CN114561351A - Differentiation of CD4 from induced pluripotent stem cells+Methods of non-T cells - Google Patents

Differentiation of CD4 from induced pluripotent stem cells+Methods of non-T cells Download PDF

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CN114561351A
CN114561351A CN202210171731.7A CN202210171731A CN114561351A CN 114561351 A CN114561351 A CN 114561351A CN 202210171731 A CN202210171731 A CN 202210171731A CN 114561351 A CN114561351 A CN 114561351A
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韦俊
季伟
王振坤
曹哲厚
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Hangzhou Yuansheng Biotechnology Co ltd
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Abstract

The invention relates to the technical field of immunology, in particular to a method for differentiating CD4 from induced pluripotent stem cells+A method of non-T cells, comprising: s1, maintaining and culturing the PSC cells; s2, preparing EB (embryoid body); s3, differentiating EB to mesoderm; s4, differentiation of mesoderm to CD34+The hematopoietic progenitor cells of (a); s5, differentiation CD34+To CD4+The invention starts from human iPSC cell, and fundamentally solves the problem of CD4+The source and quantity of non-T cells is a very important medical concern.

Description

Differentiation of CD4 from induced pluripotent stem cells+Methods of non-T cells
Technical Field
The invention relates to the technical field of immunology, in particular to a method for differentiating CD4 from induced pluripotent stem cells+A non-T cell method.
Background
Cell therapy plays an increasingly important role in current clinical therapy, and the remarkable effect of the cell therapy shows that the cell therapy cannot be worn out in the aspect of treating various diseases, particularly tumors, wherein the most widely used immune cells are immune cells, and the problems of small quantity of immune cells, batch-to-batch difference and the like are solved along with the continuous development and maturation of technologies such as CAR-T, NK, DC vaccines and the like.
Human iPSC cell is a cell with multipotential differentiation potential and can be proliferated indefinitely, it is through transferring OCT4, SOX2, c-Myc, Klf4 gene into human primary somatic cell, then through the transcriptional regulatory action of a series of proteins expressed by this gene, reprogram the cell with multipotential property, iPSC can differentiate CD34+ hematopoietic progenitor cell under the specific culture medium and many kinds of cytokine combination culture, this cell is the precursor cell of many kinds of immunocytes, can differentiate into many kinds of immunocytes further, therefore iPSC-derived immunocyte can provide the raw materials for cell therapy well, therefore the invention provides a kind of cell differentiated CD4 from induced pluripotent stem cell+A non-T cell method.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, it is a first object of the present invention to provide a method for differentiating CD4 from induced pluripotent stem cells+non-T cell approach, solving the problems of the background art described above.
In order to achieve the purpose, the invention provides the following technical scheme:
differentiation of CD4 from induced pluripotent stem cells+A method of non-T cells, comprising the steps of:
s1, maintenance culture of PSC cells: the iPSC is maintained and cultured by a commercial E8 culture medium, an E8 culture medium contains factors such as L-ascorbic acid, transferrin, insulin, FGF2 and TGF beta 1, the totipotency of the iPSC can be well maintained, and the iPSC is cultured in a 6-well plate containing Matrigel;
s2, EB (embryoid body): the embryoid body is a spherical cell aggregate formed by embryonic stem cells or iPSC cells under a certain condition in vitro, has the capacity of differentiating ectoderm, mesoderm and endoderm similar to embryos, and has faster differentiation of target cells from EB (embryonic Stem) and higher purity;
s3, differentiation of EBs into mesoderm: AggreWell is preparedTMEB ball weight in 800 platesSuspending, sucking into a 15mL centrifuge tube, standing for 3min, carefully discarding the supernatant, resuspending with 1mL EB differentiation medium I (the EB differentiation medium I adopts a stemline II medium plus 20-50ng/mL hBMP-4,10-30ng/mL hbFGF and 20-50ng/mL VEGF can well maintain the EB structure and also can ensure that the EB is directionally differentiated to mesoderm which is the origin of hematopoietic stem cells from various immunocytes), transferring the EB balls into an ultra-low adsorption 6-well plate after resuspension, and differentiating for two days by using the EB differentiation medium I;
s4, differentiation of mesoderm to CD34+The hematopoietic progenitor cells of (a): coating a 6-hole plate with Matrigel glue after two days in the steps, adding 15ul of F12 culture medium into each hole, uniformly mixing, placing the plate into a 37 ℃ CO2 incubator for 1h, removing the F12 culture medium after coating, adding 2ml of EB differentiation culture medium II into each hole, collecting EB balls in an ultra-low adsorption plate, removing supernatant, transferring the EB balls into the culture plate and the culture medium, culturing 40 EB balls in each hole for 14 days, and replacing a new EB differentiation culture medium II every 3 days;
s5, differentiation CD34+To CD4+non-T immune cells: and (3) removing the culture medium of the culture system, continuously differentiating the rest cells by using an EB differentiation culture medium III, replacing a new EB differentiation culture medium III every two days, and after differentiating for 8 days, carrying out flow detection on the expression condition of the cells CD45 CD56CD3 CD8 CD 4.
