CN115838684B - In-vitro accurate simulation method for pulmonary fibrosis - Google Patents

Abstract

The invention provides an in-vitro accurate simulation method for pulmonary fibrosis, along with continuous induction of TGF beta, the model reproduces in vitro important molecular events in a series of fibrosis processes such as occurrence of inflammatory reaction, abnormal accumulation of pro-fibrosis factors, over-expression of fibrosis related markers and the like, and also presents typical pathological characteristics of fibrosis in morphology and histopathology.

Description

In-vitro accurate simulation method for pulmonary fibrosis

Technical Field

The invention relates to the field of stem cell biology and regenerative medicine, in particular to an in-vitro accurate simulation method for pulmonary fibrosis.

Background

As the most common idiopathic interstitial lung disease, idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) is predisposed with unknown causes, with mortality higher than most tumors, with survival in patients of only 2-3 years. Over 20 years, although more than 70 anti-fibrosis drugs showed good efficacy in preclinical studies, most encountered failure in the clinical stage, and finally only two drugs, pirfenidone and nidanib, were successfully approved. The defects of the traditional preclinical drug effect evaluation model severely limit the success rate of new drug development.

In recent years, with rapid development of stem cell biology and tissue engineering, different human organ systems such as heart, brain, kidney and the like have been established successively. Compared with the traditional 2D/3D cell model, the organoids have better physiological relevance, and are more similar to the physiological original appearance of a human body in the aspects of cell types, structural characteristics, biological functions and the like; compared with an animal model, the characteristic of humanization can well overcome the prediction deviation caused by the species difference. Therefore, the organoids are expected to become a new generation of in vitro drug evaluation models to make up for the defects of the current preclinical drug evaluation models.

Currently, fibrotic disease models based on lung organoids have been reported successively, such as Kim J H, an G H, kim J Y, et al Human pluripotent stem cell-derived alveolar organoids for modeling pulmonary fibrosis and drug testing [ J ]. Cell death discovery, 2021, 7 (1): 1-12; strikousis A, cie ś lak A, loffredox L, et al Modeling of fibrotic lung disease using 3D organoids derived from human pluripotent stem cells[J, cell reports, 2019, 27 (12): 3709-3723, e5.. These models use key signaling molecules in the course of fibrosis as in vitro inducers (e.g., tgfβ, etc.), and trigger organoid high expression of fibromarkers such as a-SMA, collagen, etc. to acquire a fibrophenotype by continuous addition to the culture medium. However, these models ignore many important pathological features in disease progression. For example, at the cellular level, these models fail to mimic the occurrence of inflammation, abnormal accumulation of pro-fibrotic factors, etc.; at the tissue level, the existing model does not present the pathological features of typical fibrosis on the basis of the microscopic and pathological staining results. These information are the basis for clinical diagnosis and efficacy confirmation.

Disclosure of Invention

In view of the above, the present invention aims to provide an in vitro accurate simulation method for pulmonary fibrosis; under the induction of TGF beta, the organoid model not only expresses a series of fibrosis markers, but also reproduces the occurrence of inflammatory reaction, abnormal accumulation of pro-fibrosis factors and other events, and also presents typical fibrosis pathological characteristics in morphology and histopathology.

In order to achieve the above purpose, the implementation mode of the technical scheme of the invention is as follows:

an in vitro accurate simulation method for pulmonary fibrosis, comprising the steps of:

1) Construction of multiple lineages of lung organoids

hpscs were first induced to differentiate into definitive endoderm in a medium containing GDF8; continuously adding ATRA into a culture medium, and adding a lung epithelial cell related induced differentiation factor, a vascular endothelial cell and macrophage induced differentiation common factor and EGM2 to induce and differentiate the lung epithelial cell related induced differentiation factor and the vascular endothelial cell and macrophage induced differentiation common factor into lung buds; finally, coating the lung buds with ECM, and continuously culturing for 70-80 days to obtain the multi-lineage lung organoids;

2) Construction of pulmonary fibrosis disease model

Treating the multi-lineage lung organoid obtained in the step 1) by using a pro-fibrosis inducer, constructing a lung fibrosis disease model, and verifying fibrosis pathology related indexes of the lung fibrosis disease model.

Further, GDF8 is used in an amount of 20-500ng/ml.

Further, ATRA is used in an amount of 0.01-10 mu M, and the common factors for the induced differentiation of vascular endothelial cells and macrophages comprise EGF, VEGF, bFGF, wherein EGF is used in an amount of 1-50ng/ml, VEGF is used in an amount of 0.5-100ng/ml, and bFGF is used in an amount of 0.5-50ng/ml; the volume percentage of EGM2 added in each stage is 5-60%.

Further, lung epithelial cell related induced differentiation factors include GSK-3 inhibitors, BMP4, FGF10, KGF, ATRA; wherein, the GSK-3 inhibitor is used in an amount of 0.1-10 mu M, BMP4 is used in an amount of 1-50ng/ml, FGF10 is used in an amount of 1-50ng/ml, KGF is used in an amount of 1-50ng/ml, and ATRA is used in an amount of 0.01-10 mu M.

Further, the culture medium in the step 1) is further added with lung epithelial maturation-promoting factors, wherein the lung epithelial maturation-promoting factors comprise dexamethasone, PDE inhibitor and PKA activator.

Further, the pro-fibrosis inducer is TGF-beta 1, which is used at a concentration of 10-100ng/ml.

Further, the treatment with the pro-fibrosis inducer is carried out for 5-20 days.

