CN115992094B - Method for inducing hematopoietic stem cells by low-density monolayer and application - Google Patents
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a method for inducing hematopoietic stem cells by a low-density monolayer and application thereof. The 2D multilayer three-dimensional bionic 'hematopoietic stem cells' structure induced by the method has high yield and stable positive rate of more than 50%, has the potential of multidirectional differentiation, can realize the large-scale induction production, avoids the use of feeder cells and heterologous matrigel in the induction process, and is beneficial to clinical transformation.
Description
Technical Field
The invention belongs to the technical field of stem cell biology, and particularly relates to a method for inducing hematopoietic stem cells by a low-density monolayer and application thereof.
Background
Hematopoietic stem cells are adult stem cells in the blood system, and have the ability to self-renew for a long period of time and the potential to differentiate into various mature blood cells. The hematopoietic stem cells commonly used clinically at present mainly originate from umbilical cord blood and bone marrow, but have the following disadvantages: 1. limited availability of materials, low hematopoietic stem cell occupancy and limited number; 2. immunological rejection phenomenon exists in clinical use; 3. individual differences exist among different donors, and cell quality differences affect the therapeutic effect.
The human pluripotent stem cells have unlimited proliferation capacity and multidirectional differentiation potential, a large number of human pluripotent stem cells can be stably obtained by in-vitro large-scale culture and used as seeds, and the differentiation potential can induce the formation of hematopoietic stem cells. The current method for differentiating human pluripotent stem cells into hematopoietic stem cells mainly comprises the following steps: firstly, forming embryoid bodies to induce and differentiate into hematopoietic stem cells, but the induction efficiency is unstable, the operation is complex, and the embryoid bodies have the problems of heterogeneity and the like; and secondly, the heterogeneous stromal cells such as OP9, MEF and the like are co-cultured with the human pluripotent stem cells to simulate the hematopoietic microenvironment to induce and differentiate into hematopoietic stem cells, but the heterogeneous stromal cells are required to be relied on for inducing and differentiating, so that the clinical use has the safety problem. And the quantity is limited, the cell development is not mature enough, and the clinical use of the cells is not facilitated.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects in the prior art, and proposes a method and application of low-density monolayer induced hematopoietic stem cells.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a method for inducing hematopoietic stem cells by ultra-low density monolayer, comprising the steps of:
s1: preparing human pluripotent stem cells into cell suspension by using induced differentiation medium A according to 2×10 4 -4×10 4 /cm 2 Inoculating the culture medium into a cell culture container at the density of 0 th day, and performing low-oxygen low-density monolayer induction on the culture medium by using half-amount liquid exchange of the induction differentiation culture medium A every two days on the 0 th day to the 4 th day;
s2: half liquid exchange is carried out every two days from the 4 th day to the 6 th day by using the induced differentiation culture medium B, the induced differentiation culture medium A is replaced by the induced differentiation culture medium B, and the induction is converted into normoxic induction;
s3: half liquid exchange is carried out every two days from the 6 th day to the 15 th day by using the induced differentiation culture medium C, the induced differentiation culture medium B is replaced by the induced differentiation culture medium C, and normal oxygen induction is carried out, so that the 2D multilayer three-dimensional bionic 'hematopoietic cell' is finally obtained.
Wherein, hypoxia induction refers to induction under the environment of 2% -8% oxygen content, and normoxic induction refers to induction under the environment of the same oxygen content as the atmosphere.
In some embodiments of the invention, the induced differentiation medium A comprises one or more of GSK3 beta inhibitor, BMP signaling pathway activator, VEGF, SCF, bFGF, IGF-1.
In some embodiments of the invention, the induced differentiation medium A is a basal induced differentiation medium supplemented with one or more of GSK3 beta inhibitor, BMP signaling pathway activator, VEGF, SCF, bFGF, IGF-1.
In some embodiments of the invention, the GSK3 beta inhibitor concentration in the induced differentiation medium A is 1-10uM, the BMP signaling pathway activator concentration is 1-100ng/ml, the VEGF concentration is 1-100ng/ml, the SCF concentration is 1-200ng/ml, the bFGF concentration is 1-50ng/ml, and the IGF-1 concentration is 1-200ng/ml.
In some embodiments of the invention, the induced differentiation medium B comprises one or more of TGF-beta inhibitor, BMP4, VEGF, bFGF, SCF, IGF-1.
In some embodiments of the invention, the induced differentiation medium B is a basal induced differentiation medium supplemented with one or more of TGF-beta inhibitor, BMP4, VEGF, bFGF, SCF, IGF-1.
In some embodiments of the invention, the TGF- β inhibitor is present in the induced differentiation medium B at a concentration of 1-10uM, BMP4 at a concentration of 1-100ng/ml, VEGF at a concentration of 1-100ng/ml, bFGF at a concentration of 1-50ng/ml, SCF at a concentration of 1-200ng/ml, and IGF-1 at a concentration of 1-200ng/ml.
In some embodiments of the invention, the induced differentiation medium C comprises one or more of UM729, BMP4, VEGF, bFGF, SCF, flt3L, IL3, IL6, IGF-1.
