CN117126798B - Culture medium and culture method for multipotent stem cell derived lung organoids - Google Patents
Culture medium and culture method for multipotent stem cell derived lung organoids Download PDFInfo
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- CN117126798B CN117126798B CN202311365073.6A CN202311365073A CN117126798B CN 117126798 B CN117126798 B CN 117126798B CN 202311365073 A CN202311365073 A CN 202311365073A CN 117126798 B CN117126798 B CN 117126798B
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Abstract
The invention provides a culture medium and a culture method for a pluripotent stem cell derived lung organoid, wherein the culture medium comprises the following components: (1) Inducing the differentiation of the human pluripotent stem cells into a definitive endoderm cell culture medium; (2) Inducing the definitive endoderm cells to differentiate into a pre-intestinal germ cell culture medium; (3) inducing differentiation of the foregut embryo into a lung progenitor cell culture medium; (4) expanding the lung progenitor cell culture medium; (5) Inducing differentiation of lung progenitor cells into alveolar organoid medium; (6) Inducing differentiation of lung progenitor cells into airway organoid medium. The chemical composition of the culture medium is determined, so that higher differentiation efficiency can be achieved, the overall operation difficulty is low, and the culture medium is easy to repeat. Compared with other schemes, the adopted cytokine is simpler, the cost is low, and the lung organoid can be updated and maintained for a long time, so that the repeated induced differentiation flow can be reduced, and the method is favorable for large-scale amplification for subsequent disease model construction, drug screening and cell transplantation treatment.
Description
Technical Field
The invention relates to the field of cell engineering, in particular to a culture medium for a pluripotent stem cell derived lung organoid and a culture method.
Background
Organoids are three-dimensional in vitro culture systems derived from stem cells. They may reflect the in vivo structural, functional and genetic characteristics of the original tissue. Thus, organoid technology has been rapidly applied to stem cell biology, organogenesis and various pathological mechanism studies. Patient stem cell derived organoids enable more accurate construction of disease models, with great potential in biomedical applications, transformation medicine and personalized therapies. The present project relates to the development of a set of media and culture methods for inducing differentiation of pluripotent stem cells into lung organoids, which are capable of inducing differentiation of pluripotent stem cells into lung organoids having a three-dimensional structure in vitro, including alveolar organoids composed of alveolar type I and type II cells and airway organoids composed of ciliated cells, goblet cells, secretory cells, basal cells. The induced differentiated lung organoids can be used in the fields of lung disease simulation (cilia immobilized syndrome, pulmonary fibrosis and pulmonary infection), environmental exposure research (cigarettes, lipopolysaccharide and PM 2.5), targeted drug screening, lung injury repair and the like, and have important basic research and clinical application values.
In 2012 Rossant et al, a method for deriving lung organoids from iPS cells was first described, which was used as a model for pulmonary cystic fibrosis by inducing pluripotent stem cells to differentiate lung organoids in CFTR mutant patients. Snoeck et al devised an improved four-stage 50 day differentiation protocol. First, definitive endoderm was induced using Activin a. Subsequently, foregut endoderm was induced by sequentially inhibiting BMP, TGF- β and Wnt signaling. The procyanidin cells were then ventrally flanked by Wnt, BMP, FGF and retinoic acid to obtain lung and airway progenitor cells. Finally, lung epithelial cells (basal cells, goblet cells, clara cells, ciliated cells, type I and type II alveolar epithelial cells) were matured using Wnt, FGF, c-AMP and glucocorticoid. Spence et al also began with Activin a inducing human pluripotent stem cells, followed by the addition of tgfβ/BMP inhibitors, FGF4, and Wnt activators, indicating that the cells were developing toward the intestinal embryo. When the Hedgehog pathway is simultaneously activated, organoids move from the ventral aspect of the foregut embryo to the differentiation fate of the lung epithelium. Finally, mature lung organoids are formed by embedding in matrigel and prolonged exposure to FGF 10. The main routes for these induced differentiation protocols are: the multipotent stem cells are differentiated into lung progenitor cells from definitive endoderm and foregut embryo, and finally mature lung organoids are formed through three-dimensional culture.