Further, the fibrosis pathology related index includes pathology index detection of tissue in situ and pathology index detection of serology.

Further, the detection of pathological indexes of tissue in situ comprises histomorphology detection, pathological staining and detection of fibrosis marker gene/protein expression;

the pathological index detection of serology comprises ELISA detection of fibrosis related proteins in culture supernatants.

Compared with the prior art, the in-vitro accurate simulation method for pulmonary fibrosis has the following advantages:

the model not only expresses a series of fibrosis markers, but also reproduces important molecular events such as occurrence of inflammatory reaction, abnormal accumulation of pro-fibrosis factors and the like, and also presents typical fibrosis pathological characteristics in morphology and histopathology. The comprehensive in-vitro fibrosis simulation capability of the model enables the model to be suitable for accurate screening and evaluation of medicines.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a graph showing the results of stage S3 Day 70; wherein, the A diagram is a Day70 bionic lung organoid morphological diagram; panel B-K shows the lineage composition of lung assayed by different specific marker antibodies, epCAM, ECAD are broad-spectrum epithelial cell markers, CD31 is mature vascular endothelial cell marker, CD68 is macrophage marker, CHGA is neuroendocrine cell marker, VIL1 is ciliated marker, CC10 is rod-like cell marker, MUC5AC is goblet cell marker, P63 is basal cell marker, HOPX is alveolar I type cell marker, SPB is alveolar II type cell marker, SOX9 is lung epithelial progenitor cell marker;

FIG. 2 is a graph showing gene expression of markers of different lineages of the lung during induction of differentiation; wherein D2 (Day 2), D7 (Day 7), D17 (Day 17) and D70 (Day 70) represent definitive endoderm, lung progenitor cell sphere, lung bud and biomimetic lung organoids, respectively; panel A shows the expression of the marker SOX2, SOX2 being the original digestive tract anterior marker (from which the lung develops); panel B shows the expression of the marker NKX2.1, NKX2.1 being a lung epithelial cell marker; panel C shows the expression of the marker SOX9, SOX9 being a lung epithelial progenitor marker; panel D shows the expression of the marker CC10, CC10 being a marker for a rod-like cell; e panel shows the expression of marker MUC5AC, MUC5AC being a goblet cell marker; f, the expression situation of a marker acTUB, wherein acTUB is a cilia marker; g is the expression of the marker KRT5, KRT5 being a basal cell marker; panel H shows the expression of the marker SPB, which is an alveolar type II cell marker; FIG. I shows the expression of marker SPC, which is alveolar type II cell marker; panel J shows the expression of the marker CD144, CD144 being a mature vascular endothelial cell marker; k is the expression of marker CD68, CD68 is macrophage marker; HL (Human lung) is human primary lung, biological repetition of different induced differentiation time points n=4, p < 0.05; * P < 0.01; * P < 0.001;

FIG. 3 is a morphological alignment of the control group and the molding module before and after molding; the diagram A is before the molding, the diagram B is after the molding, the diagram C is before the comparison molding, and the diagram D is after the comparison molding;

FIG. 4 shows the expression levels of fibrosis-related markers mRNA in control and model building groups; * P is less than 0.05; * P < 0.01; the technique repeats n=3; biological repeat n=4; panel A shows the expression level of marker A-SMA, panel B shows the expression level of marker COL3A, panel C shows the expression level of marker VIM, panel D shows the expression level of marker DES, panel E shows the expression level of marker SNAIL, and panel F shows the expression level of marker FAP;

FIG. 5 shows immunofluorescence of control and model fibrosis markers and their quantitative results; * P < 0.0001; fluorescence quantification n=18 sections per group, derived from 3 organoids; A-C is the expression of marker COL1A, D-F is the expression of marker COL3A, G-I is the expression of marker FN, and J-L is the expression of marker FAP;

FIG. 6 shows ELISA results of culture supernatants before and after TGF-beta 1 modeling; * P is less than 0.05; * P < 0.01; * P < 0.001; the technique repeats n=3; biological repeat n=4; FIG. A shows the detection result of the pro-inflammatory factor IL-6, FIG. B shows the detection result of the pro-inflammatory factor TNF-alpha, FIG. C shows the detection result of the pro-fibrotic factor CCL18, FIG. D shows the detection result of the pro-fibrotic factor MMP7, and FIG. E shows the detection result of the pro-fibrotic factor PDGF;

FIG. 7 shows Masson staining results for control and model groups.

Detailed Description

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

Definitive endoderm: definitive endoderm (definitive endoderm) refers to the primary developmental site of cells that are primarily composed of internal organs such as the lungs, liver, small intestine, and large intestine in early embryonic development.

Anterior canal derivative (or anterior canal derivative): intestinal canal (gut tube) refers to the streak of tissue that develops from the definitive endoderm at the early stages of embryonic development. In vivo studies indicate that the front and rear ends are composed of different progenitor cells, which can develop into different organs respectively; wherein the lung develops from a progenitor cell population near the anterior end of the gut tube, developmentally known as the anterior gut tube or anterior part of the gut tube.

The anterior gut tube derivative (or anterior gut tube derivative) in the present patent refers to a population of cells that are SOX2 positive and possess the potential for lung differentiation.

