CN114107196A - A method for the rational design of pluripotent stem cells to differentiate natural killer cells based on single-cell sequencing and the application of CSF1R inhibitors - Google Patents
A method for the rational design of pluripotent stem cells to differentiate natural killer cells based on single-cell sequencing and the application of CSF1R inhibitors Download PDFInfo
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Abstract
本发明提供了基于单细胞测序理性设计的多能干细胞分化自然杀伤细胞的方法及CSF1R抑制剂的应用，涉及生物技术领域。发明人根据单细胞转录组数据打开了iPSC向NK分化过程中的“黑匣子”，提供了一种iPSC分化NK的理性设计方法，理性设计了小分子抑制剂在体外多能干细胞分化NK的过程中的应用。经研究发现CSF1R抑制剂处理能够通过抑制髓系细胞的分化提高NK的比例，缩短分化所需时间，因此CSF1R抑制剂能够用于促进体外多能干细胞分化制备自然杀伤细胞中。本发明提供了一种自然杀伤细胞的制备方法，该方法简单方便，能够稳定、快速的获得高纯度自然杀伤细胞。
The invention provides a method for pluripotent stem cell differentiation natural killer cells rationally designed based on single cell sequencing and the application of a CSF1R inhibitor, and relates to the field of biotechnology. Based on the single-cell transcriptome data, the inventor opened the "black box" in the process of iPSC differentiation to NK, provided a rational design method for iPSC differentiation NK, and rationally designed a small molecule inhibitor in the process of pluripotent stem cell differentiation NK in vitro. Applications. Studies have found that CSF1R inhibitor treatment can increase the proportion of NK by inhibiting the differentiation of myeloid cells and shorten the time required for differentiation. Therefore, CSF1R inhibitors can be used to promote the differentiation of pluripotent stem cells in vitro to prepare natural killer cells. The invention provides a method for preparing natural killer cells, which is simple and convenient, and can obtain high-purity natural killer cells stably and rapidly.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a high-resolution cell map for NK differentiation drawn based on a single cell sequencing technology, and relates to application of a CSF 1R-related inhibitor in preparation of natural killer cells by promoting differentiation of in vitro pluripotent stem cells.
Background
NK cells are an immune cell with the function of killing tumor cells. Engineered CAR-expressing NK cells have the ability to target killing of tumor cells expressing a certain antigen. Compared with CAR-T cells, CAR-NK cells have lower cytokine storm, so the CAR-NK cells have good application prospect in the future. NK cells (natural killer cells) differentiated monoclonally by ipscs (induced pluripotent stem cells) enable a highly homogeneous product and a sufficient number of cells are available to form a product like an off-the-shelf one. Therefore, iPSC differentiated NK cells are an ideal choice for future off-the-shelf immune cell products. However, iPSC differentiation of NK cells faces a series of challenges, firstly the system of differentiation is not stable enough, secondly the purity of the differentiated cells has not been accurately measured and thirdly the time of differentiation is too long. More importantly, the current knowledge of the black box in the process of differentiating NK cells by iPSC is very limited, and the expression of key genes and key transcription factors and the change of a gene regulation network determine that the differentiation is not systematically described, so that the traditional optimization of the differentiation method is also blind, and the NK cells obtained by differentiation can have potential clinical treatment risks caused by unknown components.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a rational design method for differentiating natural killer cells by pluripotent stem cells.
The second purpose is to provide the application of the CSF1R inhibitor in promoting the differentiation of in vitro pluripotent stem cells to prepare natural killer cells.
A third object of the present invention is to provide a method for preparing natural killer cells, which can stably and rapidly obtain high-purity natural killer cells and solve at least one of the above problems.
