CN114561335A - Method for preparing liver organoid by peripheral blood mononuclear cells - Google Patents

Method for preparing liver organoid by peripheral blood mononuclear cells Download PDF

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CN114561335A
CN114561335A CN202210130603.8A CN202210130603A CN114561335A CN 114561335 A CN114561335 A CN 114561335A CN 202210130603 A CN202210130603 A CN 202210130603A CN 114561335 A CN114561335 A CN 114561335A
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liver
cells
cell
matrigel
medium
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CN114561335B (en
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项鹏
柯琼
范明明
李伟强
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Sun Yat Sen University
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Abstract

The invention discloses a method for preparing liver organoids by Peripheral Blood Mononuclear Cells (PBMCs), which obtains a liver organoid model which has individual genetic information and fully simulates the in vitro liver development and the pathophysiological process of liver-related diseases in a relatively non-invasive way. The collected PBMCs are reprogrammed to Induce Pluripotent Stem Cells (iPSCs), and the iPSCs are further induced and differentiated into liver organoids, so that the aim is achieved through the two steps, and the iPSCs can be infinitely amplified and stored in vitro, thereby providing a basis for large-scale production of the liver organoids.

Description

Method for preparing liver organoid by peripheral blood mononuclear cells
Technical Field
The invention relates to the technical field of stem cells, in particular to a method for preparing liver organoids by peripheral blood mononuclear cells.
Background
The liver is the largest metabolic organ of human body, and the liver-based genetic metabolic diseases are various, including fatty liver, congenital biliary atresia, limonin deficiency, alpha-1 antitrypsin deficiency, type I tyrosinemia, hepatolenticular degeneration and the like. The research on the occurrence and development of the liver diseases needs good in-vivo and in-vitro models, and although various animal models are constructed at home and abroad according to various diseases, the animal models and the human beings have great differences in development, physiological structure, pathological state and the like, and the animal models are difficult to replicate the progress of the human diseases, so that a more excellent disease model is urgently needed.
The problem is solved by the appearance of the induced pluripotent stem cells, the pluripotent stem cell line with the genetic background of the patient is directionally differentiated into organoids, a complete development and pathophysiology model can be provided for the individualized research of the genetic metabolic diseases, and a safer in vitro disease model is provided for the screening of medicines and the realization of gene therapy.
Meanwhile, in the current stage, the only cure means for the end-stage liver diseases is liver transplantation, but the shortage of liver supplies, immune rejection reaction after transplantation and expensive cost keep most patients out of the doors of cure. The liver organoid may be an important breakthrough point for solving the problem, and the in vitro amplification culture of the liver organoid from the patient can solve the shortage of the liver supply and the rejection problem after transplantation. While organoids are expensive to culture at the present stage and still have many deficiencies, they are available in the future.
The principle of inducing liver organoids is to obtain liver progenitor cells or liver stem cells, and further induce and differentiate the liver progenitor cells or the liver stem cells into liver organoids with 3D structures. At present, there are two main ways to obtain human liver stem cells:
one is a primary hepatic stem cell obtained by isolation culture from human liver tissue, but obtaining liver tissue is an invasive procedure, and particularly for patients with abnormal or failing liver function, invasive procedures such as hepatic puncture or hepatectomy greatly increase the risk of bleeding or further aggravate hepatic function deterioration, and thus obtaining liver tissue is limited and difficult.
Another important way is to simulate the process of human development, which is obtained by induced differentiation of Induced Pluripotent Stem Cells (iPSCs), because the iPSCs can be obtained by reprogramming of volunteer Peripheral Blood Mononuclear Cells (PBMCs), the process of Blood collection is simple and relatively non-invasive, and therefore, the iPSCs is obtained by reprogramming of Peripheral Blood Mononuclear Cells, and then the human liver organs which can be passaged and expanded are obtained by further induced differentiation of the iPSCs, which may be an important source for replacing human liver organs in the future.
Disclosure of Invention
Liver organoid induction protocols exist today that derive from primary liver progenitor or stem cells, which, while liver organoids with the same genetic background as the donor are available, are too invasive to draw materials, especially for patients with existing liver disease or liver failure, at great risk. And the liver progenitor cells or stem cells of primary origin are difficult to stably expand in vitro and maintain the characteristics of the stem cells, and repeated material drawing is still needed if re-induction is needed. The invention aims to overcome the defects in the prior art and provides a method for preparing a liver organoid by using peripheral blood mononuclear cells. The invention solves the problem that the liver organoid model which has individual genetic information and can fully simulate the liver development and the pathophysiology process of liver related diseases in vitro is obtained in a relatively non-invasive mode, and induced pluripotent stem cells can be infinitely amplified and stored in vitro, thereby providing a foundation for the repeated and large-scale production of the liver organoids.