The invention provides a multi-lineage lung organoid constructed by a method comprising the steps of:

s1, inducing hPSC to differentiate into definitive endoderm by using a culture medium containing GDF;

s2, inducing the definitive endoderm to differentiate into lung buds, comprising two stages: a pre-stage and a post-stage;

the formation of lung progenitor cell spheres at a pre-stage, during which ATRA is continuously added; in the later stage, a saccular lung bud is formed, and on the basis of using lung epithelial cell related induced differentiation factors to conduct lung directional differentiation, common factors of vascular endothelial cells and macrophage induced differentiation are added; EGM2 is added into the culture medium in the early stage and the later stage;

s3, inducing lung buds to differentiate into multi-lineage lung organoids;

coating lung buds by adopting ECM containing type I collagen and type III collagen, wherein the ECM is used for simulating a three-dimensional microenvironment for cell growth; and then adding common factors for the induction and differentiation of vascular endothelial cells and macrophages and EGM2 into the culture medium for continuous culture to obtain the multi-lineage lung organoids.

Specifically, the culture medium used in S1 comprises a culture medium A and a culture medium B, wherein the culture medium A is added with GDF and GSK-3 inhibitor, the culture medium B is added with GDF, FGF2 and vitamin C, hPSC is sequentially cultured in the culture medium A and the culture medium B,

GDF may be used in an amount of 20-500ng/ml, preferably 50-200ng/ml, or more preferably 100-150ng/ml, within which the object of the present invention can be achieved, and specifically 100ng/ml may be used; GSK-3 inhibitors may be used in an amount of 0.1 to 10. Mu.M, preferably 0.5 to 5. Mu.M, or more preferably 2 to 5. Mu.M, within which the object of the present invention can be achieved, and specifically 3. Mu.M can be used; FGF2 may be used in an amount of 1 to 100ng/ml, preferably 2.5 to 30ng/ml, or more preferably 5 to 20ng/ml, within which the object of the present invention can be achieved, and specifically 10ng/ml may be used; the vitamin C (AA) may be used in an amount of 5 to 300. Mu.g/ml, preferably 10 to 200. Mu.g/ml, or more preferably 20 to 100. Mu.g/ml, within which the object of the present invention can be achieved, and particularly 50. Mu.g/ml can be used.

Wherein, GDF is an Nodal signal activator, and GDF can be any one of GDF3, GDF5, GDF7 and GDF8, preferably GDF8; GDF8 makes cell growth more homogeneous and lays a foundation for later increase of SOX2 expression level in S2 stage and sufficient lung progenitor cell sphere;

GSK-3 inhibitors may be CHIR99021 and its hydrochloride, BIO, SB216763, AT7519, CHIR-98014, TWS119, tidegluib, but are not always limited thereto, specifically selected as CHIR99021;

FGF2 may be replaced by FGF4, FGF10, preferably FGF2;

the culture is carried out in medium A and medium B for 1 to 3 days, preferably 1 day, respectively.

ATRA is continuously added at the early stage of S2 and maintained at a concentration of 0.01-10 μm, preferably 0.25-5 μm, or more preferably 0.5-2 μm, and may specifically be 1 μm, to ensure that sufficient lung progenitor cell spheres in suspension can be produced for collection and continued differentiation. And the common factor EGF, VEGF, bFGF for the induction and differentiation of vascular endothelial cells and macrophages is added in the later stage, and EGM2 is added in the culture medium in the whole S2 stage, so that the expression of endothelial cells and immune cell markers is facilitated.

The volume percentage of EGM2 added at each stage is 5% -60%, preferably 10-50%, or more preferably 15-30%.

In addition, before the early stage of S2, the culture medium containing BMP inhibitor and TGF beta inhibitor may be used to culture, and EGM2 may be added to the culture medium. The volume percentage of the added EGM2 is 5% -60%, preferably 23.5%.

The early addition of EGM2 was performed to generate mesodermal progenitor cells, i.e., precursor cells of macrophages and vascular endothelial cells.

BMP inhibitors are any of NOG, CC, LDN193189, DMH1, LDN-212854, UK-383367, K02288, preferably NOG; TGF-beta inhibitors are any of SB431542, repSox, A83-01, galunisertib, vactosertib, R-268712, ML347, SD-208, R-268712, LY2109761, LY-364947, AZ12601011, LY3200882, GW788388, preferably SB431542.

Specifically, the culture medium used in S2 includes culture medium C, culture medium D, culture medium E and culture medium F, wherein culture medium D-E is used in the early stage and culture medium F is used in the late stage; wherein, the culture medium C is added with BMP inhibitor and TGF beta inhibitor, the culture medium D is added with Wnt inhibitor, TGF beta inhibitor and ATRA, the culture medium E is added with lung epithelial cell related induced differentiation factors which are GSK-3 inhibitor, BMP4, keratinocyte growth factor KGF, FGF10 and ATRA; medium F increased EGF, VEGF, bFGF on medium E while decreasing the amount of ATRA.

The amount of BMP inhibitor used in medium C may be 50-500ng/ml, preferably 100-400ng/ml, or more preferably 150-300ng/ml, within which the object of the present invention can be achieved, and particularly preferably 200ng/ml; TGF-beta inhibitors may be used in an amount of 1-50. Mu.M, preferably 5-30. Mu.M, or more preferably 5-10. Mu.M, within which the object of the present invention is achieved, particularly preferably 10. Mu.M; culturing in medium C for 1-3 days, preferably 1 day.