The prior art induced differentiation scheme uses a plurality of cytokines, the differentiation efficiency is unstable, the cost is high and the differentiation steps are complicated. For example, in the first stage, differentiation of pluripotent stem cells into definitive endoderm is induced by cytokines such as WNT3a, BMP4, activin a, FGF-2, etc. or commercial culture medium, and it is difficult to achieve a stable and efficient differentiation effect for different cell lines with different cytokines and differentiation times. In order to successfully achieve differentiation efficiency of 80% and above so as to perform the next differentiation, individual regulation and control of differentiation time and cytokine ratio according to different cell lines are required, and the use is complicated. Similar problems exist in the differentiation at the later stage, so that the efficiency of finally differentiating the lung progenitor cells is unstable, and large differences exist among different cell strains, so that popularization and application are difficult. The use of numerous cytokines results in high costs due to the whole differentiation protocol being up to 35-50 days. Therefore, it is an urgent technical problem in the art to find an economical and practical and efficient solution for inducing differentiation of lung organoids.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium for inducing pluripotent stem cells to differentiate into lung organoids and a specific culture method matched with the culture medium. The culture medium and the method can improve differentiation efficiency, reduce cost and shorten culture time.
The invention adopts the following technical scheme to realize the aim of the invention:
In a first aspect, the invention provides a medium for inducing differentiation of pluripotent stem cells into a lung organoid, the medium comprising the following 6 media:
(1) Inducing the differentiation of the human pluripotent stem cells into a definitive endoderm cell culture medium;
(2) Inducing the definitive endoderm cells to differentiate into a pre-intestinal germ cell culture medium;
(3) Inducing differentiation of the foregut embryo into a lung progenitor cell culture medium;
(4) Expanding a lung progenitor cell culture medium;
(5) Inducing differentiation of lung progenitor cells into alveolar organoid medium;
(6) Or to induce differentiation of lung progenitor cells into airway organoid medium.
As a preferred embodiment of the present invention, the (1) inducing differentiation of human pluripotent stem cells into definitive endoderm cell culture media comprises cell basic medium (i) and a small molecule composition consisting of Activin A and CHIR 99021.
Optionally, the final concentration of the Activin A is 50-200 ng/ml, and the final concentration of the CHIR99021 is 1-10 uM.
Alternatively, the final concentration of Activin A is 100ng/ml and the final concentration of CHIR99021 is 3uM.
As a preferred embodiment of the present invention, the (2) inducing differentiation of definitive endoderm cells into a pre-intestinal embryonic cell culture medium comprises a cell basal medium (ii) and a small molecule composition consisting of Dorsommorphin and SB 431542.
Optionally, the Dorsomophin has a final concentration of 1-5 uM and the SB431542 has a final concentration of 5-30 uM.
Alternatively, dorsomophin is at a final concentration of 2uM and sb431542 is at a final concentration of 10uM.
As a preferred embodiment of the present invention, the (3) inducing differentiation of the foregut embryo into a lung progenitor cell culture medium comprises a cell basal medium (ii) and a small molecule composition consisting of CHIR99021, BMP4 and Retinoic acid (retinoic acid).
Optionally, the final concentration of CHIR99021 is 1-10 uM, the final concentration of BMP4 is 10-100 ng/ml, and the final concentration of Retinoic acid is 10-500 nM.
Alternatively, the final concentration of CHIR99021 is 3uM, the final concentration of BMP4 is 10ng/ml, and the final concentration of Retinoic acid is 50nM.
As a preferred embodiment of the present invention, the (4) expanded lung progenitor cell culture medium comprises cell basal medium (ii) and a small molecule composition consisting of CHIR99021 and KGF.
Optionally, the final concentration of CHIR99021 is 1-10 uM, and the final concentration of KGF is 2-100 ng/ml.
Alternatively, the final concentration of CHIR99021 is 3uM and the final concentration of KGF is 10ng/ml.
As a preferred embodiment of the invention, the (5) inducing differentiation of lung progenitor cells into alveolar organoids comprises cell basal medium (ii) and a small molecule composition consisting of CHIR99021, KGF, dexamethasone (dexamethasone), IBMX and cAMP.
