CN112226409B - Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells - Google Patents

Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells Download PDF

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CN112226409B
CN112226409B CN202010469127.3A CN202010469127A CN112226409B CN 112226409 B CN112226409 B CN 112226409B CN 202010469127 A CN202010469127 A CN 202010469127A CN 112226409 B CN112226409 B CN 112226409B
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hbfgf
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CN112226409A (en
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郭蕾蕾
刘靖
张世都
宋益哲
李景秋
欧镇生
梁德灿
李文佳
陈小锋
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Dongguan Dongyangguang Biopharmaceutical Research And Development Co ltd
Guangdong HEC Pharmaceutical
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Sunshine Lake Pharma Co Ltd
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Abstract

The present invention provides an improved Embryoid Body (EB) differentiation system that enables differentiation of Embryoid Body (EB) into cd34+ hematopoietic progenitor cells under normoxic, xeno-free conditions. The invention also provides a method for carrying out differentiation culture on the Embryoid Body (EB) by utilizing the improved Embryoid Body (EB) differentiation system, so that the Embryoid Body (EB) is efficiently formed, and the problem that the Embryoid Body (EB) differentiation balls are easy to adhere is solved.

Description

Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells
Technical Field
The invention belongs to the field of cell differentiation culture, and particularly relates to differentiation and culture of embryonic stem cells into CD34+ hematopoietic progenitor cells.
Background
Embryonic Stem Cells (ESCs) are highly undifferentiated cells with developmental totipotency that differentiate into all tissues and organs of adult animals, including germ cells. Embryonic Stem Cells (ESCs) also have multipotency and the ability to differentiate into various tissues by cells. The most attractive prospect and use of embryonic stem cells is the production of cells and tissues for "cell therapies" that provide non-immunogenic materials for cell transplantation. Cardiomyocytes, bone cells, cartilage cells, liver cells, hematopoietic cells, adipocytes, insulin cells, nerve cells, endothelial cells, etc. have been induced to differentiate from ESCs nowadays. The induced cells are expected to provide raw materials for organ transplantation and repair of damaged organs, so that the method has very broad clinical application prospect.
CD34 molecules belong to the cadherin family, which are selectively expressed on the surface of human and other mammalian hematopoietic stem/progenitor cells (HSC/HPC) and gradually fade to disappear as the cells mature. The CD34 antigen plays a certain role in the differentiation process of hematopoietic progenitor cells, and cd34+ hematopoietic progenitor cells can reconstitute and maintain normal hematopoiesis of the body.
The committed differentiation of embryonic stem cells into hematopoietic progenitor cells provides the possibility to obtain clinically therapeutic hematopoietic progenitor cells, in particular cd34+ hematopoietic progenitor cells. However, the number of hematopoietic cells obtained by the current methods is limited, the purity of the obtained hematopoietic cells is insufficient, the growth and differentiation of mouse stromal cells are often required to be maintained by fetal bovine serum, both serum and stromal cells belong to heterologous components with complex compositions, and high-concentration hematopoietic-related cytokines are used, so that the differentiation of hematopoietic progenitor cells has low reproducibility, and the industrial production of hematopoietic progenitor cells is limited, and the clinical application of the hematopoietic progenitor cells is limited.
For example, the current prior art method of differentiation from Embryonic Stem Cells (ESCs) into CD34+ hematopoietic progenitors is largely two: (1) An Embryoid Body (EB) differentiation method has the defects that a low-oxygen environment is commonly used in the differentiation process, the operation is inconvenient, excessive cell loss or severe adhesion among EBs are caused in the EB formation process, and the differentiation efficiency is low; and (2) co-culture differentiation method with OP9 cells (mouse bone marrow stromal cells), the method has the defects of containing cells from different sources, adopting low-oxygen environment, being complex in operation and high in technical requirement.
Therefore, there is a need to improve the shortcomings of the existing EB differentiation protocols, so as to obtain differentiation from Embryonic Stem Cells (ESCs) to cd34+ hematopoietic progenitor cells with simple operation and high purity.
Disclosure of Invention
The present invention provides an improved Embryoid Body (EB) differentiation system that enables differentiation of Embryoid Body (EB) into cd34+ hematopoietic progenitor cells under normoxic, xeno-free conditions. The invention also provides a method for carrying out differentiation culture on the Embryoid Body (EB) by utilizing the improved Embryoid Body (EB) differentiation system, so that the Embryoid Body (EB) is efficiently formed, and the problem that the Embryoid Body (EB) differentiation balls are easy to adhere is solved.
