CN116376808A - In vitro induction of respiratory tract organoids and uses thereof - Google Patents

In vitro induction of respiratory tract organoids and uses thereof Download PDF

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CN116376808A
CN116376808A CN202310426450.6A CN202310426450A CN116376808A CN 116376808 A CN116376808 A CN 116376808A CN 202310426450 A CN202310426450 A CN 202310426450A CN 116376808 A CN116376808 A CN 116376808A
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cells
medium
signaling pathway
organoid
lung progenitor
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刘会生
孙佳琦
孙辉
徐涛
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Guangzhou National Laboratory
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Abstract

The invention relates to in vitro induction of respiratory organoids and to the use of the obtained respiratory organoids.

Description

In vitro induction of respiratory tract organoids and uses thereof
Technical Field
The invention belongs to the field of cell differentiation and regeneration medicine, and particularly relates to in vitro culture of organoids.
Background
In the field of respiratory diseases, there are a number of refractory diseases. Because there is no effective treatment method other than surgical lung transplantation, there is an increasing demand for lung regeneration medicine for research use of cell models, organ models, cell therapy, and the like. To date, although protocols for the production of lung organoids from human Pluripotent Stem Cells (PSC), tissue-resident Adult Stem Cells (ASC), embryonic lung, embryonic stem cells, and Induced Pluripotent Stem Cells (iPSC) have been established, there remains a need for improved in vitro culture systems for lung organoids. In particular, the current differentiation and quantitative efficiency of lung progenitor cells (NKX 2.1) is very low, and the application of lung organoids in the fields of cell therapy, drug screening and the like is greatly limited. Therefore, the establishment of a rapid large number of lung progenitor cells highly expressing NKX2.1 and a differentiation system based on the lung progenitor cells has very important scientific and clinical transformation significance.
Disclosure of Invention
The present invention provides methods of differentiating embryonic stem cells to form anterior foregut endoderm, dissociating the collected or isolated anterior foregut endoderm pellets and forming lung progenitor cells, which then differentiate to form mature airway lung organoids and alveolar organoids. The method can obtain the lung progenitor cells with higher yield and specificity and high expression of NKX2.1, and is simple and easy to implement.
In a first aspect, the present invention provides a method of inducing the formation of lung progenitor cells, the method comprising the steps of:
(1) Obtaining a population of anterior foregut endoderm cells, dissociating the population of anterior foregut endoderm cells into anterior foregut endoderm cells; and
(2) Anterior foregut endoderm cells are induced to form lung progenitor cells.
In some embodiments, the anterior foregut endoderm cells induce formation of lung progenitor cells at a purity of 95% or more.
Preferably, the anterior foregut endoderm cells induce formation of lung progenitor cells at a purity of 97% or more.
In some embodiments, the lung progenitor cells express the markers NKX2.1 and/or SOX9 and/or SOX2.
In some embodiments, the anterior foregut endoderm cell mass is induced by stem cells to form definitive endoderm cells and the definitive endoderm cells are induced.
In some embodiments, the stem cell is an embryonic stem cell or an adult stem cell.
In some embodiments, the embryonic stem cells are isolated embryonic stem cells, primary embryonic stem cells, or a population or cell line thereof.
In some embodiments, the stem cell is a pluripotent stem cell, e.g., an embryonic stem cell, a mesenchymal stem cell.
In some embodiments, the stem cell is an Induced Pluripotent Stem Cell (iPSC). The iPSC cells may be commercial cell lines or may be induced from donor cells including one or more of villus cells, skin (fibroblasts and keratinocytes), amniotic fluid, extra-embryonic tissue (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, mammary epithelial cells, adipose stem cells, umbilical cord matrix and placenta.
In some embodiments, the stem cells are human stem cells or non-human mammalian stem cells.
In some embodiments, the step of inducing embryonic stem cells to form definitive endoderm cells comprises culturing the embryonic stem cells in a medium supplemented with Activin a and gsk3β signaling pathway inhibitors.
In some embodiments, the medium supplemented with an Activin a and a gsk3β signaling pathway inhibitor is a medium supplemented with an Activin a and a gsk3β signaling pathway inhibitor in embryonic stem cell medium.
In some embodiments, the gsk3β signaling pathway inhibitor includes, but is not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-a014418, LY2090314, SB216763, AZD1080.
Preferably, the gsk3β signaling pathway inhibitor is CHIR99021, is a selective inhibitor of liver glycogen synthase kinase (gsk3β) receptor.
In some embodiments, the concentration of CHIR99021 in the medium is 0.1-3 μm.
In some embodiments, the stem cells are induced to form definitive endoderm cells within 3 days.
In some embodiments, embryonic stem cells are cultured on day 0 (D0) using a medium supplemented with Activin a and gsk3β signaling pathway inhibitors; continuing to culture on day 1 (D1) using a medium supplemented with an inhibitor of the Activin A and GSK3 beta signaling pathway; culture was continued on day 2 (D2) using the medium supplemented with Activin A. Preferably, the gsk3β signaling pathway inhibitor is CHIR99021.
In some embodiments, the concentration of Activin A in the medium is 80-120ng/mL. Preferably, the concentration of Activin A in the medium is 100ng/mL.
In some embodiments, on day 0 (D0), the medium has a CHIR99021 content of 2-4. Mu.M. Preferably, the medium has a CHIR99021 content of 3. Mu.M.
In some embodiments, MCDB131 medium is used as a basal medium for inducing stem cells to form definitive endoderm cells.
In some embodiments, the step of inducing definitive endoderm cells to form a population of anterior foregut endoderm cells comprises culturing embryonic stem cells in a medium supplemented with an inhibitor of tgfβ signaling pathway, an inhibitor of BMP signaling pathway, FGF4 (fibroblast growth factor 4), sonic Hedgehog (SHH) agonist, and an inhibitor of gsk3β signaling pathway.
In some embodiments, the medium supplemented with a tgfp signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4 (fibroblast growth factor 4), sonic Hedgehog (SHH) agonist, and GSK3 beta signaling pathway inhibitor is a medium supplemented with a tgfp signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4 (fibroblast growth factor 4), sonic Hedgehog (SHH) agonist, and GSK3 beta signaling pathway inhibitor in anterior foregut endoderm cell medium.
In some embodiments, the TGF-beta signaling pathway inhibitor is, for example, LY2109761, A83-01, SB-525334, SD-208, EW-7197, disitertide, LY3200882, SM16, SB431542.
Preferably, the tgfβ signalling pathway inhibitor is SB431542.
In some embodiments, the concentration of SB431542 is between 5 and 15. Mu.M. Preferably, the concentration of SB431542 is 10. Mu.M.
In some embodiments, the inhibitor of BMP signaling pathway is, for example, noggin, dorsomorphin, DMH1, LDN-193189.
In some embodiments, the BMP signaling pathway inhibitor is Noggin.
In some embodiments, the Noggin concentration is 150ng/mL to 250ng/mL. Preferably, the concentration of Noggin is 200ng/mL.
In some embodiments, the FGF4 concentration is 400-600ng/mL. Preferably, the concentration of FGF4 is 500ng/mL.