Preferably, step S1 ensures that ipscs are stably propagated for more than 2 generations before each differentiation.
Preferably, in step S2, AggreWell is usedTMThe EB is prepared by 800 culture plates in the following specific method: removing a culture medium of ESC cells cultured by a 6-hole plate and with the cell density reaching 90%, adding 1ml of D-PBS, washing once, removing the D-PBS, adding 1ml of tryple into the washed cell culture holes, placing the cells into a 37 ℃, digesting in a CO2 incubator for 4 minutes, observing the cells under a microscope after digestion to have obvious drop traces, adding 3ml of D-PBS into the culture holes to neutralize digestive juice, slightly purging the cells which do not drop by using a pipette and a gun head, transferring the cells into a 15ml centrifuge tube, and centrifuging for 3 minutes by 400 g;
after centrifugation, the supernatant was discarded and cultured with 1mlE8Resuspending the nutrient medium, taking a cell suspension containing 40-80 ten thousand cells, and adding AggreWellTMAdding E8 culture medium to 2ml in 800 culture plate, adding 4ul iROCK, mixing with pipette (taking care not to generate bubbles), sealing with sealing film plate, centrifuging at 400g for 30s, tearing off sealing film, and mixing AggreWellTMThe 800 plates were incubated at 37 ℃ in a CO2 incubator for 1 day.
Preferably, the step S3 is AggreWell in the step S2TM800 plates were incubated for one day.
Preferably, the EB differentiation medium ii in step S4 is specifically: the combination of the cytokines and their concentrations in the stemline II medium plus 10-30ng/mL hSCF,10-30ng/mL hFlt3L and 20-40ng/mL TPO, together with the stemline II medium, was able to differentiate mesodermal cells into CD34+CD43+The hematopoietic progenitor cells of (1).
Preferably, the EB differentiation medium iii in step S5 is: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL IL-15, 10-30ng/mL hSCF,10-30ng/mL hFlt3L plus stemline II medium enables the production of CD34+CD43+Directed differentiation of hematopoietic progenitor cells into CD4+A non-T immune cell.
Advantageous effects
Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:
the invention differentiates iPSC from human blood cells into CD4 in a specific culture medium and under the combined culture of different cytokines+non-T immune cells, expressing CD4 in humans+Mainly including BDCA4+Dendritic Cells (DC), CD33+Myeloid cells and CD14+The monocyte of (B) has a very important immunoregulation function, BDCA4+DC cells are a subset of Plasmacytoid DCs (pDCs) which are differentiated from common lymphoid progenitors and have a reduced antigen presenting capacity compared to common myeloid progenitor derived cDCs, but which express TLR7 and TLR9 which recognize pathogen-associated molecular patterns (PAMPs) on DNA and RNA viruses, respectively, and activate NF-B related signal path, and secreting type I interferon and various cytokines, thereby activating NK cells and macrophages to play immune monitoring function on microorganisms, CD33+Myeloid cells are a class of Myeloid cells that can be further differentiated into monocytes, NK cell subsets, tissue macrophages and the like, CD14+The monocyte can be further differentiated into DC cells to become a good raw material of the DC vaccine.
Drawings
FIG. 1 is a morphological diagram of the 1iPSC cell in each stage of differentiation after EB production;
FIG. 2 is a graph showing the content of FITC-CD34, APC-CD43 in suspension hematopoietic progenitor cells derived from cells of the present invention by differentiation to day 18;
FIG. 3 is a graph of the fraction of cells expressing CD45 in the present invention measuring the amount of cells expressing FITC-CD45 and APC-CD 56;
FIG. 4 is a graph of the ratio of the content of cells expressing FITC-CD3 and APC-CD4 to total cells measured in accordance with the present invention;
FIG. 5 is a graph showing the detection of the content of cells expressing FITC-CD3 and APC-CD8 according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. 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.
