The cultivating system of amplifying candidate stem cell, method and application thereofTechnical field
The present invention relates to the cultivating system of technical field of cell culture more particularly to amplifying candidate stem cell, method and itsPurposes.
Background technique
Hematopoietic stem cell transplantation technology is clinical treatment leukaemia, lymthoma, alpastic anemia, thalassemiaEtc. 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 obtainsSource 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 bloodCandidate stem cell and progenitor cell population be not enough to quickly restore adult patient immune system, cause opportunistic infections lethalityIncrease.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 patientIt 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 obtainEnough 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 effectFruit.Early stage people cultivate candidate stem cell using the cell factor in blood, as a result lead to cell differentiation, and portability function subtractsIt is weak.Later, it has been found that Wnt signaling molecule, Notch ligand, the retinoic acid antagonism factor in marrow hemopoietic stem cells microenvironmentEtc. can effectively expand CD34+ hematopoietic stem/progenitor.Remain external using CHIR99021 or BIO activation Wnt signal pathThe 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 throughActivate Notch signal and appropriate amplifying candidate stem cell.Separately studies have found that, the PTN of marrow endothelial stroma cell secretion also canEnough 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 generationCompel self-renewing and portability function that meeting damages candidate stem cell by increasing ROS level；It has been found that the addition of antioxidantAnd 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 transplantationLevel, 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 notIt is enough complete.High flux screening chemical small molecule discovery aza cycle compound SR1 and indoles analog UM171 energy in recent yearsEnough more effectively amplifications have the candidate stem cell of long-term engraftment ability.Clinical trial shows the candidate stem cell tool of SR1 amplificationThe 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, HSCOptimal amplification in vitro condition is not known together explicitly still so far.
A8301 (CAS:909910-43-6 chemical formula: C25H19N5S) is I type TGF beta receptor ALK5 kinases, I typeThe selective depressant of Activin/Nodal receptor ALK4 kinases and I type Nodal receptor ALK7 kinases, can be by blocking SMAD2The phosphorylation of albumen and inhibit TGF it is beta induced epithelial-mesenchymal transformation.A8301 effect is single-minded, only in higher concentrations to ALK-1/There are faint inhibition for 2/3/6 and MAPK activity.It is reported that A8301 promotion people induces multi-potent stem cell the generation of (iPSC).
EPZ6438 (chemical formula C34H44N4O4, No. CAS is 1403254-99-8) and it is histone methyltransferase EZH2Specific inhibitor.In clinical treatment field, EPZ6438 also known as Tazemetostat are a kind of experimental stress resistance cancer drugs, are used forTreatment lymthoma, non-small cell lung cancer, carcinoma of endometrium come into I phase and II clinical trial phase.
Currently, A8301, EPZ6438 are 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 cellMethod, can to significantly improve hematopoietic stem cell population resulting studies have shown that A8301 and EPZ6438 is combined by the present inventionCell total amount.
Present invention firstly provides the compositions being made of A8301 and EPZ6438.
In composition provided by the invention, the molar ratio of the A8301 and EPZ6438 are (0.1~10): (0.1~10).In some embodiments, the molar ratio of the A8301 and EPZ6438 are 0.5:0.2.
The present invention is studies have shown that addition A8301 and EPZ6438 significantly improves umbilical cord blood hematopoietic stem cell amplification in vitro instituteThe cell total amount obtained, when the molar ratio of the two is 0.5:0.2, effect is more preferable, generates significant synergy synergistic effect.And work asWhen A8301, EPZ6438 and TPO, SCF and FLT3L are used in conjunction with, synergy synergistic effect can be generated, to preferably promote to makeThe amplification in vitro of hemocytoblast.
The present invention also provides the compositions for promoting hematopoietic stem cell expansion, by A8301, EPZ6438, TPO, SCF andFLT3L composition.
It is provided by the invention promote hematopoietic stem cell expansion composition in, described A8301, EPZ6438, TPO, SCF andThe mass ratio of FLT3L is (42.15~4215): (57.2~5720): (30~70): (80~120): (90~110).
In some embodiments, the mass ratio of A8301, EPZ6438, TPO, SCF and FLT3L are in the composition210.75:114.4:30:80:90.