In a first aspect, the present invention provides an improved Embryoid Body (EB) differentiation system, comprising the following components:
(1) An EB forming medium, wherein one or more selected from Y-27632, hBMP-4, hbFGF and hVEGF are additionally added to a fully defined StemLine II medium; and
(2) And the mesoderm differentiation medium is prepared by adding one or more of hBMP-4, hbFGF and hVEGF into a fully defined StemLine II medium.
In some embodiments, the EB formation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the mesodermal differentiation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the concentration of Y-27632 in the EB forming medium is in the range of 8-12. Mu.M, preferably 10. Mu.M; the concentration of hBMP-4 is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
In some embodiments, the concentration of hBMP-4 in the mesodermal differentiation medium is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
The differentiation of Embryoid Bodies (EBs) into CD34+ hematopoietic progenitor cells under normoxic, xeno-free conditions can be achieved using EB-forming and mesodermal differentiation media of improved composition formulations.
Preferably, the EB forming medium and the mesoderm differentiation medium are used for suspension culture of EB.
In some embodiments, the differentiation system further comprises an aggierell plate, preferably further comprising an Anti-adhesion wash (Anti-Adherence Rinsing Solution), which may remove air bubbles from the microwells of the aggierell plate. Aggresell is a special culture vessel for suspension culture. The plate is a 24-hole plate, 300 micropores are formed at the bottom of each hole, and after the multipotent stem cells are added into the plate for centrifugation, the cells are gathered in the micropores to form uniform cell clusters.
In some embodiments, the differentiation system further comprises SB431542 (Tocres), preferably the SB431542 is added at a concentration of 4-8. Mu.M, preferably 6. Mu.M.
In some embodiments, the differentiation system further comprises a progenitor differentiation medium-1, the progenitor differentiation medium-1 being one or more additional components selected from the group consisting of hbFGF, hVEGF and hsfc in fully defined stem line II. Preferably, the progenitor cell differentiation medium-1 is additionally supplemented with one or more components selected from ITS, vc and MTG. Preferably, the concentration of said hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of SCF is in the range of 45-55ng/ml, preferably 50ng/ml. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the differentiation system further comprises a progenitor differentiation medium-2, the progenitor differentiation medium-2 being the addition of one or more components selected from hbFGF, hVEGF, hSCF, MTG, flt3l and TPO to the fully defined StemLine II. Preferably, the progenitor cell differentiation medium-2 is additionally supplemented with one or more components selected from ITS, vc and MTG. Preferably, the concentration of said hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of VEGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of SCF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M; the Flt3l is in the range of 8-12ng/ml, preferably 10ng/ml; the TPO is in the range of 25-35ng/ml, preferably 30ng/ml. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
Preferably, the Embryoid Body (EB) is obtained from Embryonic Stem Cells (ESC) or induced pluripotent stem cells (iPS). In this case, the differentiation system further comprises ESCs or iPS media, preferably Accutase (StemCell), for digesting the cells in the anchorage-dependent culture to obtain a single cell suspension.
In a second aspect, the present invention provides a method for differentiating Embryoid Body (EB) using the improved Embryoid Body (EB) differentiation system of the first aspect, the method being performed under normoxic culture conditions.
Preferably, the normoxic culture conditions are carried out under conditions of 5% carbon dioxide and air, with an oxygen content of about 20%.
In some embodiments, the differentiation culture comprises the steps of:
(1) Forming an Embryoid Body (EB) using the Embryoid Body (EB) forming medium;
(2) Culturing the Embryoid Body (EB) obtained in the step (1) by using a mesoderm differentiation medium;
(3) Culturing the Embryoid Body (EB) obtained in the step (2) by using a progenitor cell differentiation medium-1; and
(4) Culturing the Embryoid Body (EB) obtained in the step (3) by using a progenitor cell differentiation medium-2,
wherein the EB forming medium is prepared by adding one or more of Y-27632, hBMP-4, hbFGF and hVEGF in a fully defined StemLine II medium; the mesoderm differentiation medium is prepared by adding one or more of hBMP-4, hbFGF and hVEGF into a StemLine II medium.
In some embodiments, the EB formation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the mesodermal differentiation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the concentration of Y-27632 in the EB forming medium is in the range of 8-12. Mu.M, preferably 10. Mu.M; the concentration of hBMP-4 is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
In some embodiments, the concentration of hBMP-4 in the mesodermal differentiation medium is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
The differentiation of Embryoid Bodies (EBs) into CD34+ hematopoietic progenitor cells under normoxic, xeno-free conditions can be achieved using EB-forming and mesodermal differentiation media of improved composition formulations.
