CN115786245B - Human bionic lung organoid construction method - Google Patents

Abstract

The invention provides a method for constructing human bionic lung organoids, which realizes the co-differentiation and co-culture of human pluripotent stem cells to different lineages of lung through different compositions and operation steps. The organoids produced have both epithelial cells, endothelial cells, and immune cells in lineage type; structurally, the device comprises a bronchus-like structure and an alveolus-like structure. The model is suitable for being used as a new generation of humanized refined lung organoid model, and provides accurate and reliable research and evaluation tools for disease simulation, mechanism analysis, drug target spot discovery and drug effect screening of lung diseases.

Description

Human bionic lung organoid construction method

Technical Field

The invention relates to the field of stem cell biology and regenerative medicine, in particular to a method for constructing human bionic lung organoids.

Background

Along with the rapid development of the fields of developmental biology, stem cell medicine, materials science and the like in basic research and technical development, the organoid induction technology makes a series of breakthroughs, and provides a brand new concept and thought for the preclinical curative effect and toxicity evaluation of the medicine. Based on research evidence accumulated in recent years, the United states (FDA), european Union (EMA), and the drug administration (CDE) in China sequentially recommend the use of organoids as supplements or substitutes for animal models, and provide demonstration support in the aspects of mechanism research, concept verification and the like.

Currently, many organoid directed differentiation techniques based on human induced pluripotent stem cells (iPS) have been successfully developed for the intestines, liver, brain, kidneys, stomach, etc.; compared with the traditional cell model, the generated organoids are closer to the physiological original appearance of the real organs of the human body in the aspects of spectrum diversity, structural imitation and functional specificity, and have great potential in the aspects of molecular mechanism analysis of diseases, treatment target spot discovery, drug effect and drug toxicity evaluation and the like. In addition, because the organoids are completely generated from the iPS reprogrammed from autologous cells, the problem of limited sources of traditional primary model samples is solved, and the pathological features of the patient can be reproduced in vitro, so that the organoids are hopefully developed into a new generation of personalized medicine screening systems.

However, compared to real organs, most organoids currently consist only of epithelial cells that develop in the endoderm; it lacks cell types that develop from other germ layers, such as endothelial cells, smooth muscle cells, immune cells, etc. that do not yet possess mesoderm origin in the lung organoids. These cells are critical not only for the morphogenesis, structural modeling and functional maintenance of the lung under physiological conditions, but also for the development and progression of various diseases such as lung cancer and interstitial lung disease. Because of the lack of these physiologically relevant lineages, research and application models/systems developed therewith also have a great discount on the loyalty of pathophysiological reduction.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for constructing a human bionic lung organoid to solve the above-mentioned problems.

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

a method for constructing a human bionic lung organoid, comprising the following steps:

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 bionic 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 induced differentiation of vascular endothelial cells and macrophages and EGM2 into the culture medium for continuous culture to obtain the bionic lung organoid.

Further, the medium used in S1 includes medium A to which GDF and GSK-3 inhibitor are added and medium B to which GDF, FGF2 and vitamin C (AA) are added, and hPSC is sequentially cultured in medium A and medium B.

Wherein, the GDF can be any one of GDF3, GDF5, GDF7 and GDF8, preferably GDF8;

the GSK-3 inhibitor can be CHIR99021 and its hydrochloride, BIO, SB216763, AT7519, CHIR-98014, TWS119, tideglusib, preferably CHIR99021;

FGF2 may also be replaced by any of FGF4, FGF10, preferably FGF2.

Further, ATRA is used in an amount of 0.01-10. Mu.M, and common factors for the induction of differentiation of vascular endothelial cells and macrophages include EGF, VEGF, bFGF.

Further, the volume percentage of EGM2 added at each stage is 5% -60%, preferably 23.5%.

Further, the culture medium containing BMP inhibitor and tgfβ inhibitor is used before the early stage of S2, and EGM2 is added to the culture medium.

Further, the BMP inhibitor is any one 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.

The volume percentage of the added EGM2 is 5% -60%, preferably 23.5%.