In a first aspect, the present invention provides a rational design method for differentiating natural killer cells from pluripotent stem cells, comprising the following steps: in the process of differentiating the natural killer cells by the pluripotent stem cells, performing single cell sequencing and bioinformatics analysis on the differentiated cells, finding out other cell subsets generated in the differentiation process except the natural killer cells, finding out genes which are not expressed in the natural killer cells but expressed in the non-natural killer cells through a gene expression profile, determining the effect of the genes in the differentiation of the pluripotent stem cells into the natural killer cells, and if the cells expressing the genes are eliminated through targeted inhibition of the genes, promoting the differentiation of the natural killer cells by adding an inhibitor of the genes in the process of differentiating the natural killer cells by the pluripotent stem cells;
the non-natural killer cells are cells except natural killer cells which are obtained by differentiation in the process of differentiating the natural killer cells by the pluripotent stem cells;
the non-natural killer cells include at least one of myeloid neutrophil precursors, monocytes, dendritic cells, mast cells, or erythroid cells.
In a second aspect, the invention provides an application of the CSF1R inhibitor in preparation of natural killer cells by promoting differentiation of pluripotent stem cells in vitro through rational design of single cell sequencing technology.
In a third aspect, the invention provides a method for preparing natural killer cells, comprising the steps of inducing and culturing pluripotent stem cells to obtain embryoid bodies, and culturing and differentiating the embryoid bodies to obtain the natural killer cells;
CSF1R inhibitor is added in the process of culturing and differentiating the embryoid bodies.
As a further aspect, the CSF1R inhibitor comprises BLZ945 and/or PLX 3397.
As a further technical scheme, the step of obtaining the embryoid body by the induced culture of the pluripotent stem cell comprises the following steps: performing first culture on the pluripotent stem cells in a first culture medium;
the first medium comprises: basic culture medium, Y276328-12 mu M, BMP 416-24 ng/mL, VEGF 16-24 ng/mL and SCF 32-48 ng/mL.
As a further technical solution, the first culture medium comprises: basal medium A, Y2763210 μ M, BMP 420 ng/mL, VEGF 20ng/mL and SCF 40 ng/mL;
preferably, the basal medium A comprises STEMdiffTM APEL TM2。
As a further technical scheme, the time for the first culture is 5-7 days.
As a further technical scheme, the step of culturing and differentiating the embryoid bodies to obtain the natural killer cells comprises the following steps: culturing the embryoid body in a second culture medium for the second time, wherein a CSF1R inhibitor is added during the second culture;
the second medium comprises: the basal medium is B, SCF 16-24 ng/mL, IL-716-24 ng/mL, IL-158-12 ng/mL, FLt3L 8-12 ng/mL and IL-34-6 ng/mL.
As a further technical solution, the second culture medium comprises: basal medium B, SCF20ng/mL, IL-720 ng/mL, IL-1510 ng/mL, FLt3L 10ng/mL, and IL-35 ng/mL;
preferably, the base medium B comprises StemSpanTM-XF。
As a further means, CSF1R inhibitor was added from day 9 to day 32 of the second culture.
As a further technical scheme, the conditions of the first culture or the second culture at least meet one of the following conditions: at a temperature of 36-38 ℃ and 5% CO2。
Compared with the prior art, the invention has the beneficial effects that:
the method can predictably find out the gene which has the effect of promoting the differentiation of the non-natural killer cells, and promote the differentiation of the natural killer cells by inhibiting the expression of the gene.
The inventor finds that the single cell sequencing result shows that in an in vitro differentiation system, more myeloid cells exist, and CSF1R is highly expressed in the myeloid cells (macrophages, monocytes, dendritic cells and the like) and hardly expressed in the lymphoid cells (natural killer cells and the like); in the results of RNA-seq of in vitro pluripotent stem cell differentiated macrophages, CSF1R is highly expressed in the late stage of macrophage differentiation; in view of this, we believe that inhibiting the expression of CSF1R during NK cell in vitro differentiation increases NK cell directed differentiation. Therefore, the CSF1R inhibitor is adopted to treat the pluripotent stem cells in vitro in the process of differentiating the natural killer cells so as to improve the proportion of the natural killer cells obtained by differentiation and shorten the time required by differentiation.