Preferably, the EB forming medium and the mesoderm differentiation medium are used for suspension culture of EB.
In some embodiments, in step (1), the culture is performed with an aggievell plate, preferably pretreated with an Anti-adhesion wash (Anti-Adherence Rinsing Solution) to remove air bubbles from the wells of the aggievell plate prior to culturing with the aggievell plate. Aggresell is a special culture vessel for suspension culture. The plate is a 24-hole plate, 300 micropores are formed at the bottom of each hole, and after the multipotent stem cells are added into the plate for centrifugation, the cells are gathered in the micropores to form uniform cell clusters.
Preferably, in step (1), 8X 10 5 Cell/well Density cell suspensions were seeded in Aggreewell, centrifuged at 100g for 3min to aggregate cells in microwells at 37℃with 5% CO 2 The culture was carried out overnight in an incubator under normoxic conditions without using hypoxic conditions.
In some embodiments, in step (2), further comprising the step of adding SB431542 (Tocres) to the mesodermal differentiation medium, preferably the SB431542 is added at a concentration of 4-8. Mu.M, preferably 6. Mu.M. Preferably, SB431542 (Tocres) is added on day 2 of step (2), and a half change is performed on day 3.
In some embodiments, in step (2), culturing is performed using an aggresell plate.
In some embodiments, the progenitor cell differentiation medium-1 is one in which one or more components selected from the group consisting of hbFGF, hVEGF and hfcf are additionally added to the fully defined StemLine II medium. Preferably, the progenitor cell differentiation medium-1 is additionally supplemented with one or more components selected from ITS, vc and MTG. Preferably, the concentration of said hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of VEGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of SCF is in the range of 45-55ng/ml, preferably 50ng/ml. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the progenitor differentiation medium-2 is one in which one or more components selected from hbFGF, hVEGF, hSCF, MTG Flt3l and TPO are additionally added to the fully defined StemLine II medium. Preferably, the progenitor cell differentiation medium-2 is additionally supplemented with one or more components selected from ITS, vc and MTG. Preferably, the concentration of said hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of VEGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of SCF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M; the Flt3l is in the range of 8-12ng/ml, preferably 10ng/ml; the TPO is in the range of 25-35ng/ml, preferably 30ng/ml. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
Preferably, the Embryoid Body (EB) is obtained from Embryonic Stem Cells (ESC) or induced pluripotent stem cells (iPS). In this case, the differentiation system further comprises ESCs or iPS media, preferably Accutase (StemCell), for digesting the cells in the anchorage-dependent culture to obtain a single cell suspension.
Preferably, the progenitor cell differentiation medium-1 is used in step (3) on an orbital shaker (80-120 rpm) in a low adsorption 6-well plate (Corning) at 37℃with 5% CO 2 Dynamic culture is carried out in an incubator without adopting low oxygen conditions.
Preferably, the progenitor cell differentiation medium-2 is used in step (3) on an orbital shaker (80-120 rpm) in a low adsorption 6-well plate (Corning) at 37℃with 5% CO 2 Dynamic culture is carried out in an incubator without adopting low oxygen conditions.
In some embodiments, the EB obtained in step (1) has a particle size of 232.22 ±15.13 (μm); the particle size of EB obtained in the step (2) is 236.41 +/-22.85 (mu m).
The cell survival rate after cell digestion in the step (2) Aggresell of the method of the invention is above 94%. The ratio detection of EB mesoderm Marker PDGFR alpha and FLK1 obtained in the step (2) of the method shows that the FLK1 positive rate is 3.70 percent and the PDGFR is negative. The cell survival rate of the cell mass in the low adsorption 6-pore plate in the step (3) after digestion is more than 78%, and the hematopoietic progenitor cell Marker CD34 ratio detection result shows that the positive rate is more than 17%.
In a third aspect, the present invention provides an EB-forming medium which is in STEMdiff TM ApEL TM 2, one or more selected from Y-27632, hBMP-4, hbFGF and hVEGF is additionally added into the culture medium.
In some embodiments, the EB formation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the concentration of Y-27632 in the EB forming medium is in the range of 8-12. Mu.M, preferably 10. Mu.M; the concentration of hBMP-4 is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
In a fourth aspect, the present invention provides a mesoderm differentiation medium, wherein one or more selected from the group consisting of hbfgf and hbgfis additionally added to a fully defined StemLine II medium.
In some embodiments, the mesodermal differentiation medium further comprises one or more components selected from MTG, ITS, and Vc. The concentration of MTG is preferably 50. Mu.g/ml in the range of 350-450. Mu.M, preferably 400. Mu.M. The concentration of Vc is in the range of 45-55. Mu.g/ml, preferably 50. Mu.g/ml.