The Wnt inhibitor in the medium D may be IWP2, IWR-1, IWP-4, CCT251545, KY1220, but is not always limited thereto, and particularly preferably IWP2 may be used in an amount of 0.1 to 10. Mu.M, preferably 0.25 to 5. Mu.M, or more preferably 0.5 to 2. Mu.M, particularly preferably 1. Mu.M; TGF-beta inhibitors may be selected from the above, particularly preferably SB431542, in an amount of 1-50. Mu.M, preferably 5-30. Mu.M, or more preferably 5-10. Mu.M, particularly preferably 10. Mu.M; culturing in medium D for 1-3 days, preferably 1 day.

The GSK-3 inhibitor in medium E may be selected from the above, particularly preferably CHIR99021, and may be used in an amount of 0.1 to 10. Mu.M, preferably 1 to 7.5. Mu.M, or more preferably 2 to 5. Mu.M, particularly preferably 3. Mu.M; the BMP4 can be used in an amount of 1 to 50ng/ml, preferably 5 to 20ng/ml, particularly preferably 10ng/ml; the keratinocyte growth factor KGF may be used in an amount of 1 to 50ng/ml, preferably 5 to 20ng/ml, particularly preferably 10ng/ml; FGF10 may be used in an amount of 1-50ng/ml, preferably 5-20ng/ml, particularly preferably 10ng/ml; culturing in medium E for 2-5 days, preferably 3 days.

EGF may be used in the medium F in an amount of 1 to 50ng/ml, preferably 5 to 20ng/ml, particularly preferably 10ng/ml; VEGF may be used in an amount of 0.5-100ng/ml, preferably 10-30ng/ml, particularly preferably 15ng/ml; bFGF may be used in an amount of 0.5-50ng/ml, preferably 2-10ng/ml, particularly preferably 5ng/ml; the ATRA is used in an amount of 0.01 to 0.5. Mu.M, preferably 0.05 to 0.2. Mu.M, particularly preferably 0.1. Mu.M; culturing in medium F for 8-12 days, preferably 10 days.

Specifically, the medium used in S3 is medium G, and medium G is supplemented with factors promoting maturation of lung organoids on the basis of medium F.

Among the factors that promote lung organoid maturation are dexamethasone, PDE inhibitors, PKA activators.

The PDE inhibitor is any one of IBMX, rolipram, sildenafil, milrinone, preferably IBMX; the amount thereof may be 0.01 to 1mM, preferably 0.05 to 0.2mM, particularly preferably 0.1mM;

the PKA activator may be any of cAMP and its salts, FSK, CW008, taxol, belinostat and its salts, preferably cAMP; the amount thereof may be 0.01 to 1mM, preferably 0.05 to 0.2mM, particularly preferably 0.1mM;

dexamethasone MK125 may be used in an amount of 10-1000nM, preferably 25-100nM, particularly preferably 50nM.

In particular, ECM is a three-layer structure, from bottom to top, a layer a, a layer B, and a layer C, respectively;

layer a is 100% Matrigel;

the B layer is lung bud formed by S2, matrigel with a volume ratio of 40%, type I collagen with a volume ratio of 40%, and type III collagen with a volume ratio of 20%;

layer C was 100% Matrigel.

When in use, the layer A, B, C is added into the 12-hole transwell upper layer cell in sequence; standing for 10min, 60min, and 10min respectively to solidify completely. Thereafter, both the upper and lower chambers were charged with stage 3 induction medium.

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

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

The relevant reagents used in the examples are shown in tables 1 and 2, and the relevant antibodies used are shown in table 3.

EXAMPLE 1 construction of multiple lineages of lung organoids

(one) generating multiple lineages of lung organoids

On the basis of lung epithelial organoid induced differentiation factors, human multilineage lung organoid induced differentiation of epithelial cells, endothelial cells and immune cells is realized through innovation of three aspects: 1) Basal medium: addition of medium suitable for mesodermal lineage growth EGM2, 2) induction factors: common factors EGF, VEGF, bFGF for vascular endothelial cells and macrophages to induce differentiation, 3) ECM (extracellular matrix): the addition of Collagen I (type I Collagen) and Collagen III (type III Collagen) promotes multiple lineage maturation.

The whole of this example is divided into 3 phases, for 70 days:

s1 (stage 1): induction of hPSC differentiation into definitive endoderm (Day 1-2)

Differentiation can be initiated when hPSC confluency reaches 40-70%. Specifically, in this example, when hPSC confluence reaches 40%, the hpscs are sequentially added into medium a and medium B in S1 stage to induce differentiation.

S11（Day 1）：

Human pluripotent stem cells were cultured in medium A of RPMI-1640 supplemented with 100ng/ml rhGDF8, 3. Mu.M CHIR99021 for 24 hours.

S12（Day 2）：

After that, the culture was continued in medium B, which was supplemented with 100ng/ml of rhGDF8, 10ng/ml of rhFGF2, 50. Mu.g/ml of vitamin C (AA), and which was RPMI-1640 containing 2% by volume of SM1, for 24 hours.

S2 (stage 2): induction of differentiation of definitive endoderm into lung buds (Day 3-17)

S21（Day 3）：

This stage was incubated for 24h with medium C, supplemented with 200ng/ml NOG, 10. Mu.M SB431542, and medium C containing 0.5% (v/v) N2 (A), 1% (v/v) SM1, 75% (v/v) IMDM, 23.5% (v/v) EGM2 and 0.05% (solid to liquid) BSA, 0.4. Mu.M MTG.

S22（Day 4）：

This stage was incubated for 24h with medium D, which was supplemented with 1. Mu.M IWP2, 10. Mu.M SB431542, 1. Mu.M ATRA, and 0.5% (v/v) N2 (A), 1% (v/v) SM1, 75% (v/v) IMDM, 23.5% (v/v) EGM2, and 0.05% (solid/liquid) BSA, 0.4. Mu.M MTG.

