CN108588024A - Induced multi-potent stem cell is divided into the culture medium and method of candidate stem cell - Google Patents
Induced multi-potent stem cell is divided into the culture medium and method of candidate stem cell Download PDFInfo
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Abstract
The present invention relates to a kind of induced multi-potent stem cells to be divided into candidate stem cell culture medium and method, and the culture medium includes first stage culture medium and second stage culture medium；The method includes being co-cultured in two stages to multipotential stem cell and OP9 cells using first stage culture medium, second stage culture successively.Compared with prior art, the present invention has following advantageous effect：The present invention is divided into the culture medium of candidate stem cell by using the induced multi-potent stem cell of above two special component and concentration, induces multipotential stem cell break up successively, improve the efficiency that multipotential stem cell breaks up to candidate stem cell.
Description
Technical field
The invention belongs to biotechnology, it is related to the culture medium that a kind of induced multi-potent stem cell is divided into candidate stem cell
And method.
Background technology
In recent years, candidate stem cell (hematopoietic stem cell, HSC) is applied to clinic more and more
On treat Malignancy, solid tumor, marrow failure disease and some congenital disorders etc., while in paediatrics
Blood disease, hereditary disease, tumour treatment in also show certain application development prospect.With including bleeding of the umbilicus and homologous donor
Stem cell transplantation inside substitutes the development in source and the utilization of new opsonic therapy, greatlys improve and improve being cured for patient
Afterwards.From bleeding of the umbilicus candidate stem cell because of rich content and amplification ability is strong, immunogenicity is low, acquisition is convenient and to donor without
The advantages such as evil have broad prospect of application in the clinical treatment of a variety of diseases in the blood system.But at present only by donor without
The supply for repaying the bleeding of the umbilicus that the mode of donating blood is provided much can not meet clinical wilderness demand, difficult because single cord blood is small
To meet the treatment needs of adult patients, the clinical application of umbilical cord blood hematopoietic stem cell is seriously constrained.In addition blood used in clinic is deposited
Prolong in the pollution risk of the pathogen such as HIV, HCV, and by graft failure, graft versus host disease(GVH disease) (GVHD) and hematopoiesis structure
Late etc. unfavorable factors influence, cause 50% or more patient's lifelong disability or can not cure.So using candidate stem cell
Treatment, is also faced with a severe problem of comparison：How there is the candidate stem cell of sufficient amount for transplanting
Induced multi-potent stem cell is to obtain one of the important sources of candidate stem cell.Induced multi-potent stem cell (iPS) is one
Class reprograms the multipotential stem cell that technology artificial induction obtains by cells such as gene transfections, first by Japanese Scientists
Yamanaka in 2006 with the method for retroviral transduction, by the transcription of Sox2, Oct3/4, c-Myc and Klf4 tetra- because
Son is transferred in mouse embryo fibroblast (Mouse Embryonic Fibrobalst) cell, and the reprogramming to induce is one
Class has the stem cell similar to embryonic stem cell (ES) differentiation versatility potentiality.Utilize mankind's inductive pluripotent stem cells
It (hiPSCs) can for Induction of committed differentiation at a variety of cells from each germinal layer, candidate stem cell be wherein one in vitro
Kind.A large amount of candidate stem cell is gone out by using hiPSCs directed differentiations and provides safe and effective cell origin for clinic, is become
Treat one of promising new method of associated hematologic disorder.
Embryonic stem cell is also the multipotential stem cell at candidate stem cell for Differentiation Induction in vitro.Such as leads to and do embryo
Cell (ES) is trained embryoid body (embryoid body, EB), then to embryoid body directional induction, to obtain candidate stem cell.
The method of inducing differentiation of candidate stem cell further includes cultivating the above multipotential stem cell, removes feeder cells or divides
After changing inhibiting factor, multipotential stem cell is co-cultured from different stroma cells, such as 0P9 cells, direct Induction of committed differentiation
For hematopoietic cell.
Although the method that Differentiation Induction in vitro obtains candidate stem cell is various, conventional method induction differentiation Hematopoietic Stem is thin
The efficiency of born of the same parents is generally relatively low.
Invention content
Based on this, it is necessary to provide a kind of culture medium and method that can improve Hematopoiesis in Vitro stem cell differentiation efficiency.
