CN104622901B - A kind of method that human embryo stem cell for directional is divided into candidate stem cell - Google Patents
A kind of method that human embryo stem cell for directional is divided into candidate stem cell Download PDFInfo
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Abstract
The invention discloses a kind of stem cell medicines and preparation method thereof for treating acute lymphocytic leukemia, the specifically described stem cell medicine is that a kind of candidate stem cell by embryonic stem cell inductive formation in vitro is prepared, and includes at least (1-5) × 10 in the stem cell medicine6A candidate stem cell derived from embryonic stem cell, a kind of preparation method of stem cell medicine that treating acute lymphocytic leukemia includes the following steps: the secondary culture of (1) human embryo stem cell, (2) formation of candidate stem cell embryoid body, (3) human embryo stem cell breaks up to candidate stem cell, (4) culture and passage and detection of candidate stem cell, the preparation of (5) stem cell medicine.
Description
Technical field
The invention belongs to bio-medical technology field, be related to a kind of stem cell medicine for treating acute lymphocytic leukemia and
Preparation method, the specifically described stem cell medicine are that a kind of Hematopoietic Stem by embryonic stem cell inductive formation in vitro is thin
Born of the same parents are prepared.
Background technique
Leukaemia is the cancer occurred in blood, can invade blood, marrow and lymphatic system.Leukaemia can be divided into
Acute leukemia and chronic leukemia, acute leukemia can be further divided into acute myelogenous leukemia (AML) and acute leaching
Bar sample leukaemia (ALL).Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL).It is anxious
Property lymphatic leukemia (ALL) be that a kind of lacked of proper care with lymphoblast (predecessor of jejune lymphocyte) and is broken up different at hyperplasia
Often or differentiation is obstructed, and is formed the rapid progress type leukaemia that a large amount of non-eukocytes are characterized, is accounted for about Chinese children phase white blood
Disease 80% and childhood whole cancer 25%；In adult, account for about the 20% of acute leukemia.
Clinically the treatment of acute leukemia requires enough satisfactory hematopoietic cells, required for obtaining medical treatment
Cell quantity, many scientists attempt to obtain red blood cell by vitro culture, mainly by be added various cells because
Son etc., but the limited amount of currently used method expanding hemopoietic cell, and obtained hematopoietic cell purity is not high.
Candidate stem cell (hematopoietic stem cell, HSC) be one kind can break up generate various haemocytes and
The cell of immunocyte, it is to be currently applied to the most a kind of cell of cell therapy.Applied to leukaemia, aregeneratory
Property hematopoiesis such as anaemia, severe immune deficiency sign, thalassemia, acute radiation sickness, malignant solid tumor or lymthoma and immune
The patient of system dysfunction disease, it can be functioned, the intracorporal hematopoiesis function of reconstruction patients and immune system, be improved
Survival rate, to achieve the purpose that treatment, here it is hematopoietic stem cell transplantation therapy, stem cell transplantation is the pernicious blood of Current therapeutic
The best means of liquid disease and immunological diseases.Candidate stem cell currently used for transplanting is there are mainly three types of source: marrow, peripheral blood
And Cord blood leads to 90% or more due to the presence for the problems such as candidate stem cell content is rare in these tissues and graft rejection
Potential patient lack the candidate stem cell of suitable distribution type.In addition the candidate stem cell in internal source cultivates a period of time in vitro
Afterwards, therapeutic efficiency can also have a greatly reduced quality.And the candidate stem cell of derived from embryonic stem cells, it is real through experiment in vitro and animal model
It tests, has sufficiently been proved with the potential for being further divided into erythroid cells, myeloid cell and lymphoid lineage cell, be treatment hematopoiesis
And the promising cell origin of immune system dysfunction disease.Research ES cell differentiation is candidate stem cell, no
It only can be used as the model that the early stage hematopoiesis of research animal occurs, and the source of candidate stem cell can be increased, and do not exempt from
Epidemic disease rejection.It does not need to solve the problems, such as graft rejection by the methods of gene knockout, therapeutic cloning, to be hematopoiesis
Obstacle has been cleared away in the development of stem cell transplantation, therefore has important researching value and application prospect.The present invention is a kind of newest
The efficient ES cell differentiation of type is the method for candidate stem cell, has remarkable efficacy to the treatment of acute lymphocytic leukemia.