The invention provides a rational and efficient preparation method of natural killer cells by utilizing a single cell sequencing technology, and the natural killer cells are prepared by inducing and culturing pluripotent stem cells to obtain embryoid bodies and then adding a CSF1R inhibitor in the process of culturing and differentiating the embryoid bodies. The method is simple and convenient, and can stably and rapidly obtain high-purity natural killer cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the results of the analysis of flow cytometry and microscopic images of induced pluripotent stem cells to differentiate into natural killer cells at different stages;
FIG. 2 is a single cell sequencing UMAP profile of pluripotent stem cell differentiation to day 24;
FIG. 3 is a comparison of single cell clustering definitions against public databases;
FIG. 4 shows the effect of CSF1R small molecule inhibitor on differentiation of natural killer cells from pluripotent stem cells in vitro.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Interpretation of terms:
natural killer cells (NK cells), which are not only part of the innate immune system, but also have the ability to present antigens and to clonally expand during infection, resulting in long-lived memory cells. In the past decade, peripheral blood NK has been used in clinical trials, and trials using adoptively transplanted allogeneic NK cells have shown that they are highly safe and have little toxic effects such as graft versus host disease and cytokine release syndrome.
A Chimeric Antigen Receptor (CAR) is composed of three parts, namely an extracellular Antigen binding region, a transmembrane region and an intracellular signaling region. The extracellular domain is a single chain variable domain (scFv) with the function of targeting and binding to the TAA of tumor specific antigen. The transmembrane region is typically composed of the immunoglobulin superfamily, such as CD8 or CD 28. The intracellular signaling region is mainly composed of an intracellular signaling domain of an activation receptor which can activate immune cells, such as a costimulatory factor (4-1BB or CD28) specific to T cells and a signaling activation region CD3 zeta. After the immune cells loaded with the chimeric antigen receptor are specifically combined with the surface antigen of the tumor cells, the extramembranous antigen binding region transmits signals to the intracellular signal activation region, and then the immune cell activation reaction is started.
Single cell RNA sequencing is a single cell-based transcriptome sequencing analysis technology, and can be used for describing the inherent dynamic changes and typing of various complex cells. It provides deep analysis of gene expression characteristics of different cell types, and is favorable to analyzing gene regulation network and dynamic change rule of cell state at single cell level.
Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells with the potential to differentiate into multiple cells by the transformation of pluripotency factors in adult cells followed by reprogramming of the initial genomic expression profile.
iNK (iPSC-derived NK) Natural killer cells induced to differentiate from iPSC.
In order to solve the problems, firstly, cells at a specific time point and state in the differentiation process are selected for single cell sequencing, and then, whether a cell can be successfully differentiated to obtain an NK cell or not and the differentiation trend of an iPSC lineage in the differentiation process are determined by researching key genes and a regulation and control network through a bioinformatics method. After finding out the key factors, small molecules targeting the key factors or the pathways are adopted, and the factors or the pathways are activated or inhibited to realize the selection optimization of the NK cell differentiation pathway, so that the aim of establishing a method for more efficiently differentiating the NK cells is fulfilled.
In the present invention, "rational design" means: based on single cell sequencing, in the process of iPSC NK differentiation, key factors for guiding NK differentiation and influencing lineage differentiation, such as certain genes expressed in differentiated myeloid cells but not expressed in NK, are found through bioinformatics analysis, and whether the genes have the actual effect of promoting NK differentiation in the process of iPSC NK differentiation is further determined through biological experiments (such as the effect of a small molecule inhibitor).
In a first aspect, the present invention provides a rational design method for differentiating natural killer cells from pluripotent stem cells, comprising the following steps: during the process of differentiating the natural killer cells by the pluripotent stem cells, performing single cell sequencing and bioinformatics analysis on the differentiated cells, finding out other cell subsets generated in the differentiation process except the natural killer cells, and finding out genes which are not expressed in the natural killer cells but expressed in the non-natural killer cells through a gene expression profile; determining the function of the gene in the differentiation of the pluripotent stem cells into natural killer cells, and if the cells expressing the gene are eliminated by targeted inhibition of the gene, promoting the differentiation of the natural killer cells by adding an inhibitor of the gene in the differentiation of the natural killer cells into the pluripotent stem cells;
the non-natural killer cells are cells except natural killer cells which are obtained by differentiation in the process of differentiating the natural killer cells by the pluripotent stem cells;
the non-natural killer cells comprise at least one of myeloid-lineage neutrophil precursors, monocytes, dendritic cells, mast cells or erythroid cells, or other non-natural killer cells obtained by differentiation during differentiation of natural killer cells from pluripotent stem cells.