Optionally, the final concentration of CHIR99021 is 1-10 uM, the final concentration of KGF is 2-100 ng/ml, dexamethasone is 10-500 nM, the final concentration of IBMX is 50-200 uM, and the final concentration of cAMP is 50-200 uM.
Alternatively, the final concentration of CHIR99021 is 3uM, the final concentration of KGF is 10ng/ml, dexamethasone is 50nM, the final concentration of IBMX is 100uM, and the final concentration of cAMP is 100uM.
As a preferred embodiment of the present invention, the (6) inducing differentiation of lung progenitor cells into airway organoids comprises cell basal medium (ii) and a small molecule composition consisting of FGF2, FGF10, dexamethasone, IBMX, cAMP and DAPT.
Optionally, the final concentration of FGF2 is 10-300 ng/ml, the final concentration of FGF10 is 10-200 ng/ml, dexamethasone is 10-100 nM, the final concentration of IBMX is 50-300 uM, the final concentration of cAMP is 50-300 uM, and the final concentration of DAPT is 0.5-20 uM.
Alternatively, the final concentration of FGF2 is 250ng/ml, FGF10 is 100ng/ml, dexamethasone is 50nM, IBMX is 100uM, cAMP is 100uM, and DAPT is 2uM.
As a preferred embodiment of the invention, 1000ml of cell basal medium (i) comprises the following final concentration of component ：MCDB131 11.6g/L,D-Glucose 0.1~5g/L,NaHCO3 1~5g/L、BSA 1~10g/L、vitamin C 50~100ug/ml、GlutaMAX 0.5~2X、Pen-Strep 0.1~1X;, where X represents the multiple of the final dilution.
Alternatively, the 1000ml of cell basal medium (i) comprises components ：MCDB131 11.6g/L,D-Glucose 0.44g/L,NaHCO3 2.68g/L、BSA 5g/L、vitamin C 80ug/ml、GlutaMAX 1X、Pen-Strep 0.2X; in a final concentration of 1X representing a final dilution to 1-fold and 0.2X representing a final dilution to 0.2-fold.
As a preferred embodiment of the invention, 500ml of cell basal medium (ii) comprises the following final concentration of component ：BSA 0.01~0.5%（w/v）,vitamin C 20~100ug/ml,GlutaMAX 0.5~2X,Pen-Strep 0.1~1X,N2 0.5~2X,B27 0.5~2X,MTG 0~5ul/ml,DMEM/F12 1X;, where X represents the multiple of the final dilution.
Alternatively, the 500ml of cell basal medium (ii) comprises a final concentration of component ：BSA 0.05%（w/v）,vitamin C 50ug/ml,GlutaMAX 1X,Pen-Strep 0.2X,N2 1X,B27 1X,MTG 0.04ul/ml,DMEM/F12 1X;, said 1X representing a final dilution of 1-fold and said 0.2X representing a final dilution of 0.2-fold.
In a second aspect, the invention also provides a kit for preparing a medium for the directed induction of differentiation of human pluripotent stem cells into alveolar or/and airway organoids, the kit comprising any of the above.
In a third aspect, the present invention also provides a method of culturing for inducing differentiation of pluripotent stem cells into a lung organoid, the method comprising the steps of:
S1, inducing the differentiation of the human pluripotent stem cells into definitive endoderm cells by adopting the cell culture medium;
s2, inducing the definitive endoderm cells to differentiate into the foregut embryo cells by adopting the cell culture medium;
s3, inducing the foregut embryo to differentiate into lung progenitor cells by adopting the cell culture medium;
s4, sorting, purifying and amplifying the lung progenitor cells by adopting the cell culture medium;
s5, inducing the lung progenitor cells to differentiate into alveolar organoids by adopting the cell culture medium;
or inducing differentiation of the lung progenitor cells into airway organoids using a cell culture medium as described above.
As a preferred embodiment of the present invention, the lung organoids include alveolar organoids and airway organoids.
As a preferred embodiment of the present invention, the alveoli comprise type I alveoli cells, type II alveoli cells; the airway organoids include basal cells, ciliated cells, club cells, goblet cells.