In some embodiments, the mass ratio of A8301, EPZ6438, TPO, SCF and FLT3L are in the composition210.75:114.4:50:100:100.
In some embodiments, the mass ratio of A8301, EPZ6438, TPO, SCF and FLT3L are in the composition210.75:114.4:70:120:110.
Each component in composition provided by the invention can be respectively individually present, and can also be mutually mixed, the present invention is to thisWithout 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-phaseIt is mutually independent.Wherein, the solution of EPZ6438 is configured with DMSO, mother liquid concentration 100mmol/L.The solution of A8301 is matched with DMSOIt sets, 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 groupClose object.
It include basal medium, A8301 and EPZ6438 in the culture medium of amplifying candidate stem cell i.e. provided by the invention.
In amplifying candidate stem cell culture medium provided by the invention, the concentration of the A8301 is 0.1 μm of μ of ol/L~10mol/L；The concentration of EPZ6438 is 0.1 μm of ol/L~10 μm ol/L.In some specific embodiments, the concentration of the A8301 is0.5μmol/L；The concentration of EPZ6438 is 0.2 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 FLT3LConcentration be 90ng/mL.
In some embodiments, the concentration of the TPO is 50ng/mL；The concentration of the SCF is 100ng/mL；The FLT3LConcentration be 100ng/mL.
In some embodiments, the concentration of the TPO is 70ng/mL；The concentration of the SCF is 120ng/mL；The FLT3LConcentration 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 SFEM II.
In some embodiments, in the culture medium:
The concentration of the A8301 is 42.15ng/mL~4215ng/mL；
The concentration of the EPZ6438 is 57.2ng/mL~5720ng/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 SFEM II culture medium, 0.5 μMol/L A8301, EPZ6438 concentration be 0.2 μm of ol/L, 50ng/mL TPO, 100ng/mL SCF and 100ng/mLFLT3L。
In some specific embodiments, culture medium provided by the invention includes StemSpan SFEM II culture medium, 0.5 μMol/L A8301, EPZ6438 concentration be 0.2 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 SFEM II culture medium, 0.5 μMol/L A8301, EPZ6438 concentration be 0.2 μm of ol/L, 70ng/mL TPO, 120ng/mL SCF and 110ng/mLFLT3L。
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 methodFor composition provided by the invention is added until the concentration of each component is the present invention in StemSpan SFEM II culture mediumThe concentration.Composition of the present invention can be dry powder, can individually exist for the mixture or each component of each component.The composition also can be solution, or be mother liquor.It include all or part of component of composition in the mother liquor.The mother liquorSolvent be DMSO.
The method of amplifying candidate stem cell provided by the invention, with culture medium of the present invention to candidate stem cell intoRow 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 A8301 and EPZ6438 is formed, and provide it and promoting hematopoietic stem cell expansionIn application.Studies have shown that being added while cell factor is added during umbilical cord blood hematopoietic stem cell amplification cultivationA8301 and EPZ6438 has reached and has not only increased candidate stem cell quantity simultaneously but also improve candidate stem cell CFU Colony forming abilityEffect so that candidate stem cell, which is at, is proliferated undifferentiated state, and then reach clinical transplantation demand.Behaviour of the inventionMake easy, low in cost, obtained candidate stem cell is more, solve hematopoietic stem cell expansion rate in the prior art it is low,The defects of easily breaking up.
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) showsCFU-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 borrowReflect 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 fieldIt 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 throughPreferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to the side of this paperMethod 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 dilutionBorn of the same parents' separating liquid takes mononuclearcell layer (PBMC) through 1500~2000rpm/min centrifugation 20min, and brine is simultaneously resuspendedObtain PBMC cell mass；Then CD34+ cell is separated with paramagnetic particle method.
