CN105368775A - A method and a system for preparing megakaryocytes and platelets by utilization of human hemopoietic stem cells - Google Patents
A method and a system for preparing megakaryocytes and platelets by utilization of human hemopoietic stem cells Download PDFInfo
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Abstract
The invention relates to a method and a system for preparing megakaryocytes and platelets by utilization of human hemopoietic stem cells. By the technical scheme, effective and large-scale amplification of CD34<+> stem cells on the base of maintaining stemness characteristics can be achieved, and mature megakaryocytes can be prepared by further adoption of a directional differentiation technique and are used for producing platelets. The problem of sources of platelets used for disease transplantation therapy and scientific research is overcome. Strategic reserves of blood cell components can be provided for national defense.
Description
Technical field
The present invention relates to biomedicine field; More specifically, the present invention relates to and utilize human hematopoietic stem cell to prepare megalokaryocyte and hematoblastic method and system.
Background technology
Hemopoietic stem cell (Hemopoieticstemcell) refers to not yet fully-developed cell, is the origin of all hematopoietic cells and immunocyte, is therefore versatile stem cell, is mainly present in marrow, peripheral blood, Cord blood.These cells can to medullary system and lymphoid progenitor cell differentiation, and wherein medullary system comprises red corpuscle, and granulocyte, monocyte and megalokaryocyte are respectively.
Megakaryocytic system comprises Megakaryoblast, inmature megalokaryocyte and megalokaryocyte and thrombocyte.There will be red-purple particle and thrombocyte in megalokaryocyte tenuigenin after hemopoietic stem cell differentiation, when after platelet development maturation, megalokaryocyte can be departed from from endochylema, until megalokaryocyte only surplus next nucleus, no longer continue to produce thrombocyte.Megalokaryocyte/hematoblastic dyspoiesis can show as the diseases such as idiopathic thrombocytopenic purpura, connective tissue disease (CTD), hypersplenism, megaloblastic anemia clinically.
At present, utilize the amplification factor combination optimized can carry out efficient amplification (see a kind of high-efficiency in-vitro human hematopoietic stem cell amplification cultivation liquid formula to human hematopoietic stem cell, 201410066590.8), on this basis, as developed differentiation-inducing technology, the directed differentiation of megakaryocytic series is carried out to the hemopoietic stem cell after amplification, solve the problem of the platelet counts in human hematopoietic stem cell source, then can be applicable to platelet function research and the exploitation of general cell preparation.
Summary of the invention
The object of the present invention is to provide and utilize human hematopoietic stem cell to prepare megalokaryocyte and hematoblastic method and system.
In a first aspect of the present invention, provide one to prepare the Megakaryocytic method of people (for non-therapeutic method), described method comprises:
(1) hematopoietic stem cell expansion substratum amplification CD34 is utilized
+stem cell (deriving from human hematopoietic stem cell);
(2) cell after step (1) being increased is transferred in megalokaryocyte inductive differentiation medium and is cultivated, and obtains people's megalokaryocyte;
Wherein, described hematopoietic stem cell expansion substratum comprises: stem cell basic medium, according to volume ratio 0.01-0.1%DMSO and following component:
STEM CELL FACTOR (SCF): 50-500ng/mL;
Flt3-part (FLT-3L): 50-500ng/mL;
Platelet factor (TPO): 5-200ng/mL;
Interleukin-13 (IL-3): 10-250ng/mL;
Sall sample albumen 4B (Sall4B): 1-10ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1 (SR1): 0.5-3 μM;
Wherein, described megalokaryocyte inductive differentiation medium comprises: stem cell basic medium and following component:
STEM CELL FACTOR (SCF): 50-500ng/mL;
Platelet factor (TPO): 5-200ng/mL;
Interleukin-13 (IL-3): 10-250ng/mL;
Interleukin 6 (IL-6): 5-100ng/mL;
Interleukin 11 (IL-11): 5-100ng/mL;
Granular leukocyte macrophage stimulus factor (GM-CSF): 5-50ng/ml; With
Low-density lipoprotein (LDL): 5-50ug/ml.
In a preference, described stem cell basic medium is selected from (but being not limited to): StemSpan substratum or ModifiedIMDM substratum (preferably wherein serum-free).
In another preference, step (1) comprising: CD34
+stem cell joins in described hematopoietic stem cell expansion substratum, makes cell concn be 0.5 × 10
4-10 × 10
4individual cell/mL cell/mL (is preferably 1 × 10
4-8 × 10
4individual cell/mL); The hematopoietic stem cell expansion substratum cultivated after 2-4 days according to the interpolation of cell number increasing amount makes cell concn be 0.5 × 10
4-10 × 10
4individual cell/mL (is preferably 1 × 10
4-8 × 10
4individual cell/mL); Step (1) carries out 5-7 days.
In another preference, step (2) comprising: after step (1) carries out 5-7 days, and transferred to by the cell of acquisition in described megalokaryocyte inductive differentiation medium, cell density is 1.0 × 10
5-10 × 10
5individual cell/mL (preferably 2.0 × 10
5-4.0 × 10
5individual cell/mL), the megalokaryocyte inductive differentiation medium continuing to cultivate after 2-4 days according to the interpolation of cell number increasing amount makes cell concn be 1.0 × 10
5-10 × 10
5individual cell/mL (is preferably 2.0 × 10
5-4.0 × 10
5individual cell/mL), continue to cultivate harvested cell after 3-5 days.