In some embodiments of the invention, the induced differentiation medium C is a basal induced differentiation medium supplemented with one or more of UM729, BMP4, VEGF, bFGF, SCF, flt3L, IL3, IL6, IGF-1.
In some embodiments of the invention, UM729 is present in the induced differentiation medium C at a concentration of 100-900nM, BMP4 is present at a concentration of 1-100ng/ml, VEGF is present at a concentration of 1-100ng/ml, bFGF is present at a concentration of 1-50ng/ml, SCF is present at a concentration of 1-200ng/ml, flt3L is present at a concentration of 1-100ng/ml, IL3 is present at a concentration of 1-50ng/ml, IL6 is present at a concentration of 1-50ng/ml, IGF-1 is present at a concentration of 1-200ng/ml.
In some embodiments of the invention, the basal induced differentiation medium contains IMDM medium 40% -50% v/v, F12 medium 40% -50% v/v, human serum albumin 0.5-20mg/ml, alpha-MTG 0-1000uM, transferrin 10-50ug/ml, ascorbic acid 10-50ug/ml, sodium selenite 5-50ng/ml, ethanolamine 5-100uM, long R3 IGF-1-100 ng/ml, glutamaxI 2mM, polyvinyl alcohol (PVA) 0-50ng/ml, protein-free hybridoma cell medium (PFHMII) 5% v/v, ferric citrate 0.5-5ug/ml.
The invention also provides the 2D multilayer three-dimensional bionic 'hematopoietic cell' prepared by the method. The invention also provides application of the 2D multilayer three-dimensional bionic 'hematopoietic cell' differentiated progenitor cells (hematopoietic stem progenitor cells, lymphoid progenitor cells, erythroid progenitor cells and the like) to differentiating NK cells, macrophages or erythrocytes through the progenitor cells.
In some embodiments of the invention, the method of differentiating the 2D multi-layered stereoscopic biomimetic "hematopoietic cells" into NK cells comprises the steps of:
performing normoxic induction after replacing the induction differentiation culture medium C with the NK induction culture medium A on the 15 th day to the 20 th day;
half liquid exchange is carried out every five days from 20 days to 30 days by using an NK induction culture medium B, the NK induction culture medium A is replaced by the NK induction culture medium B, and normal oxygen induction is carried out;
half-volume liquid exchange is carried out every three days from 30 days to 50 days by using NK maturation medium, NK induction medium B is replaced by NK maturation medium, and normoxic induction is carried out.
In some embodiments of the invention, the NK induction medium A is one or more of IL3, IL7, IL15, SCF and Flt3L added to the basal induced differentiation medium.
In some embodiments of the invention, the NK induction medium A has a concentration of IL3 of 1-100ng/ml, IL7 of 1-100ng/ml, IL15 of 1-100ng/ml, SCF of 1-100ng/ml and Flt3L of 1-100ng/ml.
In some embodiments of the invention, the NK induction medium B is one or more of IL7, IL15, SCF and Flt3L added to the basal induced differentiation medium.
In some embodiments of the invention, the NK induction medium has a concentration of IL7 of 1-100ng/ml, IL15 of 1-100ng/ml, SCF of 1-100ng/ml and Flt3L of 1-100ng/ml.
In some embodiments of the invention, the NK maturation medium is one or more of IL7, IL15, SCF, flt3L, UM729, IL2, IL21, IL12, IL18 added to the basal induced differentiation medium.
In some embodiments of the invention, the NK induction medium has IL7 concentration of 1-100ng/ml, IL15 concentration of 1-100ng/ml, SCF concentration of 1-100ng/ml, flt3L concentration of 1-100ng/ml, UM729 concentration of 100-900nM, IL2 concentration of 1-1000U/ml, IL21 concentration of 1-100ng/ml, IL12 concentration of 1-100ng/ml, IL18 concentration of 1-100ng/ml.
In some embodiments of the invention, the method of differentiating the 2D multilayer stereoscopic biomimetic "hematopoietic cells" into macrophages comprises the steps of:
on the 15 th day, the induced differentiation medium C is replaced by a macrophage induced medium, and normal oxygen induction is carried out;
half-volume exchange of the macrophage maturation medium was performed every seven days from 15 th day to 36 th day, the macrophage induction medium was exchanged for the macrophage maturation medium, and normoxic induction was performed.
In some embodiments of the invention, the macrophage induction medium is an addition of IL3 and/or M-CSF to basal induced differentiation medium.
In some embodiments of the invention, the concentration of IL3 in the macrophage induction medium is 1-100ng/ml and the concentration of M-CSF is 1-200ng/ml.
In some embodiments of the invention, the macrophage maturation medium is supplemented with M-CSF in a basal induced differentiation medium.
In some embodiments of the invention, the concentration of M-CSF in the macrophage induction medium is in the range of 1-200ng/ml.