In addition, the method for determining the effect of the gene in the differentiation of natural killer cells from pluripotent stem cells in the invention comprises the step of determining whether the gene has the effect of promoting NK differentiation in the iPSC NK differentiation process through biological experiments (such as the effect of a small molecule inhibitor).
The method can predictably find out the gene which has the effect of promoting the differentiation of the non-natural killer cells, and promote the differentiation of the natural killer cells by inhibiting the expression of the gene.
In a second aspect, the invention provides the use of an inhibitor of CSF1R in promoting differentiation of pluripotent stem cells in vitro to produce natural killer cells.
CSF1R (colony stimulating factor-1 receptor), a lineage specific cytokine receptor, is important for the proliferation, differentiation and maintenance of monocyte activity.
The inventor researches and discovers that in an in vitro differentiation system, CSF1R is highly expressed in myeloid cells (macrophages, monocytes, dendritic cells and the like) and is hardly expressed in lymphoid cells (natural killer cell precursors, natural killer cells and the like); in the results of RNA-seq of in vitro pluripotent stem cell differentiated macrophages, CSF1R is highly expressed in the late stage of macrophage differentiation; the CSF1R inhibitor is used for treatment in the process of differentiating the natural killer cells by the in vitro pluripotent stem cells, so that the proportion of the natural killer cells obtained by differentiation can be increased, and the time required by differentiation can be shortened, therefore, the CSF1R inhibitor can be used for promoting the differentiation of the in vitro pluripotent stem cells to prepare the natural killer cells.
In a third aspect, the invention provides a method for preparing natural killer cells, comprising the steps of inducing and culturing pluripotent stem cells to obtain embryoid bodies, and culturing and differentiating the embryoid bodies to obtain the natural killer cells;
CSF1R inhibitor is added in the process of culturing and differentiating the embryoid bodies.
The preparation method of the natural killer cell provided by the invention is simple and convenient, and can stably and quickly obtain the high-purity natural killer cell.
In some preferred embodiments, the CSF1R inhibitor includes, but is not limited to, BLZ945 and/or PLX3397, and can also be, for example, Linifanib (ABT-869), OSI-930, GW2580, PLX5622, and the like. In the present invention, the CSF1R inhibitor is not particularly limited, and any substance can be used which can inhibit the expression of CSF1R in cells and does not affect the survival of cells.
It is noted that "BLZ 945 and/or PLX 3397" means that the CSF1R inhibitor may include only BLZ945, only PLX3397, and also BLZ945 and PLX 3397.
In some preferred embodiments, the step of inducing culture of the pluripotent stem cells to obtain embryoid bodies comprises: performing first culture on the pluripotent stem cells in a first culture medium;
the first medium comprises: basic culture medium, Y276328-12 mu M, BMP 416-24 ng/mL, VEGF 16-24 ng/mL and SCF 32-48 ng/mL.
In the first medium, the concentration of Y27632(ROCK (Rho-associated protein kinase) inhibitor) may be, for example, but not limited to, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M, or 12. mu.M; the concentration of BMP4 (bone morphogenic protein 4) may be, for example, but is not limited to, 16ng/mL, 18ng/mL, 20ng/mL, 22ng/mL, or 24 ng/mL; the concentration of VEGF (vascular endothelial growth factor) may be, for example, but is not limited to, 16ng/mL, 18ng/mL, 20ng/mL, 22ng/mL, or 24 ng/mL; the concentration of SCF (stem cell factor) may be, for example, but not limited to, 32ng/mL, 36ng/mL, 40ng/mL, 44ng/mL, or 48 ng/mL.