In some embodiments, the gsk3β signaling pathway inhibitor includes, but is not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-a014418, LY2090314, SB216763, AZD1080.
Preferably, the gsk3β signaling pathway inhibitor is CHIR99021.
In some embodiments, the concentration of CHIR99021 in the medium is 1-3 μm, preferably 2 μm.
In some embodiments, the SHH agonist is SHH or SAG. Preferably, the SHH agonist is SAG.
In some embodiments, the concentration of SAG in the medium is 0.5-1.5. Mu.M, preferably 1. Mu.M.
In some embodiments, definitive endoderm cells are induced to form anterior foregut endoderm cell mass within 5 days.
In some embodiments, DMEM/F-12 medium or DMEM/F-12 modified medium is used as a basal medium for the induction of definitive endoderm cells to form anterior foregut endoderm cell mass.
In some embodiments, the anterior foregut endoderm cell mass is an anterior foregut endoderm pellet.
In some embodiments, the 3D anterior foregut endoderm pellet appears on day 3 of the AFE stage. Free floating anterior foregut endoderm pellets will appear on days 4-7 of the AFE stage.
In some embodiments, the collected anterior foregut endoderm pellets are dissociated into anterior foregut endoderm single cells.
The dissociation may be accomplished by conventional methods including, but not limited to, enzymatic, chemical or mechanical methods. The enzymatic cleavage can be carried out, for example, by Ackutase, dispase or trypsin.
In some embodiments, anterior foregut endoderm single cells are cultured under extracellular matrix or hydrogel conditions.
In some embodiments, anterior foregut endoderm single cells are induced into lung progenitor cells in Matrigel using Lung Progenitor Cell (LPC) medium.
Cell culture is performed by using Matrigel matrix or protein hydrogel, and specific growth factors are added to enable proliferation and differentiation of cells in a suspended and stable environment.
In some embodiments, the lung progenitor cell culture medium comprises a Notch signaling pathway inhibitor, BMP4, fibroblast growth factor 7 (FGF 7), fibroblast growth factor 10 (FGF 10), a gsk3β signaling pathway inhibitor, and Retinoic Acid (RA).
In some embodiments, the Notch signaling pathway inhibitor is DAPT (N- (3, 5-difluorophenylacetyl) -L-alanyl) -S-phenylglycine t-butyl) or Dibenzoazepine (DBZ). Preferably, the Notch signaling pathway inhibitor is DAPT.
Preferably, the concentration of DAPT in the medium is 15-25. Mu.M. More preferably, the concentration of DAPT in the medium is 20. Mu.M.
BMP4 refers to bone morphogenic protein 4, which activates BMP4 receptor signaling.
In some embodiments, the concentration of BMP4 in the lung progenitor cell culture medium is 15-25ng/mL, preferably the concentration of BMP4 is 20ng/mL.
In some embodiments, the concentration of FGF7 and/or FGF10 in the lung progenitor cell culture medium is 5-15ng/mL, respectively, preferably the concentration of FGF7 and/or FGF10 is 10ng/mL, respectively.
In some embodiments, the gsk3β signaling pathway inhibitor includes, but is not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-a014418, LY2090314, SB216763, AZD1080.
Preferably, the gsk3β signaling pathway inhibitor is CHIR99021, is a selective inhibitor of liver glycogen synthase kinase (gsk3β) receptor.
In some embodiments, the concentration of CHIR99021 in the lung progenitor cell culture medium is 1-5 μm, preferably the concentration of CHIR99021 in the lung progenitor cell culture medium is 3 μm.
In some embodiments, the concentration of RA in the lung progenitor cell culture medium is 40-60nM, preferably the concentration of RA in the lung progenitor cell culture medium is 50nM.
The RA (vitamin a) is a metabolite of vitamin a that binds to and activates retinoic acid receptors, inducing changes in gene expression, leading to cell differentiation, decreased cell proliferation and inhibition of tumorigenesis.
In some embodiments, anterior foregut endoderm cells are induced to form lung progenitor cells within 7 days.
In some embodiments, DMEM/F-12 medium or DMEM/F-12 modified medium is used as the basal medium for lung progenitor cells.
In a second aspect, the invention provides lung progenitor cells obtained by the method of the first aspect.
In some embodiments, the purity of the lung progenitor cells is above 97%.
In some embodiments, the lung progenitor cells express the markers NKX2.1 and/or SOX9 and/or SOX2.
In some embodiments, the lung progenitor cells have an onion ring morphology.
In a third aspect the present invention provides a method for inducing the formation of a respiratory organoid from a lung progenitor cell obtained by the method of the first aspect, said method comprising the step of culturing said lung progenitor cell with a medium comprising Dexamethasone, 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX) and FGF 7.
In some embodiments, the medium further comprises a tgfβ signaling pathway inhibitor.
In some embodiments, the TGF-beta signaling pathway inhibitor is, for example, LY2109761, A83-01, SB-525334, SD-208, EW-7197, disitertide, LY3200882, SM16, SB431542.
Preferably, the tgfβ signalling pathway inhibitor is SB431542.
In some embodiments, the concentration of SB431542 is between 5 and 15. Mu.M. Preferably, the concentration of SB431542 is 10. Mu.M.
In some embodiments, the medium further comprises a gsk3β signaling pathway inhibitor.
In some embodiments, the gsk3β signaling pathway inhibitor includes, but is not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-a014418, LY2090314, SB216763, AZD1080.
Preferably, the gsk3β signaling pathway inhibitor is CHIR99021.
In some embodiments, the concentration of CHIR99021 in the medium is 2-4 μm, preferably 3 μm.
In some embodiments, the respiratory tract organoid is a lung airway organoid.
In some embodiments, suspension culture is employed to obtain airway organoids.
In some embodiments, the airway organoid is an everting airway organoid.
In some embodiments, the lung airway organoids express markers TP63, SCGB1A1, CHGA, FOXJ1, and MUC5AC.
In some embodiments, the respiratory organoid is an alveolar organoid.
In some embodiments, the alveolar organoids express markers HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC, and/or SLC34A2.
In some embodiments, lung progenitor cells are induced to form airway organoids within 30 days and may be maintained in culture for a prolonged period of time.
In some embodiments, DMEM/F-12 medium or DMEM/F-12 modified medium is used as the basal medium.
In a fourth aspect, the present invention provides a respiratory organoid obtainable by the method of the third aspect.
In some embodiments, the respiratory tract organoid is a lung airway organoid.
In some embodiments, the lung airway organoid expresses markers TP63, SCGB1A1, CHGA, FOXJ1, and/or MUC5AC.
In some embodiments, the respiratory organoid is an alveolar organoid.
In some embodiments, the alveolar organoids express markers HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC, and/or SLC34A2.
In some embodiments, the lung airway organoid is an everting airway organoid.
In a fifth aspect, the present invention provides a lung progenitor cell obtained by the method of the first aspect, a lung progenitor cell of the second aspect, a respiratory tract organoid obtained by the method of the third aspect or a respiratory tract organoid of the fourth aspect, for use in the preparation of a cell or organoid for treating a lung-related disorder, or for use in the preparation of a cell model or organoid model for studying a lung-related disorder.