It is therefore a first object of the present invention to provide a medium for the propagation of hepatic progenitors.
The second purpose of the invention is to provide an induction medium for liver organoids.
The third purpose of the invention is to provide the application of the proliferation culture medium of the hepatic progenitor cell balls and/or the induction culture medium of the liver organoid in the preparation of the liver organoid.
The fourth purpose of the invention is to provide a method for preparing liver organoid by using pluripotent stem cell.
The fifth purpose of the invention is to provide the liver organoid prepared by any one of the methods.
Meanwhile, matrigel drops are mostly dropped at the bottom of a pore plate for culturing the liver progenitor cell balls at present, and a dome (doom) mode is established, but adherent cells are easily formed on the adherent surface of the gel drops, and the formation of 3D organs in matrigel is not facilitated. And the bottom area of the pore plate is limited, and the quantity of the manufactured glue drops is limited. The invention designs a glue drop manufacturing method which can be realized through a simple centrifuge tube and a gun head, the condition that cells grow adherent to the wall can not occur when 3D organs are cultured by adopting the method, and the solidified matrigel can be cultured in batches in a common bacterial dish, so that a simple mode is provided for the large-scale culture of the 3D organs.
In order to achieve the purpose, the invention is realized by the following scheme:
a propagation medium for hepatocyte progenitors, comprising DMEM/F12 basal medium containing N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), GlutaMAX, acetylcysteine, N2, B27 serum-free cell culture additive, nicotinamide, gastrin, A83-01, Forskolin, R-spondin-1 protein, Wnt3a protein, and epidermal growth factor EGF.
Preferably, the proliferation medium is a DMEM/F12 basal medium containing 9-11 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 0.9-1.1% (volume ratio) GlutaMAX, 1.20-1.3 mM acetylcysteine, 0.9-1.1 (volume ratio) N2, 0.9-1.1% (volume ratio) B27 serum-free cell culture additive, 9.5-10.5 mM nicotinamide, 9.5-10.5 mM gastrin, 4.5-5.5 μ M A83-01, 9.5-10.5 μ M Forskolin, 245-255 μ M R-spondin-1 protein, 95-105 μ M Wnt3a protein, and 45-55 μ M epidermal cell growth factor.
More preferably, the propagation medium is a DMEM/F12 basal medium containing 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 1% (by volume) GlutaMAX, 1.25mM acetylcysteine, 1% (by volume) N2, 1% (by volume) B27 serum-free cell culture additive, 10mM nicotinamide, 10mM gastrin, 5 μ M A83-01, 10 μ M Forskolin, 250 μ M R-spondin-1 protein, 100 μ M Wnt3a protein, and 50 μ M epidermal growth factor EGF.
An induction medium for liver organoids, which is an HM basal medium containing bovine serum albumin, a B27 cell culture additive, an ITS cell culture additive, L-ascorbyl acid trisodium salt, acetylcysteine, a hepatocyte growth factor HGF protein, oncostatin M, dexamethasone, an epidermal growth factor EGF protein, EGF, 8-bromo-cyclic adenosine monophosphate, vitamin K2, lithocholic acid, and Y27632.
Preferably, the induction medium is 0.9-1.1 (mass to volume ratio) HM base medium containing bovine serum albumin, 0.9-1.1% (volume ratio) B27 cell culture additive, 0.9-1.1% (volume ratio) ITS cell culture additive, 0.15-0.25 mM L-ascorbyl acid trisodium salt, 1.20-1.30 mM acetylcysteine, 19.5-20.5. mu.M hepatocyte growth factor HGF protein, 19.5-20.5. mu.M tumor suppressor M, 0.45-0.55. mu.M dexamethasone, 9.5-10.5. mu.M epidermal growth factor EGF protein, 95-105. mu.M 8-bromo-cyclic adenosine monophosphate, 9.5-10.5. mu.M vitamin K2, 9.5-10.5. mu.M lithocholic acid, and 9.5-10.5. mu. M Y27632 HM 27632.
More preferably, the induction medium is a 1% (mass to volume) HM basal medium containing bovine serum albumin, 1% (volume to volume) B27 cell culture supplement, 1% (volume to volume) ITS cell culture supplement, 0.2mM L-ascorbyl acid trisodium salt, 1.25mM acetylcysteine, 20. mu.M hepatocyte growth factor HGF protein, 20. mu.M oncostatin M, 0.5. mu.M dexamethasone, 10. mu.M epidermal growth factor EGF protein, 100. mu.M 8-bromo-cyclic adenosine monophosphate, 10. mu.M vitamin K2, 10. mu.M lithocholic acid, and 10. mu. M Y27632.