In another preference, described method also comprises be separated people's megalokaryocyte from the cell culture obtained.
In another aspect of this invention, provide a kind of for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell, comprising: stem cell basic medium and following component:
STEM CELL FACTOR: 50-500ng/mL;
Flt3-part: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Interleukin-13: 10-250ng/mL;
Sall sample albumen 4B:1-10ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1:0.5-3 μM.
In a preference, described for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell, comprising following component:
STEM CELL FACTOR: 100-300ng/mL;
Flt3-part: 100-300ng/mL;
Platelet factor: 10-150ng/mL;
Interleukin-13: 15-150ng/mL;
Sall sample albumen 4B:2-8ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1:1-2 μM.
In another aspect of this invention, provide a kind of for the Megakaryocytic substratum of inducing differentiation of human, comprising: stem cell basic medium and following component:
STEM CELL FACTOR: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Epithelical cell growth factor: 2-20ng/mL;
Interleukin-13: 10-250ng/mL;
Interleukin 6: 5-100ng/mL;
Interleukin 11: 5-100ng/mL;
Granular leukocyte macrophage stimulus factor GM-CSF:5-50ng/ml; With
Low-density lipoprotein: 5-50ug/ml.
In a preference, described for the Megakaryocytic substratum of inducing differentiation of human, described component comprises:
STEM CELL FACTOR: 80-300ng/mL;
Platelet factor: 30-150ng/mL;
Interleukin-13: 15-150ng/mL;
Interleukin 6: 20-80ng/mL;
Interleukin 11: 15-80ng/mL;
Granular leukocyte macrophage stimulus factor GM-CSF:15-40ng/ml; With
Low-density lipoprotein: 15-40ug/ml.
In another aspect of this invention, provide the purposes of described substratum, for the preparation of people's megalokaryocyte.
In another aspect of this invention, provide a kind of for the preparation of the Megakaryocytic test kit of people, described test kit comprises:
Described in (a) for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell; With
Described in (b) for the Megakaryocytic substratum of inducing differentiation of human.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, the present invention are to people's bleeding of the umbilicus CD34
+the amplification situation of stem cell.
A, 0-6 days total cell count and CD34
+the number of cell amplification;
B, 0-6 days total cell count and CD34
+the multiple of cell amplification.
Fig. 2, the present invention are divided into the situation of megakaryocytic series to human umbilical blood stem cell amplification.
The expression of A, megakaryocytic series surface markers CD41 and CD42;
B, CD41
+cell and CD42
+the number of cell amplification;
C, CD41
+cell and CD42
+the multiple of cell amplification.
Fig. 3, the present invention are divided into Megakaryocytic morphological observations to human umbilical blood stem cell amplification.
Fig. 4, the present invention are divided into Megakaryocytic polyploid analysis to human umbilical blood stem cell amplification.
Fig. 5, the present invention are to peripheral blood CD34 after people's bone marrow mobilization
+the amplification situation of stem cell.
A, 0-6 days total cell count and CD34
+the number of cell amplification;
The multiple of B, 0-6 days total cell count and CD34+ cell amplification.
Fig. 6, the present invention are divided into megakaryocytic series to peripheral hematopoietic stem cells amplification after people's bone marrow mobilization.
The expression of A, megakaryocytic series surface markers CD41 and CD42;
B, CD41
+cell and CD42
+the number of cell amplification;
C, CD41
+cell and CD42
+the multiple of cell amplification.
Fig. 7, the present invention are divided into Megakaryocytic morphological observations to peripheral hematopoietic stem cells amplification after people's bone marrow mobilization.
Fig. 8, the present invention are divided into Megakaryocytic polyploid analysis to peripheral hematopoietic stem cells amplification after people's bone marrow mobilization.
Embodiment
The present inventor is through deep research, and open one and utilize hemopoietic stem cell efficient amplification and directed differentiation to be megalokaryocyte and hematoblastic method, technical scheme of the present invention can make CD34
+stem cell carries out effective and a large amount of amplifications on the basis keeping original dryness feature, and the megalokaryocyte adopting the technology preparation of directed differentiation ripe further, for the production of thrombocyte.Technical scheme of the present invention can solve thrombocyte and carry out source problem for the transplantation treatment of disease and scientific research, can be the strategic reserves that national defence provides blood ingredient cell.
As used herein, term " contain " or " comprising " include " comprising ", " primarily of ... form (making) " " substantially by ... form " and " by ... form ".
Substratum
The present inventor, according to the different steps of cultivating, provides different substratum, comprising: hematopoietic stem cell expansion substratum and megalokaryocyte inductive differentiation medium.
Described hematopoietic stem cell expansion substratum comprises: STEM CELL FACTOR (SCF), Flt3-part (FLT-3L), platelet factor (TPO), interleukin-13 (IL-3), Sall sample albumen 4B (Sall4B) and hematopoietic stem cell expansion agent StemRegenin-1 (SR1).Above-mentioned each component is added in stem cell basic medium (containing 0.01-0.1%DMSO) with the ratio be applicable to, and can be CD34
+stem cell provides the substratum of applicable isolated growth environment, promotes CD34
+the growth of stem cell and amplification.As optimal way of the present invention,
Consumption for each component preparing hematopoietic stem cell expansion substratum of the present invention is as shown in table 1.