In some embodiments of the invention, the method of differentiating the 2D multilayer stereoscopic biomimetic "hematopoietic cells" into erythrocytes comprises the steps of:
half-volume liquid exchange is carried out on the red blood cell induction culture medium every three days from 15 th day to 28 th day, the induction differentiation culture medium C is replaced by the red blood cell induction culture medium, and normal oxygen induction is carried out;
half-volume exchange of the red blood cell maturation medium is performed every three days from 28 th day to 35 th day, the red blood cell induction medium is replaced by the red blood cell maturation medium, and normoxic induction is performed.
In some embodiments of the invention, the red blood cell induction medium is one or more of FICZ, IL6, SCF, EPO, IL3 added to the basal induced differentiation medium.
In some embodiments of the invention, the concentration of FICZ in the red blood cell induction medium is 0.1-1uM, the concentration of IL6 is 1-50ng/ml, the concentration of SCF is 1-100ng/ml, the concentration of EPO is 1-100U/ml, and the concentration of IL3 is 1-50ng/ml.
In some embodiments of the invention, the erythrocyte maturation medium is one or more of mifepristone, transferrin and EPO added to the basal induced differentiation medium.
In some embodiments of the invention, the concentration of mifepristone in the red blood cell maturation medium is 0.5-5uM, the concentration of transferrin is 1-1000ug/ml, and the concentration of EPO is 1-100U/ml.
Compared with the prior art, the invention has the following advantages:
the invention induces a 2D multilayer three-dimensional bionic 'hematopoietic cell' structure by using a monolayer ultra-low density human pluripotent stem cell, wherein the diameter of the hematopoietic cell is controlled to be 300-500 microns, the three-dimensional structure is loosely connected or suspended with a bottom stromal cell, a natural hematopoietic system is simulated, and the diameter of 300-500 microns is beneficial to the permeation of nutritional ingredients and prolongs the production time of hematopoietic progenitor cells. While maintaining a loose structure, facilitating the shedding of the progenitor cells produced from the sphere.
The hematopoietic stem cells from the hematopoietic cell sources have high yield and stable positive rate of more than 50 percent, belong to the potential of determining that hematopoietic cells have multidirectional differentiation, and can realize the induction scale production. The induction process avoids the use of feeder cells and heterologous matrigel, and is beneficial to clinical transformation.
Drawings
FIG. 1 is a cell morphology diagram of examples 1-3 and comparative examples 1-2 on the first, third, fifth, sixth, ninth and thirteenth days;
FIG. 2 is a phenotype test plot of hematopoietic mesodermal cells of examples 1-3 and comparative examples 1-2 on day four;
FIG. 3 is a phenotype test chart of hematopoietic stem/progenitor cells of examples 1-3 and comparative examples 1-2 on the sixth day;
FIG. 4 is a phenotype test chart of examples 1-3 and comparative examples 1-2 on a sixth day of blood endothelial cells;
FIG. 5 is a phenotype test chart of hematopoietic stem/progenitor cells of examples 1-3 and comparative examples 1-2 on thirteenth day;
FIG. 6 is a phenotype test chart of red marrow progenitor cells and lymphoid progenitor cells of examples 1-3 and comparative examples 1-2 on thirteenth day;
FIG. 7 is an immunofluorescence of CD34+ marker of hematopoietic stem cells at day six of example 1;
FIG. 8 is an immunofluorescence of CD34+ marker of hematopoietic stem cells of example 1 on thirteenth day;
FIG. 9 is a cell morphology graph of the NK cell induction group of example 4 on forty-day;
FIG. 10 is a graph of flow results of NK cell induction group of example 4 on forty-day;
FIG. 11 is a cell morphology graph of the macrophage induction group of example 4 on the thirty-fourth day;
FIG. 12 is a graph of flow results for the macrophage induction group of example 4 at day thirty-fourth;
FIG. 13 is a cell morphology diagram of the red blood cell-induced group of example 4 on the thirty-fifth day;
FIG. 14 is a graph of flow results on day twenty-second for the red blood cell-induced group of example 4;
FIG. 15 is a graph of the beta, gamma, epsilon-globin RNA expression levels on day nine, fifteenth, and twenty-second days of the red blood cell-induced group of example 4.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
In the following examples and comparative examples:
(1) Flow cytometry detection during induced differentiation
(1) Hematopoietic mesoderm and hematogenesis endothelial detection: anti-CD326, anti-CD56, anti-CD31, anti-CD144;
(2) hematopoietic stem progenitor cell detection: anti-CD34, anti-CD43, anti-CD45, anti-CD235a;
(3) NK cell detection: anti-CD3, anti-CD56, anti-CD16, anti-NKG2D, anti-NKG2A, anti-NKG 30, etc.;
(4) macrophage detection: anti-CD14, anti-CD206, anti-CD80, etc.;
(5) erythrocyte progenitor detection: anti-CD235, anti-CD71, etc.