The present invention will be further described with reference to the following examples.
Example 1
As shown in FIGS. 1-5, a method for differentiating CD4 from induced pluripotent stem cells+A method of non-T cells, comprising the steps of:
s1, maintenance culture of PSC cells: the iPSC is maintained and cultured by a commercial E8 culture medium, an E8 culture medium contains factors such as L-ascorbic acid, transferrin, insulin, FGF2 and TGF beta 1, the totipotency of the iPSC can be well maintained, and the iPSC is cultured in a 6-well plate containing Matrigel;
s2, EB (embryoid body): the embryoid body is a spherical cell aggregate formed by embryonic stem cells or iPSC cells under a certain condition in vitro, has the capacity of differentiating ectoderm, mesoderm and endoderm similar to embryos, and has faster differentiation of target cells from EB (embryonic Stem) and higher purity;
s3, differentiation of EBs into mesoderm: AggreWell is preparedTMThe EB balls in the 800 culture plate are resuspended, sucked into a 15mL centrifuge tube, kept stand for 3min, the supernatant is carefully discarded, 1mL of EB differentiation culture medium I is used for resuspending (the EB differentiation culture medium I adopts a stemline II culture medium and is added with 20-50ng/mL hBMP-4,10-30ng/mL hbFGF and 20-50ng/mL VEGF, not only can the EB structure be well maintained, but also the EB can be directionally differentiated to mesoderm which is the origin of hematopoietic stem cells from various immune cells), the EB balls are transferred to an ultra-low adsorption 6-well plate after resuspension, and are differentiated for two days by the EB differentiation culture medium I;
s4, differentiation of mesoderm to CD34+The hematopoietic progenitor cells of (a): coating a 6-hole plate with Matrigel glue after two days in the steps, adding 15ul of F12 culture medium into each hole, uniformly mixing, placing the plate into a 37 ℃ CO2 incubator for 1h, removing the F12 culture medium after coating, adding 2ml of EB differentiation culture medium II into each hole, collecting EB balls in an ultra-low adsorption plate, removing supernatant, transferring the EB balls into the culture plate and the culture medium, culturing 40 EB balls in each hole for 14 days, and replacing a new EB differentiation culture medium II every 3 days;
s5, differentiation CD34+To CD4+non-T immune cells: and (3) removing the culture medium of the culture system, continuously differentiating the rest cells by using an EB differentiation culture medium III, replacing a new EB differentiation culture medium III every two days, and after differentiating for 8 days, carrying out flow detection on the expression condition of the cells CD45 CD56CD3 CD8 CD 4.
Wherein, the step S1 ensures that the iPSC is stably transmitted for more than 2 generations before each differentiation.
In step S2, AggreWell is usedTM800 preparation of culture plate EThe specific method comprises the following steps: removing a culture medium of ESC cells cultured by a 6-hole plate and with the cell density reaching 90%, adding 1ml of D-PBS, washing once, removing the D-PBS, adding 1ml of tryple into the washed cell culture holes, placing the cells into a 37 ℃, digesting in a CO2 incubator for 4 minutes, observing the cells under a microscope after digestion to have obvious drop traces, adding 3ml of D-PBS into the culture holes to neutralize digestive juice, slightly purging the cells which do not drop by using a pipette and a gun head, transferring the cells into a 15ml centrifuge tube, and centrifuging for 3 minutes by 400 g;
centrifuging, removing supernatant, re-suspending with 1mlE8 culture medium, collecting cell suspension containing 40-80 ten thousand cells, and adding AggreWellTMAdding E8 culture medium to 2ml in 800 culture plate, adding 4ul iROCK, mixing with pipette (no bubble generation), sealing membrane sealing plate, centrifuging at 400g for 30s, tearing off sealing membrane, and mixing AggreWellTMThe 800 plates were incubated at 37 ℃ in a CO2 incubator for 1 day.
Wherein the step S3 is AggreWell in the step S2TM800 plates were incubated for one day.
Wherein, the EB differentiation medium II in the step S4 specifically comprises: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL hSCF,10-30ng/mL hFlt3L and 20-40ng/mL TPO was used to differentiate mesodermal cells into CD34+CD43+The hematopoietic progenitor cells of (1).