Further, the medium used in S2 includes medium C to which BMP inhibitor, tgfβ inhibitor, and ATRA are added, medium D to which Wnt inhibitor, tgfβ inhibitor, and ATRA are added, medium E to which lung epithelial cell-related induced differentiation factor including GSK-3 inhibitor, BMP4, KGF, FGF10, and ATRA are added, medium E to which lung epithelial cell-related induced differentiation factor is added; medium F increased EGF, VEGF, bFGF on medium E while decreasing the amount of ATRA.

Further, the medium used in S3 was medium G, which was supplemented with lung epithelial maturation-promoting factor based on medium F.

Further, lung epithelial maturation-promoting factors are dexamethasone, PDE inhibitors, PKA activators.

Wherein the PDE inhibitor is any one of IBMX, rolipram, sildenafil, milrinone, preferably IBMX.

The PKA activator is any one of cAMP and its hydrochloride, FSK, CW008, taxol, belinostat and its hydrochloride, preferably cAMP.

Further, in S3, ECM is of a three-layer structure, from bottom to top, being a layer a, B, and 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.

The invention also provides a bionic lung organoid constructed by any one of the above construction methods, which has a broncholike structure and an alveolar-like structure, and simultaneously has an endodermal-derived epithelial cell type: ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, and mesoderm-derived cell types: vascular endothelial cells, macrophages.

The invention also provides a kit for the construction method of any one of the above, comprising:

GDF for S1, GDF is an Nodal signal activator, any one of GDF3, GDF5, GDF7, GDF8, preferably GDF8;

ATRA for S2, common factors for vascular endothelial cells and macrophages induced differentiation, and EGM2;

common factors for induced differentiation of type I collagen, type III collagen, vascular endothelial cells and macrophages for S3 and EGM2;

among them, common factors for vascular endothelial cells and macrophages to induce differentiation include EGF, VEGF, bFGF.

The invention also provides application of the construction method in inducing the differentiation of the human pluripotent stem cells into the three-dimensional bionic lung organoids.

Compared with the prior art, the construction method of the bionic lung organoid has the following advantages:

on the premise of not adding exogenous cells and not involving genetic operation, the invention only utilizes the iPS directional induction differentiation technology to generate the human bionic lung organoid with a bronchus-like structure and an alveolus-like structure. Unlike previous studies, this type of organ is different from the existing lung organoids in that it combines mesoderm-derived physiologically relevant lineages, including endothelial cells and immune cells, such as vascular endothelial cells, macrophages, etc., with the exception of a variety of epithelial cell types derived from the endoderm, such as ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, etc. Therefore, the method is very suitable for being used as a new generation of humanized refined lung organoid model, and provides a more reliable research tool for lung disease molecular mechanism research, disease simulation, drug target spot discovery, drug effect screening and the like. In addition, the invention adopts a serum-free induced differentiation system, has definite and controllable components, is little influenced by batch-to-batch differences and has low risk of being polluted by pathogenic microorganisms.

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 schematic diagram of the induction differentiation stage of a bionic lung organoid;

FIG. 2 is a graph showing the results of cell differentiation at stage S1; wherein, the A diagram is an initial density bright field diagram before differentiation of D1 (Day 1), the B diagram is a D2 (Day 2) definitive endoderm bright field diagram, the C diagram is a D2 (Day 2) cell immunofluorescence identification result, and the SOX17 is a definitive endoderm marker;

FIG. 3 is a graph showing the results of stage S2 Day 7; wherein, the A diagram is a D7 (Day 7) derivative form diagram; panel B shows the result of immunofluorescence identification of SOX2 of the upper suspension sphere, panel C shows the result of immunofluorescence identification of NKX2.1 of the upper suspension sphere, SOX2 is the original anterior marker of the digestive tract, and NKX2.1 is the marker of lung epithelial cells;