The proliferation culture medium of the liver progenitor cell balls and/or the induction culture medium of the liver organoid are applied to the preparation of the liver organoid.
Preferably, the application of the pluripotent stem cells in preparing liver organoids.
A method for preparing liver organoids by using pluripotent stem cells comprises the following steps:
s1, inducing and differentiating pluripotent stem cells into terminal foregut endoderm cells;
s2, resuspending the end foregut endoderm cells in matrigel, and then culturing the end foregut endoderm cells by using a proliferation culture medium of the liver progenitor cell balls to obtain organoid cell balls;
and S3, dissociating and digesting the organoid cell balls obtained in the previous step, and culturing by using the induction medium of the liver organoid.
Preferably, in step S1, pluripotent stem cell planar culture induces differentiation into terminal foregut endoderm cells.
Preferably, in step S1, the pluripotent stem cell is reprogrammed from peripheral blood mononuclear cells, i.e., the pluripotent stem cell is prepared after the peripheral blood mononuclear cells are transferred into OCT4, SOX2, Klf4 and c-Myc.
More preferably, in step S1, the recombinant Sendai virus vector is used to transfer OCT4, SOX2, Klf4 and c-Myc into peripheral blood mononuclear cells, and then cultured in a cell dish plated with feeder cells until cell clones grow.
Preferably, step S1 includes the following steps:
s11, digesting the pluripotent stem cell mTeSR culture medium until the cell density reaches 80%, and inoculating the digested pluripotent stem cell mTeSR culture medium to the mTeSR culture medium containing Y27632;
s12, culturing by using an endoderm induction culture medium 1, wherein the endoderm induction culture medium 1 is an RPMI 1640 culture medium containing B27, WNT3A protein and Activin A protein;
s13, culturing by using an endoderm induction culture medium 2, wherein the endoderm induction culture medium 2 is an RPMI 1640 culture medium containing B27 and Activin A protein;
s14, culturing by using a foregut endoderm cell culture medium, wherein the foregut endoderm cell culture medium is HCM BulletKit hepatocyte culture medium (LONZA (CC-3199& CC-4182)) containing FGF2 protein and BMP4 protein.
More preferably, the endoderm induction medium 1 is RPMI 1640 medium containing 0.9-1.1% B27, 49.5-50.5 ng/ml WNT3A protein, and 95-105 ng/ml Activin A protein;
further preferably, endoderm induction medium 1 is RPMI 1640 medium containing 1% B27, 50ng/ml WNT3A protein, and 100ng/ml Activin a protein;
more preferably, the endoderm induction medium 2 is RPMI 1640 medium containing 0.9-1.1% B27, and 95-105 ng/ml Activin A protein.
Further preferably, endoderm induction medium 2 is RPMI 1640 medium containing 1% B27, and 100ng/ml Activin a protein.
More preferably, the foregut endoderm cell culture medium is HCM BulletKit hepatocyte culture medium (LONZA (CC-3199& CC-4182)) containing 9.5-10.5 ng/ml FGF2 protein and 19.5-20.5 ng/ml BMP4 protein.
Further preferably, the foregut endoderm cell culture medium is HCM BulletKit hepatocyte culture medium (LONZA (CC-3199& CC-4182)) containing 10ng/ml FGF2 protein and 20ng/ml BMP4 protein.
More preferably, in step S11, the cells are inoculated into mTeSR medium containing 9.5-10.5. mu. M Y27632.
Further preferably, in step S11, the cells are inoculated into mTeSR medium containing 10 μ M Y27632.
More preferably, in step S11, the digestion process is an Accutase digestion process.
More preferably, in step S12, the culture is performed for 0.8 to 1.2 days.
Further preferably, in step S12, the culture is performed for 1 day.
More preferably, in step S13, the culture is performed for 2 to 3 days.
Further preferably, in step S13, the culture is performed for 2 days.
More preferably, in step S14, the culture is performed for 5 to 6 days.
Further preferably, in step S14, the culture is performed for 5 days.
Preferably, in step S2, the terminal foregut endoderm cells are resuspended in matrigel, which forms gel droplets with a three-dimensional shape.
More preferably, in step S2, the formed gel droplets are cultured in suspension in a proliferation medium culture of said hepatic progenitor cells.