Summary of the invention
The object of the present invention is to provide a kind of stem cell medicine for treating acute lymphocytic leukemia, the present invention also provides
A kind of preparation method of stem cell medicine that treating acute lymphocytic leukemia.The present invention utilizes human embryo stem cell inductive formation
Hematopoietic stem cell preparation transplanted by venoclysis mode and give acute lymphocytic leukemia patient, acute lymphoblastic can be obviously improved
The survival rate of sample leukemia patient is repaired and is replaced damaged cell, enhances metabolic function；With hematopoieticmicroenviron-ment is repaired, promote
Leukaemia is pathologically restored, and the new way of cell therapy is opened up for clinical treatment leukaemia.
To realize above-mentioned technical purpose and the technique effect, the invention is realized by the following technical scheme: Yi Zhongzhi
The stem cell medicine for treating acute lymphocytic leukemia is by the candidate stem cell in human embryo stem cell source and with concentration ratio for 1-5
Sero-abluminous physiological saline or candidate stem cell is attached to the solution with microcarrier, preferably the carrier is lipid
What the carriers such as carrier, microcapsules, microballoon were formulated.
The candidate stem cell in human embryo stem cell source is according to (1-5) × 10 in the stem cell medicine6A cell, with
1-5ml bovine serum albumin(BSA) BSA or human serum albumins HAS and 95-99.5ml normal saline form.For acute lymphoblastic
The effective dose that the patient of sample leukaemia carries out peripheral vein infusion is (1-5) × 106/ time.The effective dose is by a large amount of
Animal model test obtains therapeutic effect and clinical trial and determination.
The candidate stem cell is formed by human embryo stem cell through Differentiation Induction in vitro culture.
One kind that the embryonic stem cell is body early embryo (before gastrula stage) or original sexual gland kind is separated is thin
Born of the same parents, it has the characteristic of in vitro culture infinite multiplication, self-renewing and Multidirectional Differentiation.
The candidate stem cell vigor is maintained at 90% or more.
A kind of preparation method of stem cell medicine that treating acute lymphocytic leukemia, mainly comprises the steps that embryo
The secondary culture of stem cell, the formation of hematopoietic cell embryoid body, human embryo stem cell break up to candidate stem cell, candidate stem cell
Culture and passage and detection, the preparation of stem cell medicine；Wherein the step of secondary culture of the embryonic stem cell is specific
Are as follows: routine culture embryonic stem cell is passed on and is expanded, this stage culture medium are as follows: and 85%DMEM culture medium is basic culture medium,
Add 15% fetal calf serum or serum replacement, 1mmol/L essential amino acid, 50IU/mL penicillin, 10 μ g/mL streptomysins and
4ng/mL basic fibroblast growth factor.
The formation of the hematopoietic cell embryoid body: embryonic stem cell is induced at secondary culture 6 ~ 7 days, uses 1mg/
ML type Ⅳ collagenase is blown and beaten into small cell cluster, is then suspended 7 days, is changed the liquid once using ultralow 6 well culture plates that stick within every 2 days,
Embryoid body (EBs) formation stages are basic culture medium in embryoid body formation stages 80%DMEM culture medium, add 20% fetal calf serum
Or serum replacement, 1mmol/L essential amino acid, 50IU/mL penicillin, 10 μ g/mL streptomysins, and 4 kinds are added simultaneously quantitatively
Preparation, EB culture be separately added into the mitomycin of 0.1mM, 1 × 10 within the 5th ~ 7 day4The L- of the vitamin C of mol/L, 2mM
The beta -mercaptoethanol (β-ME) of glutamine and 0.1mM.
The step of human embryo stem cell breaks up to candidate stem cell specifically: after embryoid body culture 7 days, aobvious
Red blood cell appearance is seen whether under micro mirror, the glass pipette machine cuts that red blood cell group draws and attenuates simultaneously carry out unicellular passage training
It supports.
The step of culture and passage and detection of the candidate stem cell specifically: by making for above-mentioned success inductive formation
Hemocytoblast digestion terminates digestion reaction, carries out secondary culture and carries out flow cytomery positive mark's object CD34 and reach
To 99% or more, this stage culture medium is that 85%DMEM culture medium is basic culture medium, adds 15% fetal calf serum or blood serum substituting
Product, 1mmol/L essential amino acid, 50IU/mL penicillin, 10 μ g/mL streptomysins.