Table 1
The cytokine that table 1 is filled a prescription is added in stem cell basic medium, obtains expansion of stem cells substratum, thus is CD34
+stem cell provides suitable growth and amplification environment.Described stem cell basic medium can select StemSpan substratum or ModifiedIMDM substratum etc.
Described megalokaryocyte inductive differentiation medium comprises: STEM CELL FACTOR (SCF), platelet factor (TPO), interleukin-13 (IL-3), interleukin 6 (IL-6), interleukin 11 (IL-11), granular leukocyte macrophage stimulus factor (GM-CSF) and low-density lipoprotein (LDL).Above-mentioned each component is added in stem cell basic medium with the ratio be applicable to, and obtains megalokaryocyte inductive differentiation medium of the present invention.As optimal way of the present invention, the consumption for each component preparing megalokaryocyte inductive differentiation medium of the present invention is as shown in table 2.
Table 2
| Content | Preferred amounts | More preferably measure | |
| STEM CELL FACTOR (SCF) | 50-500ng/mL | 80-300ng/mL | 100ng/mL |
| Platelet factor (TPO) | 5-200ng/mL | 30-150ng/mL | 100ng/mL |
| Interleukin-13 (IL-3) | 10-250ng/mL | 15-150ng/mL | 15ng/mL |
| Interleukin 6 (IL-6) | 5-100ng/mL | 20-80ng/mL | 50ng/mL |
| Interleukin 11 (IL-11) | 5-100ng/mL | 15-80ng/mL | 25μg/mL |
| GM-CSF | 5-50ng/ml | 15-40ng/ml | 25ng/ml |
| Low-density lipoprotein (LDL) | 5-50ug/ml | 15-40ug/ml | 25ug/mL |
The component that table 2 is filled a prescription is added in stem cell basic medium, obtains megalokaryocyte inductive differentiation medium of the present invention, thus is that Megakaryocytic preparation provides the preferred culture medium prescription simplified.
Above-mentioned substratum after the present inventor optimizes, containing the enough and composition of reasonably Promote cell's growth and amplification, maintenance dryness and be conducive to the composition that cell realizes differentiation, is conducive to Megakaryocytic cultivation.
Be all that those skilled in the art are easy to obtain for preparing the cytokines such as Sall4B, SCF, TPO, Flt-3L, IL3, GM-CSF, IL6, IL11 of substratum, such as, buy, or by synthetic or recombinant expressed acquisition by commercial sources.
Cultural method
Current clinical treatment megakaryocytic maturation obstacle/thrombocytopoiesis lacks the problem existing for class disease: thrombocyte deposit shortage makes to treat timely and effectively; The generation of thrombocyte in human body lacks effective clinical applicable stimulating factor; The hidden danger propagating blood class disease is there is in the thrombocyte offered of donating blood in infusion process.
Therefore, the invention provides one and prepare the Megakaryocytic method of people, described method comprises: (1) utilizes hematopoietic stem cell expansion substratum amplification CD34
+stem cell; (2) cell after step (1) being increased is transferred in megalokaryocyte inductive differentiation medium and is cultivated, and obtains people's megalokaryocyte.
In the present invention, described CD34
+stem cell is separable from bleeding of the umbilicus, mobilization peripheral blood and other source (such as cultivating mechanism purchased from some) afterwards.
The present invention is separation of C D34 first
+cell, then temporally the cell growth factor adjusted in substratum realizes hemopoietic stem cell and first to increase the process of breaking up afterwards.The first step uses and promotes that propagation is for main hematopoietic stem cell expansion substratum, and second step uses to be induced to differentiate into main megalokaryocyte inductive differentiation medium.Method of the present invention can obtain other megalokaryocyte of efficient clinical grade for hematoblastic generation, this precious resources that can carry out transplantation treatment of thrombocyte can fully be obtained, the multiple thrombocyte of breakthrough solution carrys out source problem, this technology solves the under-supply present situation of the clinical thrombocyte of puzzlement, prevent the propagation of the various communicate illnesss caused in composition blood infusion of therapeutic process, and the medical treatment blood ingredient that has been national defence and strategical stock.
The present invention mainly employs the hematopoietic stem cell expansion formula of SCF, Flt-3L, TPO, IL3, Sall4B-TAT and SR1 early stage, later stage have employed the induction megakaryocytopoiesis formula of SCF, TPO, IL-6, IL-11, IL-3, GM-CSF, LDL, after realizing people's bleeding of the umbilicus and bone marrow mobilization first, the hemopoietic stem cell of derived from peripheral blood is divided into megalokaryocyte after increasing in a large number in vitro, establishes and utilizes hemopoietic stem cell to prepare universal megalokaryocyte and hematoblastic technology.Several principal feature of the present invention and advantage: the first, the present invention adopts human archeocyte somatomedin at whole culture system, does not introduce expression of exogenous genes, does not therefore change the Genome stability of former stem cell, without tumorigenesis risk; The second, the present invention can make stem cell cultivate in vitro 13 days afterwards total cell count increase more than 3000 times, there is the CD41a of megakaryocytic series mark
+cell quantity increases 37000 times, wherein has the CD42 of ripe megalokaryocyte and thrombocyte mark
+cell accounts for more than 80%.3rd, the present invention adopts the bleeding of the umbilicus of Healthy People and peripheral blood to carry out differentiation-inducing, strictly controls cell derived, source abundance, safety and stability, easy to use, effectively prevents serious infectious diseases to be propagated by blood ingredient Injection fashion.