More preferably, in step S2, the method for resuspending terminal foregut endoderm cells in matrigel comprises the following steps:
s21, digesting the tail end foregut endoderm cells, carrying out solid-liquid separation, and taking cell sediment;
s22, mixing matrigel and the precipitated cells, fully mixing by blowing and sucking, avoiding mixing bubbles, and keeping to avoid the matrigel from solidifying to obtain a cell matrigel suspension;
s23, adding the cell matrix gel suspension into a container with a pointed bottom or a conical bottom, keeping the container at the pointed bottom or the conical bottom, and incubating until all the matrix gel is solidified;
s24, blowing the matrigel at the bottom of the container with the pointed bottom or the conical bottom, and transferring the matrigel into a subsequent culture container.
More preferably, in step S2, 1-2 ten thousand cells/50 μ l matrigel are resuspended in matrigel.
Preferably, in step S2, the propagation medium of the hepatocyte progenitor cells is replaced every 2-4 times.
More preferably, the proliferation medium of the hepatocyte progenitor cells is replaced every 2 days in step S2.
Preferably, in step S2, the hepatocyte progenitor cells are co-cultured in proliferation medium for 10-14 days.
More preferably, in step S2, the hepatocyte progenitor cells are co-cultured for 14 days using the propagation medium of the hepatocyte progenitor cells.
Preferably, in step S3, the tip dissociates the organoid cytosphere obtained in the previous step by blowing.
Preferably, in step S3, the organoid cell pellet obtained in the previous step is trypsinized.
Preferably, in step S3, the liver organoid is cultured in the induction medium using a culture vessel with an ultra-low adsorption surface.
Preferably, in step S3, the liver organoid medium is replaced every 1 to 3 days.
More preferably, in step S3, the liver organoid medium is replaced every 2 days.
Preferably, in step S3, co-culturing the liver organoid in the induction medium for 10-14 days.
More preferably, in step S3, the liver organoid is co-cultured in the induction medium for 10 days.
The liver organoid prepared by any method also belongs to the protection scope of the invention.
Preferably, the liver organoids have hepatocytes and cholangiocytes.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains the liver organoid model which has individual genetic information and fully simulates the in vitro physiological process of liver development and liver related diseases in a relatively noninvasive mode. The collected PBMCs are reprogrammed to induce the pluripotent stem cells, and the induced differentiation of the pluripotent stem cells into liver organoids is further induced.
Drawings
FIG. 1 shows the results of immunofluorescence identification, RT-PCR, karyotype analysis and alkaline phosphatase staining of iPSCs pluripotency markers.
FIG. 2 shows the results of immunofluorescence staining and RT-PCR of specific markers (SOX17 and FOXA2) for differentiation of iPSCs into endoderm.
FIG. 3 is a white light diagram and a schematic diagram of the differentiation pathway of iPSCs to liver organoids.
FIG. 4 shows immunofluorescence staining and RT-PCR results of terminal foregut endoderm cells.
FIG. 5 shows that the percentage of cells inducing positive HNF4a in the resulting terminal foregut endoderm cells was 93.46%, and the percentage of cells inducing double positive AFP and ALB in the resulting hepatic progenitor cells was 95.33%.
FIG. 6 is a graph comparing day1 and day12 of 3D culture of hepatocyte progenitors, tips and EP tubes for intrastromal cell resuspension in the specific procedure.
FIG. 7 shows immunofluorescence staining and RT-PCR results for hepatocyte progenitor cell-specific markers.
FIG. 8 shows the results of immunofluorescence staining and RT-PCR of liver organoid-specific markers.
Fig. 9 is a test of indocyanine green (ICG) uptake and release by liver organoids.
FIG. 10 shows staining of glycogen deposits in liver organoids (left) and staining with rhodamine 123 (right).
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
All antibody and primer information used in the examples are shown in tables 1 and 2, respectively.
Table 1:
name of antibody Company(s) Goods number
Nanog CST 4903
SOX2 Abcam ab97959
OCT4 Santa cruz sc-9081
SSEA Sigma-Aldrich MAB4304
TRA-1-60 Sigma-Aldrich MAB4360
TRA-1-80 Sigma-Aldrich MAB4381
SOX17 R&D AF1924
FOXA2 Abcam ab60721
HNF4a Abcam ab41898
Ki67 Abcam ab15580
TBX3 Abcam ab99302
AFP R&D MAB1368
CK19 Abcam ab7754
EpCAM Abcam ab223582
SOX9 Abcam ab185966
ALB Dako A 0001
ALB Sigma-Aldrich A6684
CK18 Abcam ab181597
TF Abcam ab214039
CYP3A4 Santa cruz sc-53850
ASS1 Santa cruz sc-365475
FAH ORIGENE AP51509PU-N
Table 2:
Figure BDA0003502376970000071
Figure BDA0003502376970000081
example 1 Induction of PBMCs into iPSCs with multiple differentiation potentials
This example isolated and extracted PBMCs from peripheral blood of donors (collection of specimens was approved by the ethical committee of the hospital).