The step of preparation of the stem cell medicine specifically: according to the preparation method of injection that above-mentioned Hematopoietic Stem is thin
Born of the same parents add bovine serum albumin(BSA) BSA or human serum albumins HAS and physiological saline or are attached to candidate stem cell and microcarrier
Solution, constant volume, which is formulated, according to a certain percentage is made pallium cell injection agent i.e. stem cell medicine.
The present invention has the advantages that
(1) method that embryonic stem cell forms embryoid body and then directed differentiation generates candidate stem cell is carried out in the present invention
Innovation and success rate with higher, induction success rate under this condition can reach 90% or more.
(2) embryoid body formation stages are induced with embryonic stem cell in the present invention, used inducible factor and dosage and
Induction time is pioneering.Inducible factor is mitomycin, vitamin C, L-Glutamine and beta -mercaptoethanol (β-ME), additive
Amount is mitomycin 0.1mM, 1 × 104mol/L of vitamin C, L-Glutamine 2mM and β-ME0.1mM respectively, and the addition time is
The 5th ~ 7 day of embryoid body culture.
(3) candidate stem cell of institute's inductive formation has the existence of lymphoid leukemia cell strain Jurkat in the present invention
Significant extinction effect.
(4) present invention selection embryonic stem cell, it is from a wealth of sources due to the state basic policy of state plan fertility；Preparation system
It is mature easily controllable, and inductive condition and inducible factor are clear, have stronger reproducible rate.
(5) it is injection that the present invention, which is formed by stem cell medicine, and ingredient is clear, detects through strict standard, can be used for
Clinic, securely and reliably.
(6) present invention candidate stem cell generated has very big advantage in clinical application.It had both had height certainly
My updating ability, and there is the ability for further breaking up each system progenitor cells.Due to non-immunogenicity, no immunological rejection,
Graft versus host disease(GVH disease) will not occur；Myelosuppression can be quickly repaired, blood picture is made to restore normal；Anti-radiation respond
By force；Promote immune and hematopoiesis function；High by et al. Ke rate, affected area is gone back to the nest rapidly；Intravenous injection can be easy to be connect by patient
By.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with preferable case study on implementation of the invention and cooperate attached drawing be described in detail such as
Afterwards.A specific embodiment of the invention is shown in detail by following embodiment and its attached drawing.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is a kind of preparation flow figure of stem cell medicine for treating acute lymphocytic leukemia；
Fig. 2 be hUC-HSCs and Jurkat leukaemia cell morphological feature inverted microscope (original amplification: ×
100)；Wherein Fig. 2A: hUC-HSCs initial stage of growth, the secondary culture of Fig. 2 B:hUC-HSCs, Fig. 2 C:Jurkat cell with
HUC-HSC cell symbiosis culture；
Fig. 3 is the cell cycle distribution figure for influencing hUC-HSCs cell and Jurkat leukaemia cell；Wherein Fig. 3 A: have
Or there is no hUC-HSCs and Jurkat cell culture, cell cycle progression is analyzed, Fig. 3 B: histogram represents 3 as the result is shown
The circulation figure and cell of a independent experiment are illustrated in the percentage of each growth phase, and 3 cell cycles independently measure:
Cell HSCs is individually cultivated, and cell Jurkat is individually cultivated, cell Jurkat and cell HSCs symbiosis culture；
Fig. 4 is the influence for adding various dose fetal hematopoietic stem cell to rat blood index, in which: same letter in figure
Perhaps not marking indicates that the not significant different letters of group difference indicate that group difference is significant or extremely significant；
Fig. 5 is the influence for adding various dose fetal hematopoietic stem cell to rat smear index, in which: same letter in figure
Perhaps not marking indicates that the not significant different letters of group difference indicate that group difference is significant or extremely significant.
Specific embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, next the present invention will be described in detail, but the present invention is not only restricted to following implementations
Case.
Specific embodiment 1: the preparation experiment of stem cell medicine
A kind of preparation method of stem cell medicine that treating acute lymphocytic leukemia, mainly comprises the steps that people's embryo
The secondary culture of tire stem cell, the formation of candidate stem cell embryoid body, human embryo stem cell break up to candidate stem cell, Hematopoietic Stem
The culture and passage and detection of cell, the preparation of stem cell medicine, flow chart are as shown in Figure 1.