Compared with the prior art, the invention has the beneficial effects that:
The invention adopts the culture medium with definite chemical components in the whole course, the differentiation system is stable, and higher differentiation efficiency can be achieved when different cell lines are used for inducing the differentiation of the lung organoids, the overall operation difficulty is low, and the method is easy to repeat. Compared with other schemes, the cell factor adopted by the culture scheme is simpler, the whole-process differentiation time is only 35 days, the cost is low, and the lung organoids can be updated and maintained for a long time, so that the repeated induced differentiation process can be reduced, and the method is favorable for large-scale amplification for subsequent disease model construction, drug screening and cell transplantation treatment.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a schematic diagram of example 1 for inducing human pluripotent stem cells to differentiate into SOx17+CXCR4+definitive endoderm cells (FIG. A is a fluorescent staining pattern of SOx17+CXCR4+definitive endoderm cells; FIG. B is a differentiation efficiency pattern of flow cytometry-characterized definitive endoderm cells);
FIG. 2 is a schematic diagram of inducing definitive endoderm cells to differentiate into SOX2+FOXA2+pre-intestinal embryonic cells in example 1 (FIG. A is a fluorescent staining chart of SOX2+FOXA2+pre-intestinal embryonic cells; and FIG. B is a differentiation efficiency chart of flow cytometry-characterized pre-intestinal embryonic cells);
FIG. 3 is a schematic diagram of lung progenitor cells induced by the differentiation of prointestinal blasts into NKX2.1+ in example 1 (FIG. A is a fluorescence staining pattern of lung progenitor cells of NKX2.1+; and FIG. B is a pattern of differentiation efficiency of flow cytometry-characterized lung progenitor cells);
FIG. 4 is a schematic diagram of sorting purified and expanded lung progenitor cells of example 1 (FIG. A is a schematic diagram of sorting strategy, sorting CD47 +CD26- cells, FIG. B, C is a schematic diagram of proliferation of a three-dimensional cultured lung progenitor cell pellet);
FIG. 5 is a schematic representation of immunofluorescent staining of example 1 inducing differentiation of lung progenitor cells into more mature alveoli (SPB+, SPC+ alveolar type II cells, PDPN+ alveolar type I cells);
FIG. 6 is a schematic diagram of immunofluorescent staining of airway organoids (composed of P63+KRT5+ basal cells, MUC5AC+ Goblet cells, ACT+ ciliated cells, SCGB1A1+ Club cells) for example 1 inducing differentiation of lung progenitor cells to a more mature state;
FIG. 7 is a schematic diagram of example 2 for inducing differentiation of human pluripotent stem cells into SOx17+CXCR4+definitive endoderm cells (CHIR 99021 concentration was adjusted to 6uM and still differentiate normally into definitive endoderm cells);
FIG. 8 is a schematic representation of example 3 induction of definitive endoderm cells to differentiate into SOX2+FOXA2+ foregut germ cells (adjustment of DSM concentration to 1.5uM or 3uM had no significant effect on differentiation into foregut germ cells in the second stage);
FIG. 9 is a graph showing the comparison of the induced differentiation results of example 1 and comparative example 1;
FIG. 10 is a graph showing the comparison of the induced differentiation results of the negative control group and the comparative example.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1. Resuscitating, culturing and passaging stem cells
1.1 Preparation of Medium
Table 1: stem cell maintenance medium preparation
；
1) 1Ml Matrigel was added to 500ml DMEM/F12 to a final concentration of 1ug/ml and dispensed into 50ml centrifuge tubes. The stock solution is frozen at-20 ℃ and is refrigerated at 4 ℃ for standby when in use, so that repeated freezing and thawing are avoided.
2) Spreading glue: 1ug/ML MATRIGEL of solution was added to the 6-well plate, 1ml of solution was added to each well, the 6-well plate was gently shaken to uniformly spread the solution over the plate, and the plate was coated at room temperature for 20min.
3) And taking out the 1-tube frozen hPSC cell line H9 from the liquid nitrogen. The cells were held with forceps and thawed by shaking in a 37 ℃ water bath and transferred to a 15ml centrifuge tube.
4) 10Ml of DMEM/F12 medium was pipetted into a centrifuge tube and mixed with the cell suspension. 300g was centrifuged for 3min, and the supernatant was discarded to collect the precipitated stem cells.
5) Y27632 was added to 4ml of stem cell medium at a final concentration of 5uM, and after mixing, the medium was added to a centrifuge tube to re-suspend the cells.