First, experiment method
1. Extraction of PBMCs by Ficoll-Paque gradient centrifugation
3ml of peripheral blood of the donor was collected using a heparin anticoagulant tube and placed on ice for future use. 6ml of the lymphocyte separation medium was added to a 15ml centrifuge tube, and the tube was placed at an angle of 45 ℃. And (3) uniformly mixing anticoagulation blood by using a gun head, and slowly adding the anticoagulation blood above the lymphocyte separation liquid along the wall of the test tube to form a clear boundary between the lymphocyte separation liquid and the anticoagulation blood. The tube was centrifuged at 12 ℃ at 2500rpm with 1 ramp up and 0 ramp down for 20 minutes. And (4) after centrifugation, sucking the second layer of PBMCs layer, transferring the second layer of PBMCs layer to a new centrifuge tube, adding 3ml of PBS, and washing twice to obtain PBMSc cell suspension.
2. Reprogramming PBMCs to iPSCs with multidirectional differentiation potential
The extracted PBMCs were added to a PS (polystyrene) 24-well plate (CORNING, Inc.: 3473) having an ultra-low adsorption surface to use the PBMCs as starting cells using CytotoneTMThe iPS 2.0 Sendai virus reprogramming kit (cargo number: A16517) uses Sendai virus as a vector to transfect four Yamanaka factors of OCT4, SOX2, Klf4 and c-Myc in PBMCs (three vectors in the kit are utilized), and the pluripotent stem cells growing like clones are obtained, namely iPSCs with multidirectional differentiation potential. That is, somatic cells are transformed into induced pluripotent stem cells, and have the same function as embryonic stem cells (derived from embryos).
The following operations were carried out according to the kit instructions:
(1) the extracted PBMCs were cell counted at 5X 10 per well5cells are inoculated and cultured for two days;
(2) PBMCs were transduced with three Sendai virus reprogramming vectors and incubated overnight;
(3) completely replacing PBMCs with liquid, removing the Sendai virus reprogramming carrier, and continuously culturing for two days;
(4) PBMCs treated by Sendai virus are inoculated into a cell dish paved with feeder cells, and the culture is continued for two days;
(5) half a day of fluid change with pluripotent stem cell medium (mTeSR Catalog #05825, or other pluripotent stem cell medium such as E8) followed by a full fluid change with pluripotent stem cell medium;
(6) obvious iPSCs clone growth can be seen after about one week of culture. When the iPSCs are cloned to be of proper size (the diameter of the clones is about 30-45 um), the iPSCs are picked under a mirror and transferred to a Matrigel coated culture plate, and the pluripotent stem cell culture medium is used for continuous amplification, so that the hipPSC with the multidirectional differentiation potential is obtained.
iPSCs obtained by reprogramming PBMCs are observed under a mirror, the iPSCs grow in a clone shape, and the nucleus-cytoplasm ratio is high as shown in figure 1.
3. Immunofluorescence identification of pluripotent marker by using reprogramming iPSCs
The iPSCs grown in the well plate with multi-directional differentiation potential prepared in the previous step were washed 2 times with PBS and fixed for 30min with 4% PFA. 0.3% Triton X-100 was used for 15 min of penetration and blocked with 10% BSA for 45 min at room temperature, followed by incubation with the corresponding primary antibody overnight at 4 ℃. Next, the well plates were washed 5 times with PBS for 5 minutes each, then incubated with the corresponding secondary antibody for 2 hours at room temperature, and the sections were washed 5 times with PBS for 5 minutes each. Nuclei were counterstained with DAPI for 5 minutes, washed 2 times with PBS, and observed with confocal microscopy.
4. Alkaline phosphatase staining of the reprogrammed iPSCs
The iPSCs with multi-directional differentiation potential prepared in the previous step were fixed in 4% PFA for 20 minutes and stained using alkaline phosphatase staining kit.
5. Carrying out RT-PCR identification on the reprogramming iPSCs
And (3) extracting RNA of the iPSCs with the multidirectional differentiation potential prepared in the last step by using RNAzol, and performing reverse transcription to obtain cDNA by using a reverse transcription kit. RT-PCR analysis of the cDNA was performed using SYBR RT-PCR mix kit.
6. Carrying out karyotype analysis on the iPSCs obtained by reprogramming:
treating iPSCs with multidirectional differentiation potential prepared in the last step with 20 mu g/ml colchicine for 3h, then digesting the iPSCs into single cells by using Accutase, harvesting the single cells, and detecting the gene of the gene.
