoderm is successful.

3) Inducing differentiation of the 2D definitive endoderm-like cells to form 2D hepatic progenitor-like cells (Day 3-8);

culturing the 2D definitive endoderm-like cells obtained in step 2) in a third induction medium for 6 days, wherein the third induction medium contains IMDM medium as a basal medium and is supplemented with 20% KSR, 1% DMSO, 0.1mM b-ME, 1% GlutaMAX, and 1% NEAA.

After the culture is finished, the cells present closely arranged polygons, as shown in fig. 5; typical hepatic progenitor cell morphology; the fluorescent quantitative PCR results are shown in FIG. 6, which indicates that the hepatic progenitor cell marker AFP expression exceeds that of human Fetal Liver (FL) and human Adult Liver (AL); the expression quantity of a mature hepatocyte marker ALB is similar to that of human Fetal Liver (FL); less than human Adult Liver (AL), suggesting successful differentiation of hepatic progenitors.

4) Inducing the differentiation of the 2D hepatic progenitor-like cells into 2D hepatic precursor cells and 3D hepatic epithelial-like globular structures (Day 9-17);

culturing the 2D liver progenitor-like cells obtained in step 3) in a fourth medium for 9 days, wherein the fourth induction medium contains Advanced DMEM/F12 medium as a basal medium, and 0.5. mu. M A83-01, 20. mu.M FH1, 20. mu.M FPH1, 10. mu.M HH, 0.5. mu.M DEX, 1% B27 (-insulin), 1% KSR, 1% GlutaMAX and 1% NEAA are added.

After the culture is completed, the cells contained in the upper 3D globular structure exhibit morphological characteristics similar to liver epithelium, including a cobblestone shape, tight junctions, a certain proportion of binuclear cells, etc., as shown in fig. 7A and 7B, while the lower 2D cells exhibit the morphology of hepatic precursor cells (or immature hepatocytes), including incomplete morphological transformation (from the polygonal shape of hepatic progenitors to the cobblestone shape of hepatic cells), occult nuclei, etc., as shown in fig. 7C.

The culture conditions at this stage are 37 deg.C and 5% CO₂The culture medium is replaced by new culture medium every 24 h.

And (2) stage: separating the 3D liver epithelial-like spherical structure, activating the liver double phenotype, and continuously culturing to form the liver organoid consisting of the liver double phenotype cells on the basis. (Day 18-27)

The specific scheme is as follows:

1) 3D liver epithelioid spherical structure picking and 3D culture (Day 18)

Picking up the upper 3D liver epithelium-like spherical structure by matching the ophthalmic surgical scissors with the ophthalmic surgical forceps; transferring it into cold DPBS using sterile pasteur tubes; under a microscope, further cutting the eye-drops into pieces by using an ophthalmic surgical scissors;

2) 3D culture (Day 18)

After centrifugal collection, the supernatant is discarded, and after being resuspended by using cold matrigel (GFR), the supernatant is dripped on a cell culture plate preheated at 37 ℃ until the supernatant is solidified to form a 3D water drop-like structure;

3) sufficient liver 3D activation medium was added to completely cover the 3D water droplet structure formed by solidification, and the culture was continued for 10 days, wherein the liver 3D activation medium contained Advanced DMEM/F12 medium as a basal medium, and 15mM HEPES, 5nM Gastrin I, 1.25mM NAC, 10mM NIC, 10. mu.M FSK, 5. mu. M A83-01, 1. mu.g/ml rhRSPO1, 100ng/ml FGF10, 50ng/ml rhEGF, 25ng/ml rhHGF, 1% B27 (-insulin), and 1% N2 were added.

After the culture is finished, the in vitro proliferation/differentiation of the upper layer 3D hepatic epithelial globular structure as shown in fig. 8A and 8B illustrates that the 3D hepatic epithelial-like globular structure is successfully differentiated into liver organoids composed of liver bi-phenotypic cells; as shown in fig. 8C and 8D, the lower layer of 2D hepatocyte-like cells failed to proliferate and died.

The culture conditions at this stage were 37 ℃ and 5% CO₂Only 1 medium change in the first 3 days; the medium was replaced every 2 days thereafter 1 time.

Sequentially slicing the obtained sample by a frozen slicing technology and carrying out immunofluorescence identification; the results show that these 3D spheres are hollow structures; its constituent cells express both hepatic progenitor markers, such as AFP, CK19, SOX 9; also expressing mature hepatocyte markers such as ALB, MRP2, MDR1, CYP3a 4; at the same time, epithelial markers EPCAM, ECAD positive were also present, as shown in fig. 9.

These results show that the method uses hPSC as seed cell to produce liver organoid in vitro and realizes in vitro research of liver double-phenotype cell.

TABLE 1 list of reagents

TABLE 2 primer List

TABLE 3 list of antibodies

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Claims

1. A method for obtaining a liver organoid composed of hepatic bi-phenotypic cells derived from hpscs, comprising: the method comprises the following steps:

2. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 1, characterised in that: the S1 includes the following steps:

3. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 2, characterised in that: the S11 includes the following steps:

4. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 2, characterised in that: the S12 includes the following steps:

culturing the 2D definitive endoderm-like cells obtained in the step S11 in a third induction medium containing IMDM medium as a basal medium supplemented with 10-20% KSR, 0.5-1% DMSO, 0.1-0.5mM b-ME, 0.2-1% GlutaMAX, and 0.5-2% NEAA for 4-6 days.

5. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 2, characterised in that: the S13 includes the following steps:

culturing the 2D liver progenitor-like cells obtained in the step S12 in a fourth medium for 8-10 days, wherein the fourth induction medium contains Advanced DMEM/F12 medium as a basal medium, and 0.5-5. mu. M A83-01, 5-40. mu.M FH1, 5-20. mu.M FPH1, 10-20. mu.M HH, 0.1-1. mu.M DEX, 0.5-1% B27 (-insulin), 0.5-1% KSR, 0.2-1% GlutaMAX and 1-2% NEAA are added.

6. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 2, characterised in that: the culture conditions of the S11, S12 and S13 steps are all 37 ℃ and 5% CO₂And the medium was replaced with new medium every 24 h.

7. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 1, characterised in that: the S2 includes the following steps:

8. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 7, wherein: the S22 includes the following steps:

s221, 3D culture

s222, hepatic bi-phenotypic activation

9. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 8, wherein: the culture conditions of S22 are 37 ℃ and 5% CO₂。

10. The method for obtaining liver organoids of hepatic biphenotypic cell constitution derived from hPSC according to claim 8, wherein: in the S222 culture process, only 1 culture medium is replaced in the first 3 days, and then 1 culture medium is replaced every 2 days.