A kind of induced multi-potent stem cell is divided into the culture medium of candidate stem cell, including first stage culture medium and second-order
Section culture medium；
The first stage culture medium is fetal calf serum containing 5-15%, the a-MEM of 90-110 μm of ol/L thioglycerol is trained
It supports in base added with the SCF of final concentration of 35-45ng/mL, the BMP4 of final concentration of 15-25ng/mL, final concentration of 15-25ng/
The Flt3 of the IL-3 of mL, the VEGF of final concentration of 15-25ng/mL, final concentration of 15-25ng/mL；
The second stage culture medium is fetal calf serum containing 5-15%, the a-MEM of 90-110 μm of ol/L thioglycerol is trained
It supports in base added with the SCF of final concentration of 35-45ng/mL, the BMP4 of final concentration of 15-25ng/mL, final concentration of 15-25ng/
The IL-3 of mL, the VEGF of final concentration of 15-25ng/mL, the Flt3 of final concentration of 15-25ng/mL and final concentration of 80-
The UM171 of 120ng/mL.
In wherein some embodiments, in the first stage culture medium, described SCF, BMP4, IL-3, VEGF, Flt3's
Final concentration ratio is (1.5-2.5): (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.5-1.5)；The second stage culture
In base, the final concentration ratio of described SCF, BMP4, IL-3, VEGF, Flt3, UM171 are (1.5-2.5): (0.5-1.5): (0.5-
1.5)∶(0.5-1.5)∶(0.5-1.5)∶(4.5-5.5)。
In wherein some embodiments, in the first stage culture medium, described SCF, BMP4, IL-3, VEGF, Flt3's
Final concentration ratio is 2: 1: 1: 1: 1；In the second stage culture medium, described SCF, BMP4, IL-3, VEGF, Flt3, UM171's
Final concentration ratio is 2: 1: 1: 1: 1: 5.
A kind of method that induced multi-potent stem cell is divided into candidate stem cell, including：
Multipotential stem cell and OP9 cells are first placed in above-mentioned first stage culture medium and carry out the induction point of first stage
Change, then is placed in above-mentioned second stage culture medium the induction differentiation for carrying out second stage.
In wherein some embodiments, multipotential stem cell and OP9 cells co-inoculation are denoted as in first stage culture medium
0th day, the induction of first stage differentiation terminated since s the 0th day to the 3rd day, and the induction of the second stage is broken up from the
Beginning in 4 days terminated to the 14th day.
In wherein some embodiments, the 1st day full dose of the induction differentiation of the first stage replaces fresh culture, from
The induction of the first stage, which has been broken up the 4th day, to be started, and half amount is changed to fresh culture medium every other day.
In wherein some embodiments, the OP9 cells are trained in the α-MEM culture mediums of the fetal calf serum containing 15-25%
Support what 4-5d was obtained.
In wherein some embodiments, the quantity ratio of the OP9 cells and multipotential stem cell is (12-18): 1.
In wherein some embodiments, the multipotential stem cell is induced multi-potent stem cell, embryonic stem cell, or induction
The mixture of multipotential stem cell and embryonic stem cell.
Compared with prior art, the present invention has following advantageous effect：
The present invention is divided into candidate stem cell by using the induced multi-potent stem cell of above two special component and concentration
Culture medium, successively to multipotential stem cell induce break up, improve the efficiency that multipotential stem cell breaks up to candidate stem cell.
Further, the present invention is also dense by the end to SCF, BMP4, IL-3, VEGF, Flt3 in first stage culture medium
Degree ratio and second stage culture medium in SCF, BMP4, IL-3, VEGF, Flt3, UM171 final concentration than optimization, surprisingly keep away
The generation for having exempted from contamination phenomenon during serum-containing media use solves cell cultivation process and had not only needed serum component but also difficult
To avoid the contradiction of pollution.
Description of the drawings
Fig. 1 is the result figure of the CD34+ positive rates of flow cytometer detection embodiment 1 and its contrast groups；
Fig. 2 is the result figure of the CD34+ positive rates of flow cytometer detection embodiment 2 and its contrast groups；
Fig. 3 is the result figure of the CD34+ positive rates of flow cytometer detection feminine gender group；
Fig. 4 is the positive rate block diagram of embodiment 1 and its contrast groups, embodiment 2 and its contrast groups.
Specific implementation mode
Candidate stem cell culture medium and side are divided into the induced multi-potent stem cell of the present invention below in conjunction with specific embodiment
Method is described in further detail.The cell that the present embodiment uses can carry out acquisition purchased in market.
Embodiment 1
The present embodiment provides culture mediums and method that induced multi-potent stem cell (iPS) is divided into candidate stem cell.