S23（Day 5-7）：

Culture was continued for 3 days using medium E with medium changes every 24 hours. Medium E was supplemented with 3. Mu.M of CHIR99021, 10ng/ml of rhBMP4, 10ng/ml of rhKGF, 10ng/ml of rhFGF10, 1. Mu.M of ATRA, and medium E contained 0.5% (v/v) N2 (A), 1% (v/v) SM1, 75% (v/v) IMDM, 23.5% (v/v) EGM2 and 0.05% (solid/v) BSA, 0.4. Mu.M MTG.

By Day7 (Day 7), significant amounts of SOX2 appeared on 2D cell upper layers ⁺ 、NKX2.1 ⁺ Solid spheres of lung progenitor cells (hereinafter referred to as lung progenitor cell spheres) are collected and transferred to an ultra-low adsorption plate and cultured using the medium of stage S24.

S24（Day 8-17）：

Culture medium F was used for 10 days with medium changes every 24-48 h. Medium F containing 0.5% (v/v) N2 (A), 1% (v/v) SM1, 75% (v/v) IMDM, 23.5% (v/v) EGM2 and 0.05% (solid-to-liquid) BSA, 0.4. Mu.M MTG was supplemented with 3. Mu.M of CHIR99021, 10ng/ml of rhBMP4, 10ng/ml of rhKGF, 10ng/ml of rhEGF, 15ng/ml of rhVEGF, 5ng/ml of bFGF, 0.1. Mu.M of ATRA.

By Day17 (Day 17), the lung progenitor cell sphere will differentiate to express EpCAM ⁺ 、NKX2.1 ⁺ 、FOXA1 ⁺ 、SOX9 ⁺ And (3) saccular lung buds, and entering the S3 stage.

S3: induction of differentiation of Lung buds into multiple lineages of Lung organoids (Day 18-70)

ECM production and lung bud coating:

ECM is divided into three layers, each layer being composed of:

layer A: 100% Matrigel;

layer B: day17 lung bud+40% matrigel+40% Collagen I (2.0 mg/ml) +20% Collagen III (2.0 mg/ml);

and C layer: 100% Matrigel.

Layer A, B, C was added in succession to the 12-well transwell upper cell; standing for 10min, 60min, and 10min respectively to solidify completely. Thereafter, medium G in the S2 stage was added to each of the upper and lower chambers.

Day 18-70：

Finally, culture medium G was used for 53 days, and medium was changed every 24-72 hours. Medium G containing 0.5% (volume ratio) N2 (A), 1% (volume ratio) SM1, 75% (volume ratio) AECGM, 23.5% (volume ratio) EGM2 and 0.05% (solid solution ratio) BSA, 0.4. Mu.M MTG was supplemented with 3. Mu.M of CHIR99021, 10ng/ml of rhBMP4, 10ng/ml of rhKGF, 10ng/ml of rhFGF10, 10ng/ml of rhEGF, 15ng/ml of rhVEGF, 5ng/ml of bFGF, 0.1. Mu.M ATRA, 50nM MK125, 0.1mM IBMX, 0.1mM cAMP.

By Day70 (Day 70), human multiple lineage lung organoids with both proximal and distal structures were obtained.

(II) organoid phenotype detection

The organoid phenotype was detected comprehensively using the following indicators:

1) Morphological characterization of organoids in bright field;

2) Detecting the expression level of the related marker mRNA by a fluorescent quantitative PCR (Q-PCR) technology;

3) The expression of cell specific markers was detected by frozen section immunofluorescence and panoramic immunofluorescence techniques.

Example 2 fibrosis modeling based on lung organoids

First, fiberizing die

Setting a model/control group, and continuously incubating organoids with 50ng/ml TGF-beta 1 and PBS for 10 days; during which time the medium was changed every 48 hours.

(II) fibrosis pathology related index validation

The fibrosis pathology was assessed comprehensively using the following indices:

1) Morphological characterization of organoids in bright field;

2) Detecting the expression level of the fibrosis related marker mRNA of the modeling/control group by a fluorescence quantitative PCR (Q-PCR) technology;

3) Detecting the protein expression condition of the fibrosis related markers of the modeling/control group by using a frozen section and immunofluorescence technology, and carrying out fluorescence area quantitative analysis (related antibodies are shown in an accessory list) by using imageJ software;

4) Detecting the secretion level of the fibrosis related secretion protein of the modeling/control group by ELISA technology;

5) Modeling/control collagen deposition was assessed by paraffin section and Masson staining techniques.

Results and analysis:

1. continuously differentiating to Day70 to form mature human bionic lung organoids. Morphologically present a tissue-like appearance of complex structure, up to 0.5cm in volume ³ (panel A in FIG. 1).

Immunofluorescence results after frozen sections showed that: organoids not only possess the epithelial cell type (EpCAM) ⁺ Or ECAD ⁺ Panel B of fig. 1 and panel C of fig. 1), also has endothelial and immune cell types, CD31 ⁺ Vascular endothelial cells, panel B, CD68 in fig. 1 ⁺ Macrophages, panel C in fig. 1.

