CN109797132A - A method of promotion human pluripotent stem cells directed differentiation is endothelial cell - Google Patents
A method of promotion human pluripotent stem cells directed differentiation is endothelial cell Download PDFInfo
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Abstract
The invention discloses a kind of methods that promotion human pluripotent stem cells directed differentiation is endothelial cell, the method comprise the steps that the 0th~1 day, induction differentiation is carried out using the human pluripotent stem cells that the CDM3 differential medium of 4~8 μM of GSK3I of addition reaches 95% or more to cell density；2nd day, differentiation is further induced to the cell after previous step Fiber differentiation using the CDM3 differential medium of addition 40~60ng/ml bFGF；3rd~5 day, differentiation is further induced to the cell after previous step Fiber differentiation using the CDM3 differential medium of addition 40~60ng/ml VEGF and 20~30ng/ml BMP4；Cell after previous step Fiber differentiation was digested in 6th day, culture is then continued 3~4 days using Endothelial cell culture base.The present invention breaks up the method more economical and effective of endothelial cell, and atomization is more convenient；Serum is not used in atomization, excludes the interference of many factors, and differentiated result is more reliable；And addition RA can promote the efficiency of endothelial differentiation, once break up available more endothelial cells.
Description
Technical field
The present invention relates to a kind of methods that promotion human pluripotent stem cells directed differentiation is endothelial cell, belong to cell culture skill
Art field.
Background technique
The health of vascular diseases affect millions of people.Serious vascular diseases lead to the apoplexy of amputation and threat to life.?
The endothelial cell of all Endovascular arrangements is adjusting vasopermeability, and angiogenesis and regeneration play vital work
With.In physiological conditions, blood vessel can be generated by angiogenesis and angiogenesis.Angiogenesis refers to be formed newly from progenitor cells
Blood vessel, and angiogenesis refers to the migration by existing vascular cell and is proliferated from existing vascularization new blood vessel.However, these
Process is suffering from apoplexy, is damaged in the patient of diabetes or senile dementia.In vascular diseases, due to endothelial cell
Abnormality or death cause vascular function to deteriorate, recently some researches show that, endothelial cells have to form the ability of blood vessel, and
It is very helpful for improving vascular diseases, therefore, it is necessary to obtain the endothelial cell of a large amount of source of people to treat these blood vessels
Disease.
Human pluripotent stem cells, such as the multipotential stem cell (hiPSC) of human embryo stem cell (hESCs) and people's induction, can
Self-renewing simultaneously breaks up any cell type in adult body, therefore is the ideal resource for generating endothelial cell (ECs).Many is ground
Studying carefully group, verified ECs can derive from hiPSCs and hESCs.These inductions obtain endothelial cell and can be used for self ischemic
The reconstructing blood vessel and customization artificial blood vessel and tissue in region, in addition, being also in vitro to grind using pluripotent stem cell differentiation endothelial cell
The molecular mechanism for studying carefully regulation endothelium destiny provides new approach.Facilitate to understand the morbidity machine of vascular diseases by Mechanism Study
It makes and provides effective measures for treatment vascular diseases.However, before developing clinical application, it is necessary to establish dry thin from multipotency
The inexpensive and efficient method of ECs is obtained in born of the same parents.Furthermore, it is necessary to further explore regulation pluripotent stem cell differentiation endothelial cell
Mechanism.
At present there are many kinds of the methods of differentiation endothelial progenitor cells, but the process of generally existing differentiation is complicated, and the differentiation period is long,
Higher cost breaks up the disadvantages of unstable.Firstly, in attached cell differentiation method: 2015, Gop μ Sriram etc., using
The differential mediums such as StemDiff APEL, ECGM-MV2 in the case where continuously adding BMP4, FGF2, VEGF, obtain CD31/
The cell proportion of the bis- positives of CD144 is 80.7%.But StemDiff APEL used in the method, ECGM-MV2 differential medium
Selling at exorbitant prices greatly increases differentiation cost.2017, Kit Man Tsang etc. made under continuous seven days anoxia conditions
With BMP4, FGF2, VEGF, so that the differentiation efficiency of endothelium is promoted to 29.3% by 12.4% under normal oxygen.In suspension cell point
Under conditions of change, 2010, Hongkui Deng etc., the mode broken up using EB, during seven days, using BMP4,
The method of VEGF obtains the progenitor cells of the 27% CD34 positive.