The culture medium that the present embodiment uses includes first stage culture medium, second stage culture medium：
In first stage culture medium, the final concentration ratio of SCF, BMP4, IL-3, VEGF, Flt3 are 2: 1: 1: 1: 1, are specifically matched
Fang Wei：The fetal calf serum of final concentration of 10% (v/v), the thioglycerol of final concentration of 100 μm of ol/L are added in a-MEM culture mediums
(MTG), stem cell factor (stem cell factor, SCF), the final concentration of 20ng/ of final concentration of 40ng/mL are added and
The bone morphogenetic protein 4 (BMP4) of mL, interleukin 3 (IL-3), the final concentration of 20ng/mL of final concentration of 20ng/mL
VEGF, final concentration of 20ng/mL Flt3.
The final concentration ratio of SCF, BMP4, IL-3, VEGF, Flt3, UM171 are 2: 1: 1: 1: 1: 5 in second stage culture medium,
Specific formula is：The fetal calf serum of final concentration of 10% (v/v), the sulphur of final concentration of 100 μm of ol/L are added in a-MEM culture mediums
For glycerine (MTG), and stem cell factor (stem cell factor, SCF), the final concentration of final concentration of 40ng/mL is added
For the bone morphogenetic protein 4 (BMP4) of 20ng/mL, the interleukin 3 (IL-3), final concentration of of final concentration of 20ng/mL
The UM171 of the VEGF of 20ng/mL, the Flt3 of final concentration of 20ng/mL, final concentration of 100nm/mL.
The induction iPS methods that are divided into candidate stem cell that the present embodiment uses include：
(1) with the a-MEM cultures containing 20% (v/v) fetal calf serum based on culture OP9 cell (Mouse Bones in 60mm culture dishes
Marrow stroma cell) 4-5 days, then gained OP9 cells are transferred in first stage culture medium, obtain the re-suspension liquid of OP9 cells；
(2) Dispase (dispase) is added after iPS cells being washed twice with DMEM/F12 to digest, waits for cell clone side
Inhaling after crispaturaing, which occurs, in edge abandons Dispase, is washed one time with DMEM/F12 culture mediums, adds the DMEM/F12 culture mediums of 1.5mL, used
Clone is spread to homogeneous bulky by pipette tips, is then moved in centrifuge tube and is waited for its natural subsidence, and supernatant is abandoned, and the 1mL first stage is added
Culture medium obtains the re-suspension liquid of iPS cells；
(3) the inoculation same day is denoted as to be inoculated with the re-suspension liquid for the iPS cells that step (2) obtains on the 0th day obtains into step (1)
In the re-suspension liquid of OP9 cells, the cell quantity ratio of the OP9 cells of iPS cells is 1: 15, in 37 DEG C, 5%CO2It is trained in incubator
It supports one day, the 1st day full dose changes liquid by dead cell and the removal of non-attached cell, to cultivate two days (the 2nd day, the 3rd day) later and not have to
Change liquid, start within the 4th day half amount every other day and changes liquid to improve differentiation efficiency, co-culturing the 4th day, be changed to second stage culture medium into
Row culture, candidate stem cell was collected at the 14th day.
In order to compare, the present embodiment is also provided with contrast groups, and contrast groups and the present embodiment differ only in, only with
Culture medium as following formula carries out primary induction differentiation：The tire ox blood of final concentration of 10% (v/v) is added in the addition of a-MEM culture mediums
Clearly, the thioglycerol (MTG) of final concentration of 100 μm of ol/L.
Embodiment 2
The present embodiment is the change case of embodiment 1, and variation place is only that the type of multipotential stem cell selects embryo dry thin
Born of the same parents (ES).
In order to compare, the present embodiment is also provided with contrast groups, and contrast groups and the present embodiment differ only in, only with
Culture medium as following formula carries out primary induction differentiation：The tire ox blood of final concentration of 10% (v/v) is added in the addition of a-MEM culture mediums
Clearly, the thioglycerol (MTG) of final concentration of 100 μm of ol/L.
Embodiment 3
The present embodiment is the change case of embodiment 1, and variation place is only that first stage culture medium and second stage culture
The constituent content of base：
The final concentration ratio of SCF, BMP4, IL-3, VEGF, Flt3 are 2.25: 1: 1: 1: 1 in first stage culture medium, specifically
Group is divided into：Be added in a-MEM culture mediums the fetal calf serum of final concentration of 10% (v/v), final concentration of 100 μm of ol/L it is thio sweet
Oily (MTG), and the stem cell factor (stem cell factor, SCF), final concentration of of final concentration of 45ng/mL is added
It is the bone morphogenetic protein 4 (BMP4) of 20ng/mL, the interleukin 3 (IL-3) of final concentration of 20ng/mL, final concentration of
The Flt3 of the VEGF of 20ng/mL, final concentration of 20ng/mL；
The final concentration ratio of SCF, BMP4, IL-3, VEGF, Flt3 are 2.25: 1: 1: 1: 1: 4.5 in second stage culture medium,
Specific formula is：The fetal calf serum of final concentration of 10% (v/v), the sulphur of final concentration of 100 μm of ol/L are added in a-MEM culture mediums
For glycerine (MTG), and stem cell factor (stem cell factor, SCF), the final concentration of final concentration of 45ng/mL is added
For the bone morphogenetic protein 4 (BMP4) of 20ng/mL, the interleukin 3 (IL-3), final concentration of of final concentration of 20ng/mL
The UM171 of the VEGF of 20ng/mL, the Flt3 of final concentration of 20ng/mL, final concentration of 90ng/mL.