Application of the present invention will effectively solve existing problem, compared with prior art simultaneously, advantage of the present invention and progress are embodied in two aspects, one is in the efficient amplification of hemopoietic stem cell, current existing stem cell expansion procedures has use feeder layer cells and inserts allogenic gene, the maturity state that the method for feeder layer cells can not make sub-stem cell remain identical with female stem cell, the best also can only expanding stem cells about 10 times, and introduces ectogenic cell and disturb.And the expansion of stem cells the best introduced expression of exogenous genes and carry out can reach the expanding effect of 160 times to 200 times, but interference can be caused for the dryness of hemopoietic stem cell and gene stability.The present invention before directed differentiation, can make CD34
+the amplification of cell reaches more than 100 times, and in the maintenance amplification of directed differentiation initial stage.Two is producing on thrombocyte to megakaryocytic series differentiation hemopoietic stem cell, and current existing artificial platelets method comprises carries out genetic modification to stem cell (iPS and hES) and cytokine stimulates two classes.Although genetic modification can make stem cell persistence generate thrombocyte, the tumorigenesis risk of iPS cell etc. itself and the gene stability interference caused by foreign gene all will limit the clinical application of this technology.The present invention's total cell count of differentiation after 13 days that can increase increases 3000 times, CD41a
+cell amplification 37000 times.The present invention breaches the defect of prior art, adopts other combination of cytokines of clinical grade, carries out efficient amplification and directed differentiation, have higher clinical application and scientific research value to the reliable hemopoietic stem cell of safe source.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
Embodiment 1, substratum
Culture system of the present invention comprises hematopoietic stem cell expansion substratum, megalokaryocyte inductive differentiation medium.
One, increase the phase: hematopoietic stem cell expansion substratum
Adopt substratum based on StemSpan substratum (serum-free) (Stemcell company), add cytokine and 0.05% (v/v) DMSO of being selected from arbitrary formula in table 3 wherein.
Table 3 (beyond indicating unless otherwise, unit is ng/mL)
| Formula A | Formula B | Formula C | |
| SCF | 100 | 50 | 200 |
| FLT-3L | 100 | 50 | 200 |
| TPO | 25 | 10 | 100 |
| IL-3 | 15 | 50 | 100 |
| Sall4B | 3 | 5 | 9 |
| StemRegenin-1(SR1) | 1μM | 0.5μM | 3μM |
Two, differentiation and the ripening stage: megalokaryocyte inductive differentiation medium
Adopt substratum based on StemSpan substratum (serum-free), add the cytokine being selected from arbitrary formula in table 4 and other compositions wherein:
Table 4 (beyond indicating unless otherwise, unit is ng/mL)
| Formula D | Formula E | Formula F | |
| SCF | 100 | 50 | 200 |
| TPO | 100 | 20 | 150 |
| IL-3 | 15 | 30 | 50 |
| IL-6 | 50 | 20 | 100 |
| IL-11 | 25 | 20 | 80 |
| GM-CSF | 25 | 10 | 40 |
| LDL | 25μg/mL | 10μg/mL | 40μg/mL |
Embodiment 2, amplification bleeding of the umbilicus CD34
+stem cell also differentiation-inducingly prepares megalokaryocyte (I)
0th day:
(1) hematopoietic stem cell expansion substratum is prepared
Various cytokine is added in StemSpan substratum, make cytokine final concentration be SCF100ng/mL, Flt-3L100ng/mL, IL-315ng/mL, TPO25ng/mL, Sall4B3ng/mL, SR11 μM and 0.05% (v/v) DMSO are as amplification culture medium.
(2) separation and purification bleeding of the umbilicus CD34
+stem cell
Get fresh mature healthy newborn Cord blood, use Mei Tian Ni company MACS magnetic bead sorting systematic position CD34
+mononuclearcell, obtaining cell quantity is 2 × 10
6individual cell.Adopting hematopoietic stem cell expansion to cultivate keynote cell concn is 2 × 10
4individual cell/mL, every hole 1mL are placed in 24 orifice plates and cultivate (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2).
(3) flow cytometer detection
Get 2 × 10
5cell is placed in 15ml centrifuge tube, evenly distribute 5 × 10
4cell is placed in 1.5mLEP pipe, totally 4 EP pipes, often pipe adds the PBS washings of 1mL containing 1%BSA, centrifugal 5 minutes of 1200rpm under room temperature, supernatant discarded, precipitate containing the PBS re-suspended cell of 1%BSA with 100 μ L, pipe 1-1 adds 2 μ LCD34 antibody (PE-Mouseanti-humanCD34), and pipe 1-2 adds the corresponding Isotype control of 2 μ L (PE-Mouseanti-humanIgG1 κ).Pipe 2-1 adds CD41a antibody (APC-Mouseanti-humanCD41a) and the CD42b antibody (FITC-Mouseanti-humanCD42b) of 2 μ LBD companies respectively, pipe 2-2 adds corresponding Isotype control (APC-Mouseanti-humanIgG1 κ and FITC-Mouseanti-humanIgG1 κ) antibody 2 μ L, antibody adds rear room temperature lucifuge and hatches 15 minutes, then often pipe adds 1mLPBS washing, centrifugal 5 minutes of 1200rpm, supernatant discarded, with 100 μ LPBS re-suspended cells, with flow cytometry analysis cell surface CD34, CD41a, the expression of CD42b.