Wherein the EB differentiation medium III in the step S5 is: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL IL-15, 10-30ng/mL hSCF,10-30ng/mL hFlt3L plus stemline II medium enables the production of CD34+CD43+Directed differentiation of hematopoietic progenitor cells into CD4+A non-T immune cell.
Comparative example 1
This example is substantially the same as the method of example 1 provided, with the main differences being: in step S1, stable transmission of iPSC for more than 2 generations is not ensured before each differentiation;
comparative example 2
This example is substantially the same as the method of example 1 provided, with the main differences being: in step S3, EB balls were not differentiated with EB differentiation medium I for two days.
Comparative example 3
This example is substantially the same as the method of example 1 provided, with the main differences being: step S4 was not performed by changing a new EB differentiation medium II every 3 days.
Performance testing
The differentiation of CD4 from induced pluripotent stem cells provided in example 1 and comparative examples 1 to 3 was performed in the same amount, respectively+CD4 of non-T cell method+Number of non-T cells:
CD4+number of non-T cells
Example 1 99％
Comparative example 1 87％
Comparative example 2 73％
Comparative example 3 65％
By analyzing the relevant data in the above tables, as shown in FIGS. 1 to 5, a method for differentiating CD4 from induced pluripotent stem cells+A method of non-T cells, comprising the steps of:
s1, maintenance culture of PSC cells: the iPSC is maintained and cultured by a commercial E8 culture medium, an E8 culture medium contains factors such as L-ascorbic acid, transferrin, insulin, FGF2 and TGF beta 1, the totipotency of the iPSC can be well maintained, and the iPSC is cultured in a 6-well plate containing Matrigel;
s2, EB (embryoid body): the embryoid body is a spherical cell aggregate formed by embryonic stem cells or iPSC cells under a certain condition in vitro, has the capacity of differentiating ectoderm, mesoderm and endoderm similar to embryos, and has faster differentiation of target cells from EB (embryonic Stem) and higher purity;
s3, differentiation of EBs into mesoderm: AggreWellTMThe EB balls in the 800 culture plate are resuspended, sucked into a 15mL centrifuge tube, kept stand for 3min, the supernatant is carefully discarded, 1mL of EB differentiation culture medium I is used for resuspending (the EB differentiation culture medium I adopts a stemline II culture medium and is added with 20-50ng/mL hBMP-4,10-30ng/mL hbFGF and 20-50ng/mL VEGF, not only can the EB structure be well maintained, but also the EB can be directionally differentiated to mesoderm which is the origin of hematopoietic stem cells from various immune cells), the EB balls are transferred to an ultra-low adsorption 6-well plate after resuspension, and are differentiated for two days by the EB differentiation culture medium I;
s4, differentiation of mesoderm to CD34+The hematopoietic progenitor cells of (a): coating a 6-hole plate with Matrigel glue after two days in the steps, adding 15ul of F12 culture medium into each hole, uniformly mixing, placing the plate into a 37 ℃ CO2 incubator for 1h, removing the F12 culture medium after coating, adding 2ml of EB differentiation culture medium II into each hole, collecting EB balls in an ultra-low adsorption plate, removing supernatant, transferring the EB balls into the culture plate and the culture medium, culturing 40 EB balls in each hole for 14 days, and replacing a new EB differentiation culture medium II every 3 days;
s5, differentiation CD34+To CD4+non-T immune cells: and (3) removing the culture medium of the culture system, continuously differentiating the rest cells by using an EB differentiation culture medium III, replacing a new EB differentiation culture medium III every two days, and after differentiating for 8 days, carrying out flow detection on the expression condition of the cells CD45 CD56CD3 CD8 CD 4.
Wherein, the step S1 ensures that the iPSC is stably transmitted for more than 2 generations before differentiation.
WhereinIn the step S2, AggreWell is usedTMThe EB is prepared by 800 culture plates in the following specific method: removing a culture medium of ESC cells cultured by a 6-hole plate and with the cell density reaching 90%, adding 1ml of D-PBS, washing once, removing the D-PBS, adding 1ml of tryple into the washed cell culture holes, placing the cells into a 37 ℃, digesting in a CO2 incubator for 4 minutes, observing the cells under a microscope after digestion to have obvious drop traces, adding 3ml of D-PBS into the culture holes to neutralize digestive juice, slightly purging the cells which do not drop by using a pipette and a gun head, transferring the cells into a 15ml centrifuge tube, and centrifuging for 3 minutes by 400 g;
after centrifugation, the supernatant was discarded and resuspended in 1mlE8 medium, and a cell suspension containing 40-80 ten thousand cells was taken and added to AggreWellTMAdding E8 culture medium to 2ml in 800 culture plate, adding 4ul iROCK, mixing with pipette (taking care not to generate bubbles), sealing with sealing film plate, centrifuging at 400g for 30s, tearing off sealing film, and mixing AggreWellTM800 plates were incubated for 1 day at 37 ℃ in a CO2 incubator.