In some embodiments, the concentration of hBMP-4 in the mesodermal differentiation medium is in the range of 35-45ng/ml, preferably 40ng/ml; the concentration of the hbFGF is in the range of 45-55ng/ml, preferably 50ng/ml; the concentration of hVEGF is in the range of 45-55ng/ml, preferably 50ng/ml.
In the first to fourth aspects above, the fully defined StemLine II medium is STEMdiff TM ApEL TM 2medium or StemPro34 medium.
Embryoid Bodies (EBs) are formed by embryonic stem cells (ES) or induced pluripotent stem cells (iPS) under certain culture conditions in vitro, and have an internal, middle and external three-germ layer structure, and a spherical structure which has high similarity in morphology with the early embryo development stage of mammals is called Embryoid Bodies (EBs).
STEMdiff TM ApEL TM 2 is a fully defined medium for differentiation of human embryonic stem cells (ES) and induction of pluripotent stem cells (iPS), is a serum-free and animal component-free medium. The medium is used as a basal medium in the present invention.
SB431542 is a small molecule compound that is a potent, selective anaplastic lymphoma kinase 5/transforming growth factor-beta type I Receptor (ALK 5/TGF-beta type I Receptor) inhibitor.
Y-27632 is a ROCK1 inhibitor.
hBMP-4 is human osteogenin-4.
ITS (Insulin, transferrin, selenium, i.e. insulin, transferrin, selenium) is a cell culture supplement.
Stempro34 medium, a serum-free, chemically defined, fully defined medium. The medium is used as a basal medium in the present invention.
hbFGF is a human basic fibroblast growth factor.
hVEGF is human vascular endothelial growth factor.
hSCF is a human stem cell growth factor.
Flt3l is an FMS-like tyrosine kinase 3 ligand.
TPO is thrombopoietin.
Drawings
Figure 1 shows the morphological changes over time during differentiation of EB in example 1.
FIG. 2 shows the results of the ratio detection of mesodermal Marker PDGFR to FLK1 on day 4 in example 1.
FIG. 3 shows the results of the ratio detection of Marker CD34 of hematopoietic progenitor cells at day 12 in example 1.
FIG. 4 shows the morphological changes of EBs from day 1 (D1) to day 4 (D4) of example 2 under different differentiation medium conditions.
Figure 5 shows the morphological changes of EB under different differentiation medium conditions from day 4 (D4) to day 12 (D12) in example 2.
FIG. 6 shows the results of flow assays of mesoderm at day 4 (D4) of example 2 under different differentiation medium conditions.
FIG. 7 shows the results of RT-PCR detection of mesoderm on day 4 (D4) in example 2 under different differentiation medium conditions.
FIG. 8 shows the results of flow assays of CD34+ hematopoietic progenitor cells on day 12 (D12) of example 2 under different differentiation medium conditions.
FIG. 9 shows the results of H9 differentiation into hematopoietic progenitor cells by the static system of example 3.
FIG. 10 shows the results of H1 differentiation into hematopoietic progenitor cells by the static system of example 3.
FIG. 11 shows the results of RT-PCR of the day 4 (D4) differentiated mesoderm of the static system in example 3.
FIG. 12 shows the results of a flow assay for hematopoietic progenitor cells on day 10 (D10) of static system differentiation in example 3.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that modifications and improvements made on the basis of the inventive concept will be within the scope of the present invention for those skilled in the art. The reagents used were conventional products commercially available without the manufacturer's knowledge.
EXAMPLE 1 H9 differentiation into hematopoietic progenitor cells
1. Experimental materials
And (3) cells: human embryonic stem cells H9;
reagent: mTESR1 Basal Medium, STEMdiff TM ApEL TM 2Medium、StemPro TM -34SFM、Accutase、Matrigel、PBS、SB431542、BMP4、hbFGF、hVEGF、Y27632、SCF、MTG、Flt3l、TPO；
Antibody: APC anti-human CD140a (pdgfrα) antibodies (bioleged);
CD309 (FLK 1 monoclonal antibody (Avas 12a 1), PE (eBioscience);
CD34 monoclonal antibody (4H 11), FITC (eBioscience);
the device comprises: cell incubator (Thermo 150 i), ultra clean bench (safe in Suzhou), beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), orbital shaker (Chemgless), 4℃refrigerator (sea HYC 390), 20℃refrigerator (sea), negative pressure aspirator (fish jump), pipette (eppendorf), ice maker
2. Experimental method
Preparation of ESC cells (H9 cell line)
The cells were grown at 1.8X10 5 Density of individual cells/well was passaged into Matrigel coated 6 well plates (Corning) and the cells were placed at 37℃with 5% CO 2 The wall-attached culture is carried out in an incubator. And (5) digesting the cells by using Accutase (StemCell) when the cell fusion degree reaches 70-90%, so as to obtain a single cell suspension.