3rd day:
Carry out a point hole according to cell number and (control cell density lower than 1 × 10
6individual/mL), enlarged culturing system also every hole adds fresh culture to 1mL (adding in embodiment 1 A that fills a prescription).
6th day:
Be cultured to the 6th day and be replaced by differentiation-inducing culture medium culturing.
(1) cell counting
With the expression of flow cytometry analysis cell surface CD34.And add up total cell number, CD34
+cell number and amplification times, total cell absolute number is 4.03 × 10
6± 3.03 × 10
5, CD34
+cell absolute number is 2.38 × 10
6± 4.09 × 10
5total cells expanded is 201.62 ± 15.15 times, CD34
+cells expanded is 126.34 ± 21.76 times (n=3), the results are shown in Figure 1.
(2) inductive differentiation medium is prepared
In StemSpan substratum (serum-free), add following composition, each composition final concentration is: SCF100ng/mL, IL-315ng/mL, TPO100ng/mL, IL-650ng/mL, IL-1125ng/mL, GM-CSF25ng/mL, LDL25 μ g/mL (namely fill a prescription in embodiment 1 D).
(3) cultivate: all cells being expanded to the 6th day is above changed to inductive differentiation medium, adjusts cell concn to be 2.0 × 10
5-3.0 × 10
5individual cell/mL, adds 5mL inductive differentiation medium, is placed in T-25 Tissue Culture Flask and cultivates (n=3).Then pipe is placed in incubator (37 DEG C, 5%CO2) with microscopic examination.
9th day:
(1) cell counting, with the expression of flow cytometry analysis cell surface marker CD34, CD41a, CD42b.
(2) now total cell number is 1.14 × 10
7± 8.69 × 10
5, enlarged culturing system 2 times, adds fresh culture 3mL (namely fill a prescription in embodiment 1 D) separately.
13rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface macronucleus system mark (CD41a, CD42b), adds up the CD41a of the 3rd, 6,9,13 day
+, CD42b
+cell count compares the multiple of the 0th day, the results are shown in Figure 2A-B.At the 13rd day, total cells expanded was 3007.98 ± 1230.43 times, CD41a
+cell, CD42b
+the amplification times of cell is respectively 37000 ± 1517.90 times and 33000 ± 1138.36 times (n=3), the results are shown in Figure 2C.
(2) the 13rd day cell 0.5-1 × 10 are got
6take pictures after cell PBS liquid washes 3 times, get 4 × 10
4cell is suspended from 30 μ LPBS, makes cell smear, and takes pictures after observing with Wright-Giemsa dyeing, and visible size is 40-100 μm, the results are shown in Figure 3.
(3) carry out the detection of megakaryocyte polyploid: according to the operation steps in the cell cycle test kit optimized (BD company), use the technology of the two dye of CD41a and PI, detected by flow cytometer.Specifically, 5 × 10 are got
5individual cell, add the PBS washings of 1mL containing 1%BSA, centrifugal 5 minutes of 1200rpm under room temperature, supernatant discarded, precipitates containing the PBS re-suspended cell of 1%BSA with 100 μ L, fixedly spends the night after room temperature lucifuge hatches CD41a antibody 15min with 1% paraformaldehyde 4 DEG C.After washing three times with the damping fluid containing proteinase inhibitor/rnase, 4 DEG C of lucifuges hatch Propidium iodide solution 10 minutes, carry out flow cytometer detection.Collect at least 20000 cells and carry out flow cytometer showed.Determine as megalokaryocyte group, to carry out polyploid analysis to this group using the cell of CD41a.At the 13rd day, about have the cell of 52.27% ± 7.43% to be 4 times of bodies or more cell, wherein 4N accounts for 21.94% ± 4.12%, 8N and accounts for 18.29% ± 2.38%, 16N and account for 12.04% ± 3.74% (n=3) above, the results are shown in Figure 4.
Embodiment 3, amplification people mobilize rear peripheral blood CD34
+stem cell also differentiation-inducingly prepares megalokaryocyte (I)
Peripheral blood collection after people's bone marrow mobilization: gather first 5 days, adopts 5 μ g/kg to give healthy adult volunteer G-CSF, mobilizes 3 days continuously, each collection 200-300mL peripheral blood.
0th day:
(1) amplification culture medium is prepared
Various cytokine is added in StemSpan substratum, make cytokine final concentration be SCF100ng/mL, Flt-3L100ng/mL, IL-315ng/mL, TPO25ng/mL, Sall4B3ng/mL, SR11 μM and 0.05% (v/v) DMSO are as amplification culture medium.
(2) separation and purification peripheral blood CD34
+stem cell
After adopting fresh mobilization, peripheral blood 10mL, PBS dilute 20 times, use Mei Tian Ni company MACS magnetic bead sorting systematic position CD34
+mononuclearcell, obtaining cell quantity is 2.5 × 10
6individual cell.Adopting hematopoietic stem cell expansion to cultivate keynote cell concn is 5 × 10
4individual cell/mL, every hole 1mL are placed in 24 orifice plates and cultivate (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2)
(3) flow cytometer detection: with corresponding section in embodiment 2 (the 0th day, (3)).
3rd day:
Carry out a point hole according to cell number and (control cell density lower than 1 × 10
6individual/mL), enlarged culturing system also every hole adds fresh culture to 1mL (adding in embodiment 1 A that fills a prescription).
