CN105316293A - Method for obtaining hematopoietic stem cells/hematopoietic progenitor cells in vitro - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 210000003958 hematopoietic stem cell Anatomy 0.000 title abstract description 15
- 238000000338 in vitro Methods 0.000 title abstract 2
- 230000003394 haemopoietic effect Effects 0.000 claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims abstract description 28
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 20
- SIBCJMZHJZDUNK-UHFFFAOYSA-N methyl 4-(3-piperidin-1-ylpropylamino)-9h-pyrimido[4,5-b]indole-7-carboxylate Chemical compound C=1C(C(=O)OC)=CC=C(C=23)C=1NC3=NC=NC=2NCCCN1CCCCC1 SIBCJMZHJZDUNK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 claims abstract description 9
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 claims abstract description 9
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims abstract description 7
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims abstract description 7
- 108090000695 Cytokines Proteins 0.000 claims description 18
- 102000004127 Cytokines Human genes 0.000 claims description 18
- 108010002386 Interleukin-3 Proteins 0.000 claims description 5
- 108090001005 Interleukin-6 Proteins 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 108010082117 matrigel Proteins 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 101710113649 Thyroid peroxidase Proteins 0.000 claims description 2
- 239000002473 artificial blood Substances 0.000 claims 2
- 230000004069 differentiation Effects 0.000 abstract description 27
- 238000002474 experimental method Methods 0.000 abstract description 5
- FMSSKCBYHCEFQE-UHFFFAOYSA-N 1h-indole;pyrimidine Chemical class C1=CN=CN=C1.C1=CC=C2NC=CC2=C1 FMSSKCBYHCEFQE-UHFFFAOYSA-N 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 4
- 108010023082 activin A Proteins 0.000 abstract 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 10
- 210000000130 stem cell Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 210000000601 blood cell Anatomy 0.000 description 8
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 5
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 5
- 206010043276 Teratoma Diseases 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
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- 210000001744 T-lymphocyte Anatomy 0.000 description 1
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Abstract
The invention relates to the field of biology, and discloses a method for obtaining hematopoietic stem cells/hematopoietic progenitor cells of a human pluripotent stem cell source in vitro. The method comprises the following steps: culturing human pluripotent stem cells by using a basal culture medium added with a stem line II of ITS; adding different cell factors in different periods; culturing for 4 days after the human pluripotent stem cells are added with cell factor BMP4 and Activin A and then culturing for 2 days by using cell factors VEGF and SCF; and maintaining by using hematopoietic stem cells/hematopoietic progenitor cells, amplifying related cell factors, and culturing for 8 days to obtain the hematopoietic stem cells/hematopoietic progenitor cells with hematopoietic differentiation multipotency. An experiment proves that through addition of a pyrimidine indole derivative UM729, the efficiency of differentiation from the pluripotent stem cells to the hematopoietic stem cells/hematopoietic progenitor cells is doubled at least, and application prospect is good.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method of hematopoietic stem/progenitor of external efficient acquisition human pluripotent stem cells source.
Background technology
Hemopoietic stem cell: (hematopoieticstemcell, HSC) be multipotential stem cell in marrow, the cell of formation two type can be divided: one is still as stem cell, but another kind becomes progenitor cell (progenitorcell), the i.e. stem cell of time one-level, be also called burst forming unit (burstformingunits, and colony forming unit (colony-formingunits BFUs), CFUs), they can divide the polytype cell mass of formation, comprise: granulocyte, monocyte, thrombocyte, red corpuscle, T cell and B cell etc.Hemopoietic stem cell refers to not yet fully-developed cell, is the origin of all hematopoietic cells and immunocyte.
Although teratoma green blood pattern can obtain hemopoietic stem cell in body, but this differentiated system technical sophistication, variable is too many, the hematopoietic stem/progenitor inefficiency obtained, and teratoma model separation hematopoietic stem/progenitor difficulty in body, (only teratoma formation must be consuming time more than 2 months) consuming time, easily introduces xenogenesis pathogen contamination.Differentiation in vivo teratoma method obtains blood cell and there is the problems such as inefficiency, process are loaded down with trivial details.