Second, experimental results
As shown in FIG. 1, when the amplified iPSCs are observed under a mirror, the iPSCs grow in a clone shape and have high nuclear-cytoplasmic ratio (FIG. 1 a); immunofluorescence staining results show that the obtained iPSCs express a series of pluripotency markers including OCT4, SOX2, Nanog, SSEA4, TRA-1-60 and TRA-1-81 (FIG. 1 c); RT-PCR iPSCs were compared with embryonic stem cells, and the endogenous OCT4, Nanog and SOX2 mRNA in iPSCs were highly expressed (FIG. 1 d); karyotyping suggests that the chromosomes were normal after reprogramming of the cells (FIG. 1 e); alkaline phosphatase staining suggested high activity of the reprogrammed iPSCs (fig. 1 b).
Example 2 Induction of iPSCs with multidirectional differentiation potential into terminal Foregut endoderm cells by Targeted endoderm cells (Posterior Foregut)
First, experiment method
The iPSCs with the multi-directional differentiation potential prepared in the above example 1 were cultured in mTeSR medium at 37 ℃ and 5% CO2Culturing under the condition, replacing the culture solution every 2 days, and amplifying iPSCs. When the cell density reaches 80%, the cells are digested with Accutase for 5min, the digestion is stopped by adding culture medium, and the cells are centrifuged at 1100rpm for 4 min. Counting the cells according to 0.5-1 × 105Per cm2Seeded on cell plates and cultured overnight with mteshh +10 μ M Y27632 for further endodermal induction.
Thereafter, endoderm induction medium 1 was replaced the first day (D1): RPMI 1640 medium + 1% (v/v) B27+50ng/ml WNT3A protein +100ng/ml Activin A protein, cultured for one day, and replaced by endoderm induction medium 2 for the second to third days (D2-D3): RPMI 1640 medium + 1% B27+100ng/ml Activin A. Endoderm cells were obtained by induction for three days (D1-D3) (FIG. 3, Definitive endogerm, stage1), and expression of endoderm-specific markers SOX17 and FOXA2 were detected.
Replacing the endoderm cells obtained above with foregut endoderm cell culture medium: HCM BulletKit hepatocyte medium (LONZA (CC-3199& CC-4182)) +10ng/ml FGF2 protein +20ng/ml BMP4 protein, and the cells were cultured for 5 days (D4-D8) and the cells were changed daily to obtain terminal foregut endoderm cells (FIG. 3, Posterior foregut, stage 2). Staining of terminal foregut endoderm cell specific markers (HNF4a, SOX9, TBX3, AFP, Ki67, CK19 and EpCAM) was performed on day eight (D8); RT-QPCT identified expression of terminal foregut endoderm cell specific markers (HNF4a, SOX9, TBX3, AFP, Ki67, CK19, and EpCAM); terminal foregut endoderm cells were subjected to flow analysis.
Second, experimental results
Continued endoderm development can differentiate into foregut, midgut and hindgut. The liver is derived primarily from cells at the end of the Foregut endoderm, i.e., "terminal Foregut endoderm cells," which is described in English as Posterior Foregrt.
RT-QPCT identifies endoderm specific markers SOX17 and FOXA2, and the results are shown in FIG. 2, which illustrates that iPSCs can be induced into endoderm cells with high efficiency. Results of terminal foregut endoderm cell staining are shown in fig. 4, and the results show that iPSCs-induced terminal foregut endoderm cells can express terminal foregut endoderm cell-specific markers HNF4a, SOX9, TBX3, AFP, Ki67, CK19, and EpCAM. The results of flow analysis of terminal foregut endoderm cells are shown in fig. 5, left, and it can be seen that 93.46% of HNF4a positive cells account for the cells, indicating that the induction method is efficient.
Example 3 resuspension culture of terminal foregut endoderm cells in matrigel induced hepatocyte progenitor cells
First, experiment method
1. Terminal foregut endoderm cells resuspended in matrigel
On the ninth day (D9), the planar cultured terminal foregut endoderm cells were washed 2 times with PBS, digested to single cells by addition of Accutase, then quenched by addition of medium, collected in a centrifuge tube, centrifuged at 1100rpm for 4 minutes. Counting the cells, collecting 20-40 ten thousand cells in a 1.5ml centrifuge tube according to the cell density of 1-2 ten thousand cells/50 mu l matrigel, centrifuging, discarding the supernatant, and placing the centrifuge tube on ice for later use.