(1) secondary culture of human embryo stem cell
Routine culture embryonic stem cell is passed on and is expanded, this stage culture medium are as follows: train based on 85%DMEM culture medium
Base is supported, 15% Knockout Serum Replacement (SR, Gibco/BRL), 1mmol/L essential amino acid are added
(Gibco/BRL), 50IU/mL penicillin, 10 μ g/mL streptomysins and 4ng/mL basic fibroblast growth factor (bFGF,
Invitrogen company).Cell culture is hatched under 37 DEG C, 5% carbon dioxide conditions, and passage is primary weekly.
(2) formation of candidate stem cell embryoid body
Differentiation for from inducing human embryo stem cell to hematopoietic cell induces embryonic stem cell in 6 ~ 7 days in culture.
3 ~ 5min piping and druming is digested at 37 DEG C with 1mg/mL type Ⅳ collagenase (Gibco/BRL) at small cell cluster, then using ultralow
Stick 6 well culture plates (Corning Inc) to suspend 7 days, change the liquid once within every 2 days.The culture of induction broth and culture stem cell
It is 80%DMEM, 20% fetal calf serum or serum replacement (serum replacement, SR) that liquid, which is containing culture medium, and 1mmol/L must
It needs amino acid (Gibco/BRL), 50IU/mL penicillin, bFGF is not added in 10 μ g/mL streptomysins.4 kinds of preparations are in embryoid body
(embroidbody, EB) formation stages make an addition in culture medium containing fetal calf serum, and the concentration of addition and time are shown in Table 1.
Inducer concentrations used in the 1 embryoid body stage of table and addition time
The chemical agent concentration time (EB incubation time)
Mitomycin 0.1mM the 5th ~ 7 day
Vitamin C 1 × 104Mol/L the 5th ~ 7 day
L-Glutamine 2mM the 5th ~ 7 day
β-ME 0.1mM the 5th ~ 7 day
(3) human embryo stem cell breaks up to candidate stem cell
After 7 days, the EBs of culture moves to the coated 96 hole tissue culturing plate of 0.1% gelatin, and every hole is inoculated with 1 ~ 2 EBs.Respectively
Kind condition is 3 repetitions, i.e. 3 plates are parallel.Culture solution be containing culture medium be 85%DMEM and 15% fetal calf serum or blood serum substituting
Product culture solution, wherein addition 50IU/mL penicillin, 10 μ g/mL streptomysins, replacement in two days are primary.After adhere-wall culture, exist daily
Whether red blood cell appearance is had in optical microphotograph microscopic observation culture dish, and red blood cell occurs being considered as the hematopoietic cell successfully broken up
Function mark, some red blood cells group drawing-down glass pipette machine cuts simultaneously carry out unicellular secondary culture.
(4) culture and passage and detection of candidate stem cell
By the candidate stem cell digestion of above-mentioned success inductive formation, digestion reaction is terminated, secondary culture is carried out and is flowed
Formula cell instrument detection positive mark's object CD34 simultaneously reaches 99% or more, this stage culture medium is to cultivate based on 85%DMEM culture medium
Base adds 15% fetal calf serum or serum replacement, wherein addition 50IU/mL penicillin, 10 μ g/mL streptomysins.Take growth good
Good third generation cell is detected: third generation cell is collected, 1500rpm is centrifuged 10min, and supernatant is abandoned, PBS solution is added to be resuspended,
Draw 100 μ L cell suspensions (about 3 × 105A cell) it is added in EP pipe, it is separately added into detection antibody according to quantity；It is protected from light incubation
30min, every sample cell are added 2mlPBS and wash once, and 1500rpm is centrifuged 5min；Supernatant is abandoned, every sample cell adds 300 μ LPBS solution
It is resuspended, carries out flow cytometer detection.
The result shows that the cell of culture is through flow cytomery, negative control: 0.07%；Positive expression: CD34 is
99.47%, CD90 96.32%；Feminine gender expression: CD38 1.81%, CD44 0.01%, heretofore described cell mass height expression
Refer to positive rate >=98%, does not express or low expression refers to positive rate < 2%.
(5) preparation of stem cell medicine
The successful hematopoietic cell 1 × 10 of culture is taken respectively6A cell, 3 × 106A cell, 5 × 106A cell, with
0.05ml bovine serum albumin(BSA) BSA or human serum albumins HAS and 0.95ml normal saline are at fetal hematopoietic stem cell system
Agent 1ml.Patient for acute lymphocytic leukemia carries out peripheral vein infusion, can reach impaired blood rapidly after external transplanting
Liquid tissue has remarkable efficacy to this leukemoid treatment.