EB formation (day 0-1 (D0-D1))
EB formation medium: STEMdiff TM ApEL TM 2Medium, Y-27632 (10. Mu.M), hBMP-4 (40 ng/ml), hbFGF (50 ng/ml), VEGF (50 ng/ml), MTG (thioglycerol, 400. Mu.M), ITS, vc (50. Mu.g/ml);
aggresell pretreatment: anti-adhesion wash (Anti-Adherence Rinsing Solution (StemCell)) was added to aggresell at 500 μl/well, and 1300g was centrifuged for 5min to remove air bubbles in the microwells;
the wash solution in Aggresell was aspirated and H9 cells were resuspended in EB-forming medium at 8X 10 5 Density of individual cells/well was seeded in aggresell, centrifuged at 100g for 3min to aggregate cells in microwells, 37 ℃,5% co 2 The culture was carried out overnight in an incubator, and the formation of EB was observed the next day to carry out particle size statistics.
3. Mesoderm differentiation (day 1-4 (D1-D4))
Mesoderm differentiation medium: STEMdiff TM ApEL TM 2Medium,ITS,Vc,hBMP-4(40ng/ml),hbFGF(50ng/ml),VEGF(50ng/ml),MTG(400μM)
The old medium was carefully aspirated and replaced with mesodermal differentiation medium, 6. Mu.M SB431542 (Tocres) was added on day 2 (D2), and half-changed (containing 6. Mu.M SB 431542) was performed on day 3 (D3).
4. Hematopoietic progenitor differentiation stage-1 (day 4-day 6 (D4-D6))
Progenitor cell differentiation medium-1: stempro34 Medium, ITS, vc,50ng/ml hbFGF,50ng/ml hVEGF,50ng/ml SCF, 400. Mu.M MTG;
EB spheres in aggresell plates were carefully aspirated using a 5mL pasteur pipette, passed through a 37 μm reversible filter (StemCell), turned over, EB spheres were collected using PBS into fresh 15mL centrifuge tubes (EB in each 4-well aggresell was collected into 1 centrifuge tube), centrifuged for 4min at 200g, the supernatant was discarded, EB was resuspended using 5.5mL progenitor differentiation medium-1, transferred into a low adsorption 6 well plate (Corning), the 6 well plate placed on an orbital shaker (100 rpm), 5% co at 37 °c 2 Dynamic culture in incubator.
5. Hematopoietic progenitor cell differentiation stage-2 (D6-D12)
Progenitor cell differentiation Medium-2: stempro34 Medium, ITS, vc,50ng/ml hbFGF,50ng/ml VEGF,50ng/ml SCF, 400. Mu.M MTG,10ng/ml Flt3l,30ng/ml TPO
EB spheres in the low adsorption 6 well plate were carefully aspirated using a 5mL pasteur pipette, passed through a 37 μm reversible filter (StemCell), turned over, EB spheres were collected using PBS into a new 15mL centrifuge tube, centrifuged for 4min at 200g, the supernatant was discarded, EB was resuspended using 5.5mL progenitor differentiation medium-2, transferred into a new low adsorption 6 well plate (Corning), 6 well plate placed on an orbital shaker (100 rpm), 5% co at 37% 2 Dynamic culture in incubator, changing liquid every other day.
6. Digestion of EB
EB spheres in aggresell plates were carefully aspirated using a 5mL pasteur pipette, passed through a 37 μm reversible filter (StemCell), inverted, collected into a new 15mL centrifuge tube using PBS, centrifuged at 200g for 4min, and the supernatant discarded. 2mL of TrypLE (Gibco) was added, the pellet was prevented from aggregation by shaking in a water bath at 37℃and digested for 30min, 2mL of serum-containing alpha MEM medium was added to stop the digestion, 200g was centrifuged for 4min, the supernatant was discarded, and 1mL PBS was used for resuspension counting.
7. Flow detection mesoderm Marker PDGFR alpha and FLK1 ratio
Taking 1.2X10 6 EB cells were resuspended with 300. Mu.L of Stain Buffer and divided equally into 3 parts (three groups of unstaine, isotype and Stain), with 5. Mu.L of PDGFR alpha, FLK1 antibody added to the Stain group; adding the corresponding isotype into the isotype group; unstaine was left untreated, incubated at room temperature for 1h in the dark, washed twice with 1mL of Stain Buffer, resuspended in 200. Mu.L of Stain Buffer, and assayed using BD FACSVerse.