6th day:
Be cultured to the 6th day and be replaced by inductive differentiation medium cultivation.
(1) cell counting
With flow cytometry analysis cell surface CD34, the expression of CD41a, CD42b.And add up total cell number, CD34 cell number, total cell absolute number is 4.28 × 10
6± 7.03 × 10
5, CD34 cell absolute number is 2.95 × 10
6± 4.09 × 10
5(n=3), total cells expanded is 85.65 ± 7.03 times, CD34
+cells expanded is 62.91 ± 4.36 times (n=3), the results are shown in Figure 5A-B.
(2) inductive differentiation medium is prepared
In StemSpan substratum (serum-free), add following composition, each composition final concentration is: SCF100ng/mL, IL-315ng/mL, TPO100ng/mL, IL-650ng/mL, IL-1125ng/mL, GM-CSF25ng/mL, LDL25 μ g/mL (namely fill a prescription in embodiment 1 D).
(3) cultivate: all cells being expanded to the 6th day is above changed to inductive differentiation medium, adjusts cell concn to be 2.0 × 10
5-3.0 × 10
5individual cell/mL, adds 5mL inductive differentiation medium, is placed in T-25 Tissue Culture Flask and cultivates (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2).
9th day:
(1) cell counting, with the expression of flow cytometry analysis cell surface marker CD34, CD41a, CD42b.
(2) now total cell number is 1.32 × 10
7± 1.19 × 10
6, enlarged culturing system 2 times, adds fresh culture 3mL (namely fill a prescription in embodiment 1 D) separately.
13rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface macronucleus system mark (CD41a, CD42b), adds up the CD41a of the 3rd, 6,9,13 day
+, CD42b
+cell count compares the multiple of the 0th day, the results are shown in Figure 6A-B.At the 13rd day, CD41a
+the amplification times of cell is 18666.91 ± 1264.58 times, CD42b
+the amplification times of cell is 12234.92 ± 1306.63 times (n=3), the results are shown in Figure 6C.
(2) the 13rd day cell 0.5-1 × 10 are got
6take pictures after cell PBS liquid washes 3 times, get 4 × 10
4cell is suspended from 30 μ LPBS, makes cell smear, and takes pictures after observing with Wright-Giemsa dyeing, the results are shown in Figure 7.
(3) detection of megakaryocyte polyploid is carried out: operate same embodiment 2, at the 13rd day, the cell of 46% ± 2.96% is about had to be 4 times of bodies or more cell, wherein 4N accounts for 19.45% ± 7.42%, 8N accounts for 14.16% ± 6.15%, 16N and account for 12.91% ± 0.39% (n=3) above, the results are shown in Figure 8.
Embodiment 4, amplification bleeding of the umbilicus CD34
+stem cell also differentiation-inducingly prepares megalokaryocyte (II)
0th day:
(1) hematopoietic stem cell expansion substratum is prepared
Various cytokine is added in StemSpan substratum, make cytokine final concentration be SCF50ng/mL, Flt-3L50ng/mL, IL-350ng/mL, TPO10ng/mL, Sall4B5ng/mL, SR10.5 μM (namely fill a prescription in embodiment 1 B) and 0.05% (v/v) DMSO are as amplification culture medium.
(2) separation and purification bleeding of the umbilicus CD34
+stem cell
After adopting fresh mobilization, peripheral blood 10mL, PBS dilute 20 times, use Mei Tian Ni company MACS magnetic bead sorting systematic position CD34
+mononuclearcell, obtaining cell quantity is 1.5 × 10
6individual cell.Adopting hematopoietic stem cell expansion to cultivate keynote cell concn is 3 × 10
4individual cell/mL, every hole 1mL are placed in 24 orifice plates and cultivate (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2)
(3) flow cytometer detection is with corresponding section in embodiment 2 (the 0th day, (3))
3rd day:
Carry out a point hole according to cell number and (control cell density lower than 1 × 10
6individual/mL), enlarged culturing system also every hole adds fresh culture to 1mL (adding in embodiment 1 B that fills a prescription).
6th day:
Be cultured to the 6th day and be replaced by differentiation-inducing culture medium culturing.
(1) cell counting
With the expression of flow cytometry analysis cell surface CD34.And add up total cell number, CD34
+cell number and amplification times, total cell absolute number is 3.47 × 10
6± 2.83 × 10
5, CD34
+cell absolute number is 1.78 × 10
6± 5.23 × 10
5, total cells expanded is 112.62 ± 35.34 times, CD34
+cells expanded is 54.48 ± 21.16 times (n=3).
(2) inductive differentiation medium is prepared
In StemSpan substratum (serum-free), add following composition, each composition final concentration is: SCF50ng/mL, IL-330ng/mL, TPO20ng/mL, IL-620ng/mL, IL-1120ng/mL, GM-CSF10ng/mL, LDL10 μ g/mL (namely fill a prescription in embodiment 1 E).
(3) cultivate: all cells being expanded to the 6th day is above changed to inductive differentiation medium, adjusts cell concn to be 2.0 × 10
5-3.0 × 10
5individual cell/mL, adds 5mL inductive differentiation medium, is placed in T-25 Tissue Culture Flask and cultivates (n=3).Then pipe is placed in incubator (37 DEG C, 5%CO2) with microscopic examination.