(2) Erythrocyte globin qPCR detection
The primers used for the globin qPCR assay were obtained from PrimerBank website (https:// pga.mgh.harvard.edu/PrimerBank /), the primer ID of b-globin was 28302128c1 and the primer ID of ɛ -globin was 28302129c1. The r-globin primer was obtained from the GetPrime website (https:// gecftools. Epfl. Ch/getPrime /), ID 1957102. (3) basal induced differentiation medium composition the following table:
TABLE 1 composition of basal induced differentiation medium
The present invention will be described in detail with reference to examples.
Example 1
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Based on the result of cell counting, appropriate amount of cells were taken and prepared into a cell suspension using an induced differentiation medium A in which 5uM CHIR-99021, 20ng/ml BMP4, 20ng/ml VEGF, 40ng/ml SCF, 2ng/ml bFGF,40ng/ml IGF-1 were added to the basal induced differentiation medium of Table 1 at a rate of 2.2X10 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-volume liquid exchange is carried out every two days by using an induced differentiation culture medium A, and the liquid exchange is carried out at 37 ℃ and 5% O 2 ，5% CO 2 Induction was performed in a hypoxia incubator.
(4) Day4-6: half-volume change of liquid was performed every two days using the induced differentiation medium B, which was prepared by adding 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1 to the basal induced differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
(5) Day6-15: half liquid exchange is carried out every two days, the process generates 2D multilayer three-dimensional bionic 'hematopoietic cells', hematopoietic stem progenitor cells exist in culture supernatant, and the specific operation process of half liquid exchange is as follows: collecting half culture supernatant of step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half induction differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729, and placing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
Example 2
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Based on the result of cell counting, appropriate amount of cells were taken and prepared into a cell suspension using an induced differentiation medium A in which 5uM CHIR-99021, 20ng/ml BMP4, 20ng/ml VEGF, 40ng/ml SCF, 2ng/ml bFGF,40ng/ml IGF-1 were added to the basal induced differentiation medium of Table 1 at a rate of 2.8X10 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-volume liquid exchange is carried out every two days by using an induced differentiation culture medium A, and the liquid exchange is carried out at 37 ℃ and 5% O 2 ，5% CO 2 Induction was performed in a hypoxia incubator.
(4) Day4-6: half-volume change of liquid was performed every two days using the induced differentiation medium B, which was prepared by adding 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1 to the basal induced differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Is induced by the constant oxygen incubator.
(5) Day6-15: half liquid exchange is carried out every two days, the process generates 2D multilayer three-dimensional bionic 'hematopoietic cells', hematopoietic stem progenitor cells exist in culture supernatant, and the specific operation process of half liquid exchange is as follows: collecting half amount of culture supernatant obtained in step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half amount of induced differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729 is added to the basal induced differentiation medium of Table 1, and the mixture is placed at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
Example 3
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Based on the cell count result, appropriate amount of cells were taken and prepared into cell suspension using induced differentiation medium A, which was prepared by adding 5uM CHIR-99021, 20ng/ml BMP4, 20ng to the basal induced differentiation medium of Table 1VEGF/ml, SCF 40ng/ml, bFGF 2ng/ml, IGF-1 40ng/ml, 3.3X10 g 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-volume liquid exchange is carried out every two days by using an induced differentiation culture medium A, and the liquid exchange is carried out at 37 ℃ and 5% O 2 ，5% CO 2 Induction is performed in a hypoxia incubator.
(4) Day4-6 was half-changed every two days using the induced differentiation medium B, which was prepared by adding 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1 to the basal induced differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
(5) Day6-15: half liquid exchange is carried out every two days, the process generates 2D multilayer three-dimensional bionic 'hematopoietic cells', hematopoietic stem progenitor cells exist in culture supernatant, and the specific operation process of half liquid exchange is as follows: collecting half amount of culture supernatant obtained in step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half amount of induced differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729, and placing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
Comparative example 1
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Based on the result of cell counting, appropriate amount of cells were taken and prepared into a cell suspension using an induced differentiation medium A in which 5uM CHIR-99021, 20ng/ml BMP4, 20ng/ml VEGF, 40ng/ml SCF, 2ng/ml bFGF,40ng/ml IGF-1 was added to the basal induced differentiation medium of Table 1 at a rate of 1.6X10 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-dose change every two days using induced differentiation medium ALiquid and placed at 37 ℃ and 5% O 2 ，5% CO 2 Induction was performed in a hypoxia incubator.
(4) Day4-6: half-volume change of liquid was performed every two days using the induced differentiation medium B, which was prepared by adding 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1 to the basal induced differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
(5) Day6-15: half liquid exchange is carried out every two days, and due to low cell density, hematopoietic stem progenitor cells exist in culture supernatant in the process, but no 2D multilayer three-dimensional bionic 'hematopoietic cells' are produced, and the specific operation process of half liquid exchange is as follows: collecting half amount of culture supernatant obtained in step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half amount of induced differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729, and placing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
Comparative example 2
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Based on the result of cell counting, appropriate amount of cells were taken and prepared into a cell suspension using an induced differentiation medium A in which 5uM CHIR-99021, 20ng/ml BMP4, 20ng/ml VEGF, 40ng/ml SCF, 2ng/ml bFGF,40ng/ml IGF-1 was added to the basal induced differentiation medium of Table 1 at a ratio of 4.4X10 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-volume liquid exchange is carried out every two days by using an induced differentiation culture medium A, and the liquid exchange is carried out at 37 ℃ and 5% O 2 ，5% CO 2 Induction was performed in a hypoxia incubator.