8. Ratio of marker CD34 for flow detection of hematopoietic progenitor cells
Taking 1.2X10 6 The individual cells were resuspended in 300. Mu.L of Stain Buffer and divided equally into 3 parts (divided into unstaine, isotype and Stain groups) with 5. Mu.L of CD34 monoclonal antibody added to the Stain group; adding the corresponding isotype into the isotype group; unstaine was left untreated, incubated at room temperature for 1h in the dark, washed twice with 1mL of Stain Buffer, resuspended in 200. Mu.L of Stain Buffer, and assayed using BD FACSVerse.
3. Experimental results
1. The morphological changes of EB during differentiation are shown in figure 1. Particle diameter of day 1 (D1) EB is 232.22 + -15.13 (μm), particle diameter of day 4 (D4) is 236.41 + -22.85 (μm);
2. differentiated cell digestibility on day 4 (D4): cell viability 94.97% after cell digestion in 2-well aggresell, number of viable cells 1.96×10 6 ；
3. Results of ratio detection of mesodermal Marker pdgfrα, FLK1 on day 4 (D4): FLK1 positive rate was 3.70% and PDGFR negative, see fig. 2;
4. digestion of differentiated cells on day 12 (D12): cell viability after digestion of cell mass in 1-well low-adsorption six-well plate was 78.23% and viable cell count was 1.24X10 6 A cell;
5. results of ratio detection of hematopoietic progenitor marker CD34 on day 12 (D12): the positive rate was 17.65%, see FIG. 3.
Example 2 Effect of different culture media on hematopoietic progenitor differentiation on days 1-4 (D1-D4)
1. Experimental materials
And (3) cells: human embryonic stem cells H9;
reagent: mTESR1 Basal Medium, STEMdiff TM ApEL TM 2Medium、StemPro TM -34SFM、Accutase、Matrigel、PBS、SB431542、BMP4、hbFGF、hVEGF、Y27632、SCF、MTG、Flt3l、TPO；
Antibody: APC anti-human CD140a (pdgfrα) antibodies (bioleged); CD309 (FLK 1) monoclonal antibody (Avas 12a 1), PE (eBioscience);
CD34 monoclonal antibody (4H 11), FITC (eBioscience);
the device comprises: cell incubator (Thermo 150 i), ultra clean bench (safe in Suzhou), beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), orbital shaker (Chemgless), 4℃refrigerator (sea HYC 390), 20℃refrigerator (sea), negative pressure aspirator (fish jump), pipette (eppendorf), ice maker.
2. Experimental method
The culture media according to days 1-4 (D1-D4) were divided into 4 groups as follows:
TABLE 1
Note that: apEL, i.e. STEMdiff TM ApEL TM 2Medium
Specific experimental methods refer to example 1.
3. Experimental results
1. The morphology changes of EBs from day 1 to day 4 (D1-D4) during differentiation are shown in FIG. 4. The particle sizes of the 4 groups of EB balls are larger, and the particle sizes of the D1-K1 groups are the smallest. The particle size statistics are shown in Table 2. The morphology of each group of EB is similar, and no obvious difference exists;
2. the EB morphology changes from day 4 to day 12 (D4 to D12) of each group during differentiation are shown in table 2. The cell clusters scatter in the culture process, the cell clusters are different in size, obvious cavitation bubbles are not observed, the K2-K4 have cavitation bubbles on the 12 th day (D12), the K2 cavitation bubbles are most obvious, and adhesion does not occur in the culture processes of all groups;
3. EB was digested on day 4 (D4), with higher survival rates for the 4 groups and more viable cells, see FIG. 5. The mesoderm Marker PDGFR alpha and FLK1 are detected, and the flow detection result shows that the FLK1 positive rate is low by about 3%, PDGFR K1-K3 are negative, the K4 positive rate is 4.55%, and 4 groups have no double positive cells, see Table 3. The results of detection of the RT-PCR mesoderm related genes show that the positive indexes PDGFR, MIXL1, KDR and T of the K3 group are obviously increased on the 4 th day (D4) and the negative index Oct4 is obviously reduced relative to the differentiation initial cells H9 and K3. Among the indexes of K3 group, PDGFR increases most obviously, and increases more than 50 times. The other indexes perform poorly in combination, see figure 6.
