CN113046318A - Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells - Google Patents

Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells Download PDF

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CN113046318A
CN113046318A CN202110396567.5A CN202110396567A CN113046318A CN 113046318 A CN113046318 A CN 113046318A CN 202110396567 A CN202110396567 A CN 202110396567A CN 113046318 A CN113046318 A CN 113046318A
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medium
hematopoietic
cells
differentiation
precursor cells
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CN113046318B (en
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雷晓华
张键
马驰原
赵华山
汪宝蓓
李梦霞
李荣荣
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The present invention relates to a medium and a method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells. Specifically, the method comprises 1) differentiating human pluripotent stem cells to generate hematopoietic endothelial precursor cells, and preparing to obtain the hematopoietic endothelial precursor cells; 2) hematopoietic progenitor cells are prepared by a method of promoting differentiation of hematopoietic endothelial progenitor cells into hematopoietic progenitor cells. The culture medium is adopted in the preparation process and cultured in a random rotation mode, so that excellent effective efficiency is obtained, and the components of the culture medium are clear.

Description

Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells
Technical Field
The invention belongs to the technical field of biology, and relates to a method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells.
Background
Pluripotent stem cells including embryonic stem cells of embryonic origin and induced pluripotent stem cells of in vitro reprogramming inducing origin. Pluripotent stem cells are capable of long-term culture in vitro to retain self-renewal capacity and have the potential to differentiate in multiple ways, including into almost all functional blood cells. Hematopoietic precursor cells are precursor cells that have self-renewal ability and can differentiate into various blood cells, ultimately producing various blood cell components, including red blood cells, white blood cells, and platelets, which can also differentiate into various other cells. In the clinic, hematopoietic stem/progenitor cells and various mature blood cells can serve as the major strategic demands of clinical hematopoietic stem cell transplantation and blood cell transfusion therapy. How to obtain enough hematopoietic stem cells has been a problem for researchers and medical staff. Since 2001, Kaufman et al first achieved differentiation of human embryonic stem cells into hematopoietic stem/progenitor cells of CD34+ [ Kaufman D, Hanson E, Lewis et al, hematopoetic color-forming cells derived from human embryonic stem cells, Proc Natl Acad USA.2001; 98(19) 10716-21], and research on the differentiation of human pluripotent stem cells into hematopoietic stem cells in vitro has been carried out. Although there has been some progress in this field, from the perspective of current clinical application, the research of in vitro hematopoietic differentiation of human pluripotent stem cells still faces a great challenge, for example, the number of hematopoietic endothelial precursor cells and hematopoietic stem cells and functional cells induced by human pluripotent stem cells is not enough to meet the requirement of one-time transfusion; hematopoietic stem cells induced by human pluripotent stem cells and differentiated therefrom do not have the capacity of in vivo transplantation; the conditions of in vitro culture comprise exogenous substances such as serum, feeder cells and the like, and the problems can greatly limit the clinical application of in vitro hematopoietic differentiation by adopting human pluripotent stem cells at present.
Because the existing methods have complicated culture conditions, relatively long differentiation period, low differentiation efficiency and yield and poor operability. Based on the above problems in the prior art, there is a need to find a method for inducing the generation of hematopoietic progenitor cells from vascular/hematopoietic endothelial progenitor cells.
Disclosure of Invention
The invention aims to provide a simply-operated and high-efficiency system for inducing differentiation from pluripotent stem cells to hematopoietic precursors, which comprises a system for inducing differentiation from pluripotent stem cells to hematopoietic endothelial precursors and a system for inducing differentiation from hematopoietic endothelial precursor cells to hematopoietic precursor cells. Specifically, the invention provides a culture medium for differentiating pluripotent stem cells into hematopoietic precursor cells with definite chemical components, and a method for efficiently inducing the differentiation of the pluripotent stem cells into human hematopoietic precursor cells by simulating the microgravity effect by using a random gyroscope by using the culture medium; compared with the existing method, the method has higher operability and repeatability, and lays a technical foundation for large-scale production of hematopoietic precursor cells for biomedicine and clinical treatment in the future.
In a first aspect, the present invention provides a mesoderm cell-inducing differentiation medium comprising a basal differentiation medium and bone morphogenetic protein 4(BMP4), activin a (activin a), and a GSK-3 α/β inhibitor (CHIR 99021);
the basic differentiation medium is an IF9S complete medium, and comprises an IMDM medium and an F12 medium which are mixed according to the volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, Lipids 100X additive with the volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with the volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA2P) with the final concentration of 55-70mg/L, and glutamic alanine dipeptide (GlutaMax) with the volume percentage of 0.8-1.2% of the IF9S complete mediumTM)100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 0.8% -1.2% of the IF9S complete medium.
Preferably, the IF9S complete medium comprises IMDM mixed at a volume ratio of 1:1Medium and F12 medium, as well as polyvinyl alcohol (PVA) at a final concentration of 10mg/L, Lipids 100X additive at a volume percentage of 0.1% of the medium, ITS-X100X additive at a volume percentage of 2% of the IF9S complete medium, monothioglycerol (α MTG) at a final concentration of 40 μ L/L, sodium ascorbyl phosphate (AA2P) at a final concentration of 64mg/L, GlutaMax at a volume percentage of 1% of the IF9S complete mediumTM100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 1% of the IF9S complete medium.
Preferably, the mesodermal cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL BMP4, 15-25 μ g/mL Activin A, 0.8-1.2 μ M CHIR 99021.
More preferably, the mesodermal cell differentiation medium is IF9S complete medium supplemented with 50ng/mL BMP4, 20. mu.g/mL Activin A, 1. mu.M CHIR 99021.
In a second aspect, the present invention provides a hematopoietic endothelial precursor cell-inducing differentiation medium comprising basal differentiation medium, SB431542, VEGF, bFGF and SCF;
the basic differentiation medium is an IF9S complete medium and comprises an IMDM medium and an F12 medium which are mixed according to the volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, Lipids 100X additive with the volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with the volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA2P) with the final concentration of 55-70mg/L and GlutaMax with the volume percentage of 0.8-1.2% of the IF9S complete mediumTM100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 0.8% -1.2% of the IF9S complete medium.
Preferably, the IF9S complete medium comprises IMDM medium and F12 medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) at a final concentration of 10mg/L, Lipids 100X additive at 0.1% by volume of the medium, ITS-X100X additive at 2% by volume of the IF9S complete medium, and final concentrationMonothioglycerol (α MTG) at a concentration of 40 μ L/L, sodium ascorbyl phosphate (AA2P) at a final concentration of 64mg/L, GlutaMax at 1% by volume of the complete medium of IF9STM100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 1% of the IF9S complete medium.