In the invention, the pluripotent stem cells are cultured in a first culture medium to be capable of inducing and culturing to obtain the embryoid bodies.
In some preferred embodiments, the first medium comprises: basal medium A, Y2763210 μ M, BMP 420 ng/mL, VEGF 20ng/mL and SCF 40 ng/mL;
wherein the basic culture medium A is used for the induction culture of pluripotent stem cells to embryoid bodies, and is preferably STEMdiffTMAPELTM2。
Through the optimization of the composition of the first culture medium, the induction culture of the pluripotent stem cells to the embryoid bodies is better realized.
In some preferred embodiments, the first culturing is performed for 5 to 7 days, and the embryoid body can be obtained after 5 to 7 days of culturing.
In some preferred embodiments, the step of differentiating the embryoid body culture into natural killer cells comprises: culturing the embryoid body in a second culture medium for the second time, wherein a CSF1R inhibitor is added during the second culture;
the second medium comprises: the basal medium is B, SCF 16-24 ng/mL, IL-716-24 ng/mL, IL-158-12 ng/mL, FLt3L 8-12 ng/mL and IL-34-6 ng/mL.
The concentration of SCF in the second medium can be, for example, but not limited to, 16ng/mL, 18ng/mL, 20ng/mL, 22ng/mL, or 24 ng/mL; IL-7 concentration may be, for example but not limited to, 16ng/mL, 18ng/mL, 20ng/mL, 22ng/mL or 24 ng/mL; IL-15 concentration can be, but is not limited to, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL or 8-12 ng/mL; the concentration of FLt3L (FMS-like tyrosine kinase 3 ligand) may be, for example but not limited to, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, or 8-12 ng/mL; IL-3 concentration may be, for example, but not limited to, 4ng/mL, 4.5ng/mL, 5ng/mL, 5.5ng/mL, or 6 ng/mL.
In the present invention, the embryoid body is cultured in a second medium to obtain a natural killer cell.
In some preferred embodiments, the second medium comprises: basal medium B, SCF20ng/mL, IL-720 ng/mL, IL-1510 ng/mL, FLt3L 10ng/mL, and IL-35 ng/mL;
the basic culture medium B provides basic components required for culturing and differentiating the embryoid bodies to obtain natural killer cells, and preferably is StemSpanTM-XF。
And the differentiation culture from the embryoid bodies to the natural killer cells is realized by optimizing the composition of the first culture medium.
In some preferred embodiments, the CSF1R inhibitor is added from day 9 to day 32 of the second culture. At this stage, monocytes and macrophages appear with NK in higher proportion, and CSF1R inhibitor is added to help reduce differentiation of other cells.
In some preferred embodiments, the conditions of the first or second culturing are at least one of: at a temperature of 36-38 ℃ and 5% CO2。
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
A sufficient amount of mTeSR1 was preheated (15-25 ℃).
iPS cells were digested into single cells with TrypLE.
1. 1mL of Ca-free2+And Mg2+The wells were washed with PBS.
2. PBS was aspirated and digested for 5min by addition of 200. mu.L TrypLE.
3. After termination of digestion, centrifugation was carried out at 200 Xg for 5 minutes.
4. Resuspend with the following medium:
basic culture medium: STEMdiffTMAPELTM2 contained Y27632 (10. mu.M), BMP4(20ng/mL), VEGF (20ng/mL) and SCF (40 ng/mL).
5. The cell density was adjusted to 80,000cells/mL and 100. mu.L/well of the cell suspension was added to a low adsorption 96-well plate.
6.300 Xg for 5 minutes at 37 ℃ in 5% CO2After 6 days in the cell incubator, EBs (embryoid bodies) were formed, and on day 7, 20-40 EBs were placed in 6-well plates pre-coated with matrigel (1mg/mL or 0.1% gelatin) and incubated with the following media:
basic culture medium: StemBanTMXF, containing SCF (20ng/mL), IL-7(20ng/mL), IL-15(10ng/mL), FLt3L (10ng/mL) and IL-3(5 ng/mL).