4. EB was digested on day 12 (D12), with higher survival of 4 groups of cells and more viable cells, see FIG. 7. Flow-through detection of hematopoietic progenitor cell marker CD34, see FIG. 8, shows that group K1 CD34 is negative, CD34 positive rate K3> K4> K2, and the positive rate of group K3 is up to 17.65%;
in conclusion, the K3 component has better differentiation effect than other components, and the differentiation efficiency is higher and reaches 17.65 percent.
TABLE 2 particle size statistics of EB under different differentiation Medium conditions from day 1 (D1) to day 4 (D4)
TABLE 3 statistics of EB cell viability following digestion on days 4 (D4) and 12 (D12)
EXAMPLE 3 differentiation of H9 or H1 into hematopoietic progenitor cells under static System
1. Experimental materials
And (3) cells: human embryonic stem cells H9, H1;
reagent: mTESR1 Basal Medium, STEMdiff TM ApEL TM 2Medium、StemPro TM -34SFM、Accutase、Matrigel、PBS、SB431542、BMP4、hbFGF、hVEGF、Y27632、SCF、MTG、Flt3l、TPO；
Antibody: APC anti-human CD140a (pdgfrα) antibodies (bioleged); CD309 (FLK 1) monoclonal antibody (Avas 12a 1), PE (eBioscience), CD34 monoclonal antibody (4H 11), FITC (eBioscience); the device comprises: cell incubator (Thermo 150 i), ultra clean bench (safe in Suzhou), beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), orbital shaker (Chemgless), 4℃refrigerator (sea HYC 390), 20℃refrigerator (sea), negative pressure aspirator (fish jump), pipette (eppendorf), ice maker.
2. Experimental method
EB formation (day 0-4 (D0-D1))
EB formation medium: STEMdiff TM ApEL TM 2Medium,Y-27632(10μM)；
Aggresell pretreatment: anti-adhesion wash (Anti-Adherence Rinsing Solution) (StemCell) was added to aggresell at 500 μl/well, and 1300g was centrifuged for 5min to remove air bubbles in the microwells;
the wash solution in Aggresell was aspirated and H9 cells were resuspended in EB-forming medium at 8X 10 5 Cell/well density was seeded in Aggresell, centrifuged at 100g for 3min to aggregate cells in microwells, 37℃and 5% CO 2 Culturing overnight in an incubator, observing the formation of EB the next day, and carrying out particle size statistics;
2. mesoderm differentiation (day 0-4 (D1-D4))
Mesoderm differentiation medium: stemPro TM -34SFM，hBMP-4(40ng/ml)，hbFGF(50ng/ml),VEGF(50ng/ml)，MTG(400μM)，ITS(1X)，Vc(50μg/ml)；
EB spheres in aggresell plates were carefully aspirated using a 5mL pasteur pipette, passed through a 37 μm reversible filter (StemCell), inverted the reversible filter, collected EB spheres using PBS into fresh 15mL centrifuge tubes (EB in each 4-well aggresell was collected into 1 centrifuge tube), centrifuged for 4min at 200g, the supernatant discarded, EB resuspended using 5.5mL mesodermal differentiation medium, transferred into a low-adsorption 6-well plate (Corning), shaken, and the 6-well plate placed at 37 ℃ at 5% co 2 Static culture in incubator. 6. Mu.M SB431542 (Tocris) was added on day 2 (D2) and half-changed (containing 6. Mu.M SB 431542) was performed on day 3 (D3).
The rest of the procedure is described in example 1.
3. Conclusion of the experiment
1. The bonding between EBs is very serious in the differentiation process of days 4-10 (D4-D10), the clusters are still bonded together after the clusters are quickly picked out on the 6 th day (D6), the morphology of the EBs is very poor, and the cavitation morphology does not appear (see figure 9 and figure 10);
2. the detection of the RT-PCR mesoderm related genes shows that the negative index Oct4 is obviously reduced relative to the differentiation initial cell H1. But the positive indexes PDGFR, MIXL1, KDR and T have smaller and even lower rising times on the 4 th day (D4). The comprehensive performance of each index is poor, see figure 11;
3. EB was digested on day 10 (D10) with a cell viability of 90.56% for the H9 differentiated group and 4.25X10 viable cell count 6 The method comprises the steps of carrying out a first treatment on the surface of the The cell viability of the H1 differentiated group was 86.73% and the number of viable cells was 4.12X10 6 . Flow through detection of hematopoietic progenitor marker CD34 showed 4.11% positive for CD34+ on day 10 (D10) of H1 differentiation, 5.36% positive for CD34+ on day 10 (D10) of H9 differentiation, and very low positive for all, see FIG. 12.