Specific embodiment 2:CEM cell line (human lymphocyte Leukemia Cell Lines) Leukemic Rat modeling experiment
Guidance model: male SD rat cem cell system guidance model
Animal pattern pretreatment: take week old in 5-6 weeks or so SD male rat in the laminar-flow rack mouse basket of relative clean
Raising.Mouse, which gives up air, to raise acid water to mouse after the filtering of middle effect (Ph is about 3-4).Give plyability vitamin B mark
Quasi- feed, padding etc. all contact article all passes through sterilization treatment with mouse.
Human leukemia cell line's culture: cem cell system (human lymphocyte Leukemia Cell Lines) and J6-1 cell line is taken to add
Enter RPMI1640 culture medium (newborn bovine serum containing 15% inactivation, penicillin 100U, streptomysin 100ug/ml, glutamine
30ug/ml) and it is put into 5% carbon dioxide incubator and cultivates.
The preparation of animal model: SD rat is irradiated through Ce 137,300CGy or without any processing, will transplant cem cell,
J6-1 cell line is inoculated into abdominal cavity in its logarithmic growth phase.Every is inoculated with 2,000,000 to 1,005,000,000.Inoculation front and back
Respectively at the 7th, 14,21 and 28 day to mouse peripheral blood white blood cell count(WBC), leucocyte >=3 × 109/L is calculated, neutrophil leucocyte >
1.5 × 109/L or more illustrates male SD rat cem cell system guidance model modeling success.
Specific embodiment 3: the candidate stem cell venoclysis experiment in Human embryo source
Dosage: cell input group is divided into 3 dosage groups, and every group of five experimental mouses give 0.1 × 10 respectively6A cell/
Ml/ (A group), 0.3 × 106A cell/ml/ times (B group), 0.5 × 106The cell dosage of a cell/ml/ times (C group), is 1
Secondary infusion.In addition one group of successful control group of modeling is set, control group also five experimental mouses.
Approach: tail vein
Therapic opportunity: begin to use invention formulation within modeling water 1 week to stop reference after the success of modeling in 4 weeks.
Effect assessment: (1) state of mind, capacity: the cell infusion group of model group rats and various dose.
(2) physiochemical indice: after cell transplantation at the 28th day transplantation group, model control group and cell infusion group rat whole
Extremely, right ventricle blood sampling 5-10ml detects physiochemical indice with automatic clinical chemistry analyzer.The result shows that fetal hematopoietic stem cell moves
It is implanted in after treatment 4 weeks substantially close to normal, wherein C group dosage i.e. 0.5 × 106The cell dosage of a cell/ml/ times (C group) is imitated
Fruit is best, and model control group physiochemical indice fails to restore.Specific targets are in detail as shown in Figure 4.
(3) morphological examination/cytochemistry inspection: bone marrow smear analyzes 200 cells using conventional Wright's staining, and
It does peroxidase, non-specific vinegar enzyme dyeing and sodium fluoride and inhibits the cytochemistries inspections such as test and staining for glycogen.Blood film
Using conventional Wright's staining, 100 cells are analyzed.As the result is shown: fetal hematopoietic stem cell be implanted in treatment 4 weeks after substantially close to
Normally, wherein C group dosage i.e. 0.5 × 106The cell dosage effect of a cell/ml/ times (C group) is best, model control group blood
Index fails to restore, and wherein for model group Lymphatic System (%) still 50% or more, former children's leaching (%) is still white for lymphocyte greater than 3%
Blood diseased state.Specific targets are in detail as shown in Figure 5.
(4) statistical analysis: all dosage informations are indicated with mean value ± standard error (means ± SEM), carry out ANOVE points
Analysis, attribute data are analyzed with the accurate probability inspection of Fishers.
The tail vein infused rats blood mould of the above-mentioned embryonic stem cell preparation using Human embryo source of the present invention
Type it is demonstrated experimentally that by the rat of the hematopoietic stem cell transplantation in Human embryo source to Leukemia Model, it is big model to be significantly improved
The survival ability of mouse, the recovery for improving impaired blood tissues microenvironment, promoting blood regeneration and function.It can block and reverse white
The process of blood disease, improves general body state and blood status promotes the recovery of leukaemia.