Preferably, the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 8-12 μ M SB431542, 40-60ng/mL VEGF, 40-60ng/mL bFGF and 40-60ng/mL SCF;
more preferably, the hematopoietic progenitor differentiation medium is IF9S complete medium supplemented with 10. mu.M SB431542, 50ng/mL VEGF, 50ng/mL bFGF, and 50ng/mL SCF.
Preferably, deionized water is used to dissolve polyvinyl alcohol (PVA) and make up a 5% stock solution before making up the IF9S complete medium.
Preferably, a 500 Xstock solution is prepared by 2% dilution of monothioglycerol in IMDM medium before the complete medium of IF9S is prepared.
Preferably, AA2P is dissolved in F12 medium and a 5mg/mL stock solution is prepared before the complete medium of IF9S is prepared.
Wherein the IMDM medium and the F12 base medium are conventional media, commercial culture media can be purchased, or self-prepared, and in a preferred embodiment of the invention, the IMDM medium and the F12 medium are purchased from Gibco company.
The Lipids (100X) is a chemically-defined lipid concentrate that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the invention, the Lipids additive is available from Gibco under the designation 11905031.
The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine for the reduction or replacement of fetal bovine serum in cell culture medium. In a preferred embodiment of the invention, the ITS-X additive is available from Gibico, Inc. under the designation 51500056.
The GlutaMaxTM(100X) the additive is glutamic-alanine dipeptide,is a cell culture additive and can directly replace L-glutamine in a cell culture medium. In a preferred embodiment of the present invention, the GlutaMax additive is available from Gibico as Cat 35050-.
The NEAA (100X) additive is a non-essential amino acid and is a cell culture additive. In a preferred embodiment of the present invention, the NEAA additive is available from Gibico corporation under the designation 35050-.
In a third aspect, the present invention provides a combined culture medium for inducing differentiation of hematopoietic endothelial precursor cells, which comprises the above-mentioned mesodermal cell differentiation-inducing culture medium and the above-mentioned hematopoietic endothelial precursor cell differentiation-inducing culture medium.
Preferably, the mesodermal cell differentiation-inducing medium and the hematopoietic endothelial precursor cell differentiation-inducing medium are placed or formulated separately.
In a fourth aspect, the present invention provides a medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, comprising a basal differentiation medium and Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), Stem Cell Factor (SCF), interleukin 3(IL-3), interleukin 6(IL-6), and Thrombopoietin (TPO);
the basic differentiation medium is an IF9S complete medium and comprises an IMDM basic medium and an F12 basic medium which are mixed according to the volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, Lipids (100X) additive with the volume percentage of 0.08-0.12% of the medium, ITS-X (100X) additive with the volume percentage of 1.8-2.2% of the medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, AA2P with the final concentration of 55-70mg/L and GlutaMax with the volume percentage of 0.8-1.2% of the mediumTM(100X) additives and non-essential amino acid additives (NEAA, 100X) in a volume percentage of 0.8-1.2% of the culture medium.
Preferably, the basic differentiation medium is an IF9S complete medium comprising IMDM basal medium and F12 basal medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) at a final concentration of 10mg/L, the volume percentage of the medium0.1% Lipids (100X) supplement, 2% ITS-X (100X) supplement by volume of the medium, monothioglycerol (α MTG) at a final concentration of 40 μ L/L, AA2P at a final concentration of 64mg/L, 1% GlutaMax by volume of the mediumTM(100X) additives and non-essential amino acid additives (NEAA, 100X) in a volume percentage of 1% of the culture medium.
Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL VEGF, 15-25ng/mL bFGF, 40-60ng/mL SCF, 8-12ng/mL IL-3, 40-60ng/mL IL-6, and 15-25ng/mL TPO.
Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 50ng/mL VEGF, 20ng/mL bFGF, 50ng/mL SCF, 10ng/mL IL-3, 50ng/mL IL-6 and 20ng/mL TPO.
Preferably, deionized water is used to dissolve polyvinyl alcohol (PVA) and make up a 5% stock solution before making up the IF9S complete medium.
Preferably, a 500 Xstock solution is prepared by 2% dilution of monothioglycerol in IMDM medium before the complete medium of IF9S is prepared.
Preferably, AA2P is dissolved in F12 medium and a 5mg/mL stock solution is prepared before the complete medium of IF9S is prepared.
Wherein the IMDM basal medium and the F12 basal medium are conventional media, commercial culture media can be purchased, and the culture media can be prepared by self, and in a preferred embodiment of the invention, the IMDM and the F12 culture media are purchased from Gibco company.
The Lipids (100X) is a chemically-defined lipid concentrate that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the present application, the Lipids additive is available from Gibco under the designation 11905031.
The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine for the reduction or replacement of fetal bovine serum in cell culture medium. In a preferred embodiment of the invention, the ITS-X additive is available from Gibico, Inc. under the designation 51500056.
The GlutaMaxTMThe (100X) additive is glutamine dipeptide, is a cell culture additive, and can directly replace L-glutamine in a cell culture medium. In a preferred embodiment of the present invention, the GlutaMax additive is available from Gibico as Cat 35050-.
The NEAA (100X) additive is a non-essential amino acid and is a high-grade cell culture additive. In a preferred embodiment of the present invention, the NEAA additive is available from Gibico corporation under the designation 35050-.
In a fifth aspect, the present invention provides a medium composition for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells, the medium composition comprising:
1) the mesoderm cell induction differentiation medium;
2) the hematopoietic endothelial precursor cell differentiation-inducing medium;
3) the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.
Preferably, the mesodermal cell differentiation induction medium, the hematopoietic endothelial precursor cell differentiation induction medium are separately placed or formulated and the medium for inducing differentiation of the hematopoietic endothelial precursor cells into hematopoietic precursor cells.
In a sixth aspect, the invention provides a method of promoting transformation of mesodermal cells into hematopoietic endothelial precursor cells, the method comprising the step of culturing the cells in a cell culture vessel under spinning conditions using a random three-dimensional gyroscope during mesodermal cell culture.
Preferably, the culturing under spinning conditions is performed by randomly spinning the cell culture vessel with a random three-dimensional gyroscope, more preferably, the random spinning is at a speed of 0.1-10 rpm/min; more preferably, the rotational speed is 5 rpm/min.
