CN109609455A - The cultivating system of amplifying candidate stem cell, method and application thereof - Google Patents
The cultivating system of amplifying candidate stem cell, method and application thereof Download PDFInfo
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Abstract
The present invention relates to the cultivating systems of technical field of cell culture more particularly to amplifying candidate stem cell, method and application thereof.The present invention provides the compositions that ISOPROTEREND and MS275 is formed, and provide it and promoting the application in hematopoietic stem cell expansion.Research shows that, during umbilical cord blood hematopoietic stem cell amplification cultivation, ISOPROTEREND and MS275 is added while cell factor is added, achieve the effect that not only to increase candidate stem cell quantity simultaneously but also improves candidate stem cell CFU Colony forming ability, so that candidate stem cell, which is at, is proliferated undifferentiated state, and then reach clinical transplantation demand.Of the invention is easy to operate, low in cost, and obtained candidate stem cell is more, solves the defects of low hematopoietic stem cell expansion rate in the prior art, easy differentiation.
Description
Technical field
The present invention relates to the cultivating system of technical field of cell culture more particularly to amplifying candidate stem cell, method and its
Purposes.
Background technique
Hematopoietic stem cell transplantation technology is clinical treatment leukaemia, lymthoma, alpastic anemia, thalassemia
Etc. the common and effective treatment means of a variety of blood class diseases and disease of immune system.Usually there are three sources for candidate stem cell:
Marrow, peripheral blood and Cord blood.Compared with marrow and peripheral blood hematopoietic stem cells, it is convenient, next that umbilical cord blood hematopoietic stem cell obtains
Source is abundant, not damaged to donor without side-effects, therefore becomes a big important sources of hematopoietic stem cell transplantation donor.
Currently, the bottleneck of umbilical cord blood stem cell transplantation technology is that its cell content is few, it is contained in a cord blood
Candidate stem cell and progenitor cell population be not enough to quickly restore adult patient immune system, cause opportunistic infections lethality
Increase.Strategy provisional at present is double Umbilical Cord Blood Transplantation, i.e. successively receives the shifting of two cord bloods after a clear marrow of patient
It plants, but which increase the HLA distribution type difficulty of matching of donor, therefore, the method for needing amplification umbilical cord blood hematopoietic stem cell, to obtain
Enough candidate stem cells for transplanting.
People have carried out a large amount of trials for the amplification in vitro of umbilical cord blood hematopoietic stem cell, but all without obtaining ideal effect
Fruit.Early stage people cultivate candidate stem cell using the cell factor in blood, as a result lead to cell differentiation, and portability function subtracts
It is weak.Later, it has been found that Wnt signaling molecule, Notch ligand, the retinoic acid antagonism factor in marrow hemopoietic stem cells microenvironment
Etc. can effectively expand CD34+ hematopoietic stem/progenitor.Remain external using CHIR99021 or BIO activation Wnt signal path
The transfer ability of the candidate stem cell of culture；And DLL1 is added in the cultivating system of candidate stem cell, DSL1 etc. can pass through
Activate Notch signal and appropriate amplifying candidate stem cell.Separately studies have found that, the PTN of marrow endothelial stroma cell secretion also can
Enough slight amplifying candidate stem cells.Under physiological status at candidate stem cell under low oxygen conditions, the oxygen side of body of in vitro culture generation
Compel self-renewing and portability function that meeting damages candidate stem cell by increasing ROS level；It has been found that the addition of antioxidant
And the inhibition of mTOR can offset these damages.However, above-mentioned technology can not expand umbilical cord blood hematopoietic stem cell significantly.
Accidental discovery, copper ion chelator TEPA, SIRT inhibitor Nicotinamide can significantly improve hematopoietic stem cell transplantation
Level, and preliminary efficacy is shown in clinical trial, but in the cell body after expanding the time-to-live fall short of, and break up pedigree not
It is enough complete.High flux screening chemical small molecule discovery aza cycle compound SR1 and indoles analog UM171 energy in recent years
Enough more effectively amplifications have the candidate stem cell of long-term engraftment ability.Clinical trial shows the candidate stem cell tool of SR1 amplification
The ability of standby reconstruction patients immune system, but it does not get rid of the dependence to double Umbilical Cord Blood Transplantation still.Generally speaking, HSC
Optimal amplification in vitro condition is not known together explicitly still so far.