FIG. 4 is a graph showing the results of stage S2 Day 17; wherein, the A diagram is the form diagram of the D17 (Day 17) derivative; panel B-D show immunofluorescence identification results of saccular structures, epCAM is broad-spectrum epithelial marker, NKX2.1 and FOXA1 are lung epithelial markers, and SOX9 is lung progenitor cell marker;

FIG. 5 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, SP-B is alveolar II type cell marker, SOX9 is lung epithelial progenitor cell marker;

FIG. 6 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. 7 is the expression level of Day7 derivative SOX2 under different definitive endoderm production methods of comparative example 1;

FIG. 8 is the efficiency of Day7 derivative production of comparative example 1 under different definitive endoderm production methods; panel A shows the lung progenitor cell pellet generated in scheme B, panel B shows the lung progenitor cell pellet generated in scheme C, and panel C shows the lung progenitor cell pellet generated in scheme f;

FIG. 9 is a graph comparing the effect of ATRA on Day7 suspended lung progenitor cell pellet production during stages S22-S23 of the study of comparative example 2; panel A shows the lung progenitor cell pellet generated by scheme a Day7, panel B shows the lung progenitor cell pellet generated by scheme B Day7, panel C shows the lung progenitor cell pellet generated by scheme C Day7, and panel D shows the lung progenitor cell pellet generated by scheme D Day 7;

FIG. 10 is the effect of VEGF, EGF, bFGF on expression of marker NKX2.1 in stage S24 of the study of comparative example 3;

FIG. 11 is the effect of VEGF, EGF, bFGF on the expression of marker CD31 in stage S24 of the study of comparative example 3;

FIG. 12 is the effect of VEGF, EGF, bFGF on the expression of marker CD11b in stage S24 of the study of comparative example 3;

FIG. 13 shows the medium vs. immune cells (CD 68) studied in comparative example 4 ⁺ Macrophages) of the cell surface; scale bar, 250 μm; the image A is the immunofluorescence identification result of the method a, the image B is the immunofluorescence identification result of the method B, the image C is the immunofluorescence identification result of the method C, the image D is the immunofluorescence identification result of the method D, the image E is the immunofluorescence identification result of the method E, and the image F is the immunofluorescence identification result of the method F;

FIG. 14 shows the detection of gene expression in lung organoids of each lineage by fluorescent quantitative PCR under different ECM components studied in comparative example 5; panel A shows the expression of the marker NKX2.1, NKX2.1 being a lung epithelial cell marker; panel B shows the expression of the marker CC10, CC10 being a marker for a rod-like cell; panel C shows the expression of the marker MUC5AC, MUC5AC being a goblet cell marker; panel D shows the expression of the marker acTUB, which is a ciliated cell marker; e diagram shows the expression of marker KRT5, KRT5 being basal cell marker; f, the expression condition of a marker CAV1, wherein CAV1 is an alveolar I type cell marker; g is the expression of the marker SPB, which is alveolar type II cell marker; panel H shows the expression of the marker CD31, CD31 being a mature vascular endothelial cell marker; panel I shows the expression of the marker CD68, CD68 being a macrophage marker; biological repeat n=3, p < 0.05; * P < 0.01; * P < 0.001.

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 method for constructing human bionic lung organoids, which comprises the following steps:

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 bionic 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 induced differentiation of vascular endothelial cells and macrophages and EGM2 into the culture medium for continuous culture to obtain the bionic lung organoid.

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 kept at a concentration of 0.01-10 μm, preferably 0.25-5 μm, or more preferably 0.5-2 μm, and may be specifically 1 μm, to ensure that sufficient lung progenitor cell spheres (see fig. 9) 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, 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 also provides a bionic lung organoid constructed by the construction method, such as a bionic lung organoid morphological diagram shown in a diagram A in fig. 5. The bionic lung organoid has a bronchus-like structure (see the lower right corner of the F-diagram in FIG. 5, see that there is a hole-like (circle-like) structure in the structure, i.e. a bronchus-like structure) and an alveolus-like structure (see the I-diagram in FIG. 5, J-diagram in FIG. 5, a close-up of the alveolus-like structure), and also has endodermal-derived epithelial cell types: ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, and mesoderm-derived cell types: vascular endothelial cells, macrophages.