Preferably, the culturing time under the spinning condition is 48 to 96 hours, preferably 60 to 72 hours.
Preferably, the culture medium for culturing under the rotating condition adopts the hematogenous endothelial precursor induced differentiation culture medium.
In a seventh aspect, the present invention also provides a method for differentiating and producing hematopoietic endothelial precursor cells from human pluripotent stem cells, the method comprising the steps of:
s1) inducing differentiation culture of human pluripotent stem cells into mesodermal cells:
s11). cell container pre-coating with growth factor-reduced basement membrane matrix (Matrigel matrix);
s12), adopting digestive enzyme to clone the human pluripotent stem cells, digesting and centrifuging;
s13) inoculating the digested and centrifuged small human pluripotent stem cell clone masses into a cell container obtained in S11) to obtain a pre-coated growth factor-reduced Matrigel, and adding TeSRTM-E8TMPerforming static culture on the culture medium for 24-48 hours;
s14), replacing the culture medium with the mesoderm cell induction differentiation culture medium, and statically culturing for 24-48 hours to obtain induced mesoderm cells;
s2) further differentiating and culturing the induced mesodermal cells into hematopoietic endothelial precursor cells:
s21). replacing the cell culture medium of the mesodermal cells with the above-mentioned hematopoietic endothelial precursor cell-inducing differentiation medium;
s22), mounting the cell culture container on a random three-dimensional gyroscope, setting the rotation mode to be random rotation, and adjusting the rotation speed to be 0.1-10 rpm/min;
s23) placing a random three-dimensional gyroscope into a chamber with the temperature of 37 ℃ and the CO content of 5%2Rotating and culturing in incubator for 60-96 hr.
Preferably, the Growth Factor-Reduced Matirgel matrix described in S11) is a Growth Factor Reduced Matrigel manufactured by Corning Corp, Cat 354230.
Preferably, the density of the inoculation in S13) is 50-200 clonal clumps/cm2。
Still more preferably, the density of the inoculation described in S13) is 100 clonal clumps/cm2；。
Preferably, the hematopoietic endothelial precursor cell-inducing differentiation medium described in S21) is added to 95% -100% of the culture vessel;
preferably, the rotation speed in S22) is 5 rpm/min;
preferably, S22) for 72 hours.
Preferably, S12), the human pluripotent stem cell clone is subjected to amplification culture before digestion:
s121) coating a culture cell container with extracellular matrix Matrigel of embryonic stem cell grade;
s122) inoculating the human pluripotent stem cells into the coated cell container and using TeSRTM-E8TMThe culture medium is used for culturing.
Preferably, replacing the fresh culture medium every 24 hours in S122), and performing digestion passage or induced differentiation culture after the cell clone grows to 70-80% of the density; the cell passage cycle was 3-4 days.
Preferably, the extracellular matrix of embryonic stem cell grade is Matrigel manufactured by BioCoat corporation under the code 354277.
In an eighth aspect, the present invention also provides a method for promoting differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, which comprises the step of culturing the hematopoietic endothelial precursor cells in a cell culture container under a rotating condition by using a random gyroscope.
Preferably, the culturing under the rotation condition is carried out by randomly rotating the cell culture vessel by a random gyroscope, and more preferably, the inner diameter and the outer diameter of the random rotation are both 0.1-10 rpm/min; more preferably, the rotation speed is 5 rpm/min for the inner and outer diameters.
Preferably, the culturing time under the rotation condition is 48 to 72 hours, preferably 72 hours.
Preferably, the culture medium for culturing under the rotation condition is the culture medium for inducing the differentiation of the vascular/hematopoietic endothelial precursor cells into the hematopoietic precursor cells.
Preferably, it comprises the following steps:
s3) culturing in the original medium for 72 hours, and then changing the cell culture medium to the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;
s4) the cell culture bottle is remounted on a random gyroscope to continue the rotary culture;
preferably, the medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells described in S3) is added to 95% -100% of the culture vessel;
preferably, S4), the cells are cultured for 48 to 72 hours, preferably 72 hours.
Preferably, S4), the cell culture conditions are 37 ℃ and 5% CO2。
In a ninth aspect, the present invention also provides a method of differentiating to produce hematopoietic precursor cells from human pluripotent stem cells, the method comprising the steps of:
1) the method for generating the hematopoietic endothelial precursor cells by differentiating the human pluripotent stem cells is adopted to prepare and obtain the hematopoietic endothelial precursor cells;
2) the method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is used.
In a tenth aspect, the present invention provides the use of a random three-dimensional gyroscope for promoting the transformation of mesodermal cells into hematopoietic endothelial precursor cells, or for promoting the transformation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.
The invention selects a culture medium with definite chemical components for culturing and differentiating the human pluripotent stem cells, and also describes a technology for carrying out differentiation of the pluripotent stem cells into hematopoietic endothelial precursor cells and hematopoietic precursor cells by simulating the microgravity effect by using a random rotator. In summary, compared with the prior art, the invention has the following advantages:
the culture system is characterized in that the culture system is cultured under the conditions of no serum, no feeding layer and clear chemical composition from the culture and amplification of the pluripotent stem cells to the mesendoderm differentiation, the hematopoietic endothelial precursor differentiation and the hematopoietic precursor cells. The currently reported methods for in vitro differentiation of human pluripotent stem cells into hematopoietic precursors/stem cells mainly include systems requiring co-culture with mouse bone marrow stromal cells (OP9 cell line) or mouse aortic gonadal mesonephroma stromal cells (mAGM cell line), and systems using Embryoid Bodies (EB) for differentiation. The co-culture system contains animal-derived substances, so that the instability and safety of the experiment are improved, and the complexity of the experiment steps is increased; in the EB differentiation system, the differentiation efficiency was low. The culture system with definite chemical components and the culture method of random three-dimensional rotary simulated microgravity can obtain a large amount of hematopoietic endothelial precursor cells and hematopoietic precursor cells from pluripotent stem cells in a short time, and has high differentiation efficiency which is improved by more than 2 times compared with static culture. The low-cost, safe and controllable random three-dimensional rotary culture strategy of the serum-free and feeder layer-free cells can lay a foundation for the future large-scale production of hematogenic endothelial precursor cells or hematogenic precursor cells for clinical treatment.
Drawings
FIG. 1 is a bright field picture of hematopoietic endothelial precursor cells derived from human pluripotent stem cell induction using the method of the present invention.