6) The DMEM/F12 in Matrigel coated plates was discarded and the cell suspension was added at 2ml per well to 6-well plates. The culture plate is placed in a 5% CO 2 cell incubator at 37 ℃, and the culture plate is moved up and down and left and right horizontally to uniformly plant cells on the culture plate for culture.
1.2 Culture of Stem cells
Every 24 hours, the medium in the 6-well plate was discarded, and the stem cell medium was replaced at 2ml per well.
1.3 Passage of stem cells: when the cell density reached 80-90%, passaging was performed.
1) 1Ug/ml Matrigel coated plate and left to stand at room temperature for 20min.
2) Media in 6-well plates was discarded, 1ml of 1XPBS was added to each well for washing, and PBS was subsequently discarded.
3) 0.5Ml of 0.5mM EDTA was added to each well and the cells were returned to the 37℃5% CO 2 cell incubator.
4) After digestion for 7min, the cells were removed, EDTA was removed and discarded, resuspended cells were blown with Y27632 (5 uM) in stem cell medium, and the cell suspension volume was determined according to the final desired degree of cell fusion.
5) The DMEM/F12 in the coated culture plate is removed, the cell suspension is added into a 6-hole plate with 2ml per hole, the culture plate is placed in a 5% CO 2 cell incubator at 37 ℃, and the culture plate is moved up and down and left and right so that cells are uniformly planted on the culture plate for culture.
2. Differentiation of pluripotent stem cells
S1, inducing the differentiation of the human pluripotent stem cells into definitive endoderm cells.
TABLE 2 preparation of stage 1 basal Medium (i)
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Remarks: the components of the culture medium are dissolved in ddH 2 O and fixed in 1000ml, and then filtered and sterilized by a 0.22um filter screen for standby.
Table 3: cytokines/compounds required for stage 1 differentiation
；
The operation is as follows:
1) Day0 was passaged using the methods described above to digest pluripotent stem cells and seeded on 24/12/6 well plates to achieve cell fusion of 80% -90%.
2) To 100ml of basal medium of Table 2, 10ug of Activin A was added to a final concentration of 100ng/ml, and the mixture was sub-packed in 50ml centrifuge tubes at 4℃for further use.
3) To the basal medium containing 100ng/ML ACTIVIN A, 20mM CHIR99021 stock solution was added to a final concentration of 3. Mu.M, and the medium at stage 1, day 1 was prepared. It is ready for use.
4) Stage 1 differentiation was started within 24h (day 1): the stem cell medium was discarded and replaced with the 1 st day medium at stage 1.
5) Day 2: the medium on day 1 of stage 1 was discarded, and the medium of Table 1 was replaced with the medium to which Activin A (100 ng/ml) was added.
6) Day 3: the old medium was discarded by changing the liquid, and a new medium containing Activin A (100 ng/ml) as shown in Table 1 was added.
The results are shown in fig. 1: after the differentiation of stage 1, definitive endoderm cells of SOx17+CXCR4+ can be obtained.
S2, inducing the definitive endoderm cells to differentiate into the foregut germ cells.
TABLE 4 preparation of differentiation basal Medium (ii)
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Remarks: after each component was dissolved in 500ml of DMEM/F12, it was sterilized by filtration through a 0.22um screen.
Table 5: stage 2 differentiation to formulate cytokines/Compounds
；
The operation is as follows:
1) The differentiation medium of stage 2 was prepared by adding the small molecules of Table 5 to the medium of Table 4. It is ready for use.
2) And (3) paving: 1ug/ML MATRIGEL chamber temperature coated plate for 20min.
3) After PBS washing, the cells were digested with 0.5mM EDTA for 7min.
4) The EDTA is removed, and the cell suspension is prepared by adding the EDTA into the differentiation medium of the stage 2 and blowing and mixing the EDTA.
5) According to the following steps of 1:3 cells were plated uniformly on Matrigel coated plates. The liquid is changed for 1 time every 24 hours.
The results are shown in fig. 2: after the completion of the stage 2, SOX2+FOXA2+ prointestinal blastocytes were obtained.
S3, inducing the differentiation of the foregut germ cells into lung progenitor cells.