The epithelial cells are further divided into neuroendocrine Cells (CHGA) located at the proximal end of the respiratory tract ⁺ Panel D in FIG. 1), ciliated cells (VIL 1 ⁺ FIG. 1E), rod-like cells (CC 10 ⁺ Panel F in FIG. 1), goblet cells (MUC 5AC ⁺ FIG. 1, panel G), basal cells (P63 ⁺ H in fig. 1), alveolar type I (HOPX) located at the distal end of the respiratory tract ⁺ Panel I of FIG. 1) and type II cells (SP-B ⁺ Fig. 1, J).

The lung progenitor cells exhibit a bulk distribution at both the proximal and distal ends (SOX 9) ⁺ Fig. 1, K).

On the other hand, the expression level of each lineage marker was detected by Q-PCR, and as shown in fig. 2, the results confirmed the above immunofluorescence results, and also confirmed the overall maturation of the lung organoids.

The above results demonstrate that, unlike existing lung organoids, the bionic lung organoids have physiological-related lineages derived from mesoderm, including endothelial cells and immune cells, such as vascular endothelial cells, macrophages, etc., in addition to a variety of epithelial cell types derived from endoderm, such as ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, etc., and are therefore more suitable for modeling of pulmonary fibrosis.

2. As shown in fig. 3, after 10 days of molding with TGF- β1, the lung organoids were wholly rendered solid, and the interior of the original alveolar-like structure was changed from hollow to partially solid, compared to before molding; and a large number of mesenchymal-like cells appear around the original Epithelial-like structure, suggesting an important pathological characterization of fibrosis occurrence-EMT (epigeal-mesenchymal transition), epithelial to mesenchymal transition.

3. As shown in FIG. 4, the Q-PCR results indicate that: after TGF-beta 1 modeling, the expression levels of extracellular matrix related markers (COL 3A), mesenchymal related markers (VIM, DES), EMT related markers (SNAIL) and FMT related markers (A-SMA, FAP) are fully up-regulated, suggesting the occurrence and development of fibrosis.

4. As shown in fig. 5, immunofluorescence results indicate that: compared with the control group, 10 days of TGF-beta 1 modeling enables the fibrosis markers such as COL1A, COL A, FN, FAP and the like to be expressed in a large area; the significance of these protein changes was confirmed by fluorescence area quantification results, which confirm the occurrence of fibrosis.

5. ELISA results show that TGF-beta 1 modeling leads inflammatory factors such as IL-6, TNF-alpha and the like in supernatant (A diagram-B diagram in figure 6) and CCL18, MMP7 and PDGF pro-fibrosis factors to be obviously up-regulated (C diagram-E diagram in figure 6), and the fibrosis pathological characteristics are accurately simulated.

6. As shown in fig. 7, pathological section staining (Masson staining) results showed that: the columnar epithelium which is orderly arranged in the original alveolus-like structure is subjected to TGF-beta 1 modeling, and then the tissue structure is broken, and a plurality of substantive structures are formed. The partial amplification comparison result shows that: similar to the pathological manifestations of patients with pulmonary fibrosis, on the one hand more spindle-shaped mesenchymal-like cells appear in the modeling group, indicating the occurrence of EMT; on the other hand, the obvious collagen deposition phenomenon is presented.

The results fully demonstrate that lung organoid fibrosis modeling was successful after 10 days of induction by TGF-beta 1. Also, the present model reproduces the pathological features of pulmonary fibrosis in several ways, much like in vivo pathology.

TABLE 1 list of reagents

Reagent consumable name	Company (goods number)
		mTeSR1	STEMCELL (85850)
Y-27632	Sigma (SCM075)
		RPMI 1640	Gibco (31870082)
IMDM	Sigma (I2911)
		EGM2	Lonza (CC-3156 & CC-4176)
rhGDF8	R&D (6986-PG)
		rhFGF2	R&D (233-FB)
rhNOG	R&D (6057-NG)
		rhBMP4	R&D (314-BPE)
rhKGF	R&D (251-KG)
		rhFGF10	R&D (345-FG)
rhEGF	R&D (236-EG)
		rhVEGF	R&D (DVE00)
CHIR99021	Tocris (4423/10)
		SB431542	Tocris (1614)
IWP2	Tocris (3533)
		ATRA	Tocris (0695/50)
IBMX	Tocris (2845)
		MK125	Tocris (1126)
8-Br-cAMP	Tocris (1140/10)
		N2 (A)	STEMCELL (07152)
SM1	STEMCELL (05711)
		BSA	Sigma (A1933)
MTG	Sigma (M6145)
		Matrigel (GFR)	Biocoat (356231)
Latex beads, carboxylate-modified polystyrene, fluoroscreen red (latex beads, carboxylic acid) Salt modified polystyrene, fluorescent red)	Sigma-Aldrich (L3030)
		Low Density Lipoprotein from Human Plasma, Acetylated, Alexa Fluor™ 594 Conjugate (low density lipoprotein in human plasma, acetylated, alexa Fluor ™ 594 conjugate	(Invitrogen, L35353)
Clodronate liposomes (Chlorophosphate liposome)	(Liposoma, C-005)
		LPS	Sigma-Aldrich (L2630)
IFN-γ	R&D (285-IF)
		IL-4	R&D (204-IL)
IL-13	R&D (213-ILB)
		Masson's Trichrome Stain Kit (Masson trichromatic dyeing) Color reagent box	Polysciences (25088-100)
Human TNF-alpha Quantikine ELISA Kit (of Human origin) TNF-alpha quantitative factor ELISA kit	R&D (DTA00C)
		Human IL-6 Quantikine ELISA Kit (humanized IL-6) Quantitative factor ELISA kit	R&D (D6050)
Human CCL18/PARC Quantikine ELISA Kit (Human) Source CCL18/PARC quantitative factor ELISA kit	R&D (DCL180B)
		Human Total MMP-7 Quantikine ELISA Kit (Human ELISA kit for quantitative factors of source total MMP-7	R&D (DMP700)
Human/Mouse PDGF-AA Quantikine ELISA Kit (human/mouse PDGF-AA quantitative factor ELISA kit)	R&D (DAA00B)
		Human Collagen I (Human Collagen I)	Sigma (234138)
Human Collagen III (human collagen III)	Sigma (C4407)
		Evo M-MLV RT Kit with gDNA Clean for qPCR (in vitro reverse transcription kit)	AG (AG11705)
SYBR Green Premix Pro Taq HS qPCR Kit (SYBR Green premix Pro Taq HS qPCR kit)	AG (AG11718)
		Human lung RNA (Human lung RNA)	Clontech (636524)
Normal Donkey Serum (Normal donkey serum)	Jacksonlab (017-000-121)
		DAPI	Sigma (D9542)