FIG. 2 is a photograph of fluorescent staining of hematopoietic endothelial precursor cell marker molecules CD31 and CD34 from hematopoietic endothelial precursor cells derived from human pluripotent stem cell induction using the method of the present invention.
FIG. 3 is a flow cytometric analysis of the differentiation of hematopoietic endothelial precursor cells using the random three-dimensional rotating culture of the present invention and conventional stationary culture, comparing the efficiency of expressing CD31+ CD34+ after differentiation. Wherein, the A picture is a control group, and the B picture is a random three-dimensional rotation group.
FIG. 4 is a brightfield image of hematopoietic precursor cells derived from human pluripotent stem cell induction using the method of the invention.
FIG. 5 is a photograph of fluorescent staining of hematopoietic precursor cell marker molecules CD34 and CD43 from hematopoietic precursor cells derived from human pluripotent stem cell induction using the method of the present invention.
FIG. 6 is a flow cytometric analysis of the differentiation of hematopoietic precursor cells using the random round culture of the present invention and conventional static culture, comparing the efficiency of the cells expressing CD34+ CD43+ after differentiation. Wherein, the A picture is a control group, and the B picture is a random rotation group.
Detailed Description
The present invention will now be further illustrated with reference to examples, but the practice of the invention is not limited thereto.
Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from a regular channel.
Unless otherwise specified, the human pluripotent stem cells used in the present invention are human embryonic stem cell line H1.
Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from a regular channel.
Example 1: preparation of serum-free culture medium
(1) Preparation of IF9S Medium:
the present application uses IMDM medium, F12 medium, polyvinyl alcohol (PVA), Lipids (100X), ITS-X (100X), monothioglycerol (α MTG), AA2P, GlutaMaxTM(100X), non-essential amino acids (NEAA, 100X) to prepare IF9S medium.
Wherein, each component of the culture medium is purchased from:
IMDM medium was purchased from Gibco under the accession number 21056-;
f12 medium was purchased from Gibco under the accession number 31765-027;
polyvinyl alcohol (PVA) was purchased from Sigma under the trade designation P8136;
lipids (100X) available from Gibco, Inc. under the product number 11905031;
ITS-X (100X) was purchased from Gibco corporation under the accession number 51500-;
monothioglycerol (. alpha.MTG) was purchased from Sigma under the designation M6145;
AA2P from Sigma under a cat # a 8960;
GlutaMaxTM(100X) purchased from Gibco, Inc. under the Cat No. 35050-;
nonessential amino acids (NEAA, 100X) were purchased from Gibco under the accession number 11140-035;
preparing the culture medium. Wherein, the polyvinyl alcohol (PVA) is powder, and is dissolved by deionized water before use to prepare a stock solution with the concentration of 5 percent.
Specifically, the IF9S medium was prepared by the following method:
firstly, mixing an IMDM culture medium and an F12 culture medium according to a ratio of 1:1, uniformly stirring, and adding the following reagents into the mixed culture medium, wherein the final concentrations of the reagents are as follows: 10mg/L polyvinyl alcohol (PVA), 0.1% by volume additives such as Lipids (100X), 2% by volume additives such as ITS-X (100X), 40. mu.l/L monothioglycerol (. alpha.MTG), 64mg/L AA2P, 1% by volume GlutaMaxTM(100X) additives and 1% by volume of non-essential amino acid additives (NEAA, 100X).
The addition of the reagents is not in strict order, and the preparation process is carried out at normal temperature, and the preparation process does not need heating, but the preparation of the culture medium needs sterile filtration by using a filter of 0.22 mu m, and the culture medium is stored in a refrigerator at 4 ℃.
(2) Preparation of mesoderm cell differentiation medium
The mesoderm cell differentiation medium is an IF9S medium added with 50ng/mL BMP4, 20 μ g/mL Activin A and 1 μ M CHIR 99021.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
(3) Preparation of differentiating culture medium for hematogenesis endothelial precursor
The hematopoietic endothelial precursor differentiation medium was IF9S medium supplemented with 10. mu.M SB431542, 50ng/mL VEGF, 50ng/mL bFGF, 50ng/mL SCF.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
(4) Preparation of hematopoietic precursor cell differentiation medium
The hematopoietic precursor/stem cell differentiation medium was prepared by adding 50ng/mL VEGF, 20ng/mL bFGF, 50ng/mL SCF, 10ng/mL IL-3, 50ng/mL IL-6 and 20ng/mL TPO to IF9S complete medium.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
Example 2: many peoplePreparation of hematopoietic endothelial precursor cells capable of being derived from stem cells
The human pluripotent stem cells used in this example were a human embryonic stem cell line (H1) (from university of Wisconsin, USA).
a. Firstly, coating a dish for culturing cells by adopting extracellular matrix Matrigel of embryonic stem cell grade, then inoculating human embryonic stem cells into the coated dish and using a commercial culture medium TeSR with clear chemical componentsTM-E8TMThe culture was performed with replacement of fresh medium every 24 hours. The human embryonic stem cells grow in a clone form, and when the cell clone grows to 70-80% of density, the cells need to be subjected to digestion passage or induced differentiation culture after the cell clone grows. The cell passage cycle is typically 3-4 days.
b. And (3) cloning the human embryonic stem cells grown in the step a by adopting ReLeSRTM digestive enzyme for digestion treatment, and digesting for 2-3 minutes at 37 ℃. After 2 minutes of digestion, the digestion condition of the cells is observed under a mirror, and TeSR is used when the cell clone edge is curledTM-E8TMThe digestion of the culture medium is stopped, so that excessive digestion is avoided;
c. inoculating the small digested and centrifuged human embryonic stem cell clone blocks into the culture bottle pre-coated with growth factor reduction Matrigel, wherein the inoculation density is 50-200 clone blocks/cm2. Adding a proper amount of TeSRTM-E8TMCulture medium, 5% CO at 37 ℃2Standing and culturing in an incubator for 24-48 hours; after culturing for 24-48 hours, replacing the culture medium with a mesoderm cell differentiation culture medium;
d. after addition of mesoderm cell differentiation medium, 5% CO was added at 37 deg.C2Carrying out static culture in an incubator for 48 hours;
e. culturing the cells in a mesoderm cell induction culture medium for 48 hours, then, sucking and replacing the culture medium in a culture bottle with a fresh hematogenous endothelial precursor cell induction culture medium, and filling the culture bottle with the culture medium;
f. sealing the cell culture bottle, then installing the cell culture bottle on a random three-dimensional gyroscope, setting a rotation mode as a random rotation mode, and adjusting the rotation speed to be 0.1-10 rpm/min;
g. random three-dimensional retransfer to 37 ℃ with 5% CO2Rotationally culturing for 72 hours in an incubator, and observing a vascular endothelial-like grid shape formed by cells after culturing for 72 hours, wherein the vascular endothelial-like grid shape is blood vessel/hematogenic endothelial precursor cells; the morphology of the hematopoietic endothelial precursor cells is shown in FIG. 1.