(4) Day4-6: half-volume liquid exchange is carried out every two days by using the induction differentiation culture medium B, and induction is carried outDifferentiation medium A was replaced with induced differentiation medium B, which was prepared by adding 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1 to the basal induced differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
(5) Day6-15: half liquid exchange is carried out every two days, and the uniform differentiation of cells is influenced due to high cell density, the cell development is retarded and stagnated, the generation efficiency of the 'hematopoietic cells' is not in direct proportion to the inoculation density, and the 2D multilayer three-dimensional bionic 'hematopoietic cells' are not generated. The specific operation process of the half liquid exchange is as follows: collecting half culture supernatant of step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half induction differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729, and placing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
Fig. 1 is a cell morphology diagram of examples 1-3 and comparative examples 1, 2 on the first, third, fifth, sixth, ninth and thirteenth days, and it can be seen from fig. 1 that the whole development process of 2D multi-layered biomimetic "hematopoietic cells" is induced from single-layered human pluripotent stem cells. Example 1-example 3 "hematocrit" resulted from the fifth day, and the "hematocrit" volume increased over time, whereas comparative examples 1 and 2 failed to yield this structure. The initial seeding density determines whether "hematopoietic cells" are to be produced, and too low or too high a density to produce the structure is controlled to be 2X 10 4 -4×10 4 /cm 2 Within the range of example 1 density 2.2X10 4 /cm 2 Is the optimal scheme.
Fig. 2 to 6 are graphs showing the flow cytometry results of examples 1 to 3 and comparative examples 1 and 2 on the fourth, sixth and thirteenth days, and table 2 shows the flow cytometry results of examples 1 to 3 and comparative examples 1 and 2 on the fourth, sixth and thirteenth days.
TABLE 2 flow cytometry detection results
The results of the phenotypic assay during development of "hematopoietic cells" are shown in FIGS. 2-6 and Table 2. It can be seen that the fourth day examined mesodermal markers cd56+cd326-, examples 1-3 developed to reach mesoderm on the fourth day, whereas comparative examples 1 and 2 developed less slowly and did not reach mesoderm all the way; detecting the endothelial markers CD31+CD144+ of the blood production on the sixth day, wherein the positive rate of the examples 1-3 and the comparative examples 1 and 2 are higher when all the markers develop to the endothelial stage of the blood production; the detection of hematopoietic stem cell markers cd34+, lymphoid progenitor cell antigen cd45+, and erythroid progenitor cell marker cd235a+ on the thirteenth day, with cd34+, cd45+, cd235a+ positive rates of examples 1-3 being higher than comparative examples 1 and 2, shows that hematopoietic stem cells of examples 1-3 are higher than comparative examples 1 and 2, and have the potential to differentiate towards erythroid and lymphoid lines.
FIG. 7 is an immunofluorescence of CD34+ marker of hematopoietic stem cells at day six of example 1, and from FIG. 7 it can be seen that CD34+ is enriched in 2D multi-layered three-dimensional biomimetic "hematopoietic cells", indicating the presence of hematopoietic stem cells in the three-dimensional structure.
FIG. 8 is an immunofluorescence of CD34+ marker of hematopoietic stem cells of example 1 on thirteenth day, and it can be seen from FIG. 8 that CD34+ is enriched in 2D multi-layered three-dimensional biomimetic "hematopoietic cells" that develop continuously with increasing days of induction.
Example 4
(1) ESC cells were digested into cell suspensions by accutase, stopped by adding DMEM/F12, and centrifuged.
(2) Examples 1, 2 and 3 all produced "hematopoietic cells", compared with example 1, which had a lower cell density and more homogeneous differentiation, and based on the cell count results, appropriate amount of cells were taken and prepared into cell suspensions using induced differentiation medium A, which was prepared by adding 5uM CHIR-99021, 20ng/ml BMP4, 20ng/ml VEGF, 40ng/ml SCF, 2ng/ml bFGF,4 to the basal induced differentiation medium of Table 10ng/ml IGF-1, as in example 1, 2.2X10 4 /cm 2 Density was seeded on VTN coated 12 well plates, designated Day0.
(3) Day0-4: half-volume liquid exchange is carried out every two days by using an induced differentiation culture medium A, and the liquid exchange is carried out at 37 ℃ and 5% O 2 ，5% CO 2 Induction is performed in a hypoxia incubator.
(4) Day4-6: half-volume liquid change is carried out every two days by using an induced differentiation medium B, wherein the induced differentiation medium A is replaced by the induced differentiation medium B, and 5uM SB431542, 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF and 40ng/ml IGF-1 are added to the basic induced differentiation medium in the table 1, and the mixture is placed at 37 ℃ and 5% CO 2 Induction was performed in a normoxic incubator.