MS275 also known as SNDX-275, molecular formula C21H20N4O3, it is that I type histone is gone that No. CAS, which is 209783-80-2,
The specific inhibitor of acetylase HDAC1 and HDAC3.Medical field is mostly used Entinostat to censure MS275, and the drug is white
The Several Kinds of Malignancy such as blood disease, non-Hodgkin lymphoma, Hodgkin lymphoma, breast cancer, oophoroma, lung cancer, kidney into
Enter I phase and II clinical trial phase.It is reported that MS275 can promote the generation of people's induction type pluripotent stem cell (iPSC).
ISOPROTEREND, Chinese name isopropyl (going first) adrenaline, molecular formula C8H7NOCl2, No. CAS is 105219-
27-0.Molecular weight is 204.05.It is Beta-3 adrenergic receptor non-selective activation agent, is clinically commonly used to treat mistake aroused in interest
The diseases such as slow, heart block, asthma.
Currently, ISOPROTEREND and MS275 combination is to the maintenance of human hematopoietic stem cell and amplification effect all without report.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing composition and its application and amplifying candidate stem cell
Method, the present invention studies have shown that MS275 and ISOPROTEREND combination can significantly improve hematopoietic stem cell population institute
The cell total amount obtained.
Present invention firstly provides the compositions being made of ISOPROTEREND and MS275.
In composition provided by the invention, the molar ratio of the ISOPROTEREND and MS275 are (0.1~10): (0.1
~10).In some embodiments, the molar ratio of the ISOPROTEREND and MS275 are 3:1.
The present invention studies have shown that addition ISOPROTEREND and MS275 to significantly improve umbilical cord blood hematopoietic stem cell external
Resulting cell total amount is expanded, effect is more preferable when the molar ratio of the two is 3:1, generates significant synergy synergistic effect.And work as
When ISOPROTEREND, MS275 and TPO, SCF and FLT3L are used in conjunction with, synergy synergistic effect can be generated, thus preferably
Promote the amplification in vitro of candidate stem cell.
The present invention also provides promote hematopoietic stem cell expansion composition, by ISOPROTEREND, MS275, TPO,
SCF and FLT3L composition.
It is provided by the invention promote hematopoietic stem cell expansion composition in, the ISOPROTEREND, MS275, TPO,
The mass ratio of SCF and FLT3L is (20.4~2040): (38~3800): (30~70): (80~120): (90~110).
In some embodiments, the mass ratio of ISOPROTEREND, MS275, TPO, SCF and FLT3L are in the composition
612:380:30:80:90.
In some embodiments, the mass ratio of ISOPROTEREND, MS275, TPO, SCF and FLT3L are in the composition
612:380:50:100:100.
In some embodiments, the mass ratio of ISOPROTEREND, MS275, TPO, SCF and FLT3L are in the composition
612:380:70:120:110.
Each component in composition provided by the invention can be respectively individually present, and can also be mutually mixed, the present invention is to this
Without limitation.Each component can be that solution can also be powder.In the present invention, each component exists in the form of a solution, each group split-phase
It is mutually independent.Wherein, the solution of MS275 is configured with DMSO, mother liquid concentration 100mmol/L.The solution of ISOPROTEREND with
DMSO configuration, mother liquid concentration 100mmol/L.
Additive of the composition provided by the invention as culture medium, for promoting the amplification in vitro of candidate stem cell.
The present invention also provides the culture mediums of amplifying candidate stem cell comprising basal medium and of the present invention group
Close object.