Example 3: detection of hematopoietic endothelial precursor cells
The vascular/hematopoietic endothelial precursor cells generated in example 2 were collected for identification by immunofluorescence staining.
Specifically, the method comprises the following steps:
a. removing the hematopoietic endothelial precursor cell induction medium in the culture flask, and washing the cells for 2 times by using PBS (phosphate buffer solution) to remove dead cells;
b. adding 4% paraformaldehyde for fixation, and standing and fixing for 20 minutes at room temperature;
c. removing 4% paraformaldehyde by suction, and washing with PBS for 2-3 times;
d. adding donkey serum with the concentration of 5% for sealing, and sealing at room temperature for 1 hour;
e. adding a fluorescence-labeled mouse anti-human PE-CD31 antibody (cargo number: 560983, BD company), a fluorescence-labeled mouse anti-human FITC-CD34 antibody (cargo number: 560942, BD company) and 0.1 mu g/mL Hoechst33342, wherein the dilution times of the antibodies are recommended to be diluted by a confining liquid according to the instruction number, and incubating for 1 hour at room temperature or incubating overnight at 4 ℃;
f. the staining solution containing the antibody was aspirated, and cells were washed 3 times for 5 minutes each by adding an appropriate amount of PBS.
g. And (3) detecting CD31 and CD34 proteins of the cells by adopting a confocal fluorescence microscope, wherein Hoechst33342 is used for marking cell nuclei, and light is avoided when operation is carried out to prevent fluorescence quenching. The results of FIG. 2 show the results of the staining identification.
CD31 and CD34 are important markers of blood vessel/hematogenic endothelial precursor cells, and the fluorescence color result can confirm that the mesodermal cells can be further induced into CD31 and CD34 positive cells, namely hematogenic endothelial precursor cells, by the method.
Example 4: before hematogenesis endotheliumEfficiency analysis of somatic cells
The resultant hematogenic endothelial precursor cells produced in example 2 were collected for identification by flow cytometry analysis.
Specifically, the method comprises the following steps:
a. cell digestion: the cells described in example 2 were digested into single cells using Accutase enzyme (cat. No.: A1110501, Gibco Co., Ltd.), and the digestion was terminated with IF9S medium.
b. Cell collection: the cells were transferred to a centrifuge tube at 1000 rpm and centrifuged for 5 minutes to enrich the cells.
c. Antibody incubation labeling: centrifuging, sucking and removing supernatant, resuspending cells with 0.2% BSA, and setting the grouping of cells and the labeling of antibodies according to the specific conditions of the experiment, wherein the identification and labeling antibodies for hematopoietic endothelial precursor cells are mouse anti-human PE-CD31 antibody (cat No. 560983, BD company) and mouse anti-human FITC-CD34 antibody (cat No. 560942, BD company); the marker molecule antibodies identified by the hematopoietic precursor/stem cells are mouse anti-human FITC-CD34 antibody (cat 560942, BD Co.) and mouse anti-human APC-CD43 antibody (cat 560198, BD Co.). The dilution ratio of the antibody is generally 1:200, and the antibody is labeled at room temperature for 30 minutes by horizontal shaking.
d. And (3) washing cells: adding a proper amount of PBS, rotating at 1000 rpm, centrifuging for 5 minutes to enrich cells, and sucking and removing supernatant.
e. Cell fixation and detection on a machine: resuspend cells with 1% paraformaldehyde in PBS. Sieving with cell sieve (40um) before detection. The cell suspension was transferred to a flow tube ready for flow analysis. The flow cytometer was a FACS Calibur analytical flow cytometer (BD), and after the test was completed, data analysis was performed using FlowJo 10.0.7 software.
FIG. 3 shows the flow cytometry analysis results of the hematopoietic endothelial precursor cells in the control group and the random three-dimensional rotation group.
Wherein the method for preparing the random three-dimensional rotating group cells is the method of example 2, the method for preparing the control group cells is basically the same as the method of example 2, and the difference is only that the step e is directly followed by 5% CO at 37 DEG C2And (5) performing static culture in an incubator for 72 hours to obtain control cells.
As can be seen from the results of fig. 3, the control group achieved induction of 37.6% of mesodermal cells into hematopoietic endothelial precursor cells using only the culture medium of the present invention. By combining the culture medium of the invention and random three-dimensional rotation, 71.6% of mesodermal cells are induced into hematopoietic endothelial precursor cells. The method of the invention proves that the random rotation can increase the induction efficiency to the hematopoietic endothelial precursor cells, and further realizes the high-efficiency induction of the hematopoietic endothelial precursor cells by matching with the culture medium of the invention.
Example 5: preparation of hematopoietic precursor cells derived from human pluripotent stem cells
Specifically, the method comprises the following steps:
a. discarding the hematopoietic endothelial precursor cell original culture medium in the culture flask, and replacing with the hematopoietic precursor cell induction culture medium obtained in example 1; filling the culture bottle with culture medium;
b. installing the cell culture bottle on a random gyroscope, setting a rotation mode to be random rotation, and adjusting the rotation speed to the rotation speed with the inner diameter and the outer diameter of 5 rpm/min;
c. the random gyroscope was placed at 37 ℃ and 5% CO2Continuously carrying out rotary culture in the incubator for 48-72 hours;
d. after 48 hours of cell culture, a large amount of "pebble-like" hematopoietic precursor cells appeared in the culture flask, and some of the hematopoietic precursor cells separated from the bottom of the dish to become suspended hematopoietic precursor cells. The hematopoietic precursor cells produced are shown in FIG. 4, where A is "pebble-like" hematopoietic precursor cells and B is suspended hematopoietic precursor cells with some of the hematopoietic precursor cells detached from the bottom of the dish.