Taking 1ml of Matrigel collagen liquid, putting the Matrigel collagen liquid on ice to melt into a liquid state, and placing a gun head in a refrigerator for precooling at 4 ℃. Adding 1ml of Matrigel matrix glue into a centrifuge tube containing cell sediment, fully and uniformly mixing by using a gun head, wherein the Matrigel is prevented from being solidified on ice in the whole operation process, and air bubbles are prevented from being mixed in the operation process. Sequentially sucking 50 mul of cell matrix gel suspension, adding the cell matrix gel suspension to the bottom of a prepared 1.5ml centrifuge tube, vertically placing the centrifuge tube on a centrifuge tube rack until all the cell suspension is completely subpackaged, and fig. 6b shows the cell matrix gel suspension subpackaged in one tube. And placing the centrifuge tube shelf in an incubator at 37 ℃ for incubation for 20min until all matrigel is solidified to obtain a three-dimensional matrigel drop suspended with the end foregut endoderm cells.
The matrigel at the bottom of the centrifuge tube was blown in sequence with a 1ml tip to suck the media, the solid matrigel was blown off from the bottom of the centrifuge tube, and the matrigel and media were sucked in a bacterial dish with a truncated tip (fig. 6 a).
2. 3D-induced culture of hepatocyte progenitors
The three-dimensional matrigel drops with terminal foregut endoderm cells suspended in suspension were supplemented with sufficient proliferation medium of hepatocyte progenitor cells in 5% CO at 37 deg.C2Culturing under the condition, and changing the culture solution every 2 days until the appearance of 3D organoid cytospheres.
The formula of the proliferation culture medium of the hepatic progenitor cell balls is that the following components in the table 3 are added into a DMEM/F12 basal medium.
TABLE 3
Composition (I) Final concentration
N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (GIBCO: 15630-080) 10mM
GlutaMAX (cell culture supplement) (GIBCO: 35050061) 1% (volume ratio)
Acetylcysteine (sigma: A9165) 1.25mM
N2 (cell culture additive) (GIBCO:17502-048) 1% (volume ratio)
B27 serum-free cell culture additive (GIBCO:17504- 1% (volume ratio)
Nicotinamide (sigma: N0636) 10mM
Gastrin (sigma: G9145) 10nM
A83-01(Selleck:S7692) 5μM
Forskolin(Selleck:S2449) 10μM
R-spondin-1 protein (novoprotein: CX83) 250μM
Wnt3a protein (novoprotein: C06D) 100μM
Epidermal growth factor EGF (novoprotein: C029) 50μM
Second, experimental results
Terminal foregut endoderm cells were single cells suspended in matrigel at Day one, as shown by Day1(D9) in fig. 6, and proliferation of 3D cell spheres was seen in matrigel after 11 days of culture (Day 12(D20) in fig. 6, and hepatotic promoter sphere, stage3 in fig. 3). Co-expression of AFP and ALB can reach 95.33%, as shown on right of fig. 5; the staining of the hepatocyte progenitor cell-specific marker, as shown in FIG. 7, demonstrates that the induced hepatocyte progenitor cell-specific marker is successfully expressed by the induced hepatocyte progenitor cell, and the method can successfully induce the hepatocyte progenitor cell-specific marker.
Example 4 Induction of hepatic progenitors into liver organoids
First, experiment method
1. Induction of liver organoids
After the 3D organoid cytospheres obtained from the culture in example 3 were cultured for another 3 days, i.e., about 14 days (D22), the matrigel drops containing the 3D organoid cytospheres obtained from the culture in example 3 were detached into small pieces by blowing with a gun head, while the hepatic progenitor cytospheres in the matrigel were also blown into small cell masses, which were washed twice with PBS and then digested with a suitable amount of 0.25% (mass to volume) of trypsin for 10 minutes. 1500rpm, centrifuging for 4min to obtain single cell precipitate, resuspending the cells in liver organoid induction culture medium, adding into PS (polystyrene) culture dish with ultra-low adsorption surface, changing the culture solution every 2 days, and culturing for 10 days. The formula of the liver organoid induction medium is that the following components in Table 4 are added into HM basic medium.
Table 4:
Figure BDA0003502376970000131
the individual cells were aggregated and proliferated under the culture of liver organoid induction medium to form liver organoids with liver function (fig. 3, liver organoid, stage 4), immunofluorescent staining was performed on the liver organoids, and detection was performed on specific markers ALB, CK19, CK18, TF, CYP3a4, ASS1 and FAH specific to mature hepatocytes and cholangiocytes.
2. Functional characterization of liver organoids
(1) Indolocyanine green (ICG) uptake and release assay
Removing the prepared liver organoid supernatant, washing with PBS 3 times, adding into HCM medium containing 1mg/ml indocyanine green, incubating for 30min, removing culture solution, washing with PBS 3 times, and adding HCM medium. Bright field photographs were taken using a DMI8 microscope, once per hour until indocyanine green release was complete.