In the culture medium of amplifying candidate stem cell i.e. provided by the invention include basal medium, ISOPROTEREND and
MS275。
In amplifying candidate stem cell culture medium provided by the invention, the concentration of the ISOPROTEREND is 0.1 μm of ol/L
~10 μm of ol/L；The concentration of MS275 is 0.1 μm of ol/L~10 μm ol/L.In some specific embodiments, the ISOPROTEREND
Concentration be 3 μm of ol/L；The concentration of MS275 is 1 μm of ol/L.
It further include TPO, SCF and FLT3L in the culture medium of amplifying candidate stem cell provided by the invention.
In the present invention, the concentration of the TPO is 30ng/mL~70ng/mL；
The concentration of the SCF is 80ng/mL~120ng/mL；
The concentration of the FLT3L is 90ng/mL~110ng/mL.
In some embodiments, the concentration of the TPO is 30ng/mL；The concentration of the SCF is 80ng/mL；The FLT3L
Concentration be 90ng/mL.
In some embodiments, the concentration of the TPO is 50ng/mL；The concentration of the SCF is 100ng/mL；The FLT3L
Concentration be 100ng/mL.
In some embodiments, the concentration of the TPO is 70ng/mL；The concentration of the SCF is 120ng/mL；The FLT3L
Concentration be 110ng/mL.
In the present invention, the basal medium is StemPro, RPMI1640, IMDM, α-MEM or StemSpan
SFEMII.In some embodiments, the basal medium is StemSpan SFEMII.
In some embodiments, in the culture medium:
The concentration of the ISOPROTEREND is 20.4ng/mL~2040ng/mL；
The concentration of the MS275 is 38ng/mL~3800ng/mL；
The concentration of the TPO is 30ng/mL~70ng/mL；
The concentration of the SCF is 80ng/mL~120ng/mL；
The concentration of the FLT3L is 90ng/mL~110ng/mL.
In some specific embodiments, culture medium provided by the invention includes StemSpan SFEMII culture medium, 3 μm of ol/L
The concentration of ISOPROTEREND, MS275 are 1 μm of ol/L, 50ng/mL TPO, 100ng/mL SCF and 100ng/mL FLT3L.
In some specific embodiments, culture medium provided by the invention includes StemSpan SFEMII culture medium, 3 μm of ol/L
The concentration of ISOPROTEREND, MS275 are 1 μm of ol/L, 30ng/mL TPO, 80ng/mL SCF and 90ng/mL FLT3L.
In some specific embodiments, culture medium provided by the invention includes StemSpan SFEMII culture medium, 3 μm of ol/L
The concentration of ISOPROTEREND, MS275 are 1 μm of ol/L, 70ng/mL TPO, 120ng/mL SCF and 110ng/mL FLT3L.
Culture medium of the present invention can be preceding ready-to-use in using, and may be made as finished product long term storage.Preparation method
For composition provided by the invention is added until the concentration of each component is institute of the present invention in StemSpan SFEMII culture medium
The concentration stated.Composition of the present invention can be dry powder, can individually exist for the mixture or each component of each component.Institute
Stating composition also can be solution, or be mother liquor.It include all or part of component of composition in the mother liquor.The mother liquor
Solvent is DMSO.
The method of amplifying candidate stem cell provided by the invention, with culture medium of the present invention to candidate stem cell into
Row culture.
In method of the present invention, the candidate stem cell is umbilical cord blood hematopoietic stem cell；The density of inoculation be 2 ×
104cells/mL。
The condition of the culture is 37 DEG C, 5%CO2.Fresh culture medium provided by the invention was added every 2 days.Culture 5
~10 days amplification times are 4~20 times.
The present invention provides the compositions that ISOPROTEREND and MS275 is formed, and provide it and promoting Hematopoietic Stem thin
Application in born of the same parents' amplification.Studies have shown that adding while cell factor is added during umbilical cord blood hematopoietic stem cell amplification cultivation
Enter ISOPROTEREND and MS275, has reached and not only increased candidate stem cell quantity simultaneously but also improve candidate stem cell CFU colony shape
At the effect of ability, so that candidate stem cell, which is at, is proliferated undifferentiated state, and then reach clinical transplantation demand.This hair
Bright is easy to operate, low in cost, and obtained candidate stem cell is more, solves hematopoietic stem cell expansion in the prior art
The defects of rate is low, easy differentiation.