9th day:
(1) cell counting, with the expression of flow cytometry analysis cell surface marker CD34, CD41a, CD42b.
(2) now total cell number is 5.72 × 10
6± 4.61 × 10
5, enlarged culturing system 2 times, adds fresh culture 3mL (namely fill a prescription in embodiment 1 E) separately.
13rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface macronucleus system mark (CD41a, CD42b), adds up the CD41a of the 3rd, 6,9,13 day
+, CD42b
+cell count compares the multiple of the 0th day.At the 13rd day, total cells expanded was 987.13 ± 129.49 times, CD41a
+cell, CD42b
+the amplification times of cell is respectively 17400 ± 1216.30 times and 15300 ± 1178.16 times (n=3).
(2) the 13rd day cell 0.5-1 × 10 are got
6take pictures after cell PBS liquid washes 3 times, get 4 × 10
4cell is suspended from 30 μ LPBS, makes cell smear, and takes pictures after observing with Wright-Giemsa dyeing, and visible size is 40-100 μm.
(3) carry out the detection of megakaryocyte polyploid: according to the operation steps in the cell cycle test kit optimized (BD company), use the technology of the two dye of CD41a and PI, detected by flow cytometer.Specifically, 5 × 10 are got
5individual cell, add the PBS washings of 1mL containing 1%BSA, centrifugal 5 minutes of 1200rpm under room temperature, supernatant discarded, precipitates containing the PBS re-suspended cell of 1%BSA with 100 μ L, fixedly spends the night after room temperature lucifuge hatches CD41a antibody 15min with 1% paraformaldehyde 4 DEG C.After washing three times with the damping fluid containing proteinase inhibitor/rnase, 4 DEG C of lucifuges hatch Propidium iodide solution 10 minutes, carry out flow cytometer detection.Collect at least 20000 cells and carry out flow cytometer showed.Determine as megalokaryocyte group, to carry out polyploid analysis to this group using the cell of CD41a.About have the cell of 38% ± 6.23%% to be 4 times of bodies or more cell, wherein 4N accounts for 18.27% ± 4.50%%, 8N and accounts for 10.10% ± 5.87%%, 16N and account for 9.43% ± 2.49%% (n=3) above.
Embodiment 5, amplification people mobilize rear peripheral blood CD34
+stem cell also differentiation-inducingly prepares megalokaryocyte (II)
Peripheral blood collection after people's bone marrow mobilization: gather first 5 days, adopts 5 μ g/kg to give healthy adult volunteer G-CSF, mobilizes 3 days continuously, each collection 200-300mL peripheral blood.
0th day:
(1) amplification culture medium is prepared
Various cytokine is added in StemSpan substratum, make cytokine final concentration be SCF200ng/mL, Flt-3L200ng/mL, IL-3100ng/mL, TPO100ng/mL, Sall4B9ng/mL, SR13 μM (namely fill a prescription in embodiment 1 C) and 0.05% (v/v) DMSO are as amplification culture medium.
(2) separation and purification peripheral blood CD34
+stem cell
After adopting fresh mobilization, peripheral blood 10mL, PBS dilute 20 times, use Mei Tian Ni company MACS magnetic bead sorting systematic position CD34
+mononuclearcell, obtaining quantity is 2 × 10
6individual cell.Adopting hematopoietic stem cell expansion to cultivate keynote cell concn is 5 × 10
4individual cell/mL, every hole 1mL are placed in 24 orifice plates and cultivate (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2)
(3) flow cytometer detection: with corresponding steps in embodiment 3.
3rd day:
Carry out a point hole according to cell number and (control cell density lower than 1 × 10
6individual/mL), enlarged culturing system also every hole adds fresh culture to 1mL (adding in embodiment 1 C that fills a prescription).
6th day:
Be cultured to the 6th day and be replaced by inductive differentiation medium cultivation.
(1) cell counting
With flow cytometry analysis cell surface CD34, the expression of CD41a, CD42b.And add up total cell number, CD34 cell number, total cell absolute number is 5.41 × 10
7± 8.83 × 10
5, CD34 cell absolute number is 3.39 × 10
6± 3.12 × 10
5(n=3), total cells expanded is 108.20 ± 8.35 times, CD34
+cells expanded is 69.18 ± 6.39 times (n=3)
(2) inductive differentiation medium is prepared
In StemSpan substratum (serum-free), add following composition, each composition final concentration is: SCF200ng/mL, IL-350ng/mL, TPO150ng/mL, IL-6100ng/mL, IL-1180ng/mL, GM-CSF40ng/mL, LDL40 μ g/mL (namely fill a prescription in embodiment 1 F).
(3) cultivate: all cells being expanded to the 6th day is above changed to inductive differentiation medium, adjusts cell concn to be 2.0 × 10
5-3.0 × 10
5individual cell/mL, adds 5mL inductive differentiation medium, is placed in T-25 Tissue Culture Flask and cultivates (n=3).With microscopic examination then pipe is placed in incubator (37 DEG C, 5%CO
2).
9th day:
(1) cell counting, with the expression of flow cytometry analysis cell surface marker CD34, CD41a, CD42b.
(2) now total cell number is 1.77 × 10
7± 1.31 × 10
6, enlarged culturing system 2 times, adds fresh culture 3mL (namely fill a prescription in embodiment 1 F) separately.
13rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface macronucleus system mark (CD41a, CD42b), adds up the CD41a of the 3rd, 6,9,13 day
+, CD42b
+cell count compares the multiple of the 0th day.At the 13rd day, CD41a
+the amplification times of cell is 23556 ± 3834.10 times, CD42b
+the amplification times of cell is 163661 ± 2134.41 times (n=3).
(2) the 13rd day cell 0.5-1 × 10 are got
6take pictures after cell PBS liquid washes 3 times, get 4 × 10
4cell is suspended from 30 μ LPBS, makes cell smear, and takes pictures after observing with Wright-Giemsa dyeing.
(3) detection of megakaryocyte polyploid is carried out: operate same embodiment 2, at the 13rd day, the cell of 31.68 ± 7.21% is about had to be 4 times of bodies or more cell, wherein 4N accounts for 13.15% ± 4.02%%, 8N accounts for 10.38% ± 3.97%%, 16N and accounts for 8.11% ± 3.83%% (n=3) above.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. prepare the Megakaryocytic method of people, it is characterized in that, described method comprises:
(1) hematopoietic stem cell expansion substratum amplification CD34 is utilized
+stem cell;
(2) cell after step (1) being increased is transferred in megalokaryocyte inductive differentiation medium and is cultivated, and obtains people's megalokaryocyte;
Wherein, described hematopoietic stem cell expansion substratum comprises: stem cell basic medium, according to volume ratio 0.01-0.1%DMSO and following component:
STEM CELL FACTOR: 50-500ng/mL;
Flt3-part: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Interleukin-13: 10-250ng/mL;
Sall sample albumen 4B:1-10ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1:0.5-3 μM;
Wherein, described megalokaryocyte inductive differentiation medium comprises: stem cell basic medium and following component:
STEM CELL FACTOR: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Interleukin-13: 10-250ng/mL;
Interleukin 6: 5-100ng/mL;
Interleukin 11: 5-100ng/mL;
Granular leukocyte macrophage stimulus factor: 5-50ng/ml; With
Low-density lipoprotein: 5-50ug/ml.
2. the method for claim 1, is characterized in that, described stem cell basic medium is selected from: StemSpan substratum or ModifiedIMDM substratum.
3. the method for claim 1, is characterized in that, step (1) comprising: CD34
+stem cell joins in described hematopoietic stem cell expansion substratum, makes cell concn be 0.5 × 10
4-10 × 10
4individual cell/mL cell/mL; The hematopoietic stem cell expansion substratum cultivated after 2-4 days according to the interpolation of cell number increasing amount makes cell concn be 0.5 × 10
4-10 × 10
4individual cell/mL; Step (1) carries out 5-7 days.
4. the method for claim 1, is characterized in that, step (2) comprising: after step (1) carries out 5-7 days, and transferred to by the cell of acquisition in described megalokaryocyte inductive differentiation medium, cell density is 1.0 × 10
5-10 × 10
5individual cell/mL, the megalokaryocyte inductive differentiation medium continuing to cultivate after 2-4 days according to the interpolation of cell number increasing amount makes cell concn be 1.0 × 10
5-10 × 10
5individual cell/mL, continues to cultivate harvested cell after 3-5 days.
5. one kind for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell, is characterized in that, comprising: stem cell basic medium and following component:
STEM CELL FACTOR: 50-500ng/mL;
Flt3-part: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Interleukin-13: 10-250ng/mL;
Sall sample albumen 4B:1-10ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1:0.5-3 μM.
6. as claimed in claim 5 for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell, is characterized in that, comprising following component:
STEM CELL FACTOR: 100-300ng/mL;
Flt3-part: 100-300ng/mL;
Platelet factor: 10-150ng/mL;
Interleukin-13: 15-150ng/mL;
Sall sample albumen 4B:2-8ng/mL; With
Hematopoietic stem cell expansion agent StemRegenin-1:1-2 μM.
7., for the Megakaryocytic substratum of inducing differentiation of human, it is characterized in that, comprising: stem cell basic medium and following component:
STEM CELL FACTOR: 50-500ng/mL;
Platelet factor: 5-200ng/mL;
Epithelical cell growth factor: 2-20ng/mL;
Interleukin-13: 10-250ng/mL;
Interleukin 6: 5-100ng/mL;
Interleukin 11: 5-100ng/mL;
Granular leukocyte macrophage stimulus factor GM-CSF:5-50ng/ml; With
Low-density lipoprotein: 5-50ug/ml.
8. as claimed in claim 7 for the Megakaryocytic substratum of inducing differentiation of human, it is characterized in that, described component comprises:
STEM CELL FACTOR: 80-300ng/mL;
Platelet factor: 30-150ng/mL;
Interleukin-13: 15-150ng/mL;
Interleukin 6: 20-80ng/mL;
Interleukin 11: 15-80ng/mL;
Granular leukocyte macrophage stimulus factor GM-CSF:15-40ng/ml; With
Low-density lipoprotein: 15-40ug/ml.
9. the purposes of the arbitrary described substratum of claim 5-8, for the preparation of people's megalokaryocyte.
10. for the preparation of the Megakaryocytic test kit of people, it is characterized in that, described test kit comprises:
Described in (a) claim 5 or 6 for the CD34 that increases
+the hematopoietic stem cell expansion substratum of stem cell; With
Described in (b) claim 7 or 8 for the Megakaryocytic substratum of inducing differentiation of human.
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