Current external evoked human pluripotent stem cells directed differentiation is that the method for blood cell mainly contains following several: 1) EB method; 2) multipotential stem cell and stroma cell Dual culture method; 3) 2D individual layer revulsion.Differentiation in vivo method is teratoma method mainly.
Obtain blood cell inefficiency by EB method, real HSC cannot be obtained; Obtain blood cell efficiency by stroma cell Dual culture low, the efficiency that uncontrollable many factors causes is unstable, also cannot obtain real HSC; 2D individual layer revulsion can be carried out under serum-free, the condition of inductive differentiation medium determined without feeder layer and composition by optimization system, needs the problem solved to remain and improves differentiation efficiency and obtain real HSC.
To sum up, current external evoked human pluripotent stem cells directed differentiation be hematopoietic cell exist directed differentiation inefficiency, acquisition hematopoietic stem/progenitor multipotency difference etc. bottleneck, be embodied in the efficiency that human pluripotent stem cells breaks up to blood cell low, the composition such as feeder cell and serum easily introduces pathogen contamination, clinical transplantation GMP levels necessitate cannot be met, need that to develop the external evoked human pluripotent stem cells directed differentiation that makes new advances be the method for blood cell badly.
Summary of the invention
The present invention is directed to the problems referred to above, by the combination of screening micromolecular compound and optimization serum-free, improve the efficiency of multipotential stem cell to hematopoietic differentiation without the approach of feeder cell, and obtain the percentage of the hematopoietic stem/progenitor of people and yield significantly improves, external Colony forming experiment shows the pluripotency hematopoietic stem/progenitor successfully obtaining differentiation source, and the hematopoietic stem/progenitor application of originating for clinical pluripotent stem cell differentiation is laid a good foundation.
Contriver utilizes human pluripotent stem cells 2D individual layer revulsion to find, pyrimidine indole derivatives UM729 can improve the efficiency that human pluripotent stem cells breaks up to blood cell, and CFU experiment proves that the blood cell obtained contains the pluripotency hematopoietic stem/progenitor comprising hemopoietic stem cell.
Pyrimidine indole derivatives UM729, (Methyl4-((3-(piperidin-1-yl) propyl) amino)-9H-pyrimido [4,5-b] indole-7-Carboxylate), its structural formula is as follows:
As preferably, hES clone described in step 1 is tens to clone's agglomerates of a hundreds of hES cell.
As preferably, BMP4 concentration is 10-30ng/ml, ActivinA concentration is 8-12ng/ml.More preferably, BMP4 concentration is 20ng/ml, ActivinA concentration is 10ng/ml.
As preferably, the concentration of VEGF is 30-50ng/ml, SCF concentration is 40-60ng/ml.More preferably, the concentration of VEGF is 40ng/ml, SCF concentration is 50ng/ml.
As preferably, the cytokine that described use hematopoietic stem/progenitor maintains and amplification is relevant is SCF, the combination of Flt-3L, TPO, IL-3, IL-6 and UM729.
As preferably, described cytokine concentration is SCF40-60ng/ml, FLT-3L40-60ng/ml, TPO8-12ng/ml, IL-340-60ng/ml, IL-640-60ng/ml, UM17180-120nM, UM72930-45ng/ml.
More preferably, described cytokine concentration is SCF50ng/ml), FLT-3L50ng/ml, TPO10ng/ml, IL-350ng/ml, IL-650ng/ml, UM72937ng/ml.
Pyrimidine indole derivatives UM729 can improve the efficiency of human pluripotent stem cells (ES, iPS) to hematopoietic stem/progenitor directed differentiation, and the hematopoietic stem/progenitor obtained by the method possesses hematopoietic differentiation multipotency.Improve multipotential stem cell and reach more than one times to the efficiency that hematopoietic stem/progenitor is broken up, the hematopoietic stem/progenitor that (see accompanying drawing 1) obtains possesses hematopoietic differentiation multipotency, has a good application prospect.
Term and definition
MTeSR:mTeSR is a kind of commercially available culture medium for hES/iPS maintain of serum-free, with front, 400ml basic medium (Catalog#05851) and 100ml5 × additive (Catalog#05852) are fully mixed, as the perfect medium of hES/iPS maintain.