Embodiment 4
The present embodiment is the change case of embodiment 1, and variation place is only that：
(1) constituent content of first stage culture medium and second stage culture medium
First stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 35ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 25ng/mL, final concentration of 15ng/mL leucocyte be situated between
The Flt3 of -3 (IL-3) of element, the VEGF of final concentration of 25ng/mL, final concentration of 15ng/mL.
Second stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 35ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 25ng/mL, final concentration of 15ng/mL leucocyte be situated between
Element -3 (IL-3), the VEGF of final concentration of 25ng/mL, the Flt3 of final concentration of 15ng/mL, final concentration of 80ng/mL
UM171。
(2) the cell quantity ratio of the OP9 cells of iPS cells is 1: 12.
Embodiment 5
The present embodiment is the change case of embodiment 1, and variation place is only that：
(1) constituent content of first stage culture medium and second stage culture medium
First stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 45ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 25ng/mL, final concentration of 25ng/mL leucocyte be situated between
The Flt3 of -3 (IL-3) of element, the VEGF of final concentration of 25ng/mL, final concentration of 25ng/mL.
Second stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 45ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 15ng/mL, final concentration of 25ng/mL leucocyte be situated between
The VEGF of -3 (IL-3) of element, final concentration of 15ng/mL, the Flt3 of final concentration of 25ng/mL, final concentration of 120ng/mL
UM171。
(2) the cell quantity ratio of the OP9 cells of iPS cells is 1: 18.
The result of embodiment 1-5 is tested
The detection of CD34+ positive rates：Example and its final gained cell of contrast groups, after washing 2 times with DMEM/F12
It first uses the clostridiopetidase A IV of 1mg/mL to digest 20min, then 15-20min is digested with 0.25% pancreatin, then stop digestion；Piping and druming
Cell makes it be in single cell suspension, collects 1000r/min in cell to 15mL centrifuge tubes and centrifuges 5min, is contained with 1mL after abandoning supernatant
The PBS of 2% (v/v) serum is resuspended, primary with 100 μm of steril cell screen filtration, antibody CD34-PE-Cy7 is added, 4 DEG C incubate
It is washed one time with the PBS containing 2% serum after educating 30min.Then with 5 × 106Cell is resuspended in the density of/ml, uses FACSCalibur
Streaming instrument cell instrument detects and analyzes its differentiation efficiency.In detection process, it is negative group with common iPS cells, i.e., does not carry out
Though the cell of hematopoiesis induction differentiation either carries out Analytical Chemical Experiment still and the cell of unused streaming antibody dyeing.
The statistics of pollution condition：Each embodiment and its contrast groups, comparative example 1 set 50 repetitions respectively, in incubation
The repeat number being contaminated is recorded, pollution rate is counted.
Acetonideexample 1 and its contrast groups test result are as Figure 1 and Figure 4, and flow cytometry finds embodiment 1
CD34+ positive rates rise to 32.07% (its contrast groups is 4.96%)；The test result of embodiment 2 and its contrast groups such as Fig. 2,
Shown in Fig. 4, CD34+ positive rates rise to 30.14% (its contrast groups is 6.36%), and differentiation efficiency obviously rises.Feminine gender comparison
Group is shown in Fig. 3.
For generally, the test result of the statistics of each example is shown in Table 1：
Comparative example 1
This comparative example is the comparative example of embodiment 1, and comparison place is only that first stage culture medium and second stage culture
The formula of base：
First stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 30ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 10ng/mL, final concentration of 30ng/mL leucocyte be situated between
The Flt3 of -3 (IL-3) of element, the VEGF of final concentration of 10ng/mL, final concentration of 30ng/mL.