Example 6: detection of hematopoietic precursor cells
Hematopoietic precursor cells produced in example 5 were collected for identification by immunofluorescence staining.
h. Removing the hematopoietic precursor cell induction medium in the culture flask by aspiration, and washing the cells 2 times with PBS to remove dead cells;
i. adding 4% paraformaldehyde for fixation, and standing and fixing for 20 minutes at room temperature;
j. removing 4% paraformaldehyde by suction, and washing with PBS for 2-3 times;
k. adding donkey serum with the concentration of 5% for sealing, and sealing at room temperature for 1 hour;
adding a fluorescence-labeled mouse anti-human FITC-CD34 antibody (cat No. 560942, BD company), a fluorescence-labeled mouse anti-human APC-CD43 antibody (cat No. 560198, BD company) and 0.1 mu g/mL Hoechst33342, wherein the dilution times of the antibodies are recommended to be diluted by a confining liquid according to the instruction number, and incubating for 1 hour at room temperature or overnight at 4 ℃;
m. the staining liquid containing the antibody was aspirated, and the cells were washed 3 times for 5 minutes each by adding an appropriate amount of PBS.
And n, detecting CD34 and CD43 proteins of cells by using a confocal fluorescence microscope, wherein Hoechst33342 is used for marking cell nuclei, and light is avoided during operation to prevent fluorescence quenching. The results of FIG. 5 show the results of the staining identification.
CD34 and CD43 are important markers of hematopoietic precursor cells, and the fluorescence color results can confirm that hematopoietic endothelial precursor cells can be induced to be CD34 and CD43 positive cells, i.e., hematopoietic precursor cells, by the method of the present invention.
The results in FIG. 2 show the results of the identification of the staining of hematopoietic progenitor cells.
Example 7: efficiency assay for hematopoietic precursor cells
The hematopoietic precursor cells produced in example 2 were collected and characterized by flow cytometry analysis.
Specifically, the method comprises the following steps:
f. cell digestion: the cells described in example 2 were digested into single cells using Accutase enzyme (cat. No.: A1110501, Gibco Co., Ltd.), and the digestion was stopped with IF9S medium, respectively.
g. Cell collection: the cells were transferred to a centrifuge tube at 1000 rpm and centrifuged for 5 minutes to enrich the cells.
h. Antibody incubation labeling: after centrifugation, the supernatant was aspirated, the cells were resuspended in 0.2% BSA, and the grouping of cells and labeling of antibodies were performed according to the experimental details, wherein the marker antibodies identified for hematopoietic progenitor cells were mouse anti-human FITC-CD34 antibody (cat # 560942, BD Co.) and mouse anti-human APC-CD43 antibody (cat # 560198, BD Co.), respectively. The dilution ratio of the antibody is generally 1:200, and the antibody is labeled at room temperature for 30 minutes by horizontal shaking.
i. And (3) washing cells: adding a proper amount of PBS, rotating at 1000 rpm, centrifuging for 5 minutes to enrich cells, and sucking and removing supernatant.
j. Cell fixation and detection on a machine: resuspend cells with 1% paraformaldehyde in PBS. Sieving with cell sieve (40 μm) before testing. The cell suspension was transferred to a flow tube ready for flow analysis. The flow cytometer was a FACS Calibur analytical flow cytometer (BD), and after the test was completed, data analysis was performed using FlowJo 10.0.7 software.
FIG. 6 shows the results of flow cytometry analysis of hematopoietic precursors in the control and random rotation groups. Wherein the preparation method of the cells of the random rotation group is the method of example 2, and the preparation method of the cells of the control group is substantially the same as that of example 2, except that the step a is directly followed by 5% CO at 37 ℃2And (5) performing static culture in an incubator for 72 hours to obtain control cells.
As can be seen from the results of fig. 3, the control group achieved induction of 28.9% of hematopoietic endothelial precursor cells into hematopoietic precursor cells using only the culture medium of the present invention. By combining the culture medium of the invention and random rotation, 45.9% of the hematopoietic endothelial precursor cells are induced into hematopoietic precursor cells. The method of the invention proves that random rotation can increase the induction efficiency to hematopoietic precursor cells, and further realizes high-efficiency induction by matching with the culture medium of the invention.
Example 8: other methods for obtaining hematopoietic progenitor cells from hematopoietic endothelial progenitor cells to hematopoietic progenitor cells Induction test
Hematopoietic endothelial precursor cells (described, for example, in Xu Cao, Gopala K. Yakala, Francijna E. van den Hil, Amy Cochrane, Christine L. Mummery, Valeria V. Orova. Difference and Functional comprison of Monocytes and Macrophages from PSCs with Peripheral Blood differentiation. Stem Cell reports.2019June 11; 12(6): 1282-1297) were prepared by a known method and cultured by induction to the hematopoietic precursor cells by the following method.
Specifically, the method comprises the following steps:
e. discarding the hematopoietic endothelial precursor cell original culture medium in the culture flask, and replacing with the hematopoietic precursor cell induction culture medium obtained in example 1; filling the culture bottle with culture medium;
f. installing the cell culture bottle on a random gyroscope, setting a rotation mode to be random rotation, and adjusting the rotation speed to the rotation speed with the inner diameter and the outer diameter of 5 rpm/min;
g. the random gyroscope was placed at 37 ℃ and 5% CO2Continuously carrying out rotary culture in the incubator for 48-72 hours;
h. after 48 hours of cell culture, a large amount of "pebble-like" hematopoietic precursor cells appeared in the culture flask, and some of the hematopoietic precursor cells separated from the bottom of the dish to become suspended hematopoietic precursor cells.
The culture of hematopoietic progenitor cells from hematopoietic endothelial progenitor cells of the invention may also be induced by the culture medium and the spinner culture method of the invention.

Claims (10)

1. A mesoderm cell-inducing differentiation medium comprising a basal differentiation medium and bone morphogenetic protein 4(BMP4), activin a (activin a), and a GSK-3 α/β inhibitor (CHIR 99021);
the basic differentiation medium is an IF9S complete medium, and comprises an IMDM medium and an F12 medium which are mixed according to a volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with a final concentration of 8-12mg/L, Lipids 100X additive with a volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with a volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) with a final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA2P) with a final concentration of 55-70mg/L, and glutamic-alanine dipeptide (GlutaMax) with a volume percentage of 0.8-1.2% of the IF9S complete mediumTM)100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 0.8% -1.2% of the IF9S complete medium;
preferably, the mesodermal cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL BMP4, 15-25 μ g/mL Activin A, 0.8-1.2 μ M CHIR 99021.