Wherein the step S3 is AggreWell in the step S2TM800 plates were incubated for one day.
Wherein, the EB differentiation medium II in the step S4 is specifically: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL hSCF,10-30ng/mL hFlt3L and 20-40ng/mL TPO was used to differentiate mesodermal cells into CD34+CD43+The hematopoietic progenitor cells of (1).
Wherein the EB differentiation medium III in the step S5 is: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL IL-15, 10-30ng/mL hSCF,10-30ng/mL hFlt3L plus stemline II medium enables the production of CD34+CD43+Directed differentiation of hematopoietic progenitor cells into CD4+A non-T immune cell.
In this example: the invention differentiates iPSC from human blood cells into CD4 in a specific culture medium and under the combined culture of different cytokines+non-T immune cells, expressing CD4 in humans+Mainly including BDCA4+Dendritic Cells (DC), CD33+Myeloid cells and CD14+The monocyte of (B) has a very important immunoregulation function, BDCA4+DC cells are a subset of Plasmacytoid DCs (pDCs), which are DC cells differentiated from common lymphoid progenitor cells and have a weaker antigen presenting ability than cDCs derived from common myeloid progenitor cells, but TLR7 and TLR9 expressed by pDCs recognize pathogen-associated molecular patterns (PAMP) on DNA and RNA viruses, activate NF-B-associated signaling pathways, and secrete type I interferons and various cytokines, thereby activating NK cells and macrophages to exert immune surveillance function on microorganisms, and CD33+Myeloid cells are a class of Myeloid cells that can be further differentiated into monocytes, NK cell subsets, tissue macrophages and the like, CD14+The monocyte can be further differentiated into DC cells to become a good raw material of the DC vaccine.
In the cell culture process, the form diagrams of iPSC cells at each stage after EB preparation and differentiation are shown in figure 1;
by day 18, the suspended hematopoietic progenitor cells were flow assayed for FITC-CD34, APC-CD 43. Its CD34+CD43+The cell content of (a) was 38.87% of the total cell number. As shown in particular in fig. 2.
Measuring the content of cells expressing FITC-CD45 and APC-CD56 at day 26 of differentiation, wherein the cells expressing CD45 accounted for 99.24% of the total cell count, as shown in FIG. 3;
detecting the content of cells expressing FITC-CD3 and APC-CD4, wherein the cells expressing CD4 account for 65.52 percent of the total cells, and are shown in a figure 4;
detecting the content of cells expressing FITC-CD3 and APC-CD8, as shown in FIG. 5;
as shown in FIGS. 3,4 and 5, almost none of the cells expressed markers for NK and T cells such as CD56, CD3 and CD8, and the majority of the cells differentiated from them were judged to be CD4+A non-T immune cell.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (6)

1. Differentiation of CD4 from induced pluripotent stem cells+A method of non-T cells, comprising the steps of:
s1, maintenance culture of PSC cells: the iPSC is maintained and cultured by a commercial E8 culture medium, an E8 culture medium contains factors such as L-ascorbic acid, transferrin, insulin, FGF2 and TGF beta 1, the totipotency of the iPSC can be well maintained, and the iPSC is cultured in a 6-well plate coated by Matrigel;
s2, EB (embryoid body): the embryoid body is a spherical cell aggregate formed by embryonic stem cells or iPSC cells under a certain condition in vitro, has the capacity of differentiating ectoderm, mesoderm and endoderm similar to embryos, and has faster differentiation of target cells from EB (embryonic Stem) and higher purity;
s3, differentiation of EBs into mesoderm: AggreWell is preparedTMThe EB balls in the 800 culture plate are resuspended, sucked into a 15mL centrifuge tube, kept stand for 3min, the supernatant is carefully discarded, 1mL of EB differentiation culture medium I is used for resuspending (the EB differentiation culture medium I adopts a stemline II culture medium and is added with 20-50ng/mL hBMP-4,10-30ng/mL hbFGF and 20-50ng/mL VEGF, not only can the EB structure be well maintained, but also the EB can be directionally differentiated to mesoderm which is the origin of hematopoietic stem cells from various immune cells), the EB balls are transferred to an ultra-low adsorption 6-well plate after resuspension, and are differentiated for two days by the EB differentiation culture medium I;
s4, differentiation of mesoderm to CD34+The hematopoietic progenitor cells of (a): coating 6-well plate with Matrigel gel two days later, adding 2ml of F12 culture medium 15ul of Matrigel gel into each well, mixing, placing at 37 deg.C, coating in CO2 incubator for 1h, discarding F12 culture medium after coating, adding 2ml of EB differentiation culture medium II into each well, collecting EB balls in ultra-low adsorption plate, discarding supernatant, transferring to above culture plate and culture medium, 40 EB balls per well, and culturingCulturing for 14 days under the system, and replacing a new EB differentiation culture medium II every 3 days;
s5, differentiation CD34+To CD4+non-T immune cells: and (3) removing the culture medium of the culture system, continuously differentiating the rest cells by using the EB differentiation medium III, replacing the EB differentiation medium III with new EB differentiation medium III every two days, and carrying out flow detection on the expression condition of the cells CD45 CD56CD3 CD8 CD4 after 8 days of differentiation.