The results of microscopic imaging and flow cytometry analysis of the embryoid bodies, cells cultured up to day 15, to day 22 and to day 57, respectively, are shown in FIG. 1 (A in FIG. 1 is a microscopic image and a flow cytometry analysis of the embryoid bodies from top to bottom, B in FIG. 1 is a microscopic image and a flow cytometry analysis of the cells cultured up to day 15, and C in FIG. 1 is a microscopic image and a flow cytometry analysis of the cells cultured up to day 15, respectivelyThe results of the microscopic image and the flow cytometry analysis of the cells cultured up to day 22 in the order from the top to the bottom, and the results of the microscopic image and the flow cytometry analysis of the cells cultured up to day 57 in the order from the top to the bottom in FIG. 1), as can be seen from FIG. 1, CD45 was observed up to days 15 and 22 in the order from the culture up to day 22+CD56+The proportion of NK cells was gradually increased from 19.2% to 29.7%. As differentiation proceeded, CD45 was observed on day 57 of culture+CD56+The proportion of NK cells was increased to 97.4%.
Taking cells differentiated to 24 days to perform single cell sequencing, the results are shown in FIG. 2 (A in FIG. 2 is UMAP map of single cells at the time of differentiation to 24 days, from the UMAP map of single cell sequencing grouping, we found that the cells were grouped into 10 groups (A in FIG. 2), B in FIG. 2, C are high expression of NK marker genes CD56 and KLRB1 in 0 group and 2 group, C in FIG. 2 is high expression of lysosome-related gene LYZ in 3,4,5 group, C in FIG. 2 is high expression of monocyte marker CD14 also in 3,4,5 group, by further comparing with the disclosed data of in vivo differentiation (GSE144024) (FIG. 3), we finally determined that 0 and 2 groups are both NK cells and their precursors, 3,4 and 6 groups are neutrophil cell precursors, monocytes and monocytes respectively, 5 group are multi-lymphoid progenitor cells (MLP), 1 and 7 are mast cell group, and 8 is erythroid cell group, group 9 are dendritic cells.
Example 2
The cells differentiated to day 30 in example 1 were collected, treated with CSF1R inhibitor BLZ 9450.5. mu.M and PLX 33971. mu.M, respectively, and a control group (no treatment, normal culture) was set, and after 8 days, CD45 was collected and examined+CD56+The results of the proportion of NK cells are shown in FIG. 4 (A in FIG. 4 is the expression of CSF1R on the UMAP map when NK was differentiated to day 24, B in FIG. 4 is the change of the expression amount of mRNA in the process of differentiating the CSF1R into macrophages by the pluripotent stem cells in vitro, C in FIG. 4 is the result of flow cytometry analysis of the control group, D in FIG. 4 is the result of flow cytometry analysis of the BLZ 945-treated group, and E in FIG. 4 is the result of flow cytometry analysis of the PLX 3397-treated group).
As shown in A in FIG. 4, CSF1R was in the myeloid lineage in the in vitro differentiation systemHigh expression in cells (monocytes, dendritic cells, etc.); and almost no expression in the lymphoid lineage cells (NK cell precursors, NK cells, etc.). Furthermore, in the RNA-seq data of iPSC differentiated macrophages in vitro, we also found that CSF1R was highly expressed in the late stage of macrophage differentiation (after monocyte appearance/day 18) (B in fig. 4). Thus, we treated iNK obtained on day 30 after differentiation for 8 days with the small molecule inhibitors BLZ945 and PLX3397 of CSF1R and found CD45 after treatment with the small molecule inhibitor of CSF1R+CD56+The proportion of NK cells was increased from 80% to 90% (C in FIG. 4, D in FIG. 4, E in FIG. 4), and NK cells (D in FIG. 1, 97.4%) having a proportion similar to that of the 57 th day of differentiation were obtained only at 38 days of differentiation, which indicates that the use of the CSF1R inhibitor not only increases the proportion of NK cells obtained by differentiation but also shortens the time required for differentiation.