In a sixth aspect, the invention provides a pharmaceutical formulation comprising a lung progenitor cell obtained by the method of the first aspect, a lung progenitor cell of the second aspect, a respiratory tract organoid obtained by the method of the third aspect or a respiratory tract organoid of the fourth aspect.
In a seventh aspect, the present invention provides a method of screening for a therapeutic or prophylactic agent, wherein the method comprises: a step of contacting the respiratory organoid obtained by the method of the third aspect or the respiratory organoid of the fourth aspect with a candidate molecule.
The invention establishes a rapid (< 10 days) high-purity (98.3%) NKX2.1 differentiation system of lung progenitor cells by dissociating anterior foregut endoderm pellets and maintaining cell activity and further combining with the actions of signal paths such as BMP, FGF, wnt and the like.
The obtained lung progenitor cells are further differentiated to obtain airway and alveolar organoids (including cilia swing, mucus secretion, ion channels, virus infection and the like) with mature physiological functions.
The high-throughput differentiation method of the invention can greatly improve the application of lung organoids in transformation medicine, such as disease model, drug development, cell therapy, etc.
Noun interpretation:
human life begins with the fusion of sperm and eggs to form fertilized eggs (day 0 of embryo phase; E0), which undergo cleavage to form blastocysts, which are planted around E7 to the mother uterus for further development. E14 begins, the embryo undergoes gastrulation, mass directional migration of cells in the rear of the embryo, and primitive streak cells are formed. Primitive streak cells further differentiate into mesoderm and definitive endoderm (definitive endoderm) while embryonic anterior cells differentiate into ectoderm. Based on this, the embryo develops into a tri-germ layer embryo with endoderm, mesoderm and ectoderm. Through the regulation of complex signal paths, the three-germ layer embryo further forms various organ primordia, and finally forms various organs of all systems including a nervous system, a digestive system, a respiratory system, a cardiovascular circulatory system, a genitourinary system and the like in our body.
The human respiratory tract is composed of a continuously branching airway and terminal alveoli. The airways are the passages of gas into and out of the lungs, and the alveoli are the functional units that carry out gas exchange, with the airways and alveoli being covered by the airways and alveolar epithelium, respectively.
In the present invention, "anterior foregut endoderm" refers to the endoderm that forms the forepart of the endoderm of the liver. "anterior foregut endoderm" includes, for example, pharyngeal endoderm and other more highly differentiated endoderm cell populations, and "anterior foregut endoderm" encompasses multiple cell types that exhibit different expression patterns of molecular markers. The "anterior foregut endoderm" will develop into a variety of tissues, such as tonsils, tympanic membrane, thyroid, parathyroid, thymus, trachea, esophagus, stomach, lung, larynx and/or pharynx.
"definitive endoderm cells (DE)" refers to cells that express one or more markers of the definitive endoderm lineage. Such markers include, but are not limited to CXCR4, SOX17, GATA-4, FOXA2, AFP, CER1, C-KIT, EPCAM, SNAI1, GSC, E-Cad and/or N-Cad. Definitive endoderm is functionally defined by cells that differentiate further toward one or more of the tissue derived from endoderm. This may include the lung, thyroid, liver, pancreas or intestine.
By "airway epithelial cells" is meant a layer of cell epithelial cells that make up lining (line) the large airways (bronchi) and the small airways (bronchioles). Airway epithelial cells include ciliated, secreted, basal and columnar cell types.
"Lung progenitor cells" have two important markers NKX2.1 and/or SOX9 and/or SOX2.
An "induced pluripotent stem cell (iPS cell)" is a pluripotent stem cell that is obtained by introducing a series of induction factors into mature somatic cells and reprogramming into a form having characteristics similar to embryonic stem cells. The induced pluripotent stem cells avoid ethical constraint of extracting stem cells by utilizing human embryo, avoid the risk of allograft immune rejection, and have great potential value in the fields of establishing various organ-like disease models, drug screening and development and the like related to brain, intestine, liver, pancreas, kidney, lung and the like. Induced pluripotent stem cells resemble natural pluripotent stem cells. Induced pluripotent stem cells are capable of differentiating into a variety of cell types including, but not limited to, definitive endoderm cells, foregut endoderm cells, and airway epithelial cells.
"organoids" refer to three-dimensional multicellular aggregates derived from stem cells, differentiated and self-organized, summarizing the structural features of mature tissues and cell-to-cell interactions. The organoids are three-dimensional aggregates of one or more cell types that mimic the surface appearance or true structure or function of a tissue or organ.
An "induction" or "induction" is related to a process or action that causes a specific effect on the phenotype of a cell. Such effects may be in a form that causes a phenotypic change, e.g., differentiation to another cellular phenotype; or may be in a form that maintains the cells in a particular cell, e.g., prevents dedifferentiation or promotes survival of the cells.
A "pluripotent stem cell" is a pluripotent cell that has the ability to self-renew, self-replicate, and differentiate into multiple cell types under certain conditions.
The terms "precursor cells", "progenitor cells" and "stem cells" are used interchangeably in the art and refer herein to multipotent or lineage-unfixed progenitor cells having the potential to mitotically divide unlimited numbers to renew themselves or differentiate into daughter cells of the desired cell type. Lineage-committed progenitor cells, in contrast to pluripotent stem cells, are generally thought to be incapable of generating numerous cell types that differ phenotypically from one another. Instead, the progenitor cells will be able to generate one or possibly both lineage committed cell types.
In the present invention, "differentiation" refers to the process of less specialized cells such as stem cells or inducing pluripotent stem cells into more specialized cell types so that they become a specific lineage, including but not limited to certain progenitor cells and more specialized somatic cells. The conditions under which stem cells differentiate are well known in the art.
In the present invention, "differentiation medium" refers to a cell growth medium that contains some additives or lacks some additives so that, when cultured in the medium, stem cells, induced pluripotent cells, or incompletely differentiated group cells develop into differentiated cells or develop into cells of some or all characteristics of more differentiated cells than stem cells, induced pluripotent cells, or other similar cells.
Such lung-related diseases include, but are not limited to, respiratory distress syndrome, acute respiratory distress syndrome, tuberculosis, cough, bronchial asthma, cystic fibrosis, airway hyperreactivity increasing cough (cough caused by bronchitis, influenza syndrome (fluxydrome), asthma, obstructive pulmonary disease, etc.), influenza syndrome, tuberculosis, asthma (airway inflammatory cell infiltration, increased airway hyperreactivity, bronchoconstriction, hypersecretion of mucus, etc.), chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, reversible airway obstruction, adult respiratory disease syndrome, bronchopulmonary dysplasia, airway disorders, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis, bronchiectasis, occupational asthma, reactive airway dysfunction syndrome, interstitial lung disease, parasitic lung disease, etc.
"dissociation" in this application refers to the dissociation of an aggregate or mass of cells into smaller aggregates or single cell suspensions. Dissociation of the cell aggregates may be accomplished by conventional methods including, but not limited to, enzymatic, chemical, or mechanical methods. The enzymatic cleavage can be carried out, for example, by Ackutase, dispase or trypsin.
The DMEM/F12 may also be replaced with a mixture of one or more of William's E cell culture medium, neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium, etc.