2. The method of claim 1, wherein the differentiation of CD4 from induced pluripotent stem cells is performed by+A method of non-T cells, characterized by: the step S1 ensures stable iPSC transfer for more than 2 generations before each differentiation.
3. The method of claim 1, wherein the induced pluripotent stem cells are differentiated into CD4+A method of non-T cells, characterized by: in step S2, AggreWell is usedTMThe EB is prepared by 800 culture plates in the following specific method: removing a culture medium of ESC cells cultured by a 6-hole plate and with the cell density reaching 90%, adding 1ml of D-PBS, washing once, removing the D-PBS, adding 1ml of tryple into the washed cell culture holes, placing the cells into a 37 ℃, digesting in a CO2 incubator for 4 minutes, observing the cells under a microscope after digestion to have obvious drop traces, adding 3ml of D-PBS into the culture holes to neutralize digestive juice, slightly purging the cells which do not drop by using a pipette and a gun head, transferring the cells into a 15ml centrifuge tube, and centrifuging for 3 minutes by 400 g;
centrifuging, removing supernatant, re-suspending with 1mlE8 culture medium, collecting cell suspension containing 40-80 ten thousand cells, and adding AggreWellTMAdding E8 culture medium to 2ml in 800 culture plate, adding 4ul iROCK, mixing with pipette (taking care not to generate bubbles), sealing with sealing film plate, centrifuging at 400g for 30s, tearing off sealing film, and mixing AggreWellTMThe 800 plates were incubated at 37 ℃ in a CO2 incubator for 1 day.
4. The method of claim 1, wherein the differentiation of CD4 from induced pluripotent stem cells is performed by+A method of non-T cells, characterized by: the step S3 is in step S2AggreWellTM800 plates were incubated for one day.
5. The method of claim 1, wherein the induced pluripotent stem cells are differentiated into CD4+A method of non-T cells, characterized by: the EB differentiation culture medium II in the step S4 is specifically as follows: the combination of the cytokines and their concentrations in the presence of stemline II medium plus 10-30ng/mL hSCF,10-30ng/mL hFlt3Land 20-40ng/mL TPO, together with stemline II medium, was able to differentiate mesodermal cells into CD34+CD43+The hematopoietic progenitor cells of (1).
6. The method of claim 1, wherein the induced pluripotent stem cells are differentiated into CD4+A method for non-T cells, wherein EB differentiation medium iii in step S5 is: the combination of the cytokines and their concentrations in stemline II medium plus 10-30ng/mL IL-15, 10-30ng/mL hSCF,10-30ng/mL hFlt3L plus stemline II medium enables the production of CD34+CD43+Directed differentiation of hematopoietic progenitor cells into CD4+A non-T immune cell.
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Publication number Priority date Publication date Assignee Title
CN115247152A (en) * 2022-09-21 2022-10-28 呈诺再生医学科技(北京)有限公司 Method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells and method for culturing long-term regenerative hematopoietic stem cells

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115247152A (en) * 2022-09-21 2022-10-28 呈诺再生医学科技(北京)有限公司 Method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells and method for culturing long-term regenerative hematopoietic stem cells

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