Currently, have the following deficiencies: that cost is excessively high about differentiation endothelial cell, and mTeSR culture medium, StemDiffAPEL,
ECGM-MV2 culture medium selling at exorbitant prices is not suitable for mass propgation and differentiation endothelial cell；Prior art differentiation efficiency is low, yield
It is low.
Summary of the invention
In order to solve the above technical problems, it is endothelial cell that the present invention, which provides a kind of promotion human pluripotent stem cells directed differentiation,
Method, method of the invention reduces human embryo stem cell and people's multipotency induces the cost of stem cell external evoked endothelial cell,
And the differentiation efficiency of endothelial cell is further improved with retinoic acid RA, more endothelial cells are obtained in an atomization.
It is the method for endothelial cell the first purpose of the invention is to provide a kind of promotion human pluripotent stem cells directed differentiation,
Include the following steps:
(1) at the 0th~1 day, using the CDM3 differential medium of 4~8 μM of GSK3I of addition, cell density is reached
95% or more human pluripotent stem cells carry out induction differentiation；
(2) on day 2, using the CDM3 differential medium of addition 40~60ng/ml bFGF to step (1) Fiber differentiation
Cell afterwards carries out further induction differentiation；
(3) at the 3rd~5 day, the CDM3 using addition 40~60ng/ml VEGF and 20~30ng/ml BMP4 breaks up training
It supports base and further induction differentiation is carried out to the cell after step (2) Fiber differentiation；
(4) at the 6th day, addition cell dissociation enzyme digests the cell after step (3) Fiber differentiation, then will digestion
The cell obtained afterwards continues culture 3~4 days using Endothelial cell culture base；
Contain RPMI-1640 basal medium, dual anti-, bovine serum albumin(BSA) and Vitamin C in the CDM3 culture medium
Acid.
Further, in step (2), 0.8~1.2 μM of retinoic acid (RA) is added while adding bFGF.
Further, the human pluripotent stem cells are the multipotential stem cell that human embryo stem cell (hESCs) or people induce
(hiPSC)。
It further, further include that passage training is carried out to human pluripotent stem cells using stem cell media before step (1)
It supports to cell density and reaches 95% or more.
Further, the stem cell media is PSeasy-E8 or mTeSR.
Further, the culture and passage specifically comprise the following steps: human pluripotent stem cells culture is close to cell
After degree reaches 85~90%, cell fragment is cleaned using no calcium phosphate buffer (DPBS), 0.5 μM of EDTA is then added,
5~10min is digested, is then dispelled cell, the ratio after dispelling according to 1:6~1:10 is passaged to the coated culture of Matrigel
In ware, continuing culture to cell density is 95% or more.
Further, in step (1), the GSK3I is CHIR99021.
Further, the concentration of bovine serum albumin(BSA) is 0.4~0.6mg/ml, ascorbic acid in the CDM3 culture medium
Concentration be 0.20~0.25mg/ml, it is described it is dual anti-be penicillin and streptomysin, wherein penicillin concn is 100U/ml, chain
Mycin concentration is 0.1mg/ml.
Further, the cell dissociation enzyme is pancreatin cell dissociation buffer or Accutase cell dissociation buffer.
Further, the Endothelial cell culture base is ECM culture medium.
Further, in step (4), the cell inoculation obtained after digestion is carried out into the coated culture dish of Gelatin
Culture.It is adherent that auxiliary cell is conducive to using the coated culture dish of Gelatin, is conducive to endothelial cell growth.
In the present invention, 0 is referred within the 0th day~for 24 hours, refer to 24~48h within the 1st day, and so on.
The beneficial effects of the present invention are:
The present invention breaks up the method more economical and effective of endothelial cell, and atomization is more convenient；Atomization
In do not use serum, exclude the interference of many factors, and differentiated result is more reliable；And addition RA can promote endothelial differentiation
Efficiency once breaks up available more endothelial cells.
Detailed description of the invention
Fig. 1 is the microphoto of undifferentiated embryonic stem cell；
Fig. 2 is to add RA and control group to compare in the 6th day differentiation efficiency streaming；
Fig. 3 is to add RA and control group to compare in the 10th day differentiation efficiency streaming；
Fig. 4 is the tenth day endothelial cell streaming figure after CD144 magnetic beads for purifying；
Fig. 5 is endothelial cell immunotoxin fluorescent staining after purification.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can be with
It more fully understands the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1: the inducible factor and small molecule compound of directed differentiation
Inducible factor: CHIR99021,6 μM of activity；Basic fibroblast growth factor (bFGF), activity
100ng/μl；Vascular endothelial growth factor (VEGF), activity 50ng/ μ l；Bone Morphogenetic Protein 4 (BMP4) acts on dense
Spend 25ng/ μ l；
Small molecule compound: retinoic acid (RA), 1 μM of activity.