The experimental procedure for panel B in fig. 4 is as follows:
1) induction formation of Embryoid Bodies (EB) by iPS cells
mTeSR1, DMEM/F12 and Versene were pre-warmed to 15-25 ℃ for cell passaging. Y27632 is a Rock kinase inhibitor used at a concentration of 10. mu.M. iPS cells were digested into single cells and induced the formation of EBs:
a) washing the original hole with 1mL of DPBS;
b) sucking off DPBS, adding 1mL Versene containing Y27632, and incubating at 37 deg.C for 4-5 min;
c) pipetting 1-2 times and removing cells (usually cells still in larger clumps will form better EBs);
d) the cells were immediately transferred to centrifuge tubes containing DMEM/F12 at a 1: 5-1: 9, diluting Versene in proportion; washing the primary well with 1mL DMEM/F12, collecting the remaining cells and transferring to a tube, centrifuging at 200 Xg for 5 min;
e) mTeSR1 medium containing Y27632 resuspended cells and placed the cells on an ultra-low plate with a separation ratio of 1: 1 or 2:1 (90% induced pluripotent stem cells per well).
2) Induced differentiation of Embryoid Bodies (EB) into macrophages
Step a) removing the mTeSR1 medium of the embryoid bodies in f) of 1) above, and using the first medium (STEMdiff) on day 1TM APEL TM2, 10ng/mL BMP4, 5ng/mL bFGF) for 24h, and differentiating the embryoid bodies into mesodermal cells;
step b) removing the first medium from step a) and using a second medium (STEMdiff) during the 2-7 days after inoculationTMAPELTM2, 10ng/mL BMP4, 5ng/mL bFGF, 50ng/mL VEGF and 100ng/mL SCF) while replacing the new second medium every other day to obtain hematopoietic stem cells;
step c) removing the second medium from step b) and using a third medium (STEMdiff) during the 8-10 days after inoculationTMAPELTM2, 10ng/mL bFGF, 50ng/mL VEGF, 50ng/mL SCF, 10ng/mL IGF1, 25ng/mL IL-3, 50ng/mL M-CSF and 50ng/mL GM-CSF) with replacement of fresh third medium every other day;
step d) removing the third medium from step c), inoculating the cells at a concentration of 20-25 cells/mL into a petri dish precoated with Matrigel matrix (1mg/mL) during the 11-20 days after inoculation, and using a fourth medium (StemPro)TM-34, 10ng/mL bFGF, 50ng/mL VEGF, 50ng/mL SCF, 10ng/mL IGF1, 25ng/mL IL-3, 50ng/mL M-CSF and 50ng/mL GM-CSF) incubating the cells of step c) to obtain myeloid cells;
starting on days 21-22 after inoculation in step e), the myeloid cells suspended in step d) were collected and replated onto a petri dish previously coated with matrigel, using a fifth medium (StemPro)TM34, 10ng/mL bFGF, 50ng/mL VEGF, 50ng/mL SCF, 10ng/mL IGF1, 25ng/mL IL-3, 100ng/mL M-CSF and 100ng/mL GM-CSF), and differentiating to obtain macrophages;
step f) removing the fifth medium from step e) and using the sixth medium (StemPro) during the 23-28 th day after inoculationTM-34, 10ng/mL bFGF, 50ng/mL VEGF, 50ng/mL SCF, 10ng/mL IGF1, 100ng/mL M-CSF and 100ng/mL GM-CSF) to incubate macrophages;
step g) remove the sixth medium in step f) and start maintaining mature macrophages on day 29 of inoculation with a seventh medium (RPMI-1640, 10% w/w FBS, 100ng/mL M-CSF, 100ng/mL GM-CSF).
The method can obtain mature macrophages with high quality and high purity and capable of targeting tumor cells. RNA-seq was performed on iPS cells and Embryoid Bodies (EBs) induced to differentiate at day 7 and cells at day 18, day 28 and day 38, respectively, and the results are shown as B in fig. 4.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
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