2. A hematopoietic endothelial precursor cell-inducing differentiation medium comprising basal differentiation medium, SB431542, VEGF, bFGF and SCF;
the basic differentiation medium is an IF9S complete medium and comprises an IMDM medium and an F12 medium which are mixed according to the volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, Lipids 100X additive with the volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with the volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA2P) with the final concentration of 55-70mg/L, and GlutaMax with the volume percentage of 0.8-1.2% of the IF9S complete mediumTM100X additive and non-essential amino acid additive 100X (neaa) in a volume percentage of 0.8% -1.2% of the IF9S complete medium;
preferably, the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 8-12 μ M SB431542, 40-60ng/mL VEGF, 40-60ng/mL bFGF and 40-60ng/mL SCF.
3. A medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, comprising a basal differentiation medium and Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), Stem Cell Factor (SCF), interleukin 3(IL-3), interleukin 6(IL-6), and Thrombopoietin (TPO);
the basic differentiation medium is an IF9S complete medium, and comprises an IMDM basic medium and an F12 basic medium which are mixed according to the volume ratio of 1:0.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, Lipids (100X) additive with the volume percentage of 0.08-0.12% of the medium, ITS-X (100X) additive with the volume percentage of 1.8-2.2% of the medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, AA2P with the final concentration of 55-70mg/L, and ITS-X (100X) additive with the volume percentage of 35-45 mu L/L0.8-1.2% of GlutaMax in percentage of the culture mediumTM(100X) an additive and a non-essential amino acid additive (NEAA, 100X) in a volume percentage of 0.8-1.2% of the culture medium;
preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL VEGF, 15-25ng/mL bFGF, 40-60ng/mL SCF, 8-12ng/mL IL-3, 40-60ng/mL IL-6, and 15-25ng/mL TPO.
4. A method of differentiating from human pluripotent stem cells to produce hematopoietic endothelial precursor cells, comprising the steps of:
s1) inducing differentiation culture of human pluripotent stem cells into mesodermal cells:
s11). cell containers are pre-coated with growth factor-reduced basement membrane matrix;
s12), adopting digestive enzyme to clone the human pluripotent stem cells, digesting and centrifuging;
s13) inoculating the digested and centrifuged small human pluripotent stem cell clone masses into a cell container obtained in S11) to obtain a pre-coated growth factor-reduced Matrigel, and adding TeSRTM-E8TMPerforming static culture on the culture medium for 24-48 hours;
s14) replacing the culture medium with a mesoderm cell induction differentiation culture medium according to claim 1, and statically culturing for 24-48 hours to obtain induced mesoderm cells;
s2) further differentiating and culturing the induced mesodermal cells into hematopoietic endothelial precursor cells:
s21). replacing the cell culture medium of the mesodermal cells with the hematopoietic endothelial precursor cell-inducing differentiation medium of claim 2;
s22), mounting the cell culture container on a random three-dimensional gyroscope, setting the rotation mode to be random rotation, and adjusting the rotation speed to be 0.1-10 rpm/min;
s23) placing a random three-dimensional gyroscope into a chamber with the temperature of 37 ℃ and the CO content of 5%2And rotationally culturing in an incubator for 60-96 hours to obtain the hematopoietic endothelial precursor cells.
5. The method according to claim 4, wherein the rotation speed in S22) is 5 rpm/min;
preferably, S22) for 72 hours.
6. A method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, which comprises the steps of culturing a cell culture container under a rotating condition by using a random gyroscope in the process of culturing the hematopoietic endothelial precursor cells;
the culture medium for culturing under the rotation condition is the culture medium for inducing the differentiation of the hematopoietic endothelial precursor cells into the hematopoietic precursor cells according to claim 3;
preferably, it comprises the following steps:
s3) culturing in the original culture medium for 72 hours, and then changing the cell culture medium to the culture medium for inducing the differentiation of the hematogenous endothelial precursor cells into the hematopoietic precursor cells according to claim 3;
s4) the cell culture flask is remounted on the random gyroscope for continuing the gyratory culture.
7. The method according to claim 6, wherein the random rotation of S3 is performed at a rotation speed of 0.1-10 rpm/min for both inner and outer diameters;
preferably, the culturing time under the spinning condition in S4) is 48 to 72 hours;
preferably, the medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells described in S3) is added to 95% -100% of the culture vessel;
preferably, S4), the cells are cultured for 48-72 hours;
preferably, S4), the cell culture conditions are 37 ℃ and 5% CO2。
8. A method of differentiating to produce hematopoietic precursor cells from human pluripotent stem cells, comprising the steps of:
1) preparing hematopoietic endothelial precursor cells by the method for generating hematopoietic endothelial precursor cells by differentiation from human pluripotent stem cells according to claim 4 or 5;
2) a method for producing hematopoietic precursor cells according to claim 6 or 7, which comprises promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.
9. A medium composition for inducing cell differentiation, comprising the mesodermal cell differentiation-inducing medium according to claims 1 and 2 and a hematopoietic endothelial precursor cell differentiation-inducing medium; or comprising the mesodermal cell differentiation-inducing medium, the hematopoietic endothelial precursor cell differentiation-inducing medium, and the medium for inducing differentiation of the hematopoietic endothelial precursor cells into hematopoietic precursor cells according to claims 1, 2, and 3.