The DMEM/F12 medium contains a 1:1 ratio of DMEM to Ham's F-12 mixture.
The DMEM/F12 comprises a DMEM/F-12 modified culture medium prepared by adjusting components according to practical application.
The DMEM/F12 modified medium includes, but is not limited to, DMEM-low sugar-pyruvate-glutamine-free-phenol red-free, DMEM/F-12-GlutaMAX TM 、DMEM/F-12-HEPES(DMEM/F-12with HEPES)、DMEM-low glucose-pyruvate-HEPES。
The DMEM/F12 is a DMEM/F12+HEPES culture medium, and the DMEM/F12+HEPES culture medium contains L-glutamine, HEPES and phenol red.
The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that modifications and improvements made on the basis of the inventive concept will be within the scope of the present invention for those skilled in the art. The reagents used were conventional products commercially available without the manufacturer's knowledge.
Drawings
FIG. 1 shows a flow chart of the differentiation of the method of inducing the formation of lung progenitor cells of the present application.
FIG. 2 shows the morphology of human embryonic stem cells differentiated into definitive endoderm cells and associated marker expression. Wherein a shows the morphology of definitive endoderm day 1, b shows the morphology of definitive endoderm day 3, c shows the expression of markers SOX2, foxA2 and SOX 17.
Figure 3 shows the morphology of AFE stage 2D layer and free floating 3D anterior foregut endoderm pellet and associated marker expression. Wherein a shows the 2D cell layer and 3D pellet on day 4 of anterior foregut endoderm and b shows the expression of markers SOX2 and FoxA 2.
Figure 4 shows the morphology of lung progenitor cells and associated marker expression. Wherein a shows the "onion ring morphology" of the lung progenitor cells on day 7 and b shows the expression of the lung progenitor cell-related markers.
FIG. 5 shows that example 1 obtained high purity (98.3%) NKX2.1 lung progenitor cells.
FIG. 6 shows the results of lung group cells obtained after 7 days of culture in the detached group and the control group.
Figure 7 shows the morphology of lung organoids and associated marker expression. Wherein a shows the morphology of the airway organoid after 30 days of induction, b shows the morphology of the alveolar organoid after 30 days of induction, c shows the expression of the airway organoid related marker, d shows the expression of the alveolar organoid related marker; SC group is dissociation group, 3D group is control group.
FIG. 8 shows the expression of the staining level of airway organoids.
FIG. 9 shows the expression of staining levels of alveolar organoids.
Figure 10 shows the morphology of the everting airway organoid and the associated marker expression. Wherein a shows the morphology of the everting airway organoid and b shows the expression of the everting airway organoid related marker.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below. The techniques described in the examples below are, unless otherwise indicated, conventional techniques in the various fields of cell biology, biochemistry, molecular biology and the like, which are well known to those skilled in the art.
Reagents and instruments used in the following experiments were as follows:
instrument apparatus:
biosafety cabinet (thermo Fisher, 1389), inverted fluorescence microscope (Nikon, TS 2-FL), CO 2 Incubator (ThermoFisher, HERAcell150 i), centrifuge (Hunan instrument, L600-A), cryomicrotome (Ruiwo, FS 800), laser confocal microscope (Carl ZeissLSM 980), QPCR instrument (Bio-rad, CFX-96), PCR instrument (Bio-rad, T-100), flow cytometer (Agilent Novocyte Adcanteon), medical cryorefrigerator (sea DW-25L 262)
Reagent:
MCDB131(Thermo Fisher，10372019)、Advanced DMEM/F12(Thermo Fisher，12634010)、Accutase(Stemcell，7920)、EDTA(invitrogen，AM9261)、mTeSR1(Stemcell，85850)、Matrigel(Corning，354277)、DPBS(Thermo Fisher，14190250)、Glucose(Sigma，G7528)、NaHCO 3 (Sigma, S6014), BSA (Proliant, 68700), ascobic acid (Sigma, A4544), glutaMAX (invitrogen, 35050079), penicillin (Thermo Fisher, 15140122), ITS-X (invitrogen, 51500056), activin A (MCE, HY-P70311), CHIR99021 (MCE, HY-10182), FGF7 (stemcell, 78046.2), FGF4 (MCE, HY-P7014), FGF10 (MCE, HY-P70695), NOGGIN (MCE, HY-P7051A), BMP4 (MCE, HY-P7007), SB43152 (MCE, HY-10431), SAGE (MCE, HY-12848), Y27632 (MCE, HY-10583), growth Factor Reduced (HY-GFR) Matricagel (Corning 354230), RA retinoic acid (Sigma, R2625), T (MCE, HY-P525), demethyl-P5227), KA-P7006, TAXe (HY-1308, HY-13006), TAE-1308 (HY-13052A), KA-N (K-UM-Xe, HY-UM-Xe) and XE-HY-Xe (HY-10448)A, RR 820), maxima HMinus Reverse Transcriptase (Thermo Fisher, EP 0752), random (Thermo Fisher, SO 142), oligo (Thermo Fisher, SO 132), dNTP (Thermo Fisher, RO 192), riboLock RNase (Thermo Fisher, EO 0381), RNA extraction Kit RNeasy Mini Kit (AXGEN, 74106), RNase-FreeDNase Set1 (AXGEN, 79256), 96-well QPCR reaction plate (Bio-rad, HSP 9655), 4% paraformaldehyde fixative (BBI, E672002-0500), OCT frozen section embedding agent (Biosharp, BL 557A), triton X-100 (Diambld, A110694-0500), quicklock TM Immunostaining blocking solution (Biyun, P0260), immunostaining primary anti-dilution solution (Biyun, P0103), immunofluorescent secondary anti-dilution solution (Biyun, P0108), donkey serum (Abcam, ab 63507), anti-quenching capper (ACMEC, AS 2100), SOX2 (Santa Cruz Biotechnology, sc-365823), ki67 (Abcam, ab 92742), SOX9 (Abcam, ab 76997), NKX2.1 (Abcam, ab 76013), P63 (Abcam, ab 124762), MUC5AC (Abcam, ab 3649), acetylated Tubulin (ACTTUB) (Sigma-Aldrich, T7451), aquaporin 5 (AQP 5) (Abcam, ab 92320), sufactant Protein B (SFTPB) (Santa Cruz Biotechnology, sc-133143), E-Cadherin (ECAD) (R)&D Systems, AF 748), donkey anti-Rabbit Secondary Antibody Alexa Fluor 488 (Invitrogen, A21206), donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, alexa Fluor 488 (Invitrogen, A21202), donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546 (Invitrogen, A10040), donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary AntibodyAlexa Fluor 546 (Invitrogen, A10036), donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM (Invitrogen, A21447), hoechst 33342 (Thermo Fisher, 62249), alexa Fluor 647 phalin (Thermo Fisher 22287), APC anti-huCD 47 (Bio-69), legend 26, CD 9743.
The culture conditions for cell culture in the invention are 37 ℃ and 5 percent CO 2 And will not be described in detail hereinafter. The reagents and instruments used in the invention are all commercialized and can be purchased in the market.