Table 6: cytokines/compounds required for stage 3
；
The operation is as follows:
1) The stage 3 differentiation medium was prepared by adding the small molecules of Table 6 to the medium of Table 4. It is ready for use.
2) And (3) paving: 1ug/ML MATRIGEL chamber temperature coated plate for 20min.
3) After PBS washing, the cells were digested with 0.5mM EDTA for 7min.
4) The EDTA is removed, and the cell suspension is prepared by adding the EDTA into the differentiation medium of the 3 rd stage and blowing and mixing the EDTA.
5) According to the following steps of 1:3 cells were plated uniformly on Matrigel coated plates. The liquid is changed for 1 time every 48 hours.
As a result, the NKX2.1+ lung progenitor cells were detected on days 14-16 as shown in FIG. 3.
S4, sorting, purifying and amplifying the lung progenitor cells.
TABLE 7 cytokines/small molecules required for stage 4
；
The operation is as follows:
flow sorting purified lung progenitor cells were sorted by surface marker CD47+CD26-or CPM+.
Table 8: FACS Buffer configuration:
；
remarks: after each component was dissolved in PBS, it was sterilized by filtration through a 0.22um filter screen for use.
1.1 Cells were digested with 0.25% trypsin for 10min, and after cell lysis, were blown into cell suspensions, transferred to 50ml centrifuge tubes, and washed with 20ml pbs.
1.2 300G was centrifuged for 3min, the supernatant was discarded, the cell pellet was collected and resuspended in 10ml PBS. Cell counting was performed.
1.3 300G was centrifuged for 3min, the supernatant was discarded, the cell pellet was collected and resuspended in 1ml FACS buffer.
1.4 1Ul of CD47-APC and 1ul of CD26-PE direct antibody were added to 10 6 cells, and the mixture was reacted at 4℃for 10min after homogenization.
1.5 10Ml PBS was added for washing, 300g was centrifuged for 3min, and the supernatant was discarded.
1.6 Adding 2mlFACS Buffer, blowing and mixing uniformly, filtering the cell suspension by a filter screen with a pore size of 40um, preparing single cell suspension and transferring to a flow sorting tube.
1.7 The cell suspension was inserted on ice bin and ready for loading.
1.8 Cells of CD47+CD26-, i.e., cells of APC+PE-, were sorted and the number of cells obtained by sorting was recorded.
2) The lung progenitor cells after sorting and purification are added into Growth Factor Reduced matrigel according to the density of 500/ul, and are blown and evenly mixed, and the step is carried out on an ice box.
3) Taking 12-well plate, adding 100ul of matrigel embedded with cells into each well, and placing into a cell incubator for solidification (10-20 min).
4) And adding the culture medium of stage 4a after the matrigel is solidified, namely adding the small molecules of Table 7 into the culture medium of Table 4, and preparing the matrigel. 10uM Y27632 is added for the first culture, and Y276232 is not needed, and the liquid is changed for 1 time every 72 hours.
As shown in fig. 4, this example sorts the purified and expanded lung progenitor cell sorting strategy and the proliferation process of the three-dimensionally cultured lung progenitor cell mass.
S5a. induce differentiation of lung progenitor cells into alveoli (type I alveoli, type II alveoli).
Table 9: stage 5a differentiation to formulate cytokines/Compounds
；
The operation is as follows:
1) On day 25 the medium was replaced with the 5a stage medium, i.e.the small molecules of Table 9 were added to the medium of Table 4. It is ready for use.
2) The liquid was changed 1 time every 3 days.
The results are shown in FIG. 5: immunofluorescence staining SPB, SPC and the like are carried out to identify on the 35 th day to obtain mature alveolar organoids, so that alveolar II cells of SPB+, SPC+ and alveolar I cells of PDPN+ can be obtained.
S5b. induce differentiation of lung progenitor cells into airway organoids (basal cells, ciliated cells, club cells, goblet cells).
Table 10: stage 5b differentiation to formulate cytokines/Compounds
；
The operation is as follows:
1) On day 25 the medium was replaced with the 5b stage medium, i.e.the small molecules of Table 10 were added to the medium of Table 4. It is ready for use.
2) The liquid was changed 1 time every 3 days.