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 6 and 7, and the relevant antibodies used are shown in Table 8.

On the basis of lung epithelial organoid induced differentiation factors, human bionic 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 induced differentiation is schematically shown in FIG. 1.

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%, as shown in a diagram in fig. 2 a, the hpscs are sequentially added to medium a and medium B in the 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: inducing differentiation of Lung buds into bionic 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), a human bionic lung organoid with both proximal and distal structures was obtained.

Comparative example 1

For the S1 phase, several other classical definitive endoderm differentiation protocols were used under serum-free system, as shown in table 1.

TABLE 1 classical definitive endoderm differentiation protocols

/>

Comparative example 2

For stages S22-S23, ATRA was able to affect the number of Day7 suspended lung progenitor cell spheres produced, so several sets of protocols were designed for comparison.

Table 2 comparison scheme of stages S22-S23

Comparative example 3

For differentiation stage S24, VEGF, EGF, bFGF was related to the expression of endothelial cells and immune cell markers, so several sets of protocols were designed for comparison.

Table 3 comparison scheme at stage VEGF, EGF, bFGF s24

Comparative example 4

For the S2-S3 stage, EGM2 medium was subjected to immune refinementCell (CD 68) ⁺ Macrophages) plays an important role in inducing differentiation, such as using DMEM/F12 or DMEM or imdm+dmem or imdm+f12 as basal medium in the previously reported protocols, several sets of protocols were designed for comparison.

TABLE 4 basic Medium design

Comparative example 5

For the S3 phase, the addition of human Collagen I and Collagen III to the ECM plays an important role in maturation of various cells of the lung organoids, so the following sets of protocols were designed for comparison.

Table 5 design of ECM

Results and analysis:

1. at stage S1, after 2 days, the cell population exhibited typical characteristics of endoderm (panel B in FIG. 2); immunofluorescence results indicated that: about 90% of the cells expressed the definitive endoderm marker SOX17 (panel C in fig. 2). The differentiation at stage S1 was confirmed to be successful.

2. In the S2 phase, after 5 days of addition of medium C, D, E, day7, a number of suspended solid spheres were found on top of the underlying 2D cells (FIG. 3, panel A).

The immunofluorescence test result shows that the marker expresses the digestive tract anterior marker SOX2 and also expresses the lung progenitor cell marker NKX2.1, so that the marker is lung progenitor cell sphere (shown as a graph B-C in figure 3).

3. After stage S24, the solid spheres differentiate into a number of capsular structures at Day17 (as shown in figure 4, panel a). Immunofluorescence showed that it expressed the epithelial marker EpCAM and also expressed the markers of lung bud period such as NKX2.1, FOXA1, SOX9, etc. (see B-D in fig. 4).

4. In the S3 stage, lung buds are coated in a sandwich structure of ECM and continuously differentiated to Day70, so that mature human bionic lung organoids can be formed.

Morphologically present a tissue-like appearance of complex structure, up to 0.5cm in volume ³ (panel A in FIG. 5).

Immunofluorescence results after frozen sections showed that: organoids not only possess the epithelial cell type (EpCAM) ⁺ Or ECAD ⁺ Panel B and C of FIG. 5), also has endothelial and immune cell types, CD31 ⁺ Vascular endothelial cells, panel B, CD68 in fig. 5 ⁺ Macrophages, panel C in fig. 5.

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

The lung progenitor cells exhibit a bulk distribution at both the proximal and distal ends (SOX 9) ⁺ Fig. 5, K).

On the other hand, the expression level of each lineage marker was detected by Q-PCR, and as shown in fig. 6, the results confirmed the above immunofluorescence results, and also confirmed the overall maturation of the lung organoids.

5. Fig. 7 and 8 show comparison of the results of several protocols of comparative example 1, and it can be seen that the expression level of SOX2 is low (fig. 7) in the S2 stage-Day 7 derivative SOX2 under the serum-free system conditions, such as using several other classical definitive endoderm differentiation protocols, and that fig. 8 shows the production of lung progenitor cells from protocols b, c, and f with relatively high SOX2 expression levels, and that the production of lung progenitor cells is insufficient, although SOX2 expression levels are relatively high.