(2) Glycogen deposition staining
The prepared liver organoid medium was removed, washed 3 times with PBS, and fixed with 4% PFA for 30 minutes. Periodic acid solution was added for 10min, followed by 5 washes with water. Adding Schiff dye solution and dyeing for 5 minutes in dark place, and then the liver organoids become purple. The plate was washed again 5 times with running water and photographed brightly using a DMi8 microscope.
(3) Rhodamine 123 staining
Removing the prepared culture medium of the liver organoid, washing with PBS for 3 times, adding HCM culture medium containing 100uM rhodamine 123, incubating at 37 ℃ for 5min, and shooting under a DMi8 fluorescence microscope, wherein the liver organoid is seen to present green fluorescence.
Second, experimental results
The results are shown in fig. 8, and the obtained liver organoid cells with liver function can express specific markers of hepatocytes and cholangiocytes such as: ALB, CYP3A4, ASS1, FAH, CK19, CK18 and TF.
As a result, as shown in fig. 9, it was found that indocyanine green was taken in by the induced liver organoids at 0 hour and released for 8 hours to restore the original state, which is one of the important functions of the liver.
The results are shown in fig. 10, left, and the obtained liver organoids become purple red after PAS staining, which indicates that the liver organoid cells can store glycogen, which is one of the important functions of the liver.
The results are shown in fig. 10, right, and it can be seen that the liver organoids exhibit green fluorescence, indicating that active mitochondria in the liver organoids are labeled, which is one of the important functions of liver cells.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A propagation medium for hepatocyte progenitor cells, which is a DMEM/F12 basal medium containing N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, GlutaMAX, acetylcysteine, N2, B27 serum-free cell culture additive, nicotinamide, gastrin, A83-01, Forskolin, R-spondin-1 protein, Wnt3a protein, and EGF.
2. An induction medium for liver organoids, which is an HM basal medium containing bovine serum albumin, a B27 cell culture additive, an ITS cell culture additive, L-ascorbyl acid trisodium salt, acetylcysteine, a hepatocyte growth factor HGF protein, oncostatin M, dexamethasone, an epidermal growth factor EGF protein, EGF, 8-bromo-cyclic adenosine monophosphate, vitamin K2, lithocholic acid, and Y27632.
3. Use of a propagation medium for the hepatocyte progenitor cell pellet of claim 1 and/or an induction medium for the liver organoid of claim 2 for the preparation of a liver organoid.
4. Use according to claim 3, in the preparation of liver organoids from pluripotent stem cells.
5. A method for preparing a liver organoid by using pluripotent stem cells is characterized by comprising the following steps:
s1, inducing and differentiating pluripotent stem cells into terminal foregut endoderm cells;
s2, resuspending terminal foregut endoderm cells in matrigel, and then culturing the cells by using the proliferation culture medium of the liver progenitor cell balls in the claim 1 to obtain organoid cell balls;
s3, dissociating and digesting the organoid cell balls obtained in the previous step, and culturing the organoid cell balls in the induction culture medium of the liver according to claim 2.
6. The method according to claim 5, wherein in step S1, the pluripotent stem cells are reprogrammed from peripheral blood mononuclear cells.
7. The method according to claim 5, wherein the terminal foregut endoderm cells are resuspended in matrigel, wherein the matrigel forms colloidal droplets having a three-dimensional shape in step S2.
8. The method of claim 5, wherein the step S2, the method for resuspending terminal foregut endoderm cells in matrigel comprises the steps of:
s21, digesting the tail end foregut endoderm cells, carrying out solid-liquid separation, and taking cell sediment;
s22, mixing matrigel and the precipitated cells, fully mixing by blowing and sucking, avoiding mixing bubbles, and keeping to avoid the matrigel from solidifying to obtain a cell matrigel suspension;
s23, adding the cell matrix gel suspension into a container with a pointed bottom or a conical bottom, keeping the container at the pointed bottom or the conical bottom, and incubating until all the matrix gel is solidified;
s24, blowing the matrigel at the bottom of the container with the pointed bottom or the conical bottom, and transferring the matrigel into a subsequent culture container.
9. The method according to claim 5, wherein in step S2, the hepatocyte progenitor cell pellet proliferation medium of claim 1 is used for co-culture for 10-14 days; in step S3, co-culturing the liver organoid of claim 2 in the induction medium for 10 to 14 days.
10. A liver organoid prepared by the method of any one of claims 5 to 9.
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