The detection method comprises the following steps:
differentiated cell samples were harvested the last day of each differentiation stage in the differentiation process, QPCR detected the expression level of the characteristic gene mRNA, immunofluorescence technique detected the expression level of the characteristic gene protein, and flow cytometry detected the purity of the lung progenitor cells NKX 2.1.
The operations of RNA extraction, RNA transcription, QPCR experiments, immunofluorescent staining, and flow cytometry experiments in the present invention will be described below, respectively. The experimental methods involved in the present invention are the same as those described below, and the following will be repeated for the experiments, and the specific process will not be described in detail.
(I) RNA was harvested and QPCR performed
Rna extraction:
reagents used in the RNA extraction process are all included in commercial RNA extraction kits. The culture solution in the culture plate is discarded, DPBS is washed once, and the washing solution is discarded. 500 mu LTrypLE digest was added to each well of the 24-well plate and incubated in the incubator for about 7 minutes. About 90% of the cells were observed under the mirror to be clear and transparent, and 1mL of the medium was added at this stage to terminate the digestion. The cells were collected by pipetting uniformly, 1.5mL centrifuge tubes, and centrifuged at 1450rpm for 5 minutes. The supernatant was discarded, 1mL of DPBS was added, and gently mixed. Centrifugation was carried out at 1450rpm for 5 minutes, and the supernatant was discarded. 600 μl of lysate (Buffer RLT+β -ME) was added, mixed upside down for 4-6 times, and allowed to stand for 5 minutes.
To the above sample, an equal volume (600. Mu.l) of 70% absolute ethanol was added and thoroughly mixed.
The liquid from the previous step was transferred to a filter column of the RNA extraction kit and centrifuged at 12000rpm for 1 minute. Each transfer was 600. Mu.l, and the transfer was performed in two portions.
The filtrate from the previous centrifugation step was discarded, and 350. Mu.l of wash RW1 was added to the filter column and centrifuged at 12000rpm for 1 minute.
The filtrate from the previous step was discarded, 80. Mu.LDNA digest (10. Mu.LNaseI+70. Mu.Lbuffer in RNA extraction kit) was added to the filter column, and the mixture was allowed to stand at room temperature for 20 minutes to digest and extract DNA from the cells.
After completion of the standing, 350. Mu.L W1 wash was added, and the mixture was centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.
The filter column was loaded with 700. Mu.LRW 1 wash solution, centrifuged at 12000rpm for 1 min, and the filtrate was discarded.
500. Mu.LRW 2 wash was added to the filter column, centrifuged at 12000rpm for 1 min, and the filtrate was discarded.
500. Mu.LRW 2 wash was added to the filter column, centrifuged at 12000rpm for 1 min, and the filtrate was discarded.
The filter column was placed back into the collection tube provided by the kit and centrifuged at 12000rpm for 2 minutes. The filter column is placed into a new collection tube.
60 mu LRNase Free H was added to the collection tube 2 O, standing for 5 minutes.
The filter column was placed with a new collection tube and centrifuged at 12000rpm for 3 minutes. The liquid in the collection tube is the extracted RNA solution.
cDNA transcription
RNA harvested in the previous step was added to a 0.2mL centrifuge tube in the order given in Table 1 below.
TABLE 1
Figure BDA0004190801100000111
Mix gently, spin transiently and heat on a PCR instrument at 65℃for 5 minutes. After heating, the mixture was centrifuged instantaneously and left to stand on ice for 3 minutes.
The primers of Table 2 below were added sequentially to the 0.2mL centrifuge tube described above (this was done on ice).
TABLE 2
Figure BDA0004190801100000112
The mixed liquid was heated on a PCR apparatus for reaction at 25℃for 10 minutes, 50℃for hours, and 85℃for 5 minutes.
The obtained product is the extracted RNA reverse transcription CDNA product.
c.QPCR
The QPCR reaction solution was prepared on ice in the following order of table 3:
TABLE 3 Table 3
Figure BDA0004190801100000113
After the preparation of the reaction solution, QPCR reaction is completed on a QPCR instrument, the reaction program is that the pre-denaturation is carried out at 95 ℃ for 30s, the reaction program is carried out at 95 ℃ for 5s, and the reaction program is carried out at 60 ℃ for 30s, and 40 cycles are carried out.
The QPCR reaction system and the flow were carried out according to the (TAKARA, RR 820) reagent instructions.
The data processing mode of QPCR in the experiment is a square method and the differential analysis standard is: * Indicating significant differences, p <0.05; * Representing the difference was very significant, p <0.001; * P <0.0001, which indicates that the difference is very significant; * Represents that the difference is extremely significant, p <0.00001
(II) immunofluorescent staining experiments
Immunofluorescent staining is primarily aimed at the fluorescent display of characteristic proteins by specific binding of antigen-antibody.
The specific steps of fluorescent staining are as follows:
1. organoids were collected for either natural sedimentation or low speed centrifugation (1450 rpm,5 minutes), gently mixed with 2mL DPBS, rinsed once, allowed to stand for 5 minutes, and the supernatant discarded. 2mL of 4% PFA (paraformaldehyde fixing solution) was added thereto, and the mixture was allowed to stand at 4℃for 30 minutes for fixing. After the completion of the standing, 2mL of DPBS was added to the sample, and the mixture was left at 4℃for further use.
2. The DPBS in the sample was discarded, 2mL of immunostaining blocking solution was added, incubated at room temperature and allowed to stand for 30 minutes, and the supernatant was discarded by either natural sedimentation or low-speed centrifugation (1450 rpm,5 minutes). An antibody was incubated overnight at 4 ℃.
3. The primary antibody was removed by either natural sedimentation or low speed centrifugation (1450 rpm,5 minutes) and DPBS was washed 1-2 times for 5 minutes each. Adding a secondary antibody. The secondary antibody was incubated at room temperature for 1 hour in the dark. DPBS is washed 3 times for 5 minutes after incubation. The immunofluorescence staining experiment was completed.
(III) flow cytometry
The main purpose of the flow cytometry experiments was to verify the purity of the lung progenitor cells NKX2.1 in the LPC stage.
The flow cytometry experiments were performed as follows:
1. the LPCd7-d10 organoids were collected for natural sedimentation or low-speed centrifugation (1450 rpm,5 min), added with 2mL DPBS, blown and mixed well, rinsed once, allowed to stand for 5 min, and the supernatant discarded. Ackutase was added and digested for 10 minutes at 37 ℃.
2. The sample is digested into single cells, and if the single cells are not formed, the digestion time can be prolonged.
3. After the organoids were digested into single cells, digestion was stopped by adding 3-5mL DPBS and the digests removed by low-speed centrifugation (1450 rpm,5 min).
4. 2mL of DPBS was added to resuspend cells, and the cells were divided into a negative control group and a CD26+CD47 staining group, and antibodies were prepared according to the antibody instructions.
5. DPBS was removed by low-speed centrifugation (1450 rpm,5 min), DPBS resuspended cells were added to the control, formulated antibody was added to the staining group, and incubated at 4℃for 30 min.
6. Cells incubated with the antibody were washed with DPBS and centrifuged at 1450rpm for 5min, and the supernatant was discarded.
7. A small amount of DPBS was added to resuspend the cells (500. Mu.L-1 mL), the tube was passed through, and the flow experiment was completed.