Specific embodiment 4: the candidate stem cell implantation blood evidence experiment in Human embryo source
The candidate stem cell in Human embryo source is implanted into blood evidence: 12 rats taken, are divided into 4 groups, every group 3, and 4 groups of rats
2 days, 4 days, 7 days and 14 days cellular localizations after fluorescent tracing is transplanted respectively, the cell for tracer is through carrying red fluorescence
(RFP) with 0.3 × 10 after slow-virus infection6/ ml/ dose infusion gives Leukemic Rat model.After the transfer the 2nd,
4,7,14 days, special red fluorescent label is seen in blood, it was demonstrated that the candidate stem cell in Human embryo source is
In implantation experiment animal blood.
Specific embodiment 5: the candidate stem cell and lymphoid leukemia cell strain Jurkat in-vitro multiplication in Human embryo source
With the experiment of survival ability
The fetal hematopoietic stem cell successfully turned out by embodiment 1 is thin with lymphoid leukemia as stroma cell
Born of the same parents' strain Jurkat cell carries out directly contact and co-cultures.By the Jurkat cell in immunomagnetic beads co-culture system,
Flow cytometry Jurkat cell proliferative capacity, the variation of cell cycle and spontaneous apoptosis rate；Western blotting
Method detection co-culture after in Jurkat cell Notch signal path activation situation.As a result after co-culturing 4 d, the white blood of Jurkat
The proliferation slowed down of sick cell, cell cycle were blocked in the G0/G1 phase, and its spontaneous apoptosis rate is then remarkably decreased.Conclusion embryo
The microenvironment that tire candidate stem cell is constituted has extinction effect to Jurkat leukaemia cell.Concrete outcome is as shown in Figures 2 and 3.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (1)
1. a kind of method that human embryo stem cell breaks up to candidate stem cell mainly comprises the steps that the biography of embryonic stem cell
It is commissioned to train feeding, the formation of hematopoietic cell embryoid body, human embryo stem cell breaks up to candidate stem cell, the culture and biography of candidate stem cell
Generation and detection；Wherein,
(1) the step of secondary culture of the embryonic stem cell described in specifically: routine culture embryonic stem cell is passed on and expanded
Increase, this stage culture medium are as follows: DMEM culture medium is basic culture medium, adds 15% fetal calf serum or serum replacement, 1mmol/L
Essential amino acid, 50IU/mL penicillin, 10 μ g/mL streptomysins and 4ng/mL basic fibroblast growth factor；
(2) formation of the hematopoietic cell embryoid body described in: embryonic stem cell is induced at secondary culture 6-7 days, uses 1mg/
ML type Ⅳ collagenase is blown and beaten into small cell cluster, is then sticked 6 well culture plate culture 7 days using ultralow, is changed the liquid once within every 2 days,
Embryoid body formation stages 80%DMEM culture medium is basic culture medium, adds 20% fetal calf serum or serum replacement, 1mmol/L must
Need amino acid, 50IU/mL penicillin, 10 μ g/mL streptomysins, at the 5-7 days of embryoid body EBs formation stages, in the medium
4 kinds of quantitative preparations are added, are mitomycin, concentration 0.1mM respectively；Vitamin C, concentration are 1 × 104mol/L；L- paddy ammonia
Amide, concentration 2mM；Beta -mercaptoethanol, concentration 0.1mM；
(3) the step of human embryo stem cell described in breaks up to candidate stem cell specifically: after embryoid body culture 7 days, micro-
Whether there is red blood cell appearance under the microscope, the glass pipette machine cuts that red blood cell group draws and attenuates simultaneously carry out unicellular passage training
It supports；(4) the step of culture and passage and detection of the candidate stem cell described in specifically: by making for above-mentioned success inductive formation
Hemocytoblast digestion terminates digestion reaction, carries out secondary culture, and carry out flow cytomery positive mark's object CD34 and reach
99% or more, this stage culture medium is that DMEM culture medium is basic culture medium, adds 15% fetal calf serum or serum replacement,
1mmol/L essential amino acid, 50IU/mL penicillin, 10ug/mL streptomysin；Well-grown third generation cell is taken to be examined
It surveys: collecting third generation cell, 1500rpm is centrifuged 10min, abandons supernatant, PBS solution is added to be resuspended, and draws 100 μ L cell suspensions and is added
In EP pipe, it is separately added into detection antibody according to quantity；Be protected from light and be incubated for 30min, every sample cell is added 2mlPBS and washes once, 1500rpm from
Heart 5min；Supernatant is abandoned, every sample cell adds 300 μ LPBS solution to be resuspended, and carries out flow cytometer detection.
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