StemlineII:StemlinII is as the basic medium (Catalog:#S0192 of hES/iPS hematopoietic differentiation; Sigma-Aldrich) be the commercial hemopoietic stem cell substratum of a kind of serum-free, non-animal derived property composition, can be used for the hemopoietic stem cell increased in the peripheral blood of marrow, Cord blood and mobilization, this substratum can maintain the balance of having broken up hematopoietic cell and not broken up hematopoietic cell, and realizes two type cells and farthest increase.
ITS:ITS (Cat.No:41400-045, Gibco), as a kind of conventional medium additives, can promote the growth of multiple attached cell, reduces cell to the dependency of serum.Usage: as the additive of hES/iPS hematopoietic differentiation basic medium, 100X uses.
Accompanying drawing explanation
Fig. 1 is atomization schematic diagram of the present invention.
Fig. 2 is the percentage that UM729 improves the CD34+CD45+ cell in multipotential stem cell source.
The each pedigree CFU of the plastidogenetic hematopoiesis of CD34+CD45+ that after Fig. 3 shows interpolation UM729, multipotential stem cell is originated.
Embodiment
The invention discloses a kind of method of hematopoietic stem/progenitor of external acquisition human pluripotent stem cells source, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1: the maintain of human pluripotent stem cells
Human pluripotent stem cells uses the iPS cell strain in H1, H9 cell strain or patient source, Matrigel carries out cultivate (mTeSR) without feeder layer, hES clones when degree of converging reaches about 80% and goes down to posterity, go down to posterity and use EDTA digestion method, according to cell density, according to the ratio of 1:4 ~ 1:5, within every 4 ~ 5 days, go down to posterity 1 time, for the hES of the differentiation-inducing experiment of hematopoiesis between 40 ~ 70 generations.
Embodiment 2: the 2D individual layer hematopoiesis of human pluripotent stem cells is differentiation-inducing:
Differentiation adopts the method for bibliographical information, and suitably improves.HES cell reaches about 80% at matrigel degree of converging, with the hES clone (tens to a hundreds of hES cell clone agglomerate) that the preparation of EDTA digestion method is less, plant matrigel bag by the Tissue Culture Plate crossed according to the ratio of 1:30-1:35/ hole (12 orifice plate), add mTeSR to 1ml/ hole.After cultivating 24h, start to carry out hematopoiesis differentiation-inducing, atomization divides three steps, and substratum is stemline II+ITS+ combination of cytokines (using different combination of cytokines in the different steps of differentiation), has atomization as shown in Figure 1.The first step (D0-D4), add cytokine BMP4 (20ng/ml) and ActivinA (10ng/ml), second step (D4-D6), add VEGF (40ng/ml) and SCF (50ng/ml), 3rd step (D6-D14), the cytokine used is hematopoietic stem/progenitor maintenance and amplification relevant cell factor, comprise: SCF (50ng/ml), FLT-3L (50ng/ml), TPO (10ng/ml), IL-3 (50ng/ml), IL-6 (50ng/ml).
Embodiment 3: comparative study
Experiment contrast group:
Basic medium: stemline II+ITS
Combination of cytokines: D0-D4:BMP4+ActivinA, D4-D6:vEGF+SCF, D6-D14:SCF+Flt-3L+TPO+IL-3+IL-6, described cytokine concentration is SCF50ng/ml), FLT-3L50ng/ml, TPO10ng/ml, IL-350ng/ml, IL-650ng/ml.
Experimental group:
Basic medium: stemline II+ITS
Combination of cytokines: D0-D4:BMP4+ActivinA, D4-D6:vEGF+SCF, D6-D14:SCF+Flt-3L+TPO+IL-3+IL-6+UM729.Described cytokine concentration is SCF50ng/ml), FLT-3L50ng/ml, TPO10ng/ml, IL-350ng/ml, IL-650ng/ml, UM72937ng/ml.
Compared with control group, after adding UM729, the percentage of the CD34+CD45+ hematopoietic stem/progenitor in H1 differentiation source significantly improves.The results are shown in accompanying drawing 2.Illustrate that UM729 can significantly improve the differentiation efficiency of the hematopoietic stem/progenitor in human pluripotent stem cells source.
With reference to above method, H9, humaniPS cell strain is verified, trend and H1 similar, namely add the differentiation efficiency of hematopoietic stem/progenitor that UM729 can improve human pluripotent stem cells source.