Embodiment 2: the culture and passage of embryonic stem cell
On the coated culture dish of Matrigel, using PSeasy-E8 culture medium culture human embryo stem cell (hESCs),
When cell confluency degree reaches 80% or more, culture medium is siphoned away, DPBS is added and washes once；EDTA, 37 DEG C of very steady cultures are added
It is incubated for 3-5 minutes in case；Digestive juice is sucked, fresh PSeasy-E8 culture medium is added immediately, is blown and beaten and is cultivated with liquid-transfering gun sector
Ware bottom, the cell colony for attaching ware bottom fall off, and soft slowly pressure-vaccum mixes.It then will be thin according to the ratio of 1:8 or 1:10
Born of the same parents reach in the new use coated tissue culture plate of Matrigel, and Pseasy-E8 culture medium is used to carry out secondary culture, passage
Preceding be added for 24 hours, in culture medium afterwards contains 10 μM of ROCK-I (Y27632), the culture solution then more renewed daily, until next time passes
Generation.
Undifferentiated human embryo stem cell is taken pictures under inverted fluorescence microscope, as a result such as Fig. 1, shown Human embryo is done thin
Born of the same parents H1 system is grown in the coated culture dish of Martrigel, and with PSeasy-E8 culture medium culture, cell arrangement is close, is in colony
Shape growth, there are apparent boundaries for cell clone and surrounding.
Embodiment 3: the vitro directed differentiation of endothelial cell
Continue culture to density in 95%-100% with PSeasy-E8 culture medium, replacement differential medium induction.This point
Change culture medium is CDM3 (containing 1640 basal medium of RPMI Medium, dual anti-, bovine serum albumin(BSA) and ascorbic acid).
Retinoic acid treatments group (RA group): being denoted as the 0th day, a few days ago (- the 1 day the 0th day) of differentiation from induction, is added
6 μM of CHIR99021.It 2nd day, replaces culture medium C DM3 and adds 50ng/mlbFGF and 1 μM of RA.3rd day to the 5th day, more
Changing culture medium is that CDM3 adds 50ng/ml VEGF, 25ng/mlBMP4.It 6th day, is digested with 0.1% pancreatin or Accutase, so
After be seeded to the coated 60mm culture dish of Gelatin continue culture 3-4 days, replace medium to ECM.
Control group (DMSO group): its incubation is identical as above-mentioned RA group step, and difference is the culture medium of addition in the 2nd day
That add in CDM3 is 50ng/ml bFGF and DMSO identical with RA volume in RA group.
By the endothelial cell of induction in the 6th day and the 10th day progress FCM analysis, the specific method is as follows:
1. being digested using 0.1% pancreatin, it is put into 37 DEG C of constant incubators 3-5 minutes, the termination of ECM culture medium is added and disappears
Change, blows and beats cell to single with liquid-transfering gun, cell suspension is added in 1.5ml centrifuge tube；
2. centrifugation, 300g, 5 minutes；
3. 80 μ l PBS, which are added, is resuspended cell (3*105A cell), be added 20 μ l CD34-APC or CD31-FITC or
CD31-APC or CD144-PE antibody, is protected from light incubation 30 minutes by 4 DEG C, 400 μ lPBS is added, cell, upper machine testing is resuspended.
Testing result is as shown in Figures 2 and 3, and CD34 is a kind of marker molecule of endothelial progenitor cells in Fig. 2, detects within the 6th day
CD34 positive rate (29.6%) to RA processing group increases 10.9% than the positive rate (16.0%) of DMSO group.In Fig. 3,
CD144 and CD31 is the marker molecule of endothelial cell, detects within the 10th day the bis- positive rates of CD144 and CD31 of RA processing group
(84.6%) 23.9% is increased than double positive rates (60.7%) of DMSO group.