6. FIG. 9 is a graph comparing the effect of ATRA on Day7 suspended lung progenitor cell pellet production during stages S22-S23 of the study of comparative example 2. As can be seen from the figure, the continuous addition of ATRA at stages S22 and S23 ensures that sufficient lung progenitor cells spheres are produced in Day7 in suspension for collection and continued differentiation.

7. FIGS. 10 to 12 show the effect of VEGF, EGF, bFGF on the expression of markers NKX2.1, CD31 and CD11b in the S24 phase of the study of comparative example 3, and FIGS. 10 to 12 show the detection of the gene expression of lung epithelial cells, endothelial cells and immune cells at the end of induced differentiation by fluorescent quantitative PCR detection. Where NKX2.1 is a lung epithelial cell marker, CD31 is a mature vascular endothelial cell marker, CD11b is an expression level analysis of a monocyte marker, biological repeat n=3.

It can be seen from the figure that on the basis of lung-directed differentiation using lung epithelial cell-related differentiation inducing factors (i.e. CHIR99021, BMP4, FGF10, KGF, ATRA), removal of any one of VEGF, EGF, bFGF will reduce the expression of endothelial cells and immune cell markers.

8. FIG. 13 shows the medium vs. immune cells (CD 68) studied in comparative example 4 ⁺ Macrophages), i.e., the formation of immune cells at the end of induced differentiation by immunofluorescence (CD 68 is a macrophage marker).

As can be seen from Table 4 and FIG. 13, immune cell CD68 could not be achieved without addition of EGM2 medium to the basal medium, such as DMEM/F12 or DMEM or IMDM+DMEM or IMDM+F12 as in the previously reported protocol ⁺ Macrophage induced differentiation, indicating that EGM2 plays a key role.

9. FIG. 14 shows the detection of gene expression in lung organoids of each lineage by fluorescent quantitative PCR under different ECM components studied in comparative example 5. From the results, it was found that the addition of human Collagen I and Collagen III to ECM plays an important role in maturation of various cells of the lung organoids, and that the removal of either would reduce the gene expression levels of several lineage markers.

In summary, the invention establishes a method for constructing the human bionic lung organoid, and the method generates the human bionic lung organoid with a bronchus-like structure and an alveolus-like structure by virtue of an iPS directional induction differentiation technology on the premise of not adding exogenous cells and not involving genetic operation. Unlike previous studies, this type of organ is different from the existing lung organoids in that it combines mesoderm-derived physiologically relevant lineages, including endothelial cells and immune cells, such as vascular endothelial cells, macrophages, etc., with the exception of a variety of epithelial cell types derived from the endoderm, such as ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, etc. Therefore, the method is very suitable for being used as a new generation of humanized refined lung organoid model, and provides a more reliable research tool for lung disease molecular mechanism research, disease simulation, drug target spot discovery, drug effect screening and the like. In addition, the invention adopts a serum-free induced differentiation system, has definite and controllable components, is little influenced by batch-to-batch differences and has low risk of being polluted by pathogenic microorganisms.

TABLE 6 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) (Matrigel)	Biocoat (356231)
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 7 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

Table 8 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 (4)

1. A method for constructing human bionic lung organoids is characterized by comprising the following steps: the method comprises the following steps:

s1, inducing hPSC to differentiate into definitive endoderm:

the culture medium used in the S1 is 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, human fibroblast growth factor 2 FGF2 and vitamin C, and hPSC is sequentially cultured in the culture medium A and the culture medium B for 1-3 days; the GDF is an Nodal signal activator, the GDF is any one of GDF3, GDF5 and GDF8, the GDF usage amount is 20-500ng/ml, the GSK-3 inhibitor usage amount is 0.1-10 mu M, the FGF2 usage amount is 1-100ng/ml, and the vitamin C usage amount is 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 usage amount of the BMP inhibitor is 50-500ng/ml, and the usage amount of the TGF beta inhibitor is 1-50 mu M;