10. Use of a random three-dimensional gyroscope for promoting the transformation of mesodermal cells into hematopoietic endothelial precursor cells, or for promoting the transformation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112760281A (en) * 2019-11-04 2021-05-07 北京基石生命科技有限公司 Culture medium for culturing primary cells of brain tumor solid tumors
CN114672455A (en) * 2022-03-25 2022-06-28 中山大学 Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells
CN114958718A (en) * 2022-05-20 2022-08-30 呈诺再生医学科技(北京)有限公司 Method for inducing cells with high FOXA2 expression
WO2022217955A1 (en) * 2021-04-13 2022-10-20 中国科学院深圳先进技术研究院 Culture medium and method for inducing differentiation of pluripotent stem cell into hematopoietic precursor cell
CN117305241A (en) * 2023-11-28 2023-12-29 上海兴瑞一达生物科技有限公司 Method for inducing and differentiating hiPSCs into NK cells
CN117946972A (en) * 2024-01-26 2024-04-30 中国科学院香港创新研究院再生医学与健康创新中心有限公司 Method for improving NK cell differentiation efficiency
WO2024104294A1 (en) * 2022-11-15 2024-05-23 上海交通大学 Method for preparing human ovarian somatic cell-like cells
WO2024113358A1 (en) * 2022-12-02 2024-06-06 Nuwacell Biotechnologies Co., Ltd. Methods and compositions for differentiation of pluripotent stem cells and derived natural killer cells
CN118421559A (en) * 2024-07-04 2024-08-02 成都赛济元生物医药有限公司 Small molecule combination, kit and method for directionally differentiating lymphohematopoietic precursor cells

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975925B (en) * 2023-03-21 2023-09-08 天九再生医学(天津)科技有限公司 Method for inducing human pluripotent stem cells to differentiate into natural killer cells by variable-speed suspension
CN116445408B (en) * 2023-05-22 2024-02-02 呈诺再生医学科技(北京)有限公司 Use of LSD1 inhibitors to promote iPSC differentiation to HSCs and maintenance of HSC dryness
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090285892A1 (en) * 2006-10-18 2009-11-19 Ramasamy Sakthivel Methods and systems for expanding ac133+ cells and directing differentiation
JP2012080874A (en) * 2010-09-15 2012-04-26 National Institute Of Advanced Industrial Science & Technology Three-dimensional tissue-constructing method in pseudo-microgravity environment
US20140273211A1 (en) * 2013-03-13 2014-09-18 Wisconsin Alumni Research Foundation Methods and Materials for Hematoendothelial Differentiation of Human Pluripotent Stem Cells Under Defined Conditions
CN105062957A (en) * 2015-06-05 2015-11-18 芜湖医诺生物技术有限公司 Culture method of vascular endothelial progenitor cells
JP2017023019A (en) * 2015-07-17 2017-02-02 国立大学法人京都大学 Method for inducing differentiation from pluripotent stem cells to mesodermal progenitor cells and blood vessel progenitor cells
US20190071636A1 (en) * 2016-03-23 2019-03-07 Kyoto University Mesoderm Induction Method Having High Blood Cell Differentiation Capacity
CN111808812A (en) * 2020-08-31 2020-10-23 首都医科大学附属北京友谊医院 Material and method for differentiation of pluripotent stem cells into hematopoietic stem cells
CN112041428A (en) * 2018-01-18 2020-12-04 新加坡科技研究局 Method for differentiating human pluripotent stem cell lines in suspension culture
WO2021032851A1 (en) * 2019-08-20 2021-02-25 Adaptimmune Limited Methods of producing haemogenic progenitor cells from pluripotent stem cells

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293730B (en) * 2014-09-23 2017-07-28 云南中科灵长类生物医学重点实验室 Multipotential stem cell vitro directed differentiation is the method for cardiac muscle cell
CN111575280B (en) * 2019-08-23 2022-12-23 西北工业大学 miR-194-5p sequence for promoting MEF2C gene expression in mouse myoblast and application thereof
CN113046318B (en) * 2021-04-13 2023-04-18 中国科学院深圳先进技术研究院 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090285892A1 (en) * 2006-10-18 2009-11-19 Ramasamy Sakthivel Methods and systems for expanding ac133+ cells and directing differentiation
JP2012080874A (en) * 2010-09-15 2012-04-26 National Institute Of Advanced Industrial Science & Technology Three-dimensional tissue-constructing method in pseudo-microgravity environment
US20140273211A1 (en) * 2013-03-13 2014-09-18 Wisconsin Alumni Research Foundation Methods and Materials for Hematoendothelial Differentiation of Human Pluripotent Stem Cells Under Defined Conditions
CN105062957A (en) * 2015-06-05 2015-11-18 芜湖医诺生物技术有限公司 Culture method of vascular endothelial progenitor cells
JP2017023019A (en) * 2015-07-17 2017-02-02 国立大学法人京都大学 Method for inducing differentiation from pluripotent stem cells to mesodermal progenitor cells and blood vessel progenitor cells
US20190071636A1 (en) * 2016-03-23 2019-03-07 Kyoto University Mesoderm Induction Method Having High Blood Cell Differentiation Capacity
CN112041428A (en) * 2018-01-18 2020-12-04 新加坡科技研究局 Method for differentiating human pluripotent stem cell lines in suspension culture
US20200399602A1 (en) * 2018-01-18 2020-12-24 Agency For Science, Technology And Research Method for Differentiation of Human Pluripotent Stem Cell Lines in Suspension Culture
WO2021032851A1 (en) * 2019-08-20 2021-02-25 Adaptimmune Limited Methods of producing haemogenic progenitor cells from pluripotent stem cells
CN111808812A (en) * 2020-08-31 2020-10-23 首都医科大学附属北京友谊医院 Material and method for differentiation of pluripotent stem cells into hematopoietic stem cells

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CAO X 等: "Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives", 《STEM CELL REPORTS》 *
CHIU B 等: "Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity", 《ACTA ASTRONAUT》 *
DEMIRCI S 等: "Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation", 《STEM CELL RES THER》 *
刘竞争 等: "微重力对造血干/祖细胞生物学特性的影响", 《微循环学杂志》 *
孟芮 等: "三维回转骨细胞条件培养基对成骨细胞功能的调节作用", 《中国生物化学与分子生物学报》 *
戴建武 等: "微重力下造血与神经干细胞三维培养与组织构建研究", 《力学与实践》 *
王哲哲 等: "微重力培养条件对造血干祖细胞分化为功能成熟的中性粒细胞的影响", 《中国细胞生物学学报》 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN112760281B (en) * 2019-11-04 2022-05-20 北京基石生命科技有限公司 Culture medium for culturing primary cells of brain tumor solid tumors
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CN114958718A (en) * 2022-05-20 2022-08-30 呈诺再生医学科技(北京)有限公司 Method for inducing cells with high FOXA2 expression
WO2024104294A1 (en) * 2022-11-15 2024-05-23 上海交通大学 Method for preparing human ovarian somatic cell-like cells
WO2024113358A1 (en) * 2022-12-02 2024-06-06 Nuwacell Biotechnologies Co., Ltd. Methods and compositions for differentiation of pluripotent stem cells and derived natural killer cells
CN117305241A (en) * 2023-11-28 2023-12-29 上海兴瑞一达生物科技有限公司 Method for inducing and differentiating hiPSCs into NK cells
CN117305241B (en) * 2023-11-28 2024-03-19 上海兴瑞一达生物科技有限公司 Method for inducing and differentiating hiPSCs into NK cells
CN117946972A (en) * 2024-01-26 2024-04-30 中国科学院香港创新研究院再生医学与健康创新中心有限公司 Method for improving NK cell differentiation efficiency
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