(5) Day6-15: half liquid exchange is carried out every two days, the process generates 2D multilayer three-dimensional bionic 'hematopoietic cells', hematopoietic stem progenitor cells exist in culture supernatant, and the specific operation process of half liquid exchange is as follows: collecting half amount of culture supernatant obtained in step (4), collecting precipitate by centrifugation, adding the collected precipitate back to the original induction system, and adding half amount of induced differentiation medium C, wherein 20ng/ml BMP4, 20ng/ml VEGF,2ng/ml bFGF,40ng/ml SCF,40ng/ml IGF-1, 10ng/ml Flt3L,5ng/ml IL3, 10ng/ml IL6, 750nM UM729, and placing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
(6) Differentiation of "hematopoietic cells" into a variety of cells is divided into three groups:
(1) NK cell induction group:
day15-20: the induced differentiation culture medium C is completely replaced by NK induction culture medium A, and the specific operation process is as follows: collecting the whole culture supernatant of step (5), collecting the precipitate by centrifugation, adding the collected precipitate to fresh NK induction medium A, wherein NK induction medium A is prepared by adding 5ng/ml IL3, 20ng/ml IL7, 10ng/ml IL15, 20ng/ml SCF,10ng/ml Flt3L to the basal induction differentiation medium of Table 1, and placing at 37℃and 5% CO 2 Induction was performed in a normoxic incubator.
Day20-30: half-volume liquid change is carried out every five days, and the deviceThe body operation process is as follows: collecting half culture supernatant, centrifuging to collect precipitate, adding the collected precipitate back to the original induction system, and adding half NK induction medium B, wherein NK induction medium B is prepared by adding 20ng/ml IL7, 10ng/ml IL15, 20ng/ml SCF,10ng/ml Flt3L into the basal induction differentiation medium of table 1, and placing at 37deg.C, 5% CO 2 Induction was performed in a normoxic incubator.
Day30-50: the liquid is changed in half a dose every three days, and the specific operation process is as follows: collecting half-volume culture supernatant, centrifuging to collect precipitate, adding the collected precipitate back to the original induction system, adding half-volume NK maturation medium, wherein the NK maturation medium is prepared by adding 20ng/ml IL7, 10ng/ml IL15, 20ng/ml SCF,10ng/ml Flt3L,750nM UM729, 100U/ml IL2, 10ng/ml IL21, 10ng/ml IL12, 10ng/ml IL18 to the basal induced differentiation medium of table 1, and placing at 37deg.C, 5% CO 2 Induction was performed in a normoxic incubator.
The phenotype is detected after NK cells are harvested, the cell morphology diagram of forty days is shown in FIG. 9, the flow type result diagram is shown in FIG. 10, the cells are induced to show obvious NK cell morphology after forty days in FIG. 9 and FIG. 10, and the flow type result shows that the positive rate of an NK cell marker CD3-CD56+ is more than 90%, the NKG2A, NKG2D, CD and the NKP30 are well expressed, and the NK cells are successfully induced.
(2) Macrophage induction group:
day15: the induced differentiation medium C is completely replaced by macrophage induced medium, and the specific operation process is as follows: collecting all culture supernatant of step (5), collecting precipitate by centrifugation, adding the collected precipitate into fresh macrophage inducing culture medium, adding 25ng/ml IL3 and 50ng/ml M-CSF to basal induced differentiation culture medium of table 1, and standing at 37deg.C and 5% CO 2 Induction was performed in a normoxic incubator.
Day15-36: half liquid change is carried out every seven days, and the specific operation process is as follows: collecting half culture supernatant, centrifuging to collect precipitate, adding the collected precipitate back to the original induction system, adding half macrophage maturation medium, adding 100ng/ml M-CSF to basal induction differentiation medium shown in Table 1, and standingAt 37 ℃,5% CO 2 Induction was performed in a normoxic incubator.
Phenotypes were examined after harvesting macrophages. The cell morphology on thirty-fourth day is shown in fig. 11, the flow result is shown in fig. 12, it can be seen from fig. 11 and 12 that cells exhibit obvious macrophage morphology after thirty-four days of induction, and the flow result shows that the positive rate of the M1 type macrophage marker cd14+cd80+ is greater than 60%, the positive rate of the M2 type macrophage marker cd14+cd206+ is greater than 80%, and macrophages are successfully induced.
(3) Red blood cell induction group:
day15-28: the liquid is changed in half a dose every three days, and the specific operation process is as follows: collecting half culture supernatant, centrifuging to collect precipitate, adding the collected precipitate back to the original induction system, adding half erythrocyte induction medium, wherein 0.2uM FICZ,10ng/ml IL6, 50ng/ml SCF,3U/ml EPO,10ng/ml IL3, and 5% CO are added to the basic induction differentiation medium of table 1, and standing at 37deg.C 2 Induction was performed in a normoxic incubator.