Detailed description of the invention
Fig. 1 shows surface antigen CD45, CD34 expression of the group 1 with the candidate stem cell of group 4~6 at the 5th day；
Fig. 2 shows surface antigen CD90, CD34 expression of the group 1 with the candidate stem cell of group 4~6 at the 5th day；
Fig. 3 shows under inverted microscope that each pedigree Colony forming representative figure (b) shows BFU-E wherein (a) shows CFU-E, (c) shows
CFU-G (d) shows CFU-M, (e) shows CFU-GM, (f) shows CFU-GEMM.
Specific embodiment
The present invention provides compositions and its method of application and amplifying candidate stem cell, those skilled in the art to borrow
Reflect present disclosure, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.Method and application of the invention has passed through
Preferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to the side of this paper
Method and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Test material, reagent or the experiment equipment that the present invention uses are all common commercially available product, can all be bought in market.
The preparation of the umbilical hemopoietic stem cell includes: that Cord blood is thin lymph to be added after 2~3 times of normal saline dilution
Born of the same parents' separating liquid takes mononuclearcell layer (PBMC) through 1500~2000rpm/min centrifugation 20min, and brine is simultaneously resuspended
Obtain PBMC cell mass；Then CD34+ cell is separated with paramagnetic particle method.
StemSpanSFEMII is serum free medium, and production firm is StemCell Technologies, and article No. is
09655；
RhMGF rhSCF (recombined human stem cell factor), production firm is
Stemimmune LLC, article No. HHM-SF-1000；
Recombined human thrombopoietin rhTPO (recombined human thrombopoietin), production firm is
Stemimmune LLC, article No. HHM-TP-0100；
Recombined human FMS-like tyrosine kinase 3 ligand rhFLT3L (recombined human FMS-like tyrosine
Kinase 3ligand), production firm is Stemimmune LLC, article No. HHM-FT-1000；
ISOPROTEREND and MS-275, production firm Sigma-Aldrich；
Peripheral blood mononuclear cells PBMC (peripheral blood mononuclear cell)
MACS: magnetic bead sorting；
DMSO: dimethyl sulfoxide；
PBS: phosphate buffer；
MethoCultTMGF H4435 is semisolid culturemedium；
CFU-E full name Colony Forming Unit of Erythrocyte, the entitled erythroid cell colonies of Chinese form list
Position；
BFU-E full name Burst Forming Unit of Erythrocyte, the entitled explosion type erythroid cell colonies shape of Chinese
At unit；
CFU-G full name ColonyForming Unit of Granulocyte, the entitled granular leukocyte colony of Chinese form list
Position；
CFU-M full name Colony Forming Unit of Macrophage, the entitled macrophage colony of Chinese form list
Position；
CFU-GM full name Colony Forming Unit of Granulocyte-Macrophage, the entitled grain of Chinese are thin
Born of the same parents-macrophage colony forms unit；
CFU-GEMM full name Colony Forming Unit of granulocyte, erythrocyte,
Macrophage/monocyte, megakaryocyte, mix colony, and Chinese name is granulocyte, red blood cell, macrophage/monokaryon
Cell, megakaryocyte colony forming unit；
Composition or culture medium provided by the invention can be suitable for the amplification in vitro of candidate stem cell, and the Hematopoietic Stem is thin
Born of the same parents can derive from experimental animal (such as mouse etc.) or the mankind.Mankind hemopoietic stem cell can derive from marrow, peripheral blood, Cord blood
And placental blood, in embodiments of the present invention, by taking umbilical cord blood hematopoietic stem cell as an example, wherein Cord blood is through detecting hepatitis B, third
Type hepatitis, syphilis, AIDS, cytomegalovirus, TORCH detection, mycoplasma, Chlamydia, G-6PD and ground it is poor be feminine gender, warp
Detection, isolated human cord blood candidate stem cell are expressed as follows several membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation
Antigens CD34, leukocyte differentiation antigen CD90, leukocyte differentiation antigen CD49f.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
1, human umbilical cord blood mononuclear cell is obtained；
(1) Cord blood is added 2~3 times of normal saline dilution, 0.4 times of volume lymphocyte separation medium is added dropwise after mixing
In, it is careful not to destroy interface；
(2) it is centrifuged 20min using 1500~2000rpm/min, because being divided into four layers from top to bottom in density difference centrifuge tube:
First layer is plasma layer, the second layer is cyclic annular milky mononuclearcell layer (PBMC), third layer is transparent separation liquid layer, the 4th
Layer is red blood cell layer；
(3) second layer ring-type milky mononuclearcell layer (PBMC) is carefully drawn with suction pipe to another 50ml centrifuge tube
In, physiological saline is added, 1500~2000rpm/min is reused and is centrifuged 5~10min；
(4) it abandons and resets and add physiological saline resuspension, be finally centrifuged 5~10min using 1500~2000rpm/min, abandon again
Supernatant obtains PBMC cell mass.