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 (10)

1. A method of differentiating embryoid bodies under normoxic culture conditions, comprising the steps of:
(1) Forming an embryoid-like body using an embryoid-like body forming medium;
(2) Culturing the embryoid body obtained in the step (1) by using a mesoderm differentiation medium;
(3) Culturing the embryoid body obtained in the step (2) by using a progenitor cell differentiation medium-1; and
(4) Culturing the embryoid body obtained in the step (3) by using a progenitor cell differentiation medium-2;
the embryoid body-forming medium is prepared by adding Y-27632, hBMP-4, hbFGF, hVEGF and MTG into a fully defined StemLine II medium;
in the embryoid body forming medium, Y-27632 is at a concentration in the range of 8-12. Mu.M, hBMP-4 is at a concentration in the range of 35-45ng/ml, hbFGF is at a concentration in the range of 45-55ng/ml, hVEGF is at a concentration in the range of 45-55ng/ml, and MTG is at a concentration in the range of 350-450. Mu.M;
the mesoderm differentiation medium is prepared by adding hBMP-4, hbFGF, hVEGF, MTG, ITS and Vc into a fully defined StemLine II medium;
in the mesoderm differentiation medium, the concentration of hBMP-4 is in the range of 35-45ng/ml, the concentration of hbFGF is in the range of 45-55ng/ml, the concentration of hVEGF is in the range of 45-55ng/ml, the concentration of MTG is in the range of 350-450 mu M, and the concentration of Vc is in the range of 45-55 mu g/ml;
the progenitor cell differentiation medium-1 is prepared by adding hbFGF, hVEGF, hSCF, ITS, vc and MTG in a fully defined StemLine II medium;
in the progenitor cell differentiation medium-1, the concentration of hbFGF is in the range of 45-55ng/ml, the concentration of hVEGF is in the range of 45-55ng/ml, the concentration of hSCF is in the range of 45-55ng/ml, the concentration of MTG is in the range of 350-450. Mu.M, and the concentration of Vc is in the range of 45-55. Mu.g/ml;
the progenitor cell differentiation medium-2 is prepared by adding hbFGF, hVEGF, hSCF, flt3l, TPO, ITS, vc and MTG into a fully defined StemLine II medium;
in the progenitor cell differentiation medium-2, the concentration of hbFGF is in the range of 45-55ng/ml, the concentration of hVEGF is in the range of 45-55ng/ml, the concentration of hSCF is in the range of 45-55ng/ml, flt3l is in the range of 8-12ng/ml, TPO is in the range of 25-35ng/ml, MTG is in the range of 350-450. Mu.M, and Vc is in the range of 45-55. Mu.g/ml.
2. The method of claim 1, wherein the embryoid body forming medium has a concentration of Y-27632 of 10. Mu.M, a concentration of hBMP-4 of 40ng/ml, a concentration of hbFGF of 50ng/ml, a concentration of hVEGF of 50ng/ml, and a concentration of MTG of 400. Mu.M.
3. The method of claim 1, wherein the mesodermal differentiation medium has a concentration of hbFGF-4 of 40ng/ml, hbFGF of 50ng/ml, hbvegf of 50ng/ml, MTG of 400 μΜ and Vc of 50 μg/ml.
4. The method of claim 1, wherein the progenitor cell differentiation medium-1 has a concentration of hbFGF of 50ng/ml, a concentration of hvgfs of 50ng/ml, a concentration of hfcf of 50ng/ml, a concentration of MTG of 400 μΜ, and a concentration of Vc of 50 μg/ml.
5. The method of claim 1, wherein the progenitor cell differentiation medium-2 has a concentration of hbFGF of 50ng/ml, a concentration of hvgff of 50ng/ml, a concentration of hfcf of 50ng/ml, flt3l of 10ng/ml, TPO of 30ng/ml, MTG of 400 μΜ, and Vc of 50 μg/ml.
6. The method of claim 1, further comprising the step of obtaining embryoid bodies from embryonic stem cells or induced pluripotent stem cells.
7. The method according to any one of claims 1 to 6, wherein the step (1) and the step (2) are suspension culture and the step (3) and the step (4) are dynamic culture.
8. The method of any one of claims 1-6, wherein the embryoid-forming medium and the mesodermal differentiation medium are used for suspension culture of embryoid-like bodies
9. The method of any one of claims 1-6, wherein the fully defined StemLine II cultureThe base being STEMdiff TM ApEL TM 2medium or stemro 34 medium.
10. The method of any one of claims 1-6, wherein the normoxic culture conditions are at about 20% oxygen in the presence of 5% carbon dioxide and air.
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