Day28-35: the liquid is changed in half a dose every three days, and the specific operation process is as follows: collecting half culture supernatant, centrifuging to collect precipitate, adding the collected precipitate back to the original induction system, adding half erythrocyte maturation medium, which is prepared by adding 1uM mifepristone, 500ug/ml transferrin, 6U/ml EPO, and placing at 37deg.C and 5% CO into the basic induction differentiation medium of table 1 2 Induction was performed in a normoxic incubator.
Phenotype was detected after harvesting the erythrocytes. The cell morphology on thirty-fifth days is shown in fig. 13, the flow result on twenty-second days is shown in fig. 14, the RNA expression levels of beta, gamma and epsilon-globin on the ninth, fifteenth and twenty-second days are shown in fig. 15, the cells can be seen to show obvious erythrocyte morphology from fig. 13 and 14, the flow result shows that the erythrocyte marker cd235a+cd71+ positive rate is more than 70%, and the change of globin can be seen from fig. 15.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. A method for inducing hematopoietic stem cells by a low-density monolayer, characterized by: the method comprises the following steps:
s1: ESC cells were prepared as a cell suspension using induction differentiation medium A at a ratio of 2X 10 4 -4×10 4 /cm 2 Inoculating the culture medium into a cell culture container at the density of 0 th day, and performing low-oxygen low-density monolayer induction on the culture medium by using half-amount liquid exchange of the induction differentiation culture medium A every two days on the 0 th day to the 4 th day;
the induced differentiation medium A is a basal induced differentiation medium added with CHIR-99021, BMP4, VEGF, SCF, bFGF and IGF-1, wherein the concentration of the CHIR-99021 is 1-10uM, the concentration of the BMP4 is 1-100ng/ml, the concentration of the VEGF is 1-100ng/ml, the concentration of the SCF is 1-200ng/ml, the concentration of the bFGF is 1-50ng/ml, and the concentration of the IGF-1 is 1-200ng/ml; s2: half liquid exchange is carried out every two days from the 4 th day to the 6 th day by using the induced differentiation culture medium B, the induced differentiation culture medium A is replaced by the induced differentiation culture medium B, and the induction is converted into normoxic induction;
the induced differentiation medium B is a basic induced differentiation medium added with SB431542, BMP4, VEGF, bFGF, SCF and IGF-1, wherein the concentration of SB431542 is 1-10uM, the concentration of BMP4 is 1-100ng/ml, the concentration of VEGF is 1-100ng/ml, the concentration of bFGF is 1-50ng/ml, the concentration of SCF is 1-200ng/ml, and the concentration of IGF-1 is 1-200ng/ml;
s3: half liquid exchange is carried out every two days from the 6 th day to the 15 th day by using the induced differentiation culture medium C, the induced differentiation culture medium B is replaced by the induced differentiation culture medium C, and normal oxygen induction is carried out, so that the 2D multilayer three-dimensional bionic 'hematopoietic cell' is finally obtained; the diameter of the 2D multilayer three-dimensional bionic 'hematopoietic cell' is 300-500 micrometers, and the 2D multilayer three-dimensional bionic 'hematopoietic cell' is loosely connected with or suspended in the bottom matrix cells;
the induced differentiation medium C is a basic induced differentiation medium added with UM729, BMP4, VEGF, bFGF, SCF, flt3L, IL3, IL6 and IGF-1; wherein UM729 has a concentration of 100-900nM, BMP4 has a concentration of 1-100ng/ml, VEGF has a concentration of 1-100ng/ml, bFGF has a concentration of 1-50ng/ml, SCF has a concentration of 1-200ng/ml, flt3L has a concentration of 1-100ng/ml, IL3 has a concentration of 1-50ng/ml, IL6 has a concentration of 1-50ng/ml, IGF-1 has a concentration of 1-200ng/ml;
the basal induced differentiation culture medium comprises 40% -50% v/v of IMDM culture medium, 40% -50% v/v of F12 culture medium, 0.5-20mg/ml of human serum albumin, 0-1000uM of alpha-MTG, 10-50ug/ml of transferrin, 10-50ug/ml of ascorbic acid, 5-50ng/ml of sodium selenite, 5-100uM of ethanolamine, 20-100ng/ml of Long R3 IGF-1, 2mM of GlutamaxI, 0-50ng/ml of polyvinyl alcohol, 5% v/v of protein-free hybridoma cell culture medium and 0.5-5ug/ml of ferric citrate.
2. The 2D multi-layered three-dimensional biomimetic "hematopoietic cell" prepared by the method of claim 1.
3. Use of the 2D multilayer stereoscopic biomimetic "hematopoietic cells" of claim 2 to differentiate into NK cells, macrophages or erythrocytes.