Example 1: differentiation of human embryonic stem cells into lung progenitor cells and lung organoids
This example describes the method of differentiating human embryonic stem cells (hereinafter referred to as stem cells) into lung progenitor cells and lung organoids, and describes the basic flow of stem cell culture such as subculture of stem cells and plating operation before differentiation before stem cells enter differentiation, and then describes stem cell differentiation.
In this example, six-well plates and 24-well plates used for stem cell culture were used without any special explanation by incubating Matrigel (Matrigel) in advance. The specific operation is as follows: commercial Matrigel was mixed with DMEM F12 according to the dilution factor supplied by the manufacturer, and then the plates were incubated with 1mL per well of six well plates and 300 μl per well of 24 well plates for 2 hours at 37 ℃. And (3) passage: stem cells were cultured in six well plates with mTesR1 as medium, 2mL of medium per well, and medium was changed every 24 h. Stem cells were grown in six well plates to a cell density of 80-90%, the culture broth was discarded, DPBS was washed once, the washing solution was discarded, 1mL of 0.5mM EDTA digest was added to each well, and digestion was performed for 5-10 minutes at room temperature. The cells were observed under a microscope to be 80-90% bright, the digestate was discarded, the cells were gently purged with mTESR1, and the cells were uniformly inoculated in the next six well plate at a ratio of 1/10. Stem cell passaging was completed. After 4-5 days, the stem cell density reaches 80-90%, and the next passage or plating is carried out.
And (3) paving: stem cells were replaced every 24h in six well plates with mTesR1 as medium, 2mL per well. The stem cells are grown in six-hole plates until the density reaches about 80-90%, the culture solution is discarded, the DPBS is cleaned once, and the cleaning solution is discarded. 500. Mu.L of Ackutase was added to each well and incubated in an incubator at 37℃for 3-5 minutes. 80-90% of the cells were observed under the microscope to be bright, and digestion was stopped by adding 2mL of DPBS per well. The cells were collected by pipetting uniformly, a 15mL centrifuge tube, and centrifuged at 1450rpm for 5 minutes. The supernatant was discarded, 1mL of mTesR1 (10. Mu. M Y27632) was added to blow the cells evenly, and counted. Cells at 0.8X10 5 Each well of cells was inoculated uniformly into 24-well plates, and cells were grown to 90% or so to enter the differentiation stage 48-72 hours.
Referring to fig. 1, the human embryonic stem cells differentiate into a large number of lung progenitor cells and highly express NKX2.1, which is carried out by the steps of:
1) Obtaining anterior foregut endoderm pellets
1.1 Differentiation of human embryonic stem cells into definitive endoderm cells
The DE medium differentiated human embryonic stem cells into definitive endoderm cells.
The DE medium content is: MCDB131 culture medium based, 5mg/mL BSA, 10-20mM Glucose, 1.5mg/mL NaHCO 3 0.25mM Vitamin C, 1% Glutamax, 1% Streptomyces lividans, 100ng/mL Activin A, 0.1-3. Mu.M ChIR99021, insulin-transferrin-selenium-aminoethanol (ITS-X) (1:50000), DE medium was co-cultured for 3 days with medium changes every 24 hours. The morphology of human embryonic stem cells differentiated into definitive endoderm cells is shown in figures 2a-b and the associated marker expression is shown in figure 2 c.
1.2 Differentiation of definitive endoderm cells into anterior foregut endoderm pellets
The AFE medium content is:
based on Advanced DMEM/F12 medium, 10mM HEPES, N2 (1:100), B27 (1:50), 1% GlutaMAX, 1% penicillin, 10. Mu.M SB431542, 200ng/mL Noggin, 1. Mu.M SAG, 50-500ng/mL FGF4, 2. Mu.M Chur 99021, and spheres were completely exchanged every 24h and observed as follows. The morphology of the AFE stage 2D layer and free floating 3D anterior foregut endoderm pellet is shown in figure 3a and 3D anterior foregut endoderm pellet related marker expression is shown in figure 3 b.
a) The monolayers were observed under a microscope. The 3D anterior foregut endoderm pellet may be seen at the earliest days 2-3 of AFE stage. Free floating anterior foregut endoderm pellets will appear on days 4-7 of the AFE stage.
b) The culture supernatant and floating 3D foregut endoderm pellets were collected into a 15mL centrifuge tube using a 1 mL pipette.
Compared with the prior art that the 2D enzymolysis is directly carried out or the 3D pellets are directly inoculated after the induction of DE and AFE for 7 days, the suspension or floating anterior foregut endoderm pellets are collected first and then subjected to enzymolysis, so that the yield and purity of the lung progenitor cells obtained by final culture are higher.
2) Obtaining lung progenitor cells
2.1 Obtaining anterior foregut endoderm pellet single cells
Control group: direct inoculation of anterior foregut endoderm pellets without dissociation
Rinsing the pellets collected in step 1.2) with DPBS, naturally settling for 5-10min, and carefully sucking off the supernatant. Proper volume of GFR Matrigel was added using a tip pre-chilled (-20 ℃) in advance, gently swirled and mixed.
Dissociation group: dissociation of anterior foregut endoderm pellets
Rinsing the pellets collected in step 1.2) with DPBS, digesting for 5 minutes at 37 ℃ by Ackutase, gently beating to dissociate the pellets into single cells, terminating digestion with 3mL DPBS, and gently mixing. Centrifugation was performed at 1450rpm for 5 minutes, and the supernatant was carefully aspirated. Proper volume of GFR Matrigel was added using a tip pre-chilled (-20 ℃) in advance, gently swirled and mixed.
2.2 Inoculating anterior foregut endoderm pellet single cells
GFR Matrigel is gently dispensed using a pre-chilled (-20 ℃) tip in advance to the center of each well of a 24-well plate, preferably 25 μl to 30 μl drop volume per GFR Matrigel. The incubator was allowed to stand at 37℃for 10 minutes to solidify GFR Matrigel, and LPC medium containing 10. Mu.MY 27632 small molecules was added.
The LPC culture medium comprises the following components:
based on Advanced DMEM/F12 medium, 10mM HEPES, N2 (1:100), B27 (1:50), 1% GlutaMAX, 1% penicillin, 20. Mu.M DAPT, 20ng/mL BMP4, 10ng/mL FGF7, 10ng/mL FGF10, 3. Mu.M Chur 99021, 50nM RA, the medium was changed every 2-3 days for a total of 7 days. The morphology of lung progenitor cells after 7 days of dissociated group culture is shown in FIG. 4a, and the expression of relevant markers is shown in FIG. 4 b. The above method resulted in high purity (98.3%) of NKX2.1 lung progenitor cells (see fig. 5).
From the results of fig. 6, it can be found that, after 7 days of culture, the lung group cell structure with the form of "onion ring" obtained by dissociation group has almost 5 times of expansion, and thus the expansion ratio of lung progenitor cells after dissociation is obviously improved.