The results are shown in FIG. 6: immunofluorescent staining of P63, KRT5, MUC5AC, ACT, etc. was performed to identify day 35 to obtain a more mature airway organoid consisting of P63+KRT5+ basal cells, MUC5AC+ Goblet cells, ACT+ ciliated cells, and SCGB1A1+ Club cells.
3. Long-term culture and passage of lung organoids
Alveolar and airway organoids were passaged every 2-3 weeks.
The operation is as follows:
1) The medium in the well plate was discarded.
2) Precooled PBS was added to blow the organoid-containing matrigel, and after blowing off, transferred to a 15ml centrifuge tube.
3) The matrigel was washed by adding 12ml pre-chilled PBS.
4) 300G of the supernatant and the upper matrigel, which contained some of the miscellaneous cells, were centrifuged for 5min and discarded.
5) TYPLE was added to the bottom precipitated lung organoid mass and the mass was repeatedly blown with a 1ml gun head for 3min to disperse the mass into single cells.
6) 10Ml PBS was added for washing, 300g was centrifuged for 3min, and the supernatant was discarded.
7) New matrigel is added into the cells precipitated at the lower layer for embedding and mixing uniformly, and the passage is carried out according to the proportion of 1:3, and the process is carried out on an ice box.
8) Taking 12-well plate, adding 100ul of matrigel embedded with cells into each well, and placing into a cell incubator for solidification (10-20 min).
9) Alveolar organoids were added to alveolar differentiation medium for further culture (small molecules of Table 7 were added to the medium of Table 3). Airway organoids were added to airway differentiation medium (small molecules of table 8 were added to the medium of table 3) and culture continued. The first culture after passage requires 10uM Y27632 to be added to the medium, after which Y276232 is not required, and 1 change of liquid every 72 hours.
Example 2
The only difference between example 2 and example 1 is as follows:
Inducing the differentiation of human pluripotent stem cells into definitive endoderm cell culture media: the final concentration of Activin A was 100ng/ml and that of CHIR99021 was 6uM. The differentiation efficiency is shown in FIG. 7.
Example 3
The only difference between example 3 and example 1 is as follows:
Inducing the definitive endoderm cells to differentiate into a pre-intestinal germ cell culture medium: dorsommorphin had a final concentration of 1.5uM or 3uM and SB431542 (SB) had a final concentration of 10uM. The differentiation efficiency is shown in FIG. 8.
The culture medium of example 2 and example 3 can induce pluripotent stem cells to differentiate into alveolar organoids and/or airway organoids in the same manner as the small molecule mixture ratio of different concentrations, and will not be described in detail here.
Comparative example 1
The only difference between comparative example 1 and example 1 is that only small SB431542 (10 uM) molecules were added to the medium that induced the definitive endoderm to differentiate into the foregut embryo. The differentiation efficiency is shown in FIG. 9.
Comparative example 2
The only difference between comparative example 2 and example 1 is that only CHIR99021 (3 uM) and Retinoic acid (50 nM) small molecules were added to the medium that induced differentiation of the foregut germ cells into lung progenitor cells. The differentiation efficiency is shown in FIG. 10.