4. A use according to claim 3, characterized in that: the method for differentiating the 2D multilayer three-dimensional bionic 'hematopoietic cells' into NK cells comprises the following steps:
performing normoxic induction after replacing the induction differentiation culture medium C with the NK induction culture medium A on the 15 th day to the 20 th day;
the NK induction medium A is obtained by adding IL3, IL7, IL15, SCF and Flt3L into a basic induction differentiation medium; wherein, the concentration of IL3 is 1-100ng/ml, the concentration of IL7 is 1-100ng/ml, the concentration of IL15 is 1-100ng/ml, the concentration of SCF is 1-100ng/ml, and the concentration of Flt3L is 1-100ng/ml;
half liquid exchange is carried out every five days from 20 days to 30 days by using an NK induction culture medium B, the NK induction culture medium A is replaced by the NK induction culture medium B, and normal oxygen induction is carried out;
the NK induction medium B is obtained by adding IL7, IL15, SCF and Flt3L into a basic induction differentiation medium; wherein, the concentration of IL7 is 1-100ng/ml, the concentration of IL15 is 1-100ng/ml, the concentration of SCF is 1-100ng/ml, and the concentration of Flt3L is 1-100ng/ml;
half-volume liquid exchange is carried out on the NK maturation medium every three days from 30 days to 50 days, the NK induction medium B is replaced by the NK maturation medium, and normoxic induction is carried out;
the NK maturation medium is obtained by adding IL7, IL15, SCF, flt3L, UM729, IL2, IL21, IL12 and IL18 into a basal induced differentiation medium; wherein, the concentration of IL7 is 1-100ng/ml, the concentration of IL15 is 1-100ng/ml, the concentration of SCF is 1-100ng/ml, the concentration of Flt3L is 1-100ng/ml, the concentration of UM729 is 100-900nM, the concentration of IL2 is 1-1000U/ml, the concentration of IL21 is 1-100ng/ml, the concentration of IL12 is 1-100ng/ml, and the concentration of IL18 is 1-100ng/ml.
5. A use according to claim 3, characterized in that: the method for differentiating the 2D multilayer three-dimensional bionic 'hematopoietic cells' into macrophages comprises the following steps:
on the 15 th day, the induced differentiation medium C is replaced by a macrophage induced medium, and normal oxygen induction is carried out;
the macrophage induction culture medium is characterized in that IL3 and M-CSF are added into a basal induction differentiation culture medium, wherein the concentration of IL3 is 1-100ng/ml, and the concentration of M-CSF is 1-200ng/ml;
half-volume liquid exchange is carried out on the macrophage maturation medium every seven days from 15 th day to 36 th day, the macrophage induction medium is replaced by the macrophage maturation medium, and normal oxygen induction is carried out;
the macrophage maturation medium is prepared by adding M-CSF at a concentration of 1-200ng/ml to basal induced differentiation medium.
6. A use according to claim 3, characterized in that: the method for differentiating the 2D multilayer three-dimensional bionic 'hematopoietic cells' into erythrocytes comprises the following steps:
half-volume liquid exchange is carried out on the red blood cell induction culture medium every three days from 15 th day to 28 th day, the induction differentiation culture medium C is replaced by the red blood cell induction culture medium, and normal oxygen induction is carried out;
the red blood cell induction culture medium is prepared by adding FICZ, IL6, SCF, EPO and IL3 into a basic induction differentiation culture medium; wherein the concentration of FICZ is 0.1-1uM, the concentration of IL6 is 1-50ng/ml, the concentration of SCF is 1-100ng/ml, the concentration of EPO is 1-100U/ml, and the concentration of IL3 is 1-50ng/ml;
half-volume liquid exchange is carried out on the red blood cell maturation medium every three days from 28 th day to 35 th day, the red blood cell induction medium is replaced by the red blood cell maturation medium, and normal oxygen induction is carried out;
the erythrocyte maturation medium is prepared by adding mifepristone, transferrin and EPO into a basal induced differentiation medium; wherein the concentration of mifepristone is 0.5-5uM, the concentration of transferrin is 1-1000ug/ml, and the concentration of EPO is 1-100U/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834224A (en) * | 2016-11-18 | 2017-06-13 | 西北农林科技大学 | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell |
| CN112608895A (en) * | 2020-12-18 | 2021-04-06 | 深圳市安棣生物科技有限责任公司 | Natural killer cell differentiated from human pluripotent stem cell and preparation method and application thereof |
| CN114774365A (en) * | 2022-06-16 | 2022-07-22 | 呈诺再生医学科技(北京)有限公司 | Method for obtaining CD34+ cells and NK cells by inducing iPSC differentiation and application thereof |
| CN115029314A (en) * | 2022-06-06 | 2022-09-09 | 北京呈诺医学科技有限公司 | CD34 + Cell differentiation medium, method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834224A (en) * | 2016-11-18 | 2017-06-13 | 西北农林科技大学 | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell |
| CN112608895A (en) * | 2020-12-18 | 2021-04-06 | 深圳市安棣生物科技有限责任公司 | Natural killer cell differentiated from human pluripotent stem cell and preparation method and application thereof |
| CN115029314A (en) * | 2022-06-06 | 2022-09-09 | 北京呈诺医学科技有限公司 | CD34 + Cell differentiation medium, method and application |
| CN114774365A (en) * | 2022-06-16 | 2022-07-22 | 呈诺再生医学科技(北京)有限公司 | Method for obtaining CD34+ cells and NK cells by inducing iPSC differentiation and application thereof |
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