s2, dividing the process 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, and all-trans retinoic acid ATRA is continuously added in the pre-stage, and the use amount of the ATRA is 0.01-10 mu M; culturing in medium D for 1-3 days, wherein Wnt inhibitor, TGF beta inhibitor and all-trans retinoic acid ATRA are added into the medium D, the Wnt inhibitor dosage is 0.1-10 mu M, and the TGF beta inhibitor dosage is 1-50 mu M; culturing in a culture medium E for 2-5 days, wherein a lung epithelial cell related induced differentiation factor GSK-3 inhibitor, human bone morphogenetic protein 4 BMP4, human keratinocyte growth factor KGF, human fibroblast growth factor 10 FGF10 and all-trans retinoic acid ATRA are added into the culture medium E; wherein, the using amount of the GSK-3 inhibitor is 0.1-10 mu M, the using amount of BMP4 is 1-50ng/ml, the using amount of FGF10 is 1-50ng/ml, and the using amount of KGF is 1-50ng/ml; culturing in a culture medium F for 8-12 days, wherein the culture medium F increases the common factors of vascular endothelial cells and macrophages induced differentiation, namely human epithelial growth factor EGF, human vascular endothelial growth factor VEGF and basic fibroblast growth factor bFGF, on the basis of the culture medium E, and simultaneously reduces the dosage of all-trans retinoic acid, and EGM2 is added into the culture medium at the early stage and the later stage, wherein the dosage of EGF is 1-50ng/ml, the dosage of VEGF is 0.5-100ng/ml, the dosage of bFGF is 0.5-50ng/ml, and the volume percentage of the added EGM2 at 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; and then culturing by using a culture medium G, wherein a lung epithelial maturation promoting factor is added into the culture medium G on the basis of the culture medium F, the lung epithelial maturation promoting factor is dexamethasone, a PDE inhibitor and a PKA activator, the dosage of dexamethasone is 10-1000nM, the dosage of the PDE inhibitor is 0.01-1mM, the dosage of the PKA activator is 0.01-1mM, and finally the human bionic lung organ is obtained by culturing.

2. A human pluripotent stem cell-based biomimetic lung organoid constructed by the construction method of claim 1, wherein: the bionic lung organoid has a bronchus-like structure and an alveolus-like structure, and simultaneously has an epithelial cell type derived from endoderm: ciliated cells, neuroendocrine cells, basal cells, alveolar type I cells, alveolar type II cells, and mesoderm-derived cell types: vascular endothelial cells, macrophages.

3. A kit for use in the construction method of claim 1, characterized in that: comprising the following steps:

human myostatin GDF8, GSK-3 inhibitor CHIR99021, human fibroblast growth factor 2 and vitamin C for use in S1;

BMP inhibitors NOG, tgfβ inhibitor SB431542, all-trans retinoic acid, wnt inhibitor IWP2, common factors for vascular endothelial cells and macrophage induced differentiation, lung epithelial cell related induced differentiation factor and Endothelial Cell Growth Medium 2 for use in S2;

type I collagen, type III collagen, matrigel, common factors for vascular endothelial cells and macrophage induced differentiation, lung epithelial cell related induced differentiation factor, lung epithelial maturation promoting factor and Endothelial Cell Growth Medium for S3;

wherein, the common factors of vascular endothelial cells and macrophages induced differentiation are human epithelial growth factors, human vascular endothelial growth factors and basic fibroblast growth factors;

the lung epithelial cell related induced differentiation factors are GSK-3 inhibitor CHIR99021, human bone morphogenetic protein 4, human keratinocyte growth factor, human fibroblast growth factor 10 and all-trans retinoic acid;

the lung epithelial maturation-promoting factor is dexamethasone, PDE inhibitor IBMX, PKA activator cAMP.

4. Use of the construction method according to claim 1 for inducing differentiation of human pluripotent stem cells into three-dimensional bionic lung organoids.

Images