Embodiment 4: the purifying of endothelial cell
1. being digested 3-5 minutes using 0.1% pancreatin at the 10th day, and using 10mLBuffer (PBS+0.5%BSA+2mM
EDTA cell) is resuspended；
2. cell suspension passes through 30 microns of filter screen, cell mass is removed；
3.300g, 10 minutes, centrifugation；
Cell is resuspended in 4.80 μ L Buffer, and 20ml CD144 magnetic bead is added and is put into 4 DEG C, is incubated for 15 minutes；
5.1-2mL Buffer washes away magnetic bead, and 300g 5 minutes, is centrifuged；
6. 300 μ l Buffer, which are added, is resuspended cell, cell is made to pass through LS Filter column (Miltenyi Biotec),
5mlBuffer crosses column, altogether three times；
7. 5ml Buffer is added, LS filters post separation magnet, flows out the cell of CD144 marked by magnetic bead, 300g, 5 points
Zhongli's heart；
8. ECM culture medium, which is added, is resuspended cell, make cell continued growth in the coated ware of Fibronectin.
Endothelial cell after purifying is detected using FCM analysis method in the same manner as in Example 3, is tied
After fruit sees that attached drawing 4, the endothelial cell of induction in the 10th day use CD144 magnetic beads for purifying, the bis- positive rates of flow cytometer detection CD144 and CD31
It is 98.3%.
Immunofluorescence dyeing is carried out using CD144 antibody, as a result as shown in figure 5, cell boundaries are irregular threadiness, carefully
It is regular ellipticity that karyon is dyed with Hoechst.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of method for promoting human pluripotent stem cells directed differentiation as endothelial cell, which comprises the steps of:
(1) at the 0th~1 day, using the CDM3 differential medium of 4~8 μM of GSK3I of addition, 95% or more is reached to cell density
Human pluripotent stem cells carry out induction differentiation；
(2) on day 2, after using the CDM3 differential medium of addition 40~60ng/ml bFGF to step (1) Fiber differentiation
Cell carries out further induction differentiation；
(3) at the 3rd~5 day, the CDM3 differential medium of addition 40~60ng/ml VEGF and 20~30ng/ml BMP4 is used
Further induction differentiation is carried out to the cell after step (2) Fiber differentiation；
(4) at the 6th day, addition cell dissociation enzyme digests the cell after step (3) Fiber differentiation, then will obtain after digestion
To cell culture is continued 3~4 days using Endothelial cell culture base；
Contain RPMI-1640 basal medium, dual anti-, bovine serum albumin(BSA) and ascorbic acid in the CDM3 culture medium.
2. the method according to claim 1, wherein being added 0.8 while adding bFGF in step (2)
~1.2 μM of retinoic acids.
3. the method according to claim 1, wherein the human pluripotent stem cells are human embryo stem cell or people
The multipotential stem cell of induction.
4. the method according to claim 1, wherein further including using stem cell media before step (1)
Secondary culture to cell density is carried out to human pluripotent stem cells and reaches 95% or more.
5. according to the method described in claim 4, it is characterized in that, the stem cell media be PSeasy-E8 or
mTeSR。
6. according to the method described in claim 4, it is characterized in that, the secondary culture specifically comprises the following steps: people
After multipotential stem cell culture reaches 85~90% to cell density, using no calcium phosphate buffer solution for cleaning cell fragment, then
0.5 μM of EDTA is added, digests 5~10min, then cell is dispelled, is passaged to after dispelling according to the ratio of 1:6~1:10
In the coated culture dish of Matrigel, continuing culture to cell density is 95% or more.
7. the method according to claim 1, wherein the GSK3I is CHIR99021 in step (1).
8. the method according to claim 1, wherein in the CDM3 culture medium bovine serum albumin(BSA) concentration
For 0.4~0.6mg/ml, the concentration of ascorbic acid is 0.20~0.25mg/ml, it is described it is dual anti-be penicillin and streptomysin,
Middle penicillin concn is 100U/ml, and streptomysin concentration is 0.1mg/ml.
9. the method according to claim 1, wherein the cell dissociation enzyme be pancreatin cell dissociation buffer or
Accutase cell dissociation buffer.
10. the method according to claim 1, wherein the Endothelial cell culture base is ECM culture medium.
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| CN116574672A (en) * | 2023-07-11 | 2023-08-11 | 北京北启生物医药有限公司 | Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells |
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| CN113897331A (en) * | 2021-09-29 | 2022-01-07 | 苏州大学 | Differentiation method for inducing human artery endothelial cells |
| CN116574672A (en) * | 2023-07-11 | 2023-08-11 | 北京北启生物医药有限公司 | Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells |
| CN116574672B (en) * | 2023-07-11 | 2023-10-20 | 北京北启生物医药有限公司 | Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells |
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