TABLE 2 reagent correspondence names

rhGDF8	Recombinant human myostatin 8
		CHIR99021	GSK-3 inhibitors
rhFGF2	Recombinant human fibroblast growth factor 2
		AA	Vitamin C
rhBMP4	Recombinant human bone morphogenetic protein 4
		rhKGF	Recombinant human keratinocyte growth factor
rhFGF10	Recombinant human fibroblast growth factor 10
		rhEGF	Recombinant human epithelial growth factor
rhVEGF	Recombinant human vascular endothelial cell growth factor
		bFGF	Basic fibroblast growth factor
rhNOG	Recombinant human noggin, BMP inhibitors
		SM1	Serum substitute with definite components
N2(A)	Neural differentiation additive
		BSA	Bovine serum albumin
MTG (1-Thioglycerol)	1-thioglycerol
		SB431542	TGF beta inhibitor
IWP2	Wnt inhibitor
		Matrigel (GFR)	Matrigel (growth factor reduction type)
ATRA (All-trans-Retinoic acid)	Natural agonists of all-trans retinoic acid, RAR nuclear receptors; an inhibitor;
		IBMX	a broad spectrum Phosphodiesterase (PDE) inhibitor
cAMP	Cyclic AMP analogs, PKA activators
		MK125 (Dexamethasone)	Dexamethasone
IMDM (Iscove's Modified Dulbecco's Medium)	Serum-free basal medium suitable for epithelial cells or blood cells
		EGM2 (Endothelial Cell Growth Medium 2)	Serum-free culture medium suitable for endothelial growth
AECGM (Airway Epithelial Cell Growth Medium)	Serum-free culture medium suitable for lung epithelial cells
		DAPI	Cell nucleus dye
ECM	Extracellular matrix
		Collagen I	Type I collagen
Collagen III	Type III collagen
		MUC5AC (Mucin 5AC)	Mucin 5AC, goblet cell markers
CC100 (Clara cell 10 kDa proteins)	Clara cell 10-kDa protein, rod-like cell marker
		acTUB (Acetylated α-tubulin)	Acetylated-alpha tubulin, ciliated cell markers
CHGA (Chromogranin A)	Human chromoprotein a, neuroendocrine cell markers
		P63 (Tumor protein p63)	Transcription factor p63, lung basal cell marker
PDPN (Podoplanin)	Flat foot protein, type I alveolar cell marker
		SP-B (Surfactant protein B)	Lung surfactant protein B, II type alveolus cell marker
CD68	Differentiation antigen cluster 68, universal macrophage marker
		CD206	Differentiation antigen cluster 206, M2-macrophage markers
CD86	Differentiation antigen cluster 86, M1 macrophage marker
		TGF-β1 (Transforming growth factor beta 1)	Transforming growth factor-beta 1, central fibrosis regulatory signals
COL1A (Collagen 1A)	Type I collagen alpha chain, fibrosis pathology related marker (ECM constituting protein)
		COL3A (Collagen 3A)	Type III collagen alpha chain, fibrosis pathology related markers (ECM constituting proteins)
FN (Fibronectin)	Fibronectin, fibrosis pathology-related markers (ECM-constituting proteins)
		FAP (Fibroblast activation protein)	Fibroblast activation protein, fibrosis pathology-related markers (characterizing activated fibroblasts)
A-SMA (α-Smooth muscle actin)	Alpha-smooth muscle actin, fibrosis pathology-related markers (characterizing myofibroblasts)
		VIM (Vimentin)	Wave form fibrin, fibrosis pathology related marker (interstitial cell marker)
DES (Desmin)	Connexin, fibrosis pathology related marker (characterising interstitial cells)
		SNAIL	Zinc finger protein transcription factor, fibrosis pathology related marker (characterization of interstitial cells)
IL-6	Interleukin-6, an inflammatory factor
		TNF-α(Tumour necrosis factor α)	Tumor necrosis factor-alpha, an inflammatory factor
MMP7 (Matrix metallopeptidase 7)	Human matrix metalloproteinase 7, a potential fibrosis marker
		CCL18 (C-C motif chemokine ligand 18)	Chemokine CC ligand 18, a potential fibrosis marker
PDGF (Platelet derived growth factor)	Platelet growth factor, a potential fibrosis marker

TABLE 3 list of antibodies

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (3)

1. An in vitro accurate simulation method for pulmonary fibrosis is characterized in that: the method comprises the following steps:

1) Construction of multiple lineages of lung organoids

S1, inducing hPSC to differentiate into definitive endoderm:

the culture mediums used in the S1 are a culture medium A and a culture medium B, wherein the culture medium A is added with GDF and a GSK-3 inhibitor, the culture medium B is added with GDF, FGF2 and vitamin C, and hPSC is sequentially and respectively cultured in the culture medium A and the culture medium B for 1-3 days; GDF is an Nodal signal activator, GDF is any one of GDF3, GDF5, GDF7 and GDF8, and the using amount of GDF is 20-500ng/ml; the GSK-3 inhibitor is any one of CHIR99021 and its hydrochloride, BIO, SB216763, CHIR-98014, TWS119 and Tidegluib, the GSK-3 inhibitor is used in an amount of 0.1-10 mu M, FGF2 is used in an amount of 1-100ng/ml, and vitamin C is used in an amount of 5-300 mu g/ml;

s2, inducing differentiation of definitive endoderm into lung buds:

culturing for 1-3 days in culture medium C before the S2 is subjected to the early stage, wherein the culture medium contains BMP inhibitor and TGF beta inhibitor, and EGM2 is added into the culture medium; the BMP inhibitor is any one of NOG, CC, LDN193189, DMH1, LDN-212854, UK-383367 and K02288, and the dosage of the BMP inhibitor is 50-500ng/ml; the TGF-beta inhibitor is any one of SB431542, repSox, A83-01, galunisertib, vactosertib, R-268712, ML347, SD-208, LY2109761, LY-364947, AZ12601011, LY3200882 and GW788388, and the use amount of the TGF-beta inhibitor is 1-50 mu M;

s2 is divided into a pre-stage and a post-stage, wherein the pre-stage uses a culture medium D and a culture medium E, the post-stage uses a culture medium F, ATRA is continuously added in the pre-stage, and the concentration of ATRA is kept to be 0.01-10 mu M; culturing in a culture medium D for 1-3 days, wherein the culture medium D is added with Wnt inhibitor, TGF beta inhibitor and ATRA, the Wnt inhibitor is any one of IWP2, IWR-1, IWP-4, CCT251545 and KY1220, the Wnt inhibitor is used in an amount of 0.1-10 mu M, the TGF beta inhibitor is any one of SB431542, repSox, A83-01, galunisertib, vactosertib, R-268712, ML347, SD-208, LY2109761, LY-364947, AZ12601011, LY3200882 and GW788388, and the TGF beta inhibitor is used in an amount of 1-50 mu M; culturing in a culture medium E for 2-5 days, wherein a GSK-3 inhibitor, BMP4, KGF, FGF10 and ATRA are added into the culture medium E; wherein the GSK-3 inhibitor is any one of CHIR99021 and its hydrochloride, BIO, SB216763, CHIR-98014, TWS119, tidegrouib, GSK-3 inhibitor is used in an amount of 0.1-10 mu M, BMP4 is used in an amount of 1-50ng/ml, FGF10 is used in an amount of 1-50ng/ml, KGF is used in an amount of 1-50ng/ml; culturing in a culture medium F for 8-12 days, wherein the culture medium F is increased by EGF, VEGF, bFGF on the basis of the culture medium E, meanwhile, the ATRA dosage is reduced, and EGM2 is added into the culture medium in the early stage and the later stage, wherein the EGF dosage is 1-50ng/ml, the VEGF dosage is 0.5-100ng/ml, and the bFGF dosage is 0.5-50ng/ml; the volume percentage of EGM2 added in each stage is 5-60%;

s3, inducing lung buds to differentiate into multi-lineage lung organoids;

coating lung buds by adopting an ECM (electro-magnetic control module) containing I-type collagen and III-type collagen, wherein the ECM is used for simulating a three-dimensional microenvironment for cell growth, and has a three-layer structure, namely an A layer, a B layer and a C layer from bottom to top; layer a is 100% Matrigel; the B layer is lung bud formed by S2, matrigel with a volume ratio of 40%, type I collagen with a volume ratio of 40%, and type III collagen with a volume ratio of 20%; layer C is 100% Matrigel; then culturing with a culture medium G, wherein dexamethasone, a PDE inhibitor and a PKA activator are added into the culture medium G on the basis of the culture medium F, the dosage of dexamethasone is 10-1000nM, the dosage of the PDE inhibitor is any one of IBMX, rolipram, sildenafil, milrinone, the dosage of the PDE inhibitor is 0.01-1mM, the dosage of the PKA activator is any one of cAMP and salified compound thereof, FSK, CW008 and Taxol, and the dosage of the PKA activator is 0.01-1mM; culturing for 70-80 days to obtain multiple lineages of lung organoids;

2) Construction of pulmonary fibrosis disease model

Treating the multi-lineage lung organoid obtained in the step 1) for 5-20 days by using a fibrosis inducing agent TGF-beta 1 with the concentration of 10-100ng/ml, constructing a lung fibrosis disease model, and verifying fibrosis pathology related indexes of the lung fibrosis disease model.

2. The method for in vitro accurate simulation of pulmonary fibrosis of claim 1 wherein: the fibrosis pathology related index comprises pathology index detection of tissue in situ and pathology index detection of serology.

3. The method for in vitro accurate simulation of pulmonary fibrosis according to claim 2, wherein: the detection of pathological indexes of tissue in situ comprises the detection of histomorphology, pathological staining and the detection of fibrosis marker gene/protein expression;

the pathological index detection of serology comprises ELISA detection of fibrosis related proteins in culture supernatants.

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