StemSpanSFEM II is serum free medium, and production firm is StemCell Technologies, and article No. is09655；
RhMGF rhSCF (recombined human stem cell factor), production firm isStemimmune LLC, article No. HHM-SF-1000；
Recombined human thrombopoietin rhTPO (recombined human thrombopoietin), production firm isStemimmune LLC, article No. HHM-TP-0100；
Recombined human FMS-like tyrosine kinase 3 ligand rhFLT3L (recombined human FMS-liketyrosineKinase 3ligand), production firm is Stemimmune LLC, article No. HHM-FT-1000；
A8301, production firm Sigma-Aldrich；
EPZ6438, 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 listPosition；
BFU-E full name Burst Forming Unit of Erythrocyte, the entitled explosion type erythroid cell colonies shape of ChineseAt unit；
CFU-G full name ColonyForming Unit of Granulocyte, the entitled granular leukocyte colony of Chinese form listPosition；
CFU-M full name Colony Forming Unit of Macrophage, the entitled macrophage colony of Chinese form listPosition；
CFU-GM full name Colony Forming Unit of Granulocyte-Macrophage, the entitled grain of Chinese are thinBorn 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/monokaryonCell, 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 thinBorn 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 bloodAnd 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, thirdType hepatitis, syphilis, AIDS, cytomegalovirus, TORCH detection, mycoplasma, Chlamydia, G-6PD and ground it is poor be feminine gender, warpDetection, isolated human cord blood candidate stem cell are expressed as follows several membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiationAntigens 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 mixingIn, 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 4thLayer is red blood cell layer；
(3) second layer ring-type milky mononuclearcell layer (PBMC) is carefully drawn with suction pipe to another 50ml centrifuge tubeIn, 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 againSupernatant 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 blockerreagent and 150 μ LThe 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 addedTube 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 mixingIt 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 cordBlood 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
| A8301 | EPZ6438 | SCF | TPO | FLT3 |
| Group 1 | 0.5μM | 0.2μM | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 2 | 0.5μM | 0.2μM | 100ng/ml | 50ng/ml | 100ng/ml |
| Group 3 | 0.5μM | 0.2μM | 120ng/ml | 70ng/ml | 110ng/ml |
| Group 4 | 0.5μM | 0 | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 5 | 0 | 0.2μM | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 6 | 0 | 0 | 80ng/ml | 30ng/ml | 90ng/ml |
Each substance is added into StemSpan SFEM II 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 trainedIt 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 cultureState 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 embodimentPosition 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 dayAmplification 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: the group 1~3 of addition A8301 and EPZ6438 is relative to group 4~6, the CD34+CD90+ cell of acquisitionMore, amplification times are bigger, through statistical analysis, the expanding effect of group 1~3 and group 4~6 there are significant difference, p <0.05.In group 1~3,2 expanding effect is organized more preferably.
2, cell flow cytometer showed
Flow cytometer showed is carried out to the CD34+ cell of the 0th day, A8301, EPZ6438 or DMSO culture in the 5th day respectively.It adoptsWith BD company FACS Verse flow cytometer detection instrument, 20 μ L of cell suspension is taken, the FITC label being dissolved in 0.5%BSA is addedThe CD45RA of the CD38 of CD34, PE label, APC-Cy7 label, the CD90 of APC label.Each pipe is protected from light incubation after being vortexed at room temperatureAppropriate PBS, 1600rpm room temperature horizontal centrifugal 5min is added in 15min, abandons supernatant, and 200 μ L of PBS is added, then upper machine pointAnalysis.Testing result such as Fig. 1~2 of group 2 and group 4~6.The result shows that relative to the group 4 for being not added with A8301 and EPZ6438 combination~6, the CD34+CD90+ cell proportion that 2 amplification of group obtains is higher, this explanation shows these candidate stem cells that 2 amplification of group obtainsIt is more original, with the stronger differentiation potential for rebuilding hematological system, more effectively clinical treatment can be supported to need.Group 1,3It expands in the cell obtained, 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.UsingThe hole culture medium 1ml/, CD34+ cell-seeding-density is added in MethoCultTMGF H4435 semisolid culturemedium in six orifice platesFor 500 cells/wells, it is placed in 37 DEG C of 5%CO2After incubator culture 14 days, each pedigree colony number is calculated, and shoot photo.?Each pedigree Colony forming representative figure such as 3 under microscope is set, 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 comeIt 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 asProtection scope of the present invention.