2, CD34+ umbilical cord blood hematopoietic stem cell is obtained from above-mentioned PBMC using MACS；
(1) every cord blood PBMC uses 50 μ L people CD34+ magnetic beads and 50 μ L FcR blocker reagent and 150 μ L
The mixed liquor of 0.5%BSA is resuspended, 4 DEG C of incubation 30min；
(2) at the same time, magnet and magnetic frame are irradiated into 30min as super-clean bench middle-ultraviolet lamp；
(3) the sterile PBS of 10ml is added, after mixing, abandons supernatant after being centrifuged 5~10min using 1500~2000rpm/min；
(4) the dedicated adsorption column of MACS is put into magnet, 500ul 0.5%BSA rinse, the liquid 15ml of outflow is added
Tube is caught；
PBMC agglomerate in the step 3) for obtaining human umbilical cord blood mononuclear cell is resuspended in (5) 500 μ L 0.5%BSA, turns after mixing
It moves on in the dedicated adsorption column of MACS, is flowed completely out to liquid；
(6) 500 μ L 0.5%BSA are washed 3 times, are removed adsorption column, are placed in 15ml tube；
(7) 1ml 0.5%BSA is added, with piston in liquid push-in 15ml tube, gained liquid contains CD34+ umbilical cord
Blood candidate stem cell.
Embodiment 2
The content of the factor such as table 1 in each group culture medium:
The content of the factor in 1 each group culture medium of table
| ISOPROTEREND | MS275 | SCF | TPO | FLT3 | |
| Group 1 | 3μM | 1μM | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 2 | 3μM | 1μM | 100ng/ml | 50ng/ml | 100ng/ml |
| Group 3 | 3μM | 1μM | 120ng/ml | 70ng/ml | 110ng/ml |
| Group 4 | 3μM | 0 | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 5 | 0 | 1μM | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 6 | 0 | 0 | 80ng/ml | 30ng/ml | 90ng/ml |
Each substance is added into StemSpan SFEMII serum free medium with 1 concentration of table.
The suspension of CD34+ umbilical cord blood hematopoietic stem cell made from embodiment 1 is inoculated in group of cells culture medium and is trained
It supports.Cell-seeding-density is 1 × 10 in 24 orifice plates4The hole cells/ is placed in 37 DEG C, 5%CO2Incubator culture.According to cell culture
State added the fresh 500 μ L of cell culture medium of each group every 2 days, can get a fairly large number of candidate stem cell within 5~10 days,
Amplification times are about 4~20 times.
Effect detection
Cell count, phenotypic evaluation and Colony forming list are carried out to the umbilical cord blood hematopoietic stem cell of 2 each group culture of embodiment
Position analysis.