3) Obtaining lung organoids
3.1 Induction of lung progenitor cells into lung airway organoids using airway organoid (HAWO) medium
The HAWO medium content is:
the culture medium is replaced every 48-72 hours based on Advanced DMEM/F12 culture medium, 2.5mg/mL BSA, 10mM HEPES, B27 (1:100), 1% Glutamax, 1% penicillin, insulin-transferrin-selenium-amino alcohol (ITS-X) (1:1000), 50nM Dexamethasone, 100nM 8Br-cAMP, 100nM 3-isobutyl-1-methyl xanthone, 10ng/mL FGF7, and the culture can be maintained for a long period of time every 48-72 hours. The morphology of airway organoids 30 days after induction is shown in FIG. 7a, the expression of QPCR detection markers is shown in FIG. 7c, and the identification of staining levels is shown in FIG. 8.
3.2 Induction of lung progenitor cells into alveolar organoids using alveolar organoid (HALO) medium
The HALO medium content is:
the culture medium was replaced with 2.5mg/mL BSA, 10mM HEPES, B27 (1:100), 1% Glutamax, 1% penicillin, insulin-transferrin-selenium-aminoethanol (ITS-X) (1:1000), 50nM Dexamethasone, 100nM 8Br-cAMP, 100nM 3-isobutyl-1-methyl xanthone, 10ng/mL FGF7, 3. Mu.M ChIR99021, 10. Mu.M SB431542 based on Advanced DMEM/F12 medium, every 48-72 hours for 30 days, and the culture was maintained for a long period. The morphology of alveolar organoids after 30 days of induction and the expression of QPCR detection markers as shown in fig. 7b and the identification of staining levels as shown in fig. 9 d.
It can also be seen from the results of FIG. 7 that the dissociative group has an advantage at the level of the relevant genes in the late organoid induced differentiation maturation stage.
Example 2: everting airway organoids
Normal airway organoids are cultured in the following manner, using HAWO medium to induce lung progenitor cells into lung airway organoids.
The HAWO medium content is:
the culture medium is replaced every 48-72 hours based on Advanced DMEM/F12 culture medium, 2.5mg/mL BSA, 10mM HEPES, B27 (1:100), 1% Glutamax, 1% penicillin, insulin-transferrin-selenium-amino alcohol (ITS-X) (1:1000), 50nM Dexamethasone, 100nM 8Br-cAMP, 100nM 3-isobutyl-1-methyl xanthone, 10ng/mL FGF7, and the culture can be maintained for a long period of time every 48-72 hours.
The everting airway organoid culture mode comprises the following steps: the culture medium was changed every 48-72 hours, incubated for two weeks, organoids were peeled from Matrigel droplets, DPBS rinsed once, and suspended in a low-adhesion six-well plate. The HAWO culture medium is continuously used for maintenance culture, and the culture solution is replaced every 48-72 hours, so that the culture can be maintained for a long time. The only difference is whether the organoids are cultured in Matrigel droplets or in suspension, otherwise as in example 1. The morphology and marker expression of the everting airway organoids are shown in figure 10.
In this embodiment, the airway organoid may be everted spontaneously after suspension culture to form an everting airway organoid. The airway organoid with airway epithelial eversion can be obtained by adopting the method, can be used for constructing an in vitro three-dimensional model of the lung organoid, is used for pathogen infection modeling, high-flux drug screening and pulmonary diseases, such as chronic obstructive pulmonary disease, interstitial pulmonary disease, inflammatory pulmonary disease, asthma, emphysema and other researches, or pulmonary function researches, such as pulmonary alveoli, airways, pulmonary lobe function researches and the like, and has important significance.

Claims (21)

1. A method of inducing the formation of lung progenitor cells, the method comprising the steps of:
(1) Obtaining a population of anterior foregut endoderm cells, dissociating the population of anterior foregut endoderm cells into anterior foregut endoderm cells; and
(2) Anterior foregut endoderm cells are induced to form lung progenitor cells.
2. The method according to claim 1, wherein the anterior foregut endoderm cells induce formation of lung progenitor cells at a purity of 95% or more, preferably 97% or more.
3. The method of claim 1, wherein the lung progenitor cells express the markers NKX2.1 and/or SOX9 and/or SOX2.
4. The method of claim 1, wherein the anterior foregut endoderm cell mass is induced from stem cells to form definitive endoderm cells and the definitive endoderm cells are induced,
Preferably, the stem cells are embryonic stem cells or adult stem cells,
preferably, the embryonic stem cells are isolated embryonic stem cells, primary embryonic stem cells, or a population or cell line thereof,
preferably, the stem cells are pluripotent stem cells,
preferably, the stem cells are Induced Pluripotent Stem Cells (iPSCs),
preferably, the stem cells are human or non-human mammalian stem cells.
5. The method of claim 4, wherein the inducing stem cells to form definitive endoderm cells comprises culturing embryonic stem cells with a medium supplemented with Activin A and GSK3 beta signaling pathway inhibitors,
preferably, the culture medium added with the actin A and the GSK3 beta signal pathway inhibitor is a culture medium added with the actin A and the GSK3 beta signal pathway inhibitor in an embryonic stem cell culture medium;
preferably, the gsk3β signaling pathway inhibitor includes, but is not limited to: CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-a014418, LY2090314, SB216763, AZD1080,
more preferably, the GSK3 beta signaling pathway inhibitor is CHIR99021,
more preferably, the concentration of CHIR99021 in the medium is 0.1-3 μm;
More preferably, the concentration of Activin A in the medium is 80-120ng/mL;
more preferably MCDB131 medium is used as the basal medium for inducing stem cells to form definitive endoderm cells.
6. The method of claim 5, wherein the stem cells are induced to form definitive endoderm cells within 3 days,
preferably, the step of inducing stem cells to form definitive endoderm cells comprises: culturing embryonic stem cells on day 0 (D0) using a medium supplemented with Activin a and gsk3β signaling pathway inhibitor; continuing to culture on day 1 (D1) using a medium supplemented with an inhibitor of the Activin A and GSK3 beta signaling pathway; culture was continued on day 2 (D2) using the medium supplemented with Activin A.
7. The method of claim 4, wherein the inducing definitive endoderm cells to form anterior foregut endoderm cell mass comprises culturing embryonic stem cells in a medium supplemented with an inhibitor of tgfβ signaling pathway, an inhibitor of BMP signaling pathway, FGF4, an SHH agonist, and an inhibitor of gsk3β signaling pathway;
preferably, the medium supplemented with tgfp signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist, and gsk3β signaling pathway inhibitor is a medium supplemented with tgfp signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist, and gsk3β signaling pathway inhibitor in anterior foregut endoderm cell culture medium;
Preferably, the TGF-beta signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, disitertide, LY3200882, SM16 or SB431542,
more preferably, the TGF-beta signaling pathway inhibitor is SB431542,
more preferably, the concentration of SB431542 is from 5 to 15. Mu.M;
preferably, the inhibitor of BMP signalling pathway is selected from Noggin, dorsomorphin, DMH or LDN-193189,
more preferably, the inhibitor of BMP signaling pathway is Noggin,
more preferably, the concentration of Noggin is 150ng/mL-250ng/mL;
preferably, the concentration of FGF4 is 400-600ng/mL;
preferably, the GSK3 beta signaling pathway inhibitor is selected from the group consisting of CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-A014418, LY2090314, SB216763, AZD1080,
more preferably, the GSK3 beta signaling pathway inhibitor is CHIR99021,
more preferably, the concentration of CHIR99021 in the medium is 1-3 μm;
preferably, the SHH agonist is selected from SHH or SAG,
more preferably, the SHH agonist is SAG,
more preferably, the concentration of SAG in the medium is 0.5-1.5. Mu.M;
preferably, DMEM/F-12 medium or DMEM/F-12 modified medium is used as a basal medium for the induction of the formation of anterior foregut endoderm cell mass by definitive endoderm cells.