Embodiment 4:
After adding UM729, the CD34+CD45+ hematopoietic stem/progenitor in H1 differentiation source can form CFU-GEMM, multiple pedigree colony such as BFU-E, CFU-G, CFU-GM on CFU substratum, shows the hematopoietic stem/progenitor (see accompanying drawing 3) containing pluripotency.
Accompanying drawing 3: under interpolation UM729 condition, the CD34+CD45+ hematopoietic stem/progenitor in H1 differentiation source cultivates the typical hematopoietic lineage colony of 14d formation on H4434 semisolid medium.Comprise and confirm that cultivating initiator cell exists the colony CFU-GEMM of hematopoietic stem/progenitor, and confirm to cultivate that initiator cell exists red system respectively, grain system, monokaryon/huge bite be, grain-monokaryon/huge bites colony B/CFU-E, CFU-G, CFU-M, CFU-GM of being.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a method for external acquisition artificial blood ancestral cells, comprises following steps:
Step 1: hES clone prepared by the human pluripotent stem cells EDTA digestion method without feeder layer subculture 40-70 generation, plants matrigel bag by the Tissue Culture Plate crossed, adds mTeSR and cultivate 24 hours;
Step 2: the basic medium using the stemline II that with the addition of ITS instead is cultivated, and adds different cytokines at different times; After interpolation cytokine BMP4 and ActivinA cultivates 4 days, use cytokine VEGF and SCF instead and cultivate 2 days; Use the cytokine that hematopoietic stem/progenitor maintains and amplification is relevant instead and cultivate 8 days, obtain artificial blood ancestral cells.
2. method according to claim 1, is characterized in that, hES clone described in step 1 is the clone's agglomerate arriving a hundreds of hES cell containing tens.
3. method according to claim 1, is characterized in that, BMP4 concentration is 10-30ng/ml, ActivinA concentration is 8-12ng/ml.
4. method according to claim 1, is characterized in that, BMP4 concentration is 20ng/ml, ActivinA concentration is 10ng/ml.
5. method according to claim 1, is characterized in that, the concentration of VEGF is 30-50ng/ml, SCF concentration is 40-60ng/ml.
6. method according to claim 1, is characterized in that, the concentration of VEGF is 40ng/ml, SCF concentration is 50ng/ml.
7. method according to claim 1, is characterized in that, the cytokine that described use hematopoietic stem/progenitor maintains and amplification is relevant is SCF, the combination of Flt-3L, TPO, IL-3, IL-6 and UM729.
8. method according to claim 1, is characterized in that, described cytokine concentration is SCF40-60ng/ml, FLT-3L40-60ng/ml, TPO8-12ng/ml, IL-340-60ng/ml, IL-640-60ng/ml, UM72930-45ng/ml.
9. method according to claim 1, is characterized in that, described cytokine concentration is SCF50ng/ml, FLT-3L50ng/ml, TPO10ng/ml, IL-350ng/ml, IL-650ng/ml, UM72937ng/ml.
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Cited By (4)
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| CN108300695A (en) * | 2018-02-07 | 2018-07-20 | 清华大学 | A kind of method that human pluripotent stem cell breaks up to candidate stem cell and culture additive |
| CN113215086A (en) * | 2021-05-26 | 2021-08-06 | 杭州原生生物科技有限公司 | Culture medium and method for induced differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN113373114A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN115927169A (en) * | 2022-10-11 | 2023-04-07 | 再造再生医学科技(杭州)有限公司 | For amplifying CD34 + Culture solution for hematopoietic stem cells and in vitro amplification of CD34 + Method for hematopoietic stem cells |
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| CN113215086A (en) * | 2021-05-26 | 2021-08-06 | 杭州原生生物科技有限公司 | Culture medium and method for induced differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN113373114A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN115927169A (en) * | 2022-10-11 | 2023-04-07 | 再造再生医学科技(杭州)有限公司 | For amplifying CD34 + Culture solution for hematopoietic stem cells and in vitro amplification of CD34 + Method for hematopoietic stem cells |
| CN115927169B (en) * | 2022-10-11 | 2023-08-11 | 再造再生医学科技(杭州)有限公司 | For amplification of CD34 + Culture medium for hematopoietic stem cells and in vitro amplification of CD34 + Methods of hematopoietic stem cells |
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