1, cell count
The cell cultivated respectively the 5th day group 1~3 or 4~6 counts, and calculates the cell number compared to the 0th day
Amplification times.Each group culture medium cultivation results such as table 2:
Table 2: each group conditioned cell number amplification times statistical form
The result shows that: group 1~3 is relative to group 4~6, and the CD34+CD90+ cell quantity of acquisition is more, and amplification times are more
Greatly, through statistical analysis, there are significant difference, p < 0.05 for the expanding effect and group 4~6 of group 1~3.In group 1~3,2 are organized
Expanding effect is more preferably.
2, cell flow cytometer showed
The CD34+ cell cultivated respectively the group 1~6 of the 0th day, the 5th day carries out flow cytometer showed.Using BD company FACS
Verse flow cytometer detection instrument takes 20 μ L of cell suspension, and the CD34 for the FITC label being dissolved in 0.5%BSA is added, PE label
The CD90 of the CD45RA of CD38, APC-Cy7 label, APC label.Each pipe is protected from light at room temperature after being vortexed is incubated for 15min, is added appropriate
PBS, 1600rpm room temperature horizontal centrifugal 5min abandon supernatant, and 200 μ L of PBS is added, then upper machine analysis.The inspection of group 2 and group 4
Survey result such as Fig. 1~2.The result shows that 2 amplification of group obtains relative to the group 4~6 for being not added with ISOPROTEREND and MS275 combination
The CD34+CD90+ cell proportion obtained is higher, these candidate stem cells that this explanation shows that 2 amplification of group obtains are more original, has
The stronger differentiation potential for rebuilding hematological system, more effectively can support clinical treatment to need.The cell that 1,3 amplification of group obtains
In, CD34+CD90+ cell proportion is similar to group 2.
3, colony forming unit is analyzed
The CD34+ cell cultivated respectively the group 1~6 of the 0th day, the 5th day carries out colony forming unit analysis.Using
MethoCultTMGF H4435 semisolid culturemedium, is added the hole culture medium 1ml/ in six orifice plates, and CD34+ cell-seeding-density is
500 cells/wells are placed in 37 DEG C of 5%CO2After incubator culture 14 days, each pedigree colony number is calculated, and shoot photo.It is inverted
Each pedigree Colony forming represents figure such as 3 under microscope, Colony forming number statistical such as table 3:
Colony forming number after 3 each group culture of table
As shown in table 3, for addition group 1~3 relative to group 4~6, the cell colony number of acquisition is more, through statistical analysis,
There are significant difference, p < 0.05 for the colony number and group 4~6 of group 1~3.Group 1~3 in, organize 2 group's number it is most.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. the composition being made of ISOPROTEREND and MS275.
2. composition according to claim 1, which is characterized in that the molar ratio of the ISOPROTEREND and MS275 is
(0.1~10): (0.1~10).
3. composition of any of claims 1 or 2 is promoting the application in hematopoietic stem cell expansion.
4. a kind of composition for promoting hematopoietic stem cell expansion, which is characterized in that by ISOPROTEREND, MS275, TPO, SCF
It is formed with FLT3L.
5. composition according to claim 2, which is characterized in that described ISOPROTEREND, MS275, TPO, SCF and
The mass ratio of FLT3L is (20.4~2040): (38~3800): (30~70): (80~120): (90~110).
6. amplifying candidate stem cell culture medium, which is characterized in that including basal medium and combination of any of claims 1 or 2
Object.
7. culture medium according to claim 6, which is characterized in that wherein further include TPO, SCF and FLT3L.
8. culture medium according to claim 7, which is characterized in that wherein:
The concentration of the ISOPROTEREND is 20.4ng/mL~2040ng/mL；
The concentration of the MS275 is 38ng/mL~3800ng/mL；
The concentration of the TPO is 30ng/mL~70ng/mL；
The concentration of the SCF is 80ng/mL~120ng/mL；
The concentration of the FLT3L is 90ng/mL~110ng/mL.
9. according to the described in any item culture mediums of claim 4~7, which is characterized in that the basal medium be StemPro,
RPMI1640, IMDM, α-MEM or StemSpan SFEM II.
10. a kind of method of amplifying candidate stem cell, which is characterized in that with the described in any item culture mediums pair of claim 6~9
Candidate stem cell is cultivated.
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