8. The method of claim 7, wherein definitive endoderm cells are induced to form a anterior foregut endoderm cell mass within 5 days;
preferably, 3D anterior foregut endoderm pellets appear on day 3 of the AFE stage, and free floating anterior foregut endoderm pellets will appear on days 4-7 of the AFE stage.
9. The method of claim 1, wherein the anterior foregut endoderm cell mass is an anterior foregut endoderm pellet,
preferably, the collected anterior foregut endoderm pellets are dissociated into anterior foregut endoderm single cells.
10. The method of claim 1, wherein the inducing the anterior foregut endoderm cells to form lung progenitor cells comprises culturing the anterior foregut endoderm single cells under extracellular matrix or hydrogel conditions,
preferably, anterior foregut endoderm single cells are induced in Matrigel using Lung Progenitor (LPC) medium to lung progenitor cells;
preferably, the Matrigel matrix or protein hydrogel is used for cell culture, and growth factors are added to enable the cells to proliferate and differentiate in a suspended and stable environment,
preferably, the lung progenitor cell culture medium comprises Notch signaling pathway inhibitors, BMP4, fibroblast growth factor 7 (FGF 7), fibroblast growth factor 10 (FGF 10), GSK3 beta signaling pathway inhibitors, and Retinoic Acid (RA),
More preferably, the Notch signaling pathway inhibitor is selected from DAPT (N- (3, 5-difluorophenylacetyl) -L-alanyl) -S-phenylglycine t-butyl) or Dibenzoazepine (DBZ), preferably selected from DAPT,
more preferably, the concentration of Notch signaling pathway inhibitor in the lung progenitor cell culture medium is 15-25 μm;
more preferably, the concentration of BMP4 in the lung progenitor cell culture medium is 15-25ng/mL;
more preferably, the concentration of FGF7 and/or FGF10 in the lung progenitor cell culture medium is 5-15ng/mL, respectively,
more preferably, the GSK3 beta signaling pathway inhibitor is selected from the group consisting of CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-A014418, LY2090314, SB216763 or AZD1080, preferably selected from the group consisting of CHIR99021,
more preferably, the concentration of GSK3 beta signaling pathway inhibition in the lung progenitor cell culture medium is 1-5 mu M,
more preferably, the concentration of RA in the lung progenitor cell culture medium is 40-60nM,
more preferably, DMEM/F-12 medium or DMEM/F-12 modified medium is used as the basic medium for lung progenitor cells,
more preferably, anterior foregut endoderm cells are induced to form lung progenitor cells within 7 days.
11. A lung progenitor cell obtained by the method of any one of claims 1-10.
12. The lung progenitor cell according to claim 11, wherein the purity of the lung progenitor cell is above 97%,
preferably, the lung progenitor cells express markers NKX2.1 and/or SOX9 and/or SOX2;
preferably, the lung progenitor cells have an onion ring morphology.
13. A method of inducing the formation of a respiratory organoid from a lung progenitor cell obtained by the method of any one of claims 1-10, said method comprising the step of culturing said lung progenitor cell with a medium comprising Dexamethasone, 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX) and FGF 7.
14. The method of claim 13, wherein the medium further comprises a TGF-beta signaling pathway inhibitor and/or a GSK3 beta signaling pathway inhibitor,
preferably, the TGF-beta signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, disitertide, LY3200882, SM16 or SB431542,
preferably, the TGF-beta signaling pathway inhibitor is selected from SB431542,
more preferably, the concentration of SB431542 is from 5 to 15. Mu.M;
preferably, the GSK3 beta signaling pathway inhibitor is selected from the group consisting of CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, tideglusib, AR-A014418, LY2090314, SB216763 or AZD1080,
Preferably, the gsk3β signaling pathway inhibitor is CHIR99021;
more preferably, the concentration of CHIR99021 in the medium is 2-4 μm;
preferably, DMEM/F-12 medium or DMEM/F-12 modified medium is used as a basal medium;
preferably, the lung progenitor cells are induced to form the airway organoids within 30 days.
15. The method of claim 13, wherein the airway organoid is a lung airway organoid and/or an alveolar organoid,
preferably, the lung airway organoid expresses markers TP63, SCGB1A1, CHGA, FOXJ1 and/or MUC5AC;
preferably, the alveolar organoid expresses markers HOPX, AGER, CAV, ABCA3, LAMP3, SFTPC and/or SLC34A2.
16. The method of claim 15, wherein the airway organoid is obtained by suspension culture,
preferably, the airway organoid is an everting airway organoid.
17. A respiratory organoid obtainable by the method of any of claims 13-16.
18. The respiratory organoid according to claim 17, wherein said respiratory organoid is a pulmonary airway organoid and/or an alveolar organoid,
preferably, the lung airway organoid expresses markers TP63, SCGB1A1, CHGA, FOXJ1 and/or MUC5AC;
Preferably, the alveolar organoid expression markers HOPX, AGER, CAV, ABCA, LAMP3, SFTPC and/or SLC34A2;
preferably, the lung airway organoid is an everting airway organoid.
19. Use of a lung progenitor cell obtained by the method according to any of claims 1-10, a lung progenitor cell according to claim 11 or 12, a respiratory tract organoid obtained by the method according to any of claims 13-16, or a respiratory tract organoid according to claim 17 or 18, for the preparation of a cell or organoid for the treatment of a lung-related disease, or for the preparation of a cell model or organoid model for the study of a lung-related disease.
20. A pharmaceutical formulation, wherein the pharmaceutical formulation comprises a lung progenitor cell obtained by the method of any one of claims 1-10, a lung progenitor cell of claim 11 or 12, a respiratory tract organoid obtained by the method of any one of claims 13-16, or a respiratory tract organoid of claim 17 or 18.
21. A method of screening for a therapeutic or prophylactic agent, wherein the method comprises: a step of contacting a respiratory organoid obtained by the method of any of claims 13-16 or a respiratory organoid of claim 17 or 18 with a candidate molecule.
CN202310426450.6A 2023-04-19 2023-04-19 In vitro induction of respiratory tract organoids and uses thereof Pending CN116376808A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126798A (en) * 2023-10-20 2023-11-28 北京大学第三医院（北京大学第三临床医学院） Culture medium and culture method for multipotent stem cell derived lung organoids

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126798A (en) * 2023-10-20 2023-11-28 北京大学第三医院（北京大学第三临床医学院） Culture medium and culture method for multipotent stem cell derived lung organoids
CN117126798B (en) * 2023-10-20 2024-05-03 北京大学第三医院（北京大学第三临床医学院） Culture medium and culture method for multipotent stem cell derived lung organoids

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