The results show that the induced differentiation medium of comparative examples 1-2 can not successfully induce qualitative differentiation of pluripotent stem cells into lung organoids or airway organs. This is due to the lack of the small molecule component of the present invention in the culture media of comparative examples 1-10. Thus, the synergistic effect among the components in each cultivated small molecule composition of the invention is demonstrated by the above comparative examples that the effect of directional differentiation cannot be achieved by breaking this specific compatibility.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (1)
1. A culture method for inducing differentiation of pluripotent stem cells into alveolar organoids or airway organoids, comprising the steps of:
S1, inducing the differentiation of human pluripotent stem cells into definitive endoderm cells, wherein the specific steps are as follows:
s1a, adding a basic culture medium (i), activin A with a final concentration of 100ng/ml and CHIR99021 with a final concentration of 3uM into a culture medium of the stem cells, and culturing for 1 day;
S1b, discarding the culture medium of the a, adding a basic culture medium (i) and Activin A with the final concentration of 100ng/ml, and culturing for 1 day;
S1c, discarding the culture medium of the b, adding a basic culture medium (i) and Activin A with the final concentration of 100ng/ml, and culturing for 1 day; obtaining definitive endoderm cells of SOX17+ cxcr4+;
1000ml of said basal medium (i) consisting of components at the final concentrations ：MCDB131 11.6g/L,D-Glucose 0.1~5g/L,NaHCO3 1~5g/L、BSA 1~10g/L、vitamin C 50~100ug/ml、GlutaMAX 0.5~2X、Pen-Strep 0.1~1X; wherein X represents the multiple of the final dilution;
s2, inducing the definitive endoderm cells to differentiate into the foregut germ cells, wherein the specific steps are as follows;
Removing the culture medium in the step S1c, adding a basic culture medium (ii), dorsomophin with a final concentration of 2uM and SB431542 with a final concentration of 10uM, and culturing for 3 days, wherein the liquid is changed 1 time every 24 hours to obtain SOX2+FOXA2+ foregut embryo cells;
s3, inducing the differentiation of the foregut embryo into lung progenitor cells, wherein the specific steps are as follows:
discarding the culture medium of the step S2, adding a basic culture medium (ii), CHIR99021 with a final concentration of 3uM, BMP4 with a final concentration of 10ng/ml and Retinoic acid with a final concentration of 50nM, and culturing for 9 days, wherein the culture medium is changed for 1 time every 48h to obtain NKX2.1+ lung progenitor cells;
s4, sorting, purifying and amplifying lung progenitor cells, wherein the specific steps are as follows:
S4a, adopting flow separation to purify the lung progenitor cells, and separating the cells of CD47+CD26-through surface markers CD47+CD26-or CPM+;
S4b, adding Growth Factor Reduced matrigel into the sorted and purified CD47+CD26-lung progenitor cells according to the density of 500 per ul, and blowing and mixing uniformly;
s4c, taking a 12-hole plate, adding 100ul of matrigel embedded with cells into each hole, and placing the mixture into a cell culture box for solidification for 10-20min;
S4d, adding a basic culture medium (ii), CHIR99021 with a final concentration of 3uM and KGF with a final concentration of 10ng/ml after the matrigel is solidified, and culturing for 10 days, wherein 10uM Y27632 is needed to be added for the first time, Y276232 is not needed, and liquid is changed for 1 time every 72 hours;
s5, inducing the differentiation of the lung progenitor cells into alveolar organoids, wherein the alveolar organoids comprise type II alveolar cells of SPB+SPC+ and type I alveolar cells of PDPN+, and the specific steps are as follows:
Discarding the culture medium of the S4d step, adding a basal medium (II), CHIR99021 with a final concentration of 3uM, KGF with a final concentration of 10ng/ml, dexamethasone with a final concentration of 50nM, IBMX with a final concentration of 100uM and cAMP with a final concentration of 100uM, and culturing for 10 days, wherein the culture medium is changed for 1 time every 3 days to obtain alveolar II cells of SPB+SPC+ and alveolar I cells of PDPN+.
Or S5, inducing the differentiation of the lung progenitor cells into airway organoids, wherein the airway organoids consist of basal cells of P63+KRT5+, goblet cells of MUC5AC+, ciliated cells of ACT+ and Club cells of SCGB1A1+, and the specific steps are as follows:
Removing the culture medium in the step S4d, adding a basal medium (ii), FGF2 with a final concentration of 250ng/ml, FGF10 with a final concentration of 100ng/ml, dexamethasone with a final concentration of 50nM, IBMX with a final concentration of 100uM, cAMP with a final concentration of 100uM and DAPT with a final concentration of 2uM, and culturing for 10 days, wherein liquid is changed 1 time every 3 days to obtain basal cells of P63+KRT5+, goblet cells of MUC5AC+, ciliated cells of ACT+ and Club cells of SCGB1A 1+.
500Ml of the basal medium (ii) consists of components at the final concentrations ：BSA 0.01~0.5%（w/v）,vitamin C 20~100ug/ml,GlutaMAX 0.5~2X,Pen-Strep 0.1~1X,N2 0.5~2X,B27 0.5~2X,MTG 0~5ul/ml,DMEM/F12 1X;. X represents the multiple of the final dilution.
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