Second stage culture medium：The fetal calf serum, final concentration of of final concentration of 10% (v/v) is added in a-MEM culture mediums
The thioglycerol (MTG) of 100 μm of ol/L, and stem cell factor (the stem cell of final concentration of 30ng/mL are added
Factor, SCF), the bone morphogenetic protein 4 (BMP4) of final concentration of 10ng/mL, final concentration of 30ng/mL leucocyte be situated between
Element -3 (IL-3), the VEGF of final concentration of 10ng/mL, the Flt3 of final concentration of 30ng/mL, final concentration of 130ng/mL
UM171。
As a result：The CD34+ positive rates that this comparative example measures are 21.62%.
Comparative example 2
This comparative example is the comparative example of embodiment 1, and comparison place is only that：Only with second stage medium culture 15
It.
As a result：The CD34+ positive rates that this comparative example measures are 15.54%.
Comparative example 3
This comparative example is the comparative example of embodiment 1, and comparison place is only that：Only with first stage medium culture D14
It.
As a result：The CD34+ positive rates that this comparative example measures are 26.42%.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
1. a kind of induced multi-potent stem cell is divided into the culture medium of candidate stem cell, which is characterized in that cultivated including the first stage
Base and second stage culture medium；
The first stage culture medium is the a-MEM culture mediums of fetal calf serum containing 5-15%, 90-110 μm of ol/L thioglycerol
In added with the SCF of final concentration of 35-45ng/mL, the BMP4 of final concentration of 15-25ng/mL, final concentration of 15-25ng/mL
The Flt3 of IL-3, the VEGF of final concentration of 15-25ng/mL, final concentration of 15-25ng/mL；
The second stage culture medium is the a-MEM culture mediums of fetal calf serum containing 5-15%, 90-110 μm of ol/L thioglycerol
In added with the SCF of final concentration of 35-45ng/mL, the BMP4 of final concentration of 15-25ng/mL, final concentration of 15-25ng/mL
IL-3, the VEGF of final concentration of 15-25ng/mL, the Flt3 of final concentration of 15-25ng/mL and final concentration of 80-120ng/
The UM171 of mL.
2. induced multi-potent stem cell according to claim 1 is divided into the culture medium of candidate stem cell, which is characterized in that institute
It states in first stage culture medium, the final concentration ratio of described SCF, BMP4, IL-3, VEGF, Flt3 are (1.5-2.5): (0.5-1.5)
∶(0.5-1.5)∶(0.5-1.5)∶(0.5-1.5)；In the second stage culture medium, the SCF, BMP4, IL-3, VEGF,
The final concentration ratio of Flt3, UM171 are (1.5-2.5): (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.5-1.5): (4.5-
5.5)。
3. induced multi-potent stem cell according to claim 1 or 2 is divided into the culture medium of candidate stem cell, feature exists
In in the first stage culture medium, the final concentration ratio of described SCF, BMP4, IL-3, VEGF, Flt3 are 2: 1: 1: 1: 1；It is described
In second stage culture medium, the final concentration ratio of described SCF, BMP4, IL-3, VEGF, Flt3, UM171 are 2: 1: 1: 1: 1: 5.
4. a kind of method that induced multi-potent stem cell is divided into candidate stem cell, which is characterized in that including：
Multipotential stem cell and OP9 cells are first placed in claims 1 to 3 any one of them first stage culture medium and carry out
The induction in one stage is broken up, then is placed in claims 1 to 3 any one of them second stage culture medium and carries out second stage
Induction differentiation.
5. the method that induced multi-potent stem cell according to claim 4 is divided into candidate stem cell, which is characterized in that will be more
Can stem cell and OP9 cells co-inoculation be denoted as the 0th day in first stage culture medium, the induction differentiation of the first stage is from the
Beginning in 0 day terminated to the 3rd day, and the induction differentiation of the second stage terminated since the 4th day to the 14th day.
6. the method that induced multi-potent stem cell according to claim 4 or 5 is divided into candidate stem cell, which is characterized in that
1st day full dose of the induction differentiation of the first stage replaces fresh culture, the 4th from the induction of first stage differentiation
It starts, and half amount is changed to fresh culture medium every other day.
7. the method that induced multi-potent stem cell according to claim 4 or 5 is divided into candidate stem cell, which is characterized in that
The OP9 cells are to cultivate 4-5d in the α-MEM culture mediums of the fetal calf serum containing 15-25% to obtain.
8. the method that induced multi-potent stem cell according to claim 4 or 5 is divided into candidate stem cell, which is characterized in that
The quantity ratio of the OP9 cells and multipotential stem cell is (12-18): 1.
9. the method that induced multi-potent stem cell according to claim 4 or 5 is divided into candidate stem cell, which is characterized in that
The multipotential stem cell is the mixed of induced multi-potent stem cell, embryonic stem cell or induced multi-potent stem cell and embryonic stem cell
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