CN105331583B - A method of promote people to induce multi-potent stem cell and breaks up to Neuronal induction - Google Patents
A method of promote people to induce multi-potent stem cell and breaks up to Neuronal induction Download PDFInfo
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Abstract
The invention discloses a kind of promotion people to induce multi-potent stem cell the method broken up to Neuronal induction, method includes the following steps: pressure-vaccum after iPS cell dissociation is dispersed into individual cells suspension, for point kind in culture hole, ROCK inhibitor is added in every hole；It first day after dividing kind, inhales and abandons supernatant, nerve-inducing complete medium is added, cultivate the neural stem cell up to obtaining P1 generation；The neural stem cell in P1 generation is inoculated into Neuronal induction culture medium and is cultivated, rapamycin is added in culture medium to promote the generation of autophagy；After 2 days, culture medium is changed to the rapamycin of addition low concentration to promote the Neural differentiation culture medium of autophagy, culture can induce to 12-15 days and be divided into neuronal cell.The method of the present invention can accelerate being induced to differentiate to form for neuron, and improve the neure growth state of three type iPS of spinocerebellar ataxia.
Description
Technical field
The present invention relates to a kind of promotion people to induce multi-potent stem cell the method broken up to Neuronal induction, and this method passes through increasing
Strong autophagy improves induction differentiation efficiency.
Background technique
Neuron is a kind of non-renewable cell, therefore in the disease of some injured neurons of the mankind, can generate one
A little irreversible consequences.With the extension of human longevity, the disease of the neurons such as Alzheimer disease, cerebrovascular accident involvement increases
Add, and people induce multi-potent stem cell can induce be divided into functional neuron, the technology be treatment this kind diseases provide
Possibility.But from induce multi-potent stem cell or induced differentiation of embryonic stem cells be neuron need many and diverse process, such as
The method for being related to suspension culture or foreign cell co-cultivation.These methods have the shortcomings that cumbersome and time-consuming.And
The problems such as Drury-Stewart etc. uses some small molecules, has well solved suspension and foreign cell co-cultivation.Life company
Also relevant reagent kit product solves the problems, such as this.But from induce multi-potent stem cell or embryonic stem cell induction for mind
Several weeks are needed through member, the problem of time-consuming is not well solved yet.Therefore a kind of simple and efficient method progress mind is needed
Through breaking up.
Autophagy (autophagy) be a phagocytosis own cells matter albumen or organelle and make its coating enter vesica, and
Autophagy lysosome is formed with lysosome fusion, the process for the content that it is wrapped up of degrading realizes the metabolism of cell itself whereby
Need the update with certain organelles.
Spinocebellar ataxia (Spinocerebellar Ataxia, SCA) is that one group of genetic heterogeneity is very big
Nervous system degeneration disease.ATXN3 gene encodes ATAXIN-3 albumen, when gene C AG copy number dynamic mutation, coding
Protein carboxyl terminal poly glumine (polyglutamine, PolyQ) chain abnormal aggregation of extension and protein extremely, draws
Cell dysfunction is played, is caused a disease so as to cause neurotoxicity.In addition to traditional ubiquitin protein enzyme degeneration system, autophagy is eukaryon
A kind of highly conserved, degradation access for being played a role by lysosome mechanism pathway in cell, with neurodegenerative disease etc.
Disease is closely related.The ATAXIN-3 albumen of aggregation mutation research shows that induction autophagy can degrade, to prevent SCA3/MJD
And the generation of other neurodegenerative diseases.It is the good tool for studying SCA3 morbidity that SCA3, which is induced multi-potent stem cell,.But
To be autophagy induce multi-potent stem cell to be induced to differentiate into neuron in SCA3 plays the role of that it is still necessary to clear.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology with it is insufficient, a kind of promotion people is provided and is induced multi-potent stem cell
The method broken up to Neuronal induction.
The purpose of the invention is achieved by the following technical solution:
A method of promote people to induce multi-potent stem cell and break up to Neuronal induction, comprising the following steps:
(1) people of degrees of fusion 70-80% induces multi-potent stem cell (iPS cell), is added 37 after the cell dissociation buffer of preheating
It DEG C is incubated for 5 minutes, then several times with pipette pressure-vaccum, cell is made to be dispersed into individual cells suspension, then cell point kind exists
In culture hole；Every hole is put into 37 DEG C, 5%CO after ROCK inhibitor is added2Incubator in cultivate；
(2) first day after dividing kind, the supernatant for abandoning culture hole was inhaled to remove not adherent cell, preheating is addedNerve-inducing complete medium is placed in and continues to cultivate in incubator；Hereafter liquid is changed every other day, can be obtained within the 7th day after point kind
The neural stem cell (NSCs) for obtaining P0 generation continues the neural stem cell that culture obtains P1 generation for 3-4 days；
(3) poly ornithine and the coated culture dish of laminin are placed in 37 DEG C, 5%CO2Incubator in cultivate 1h,
Then Neuronal induction culture medium is added in culture dish, then the neural stem cell in the P1 generation after step (2) passage is inoculated into
In culture dish, inoculum density is 2.5 × 104–5×104A cell/cm2；Rapamycin is added in Neuronal induction culture medium
To promote the generation of autophagy；After culture 2 days, culture medium is changed to Neural differentiation culture medium, replacement in every 3-4 days is primary
Culture medium, culture can induce to 12-15 days and be divided into neuronal cell；
People described in step (1) induce multi-potent stem cell be under the conditions of non-trophoblast culture obtain without differentiation or only
Containing the clone broken up on a small quantity；The non-trophoblast condition such as uses Essential 8TMCulture medium with Vitronectin orAs matrix；
It is to utilize episomal using the Skin Cell of human body as material that people of the present invention, which induces multi-potent stem cell,
Reprogramming assay (Invitrogen, USA) reprogramming kit induces；
Cell dissociation buffer described in step (1) is preferredCell dissociation buffer；
Point kind of density in step (1), preferably each culture hole inoculation 2.5 × 105–3.0×105A cell；
The preferred ROCK inhibitor Y27632 of the ROCK inhibitor；
The composition and proportion of Neuronal induction culture medium described in step (3) are as follows:
1 × KnockOutTMDMEM/F-12 culture medium 97mL；
The GlutaMAX of 2mMTM- I additive 1mL；
The 20 μ L of bFGF of 20ng/mL；
The 20 μ L of EGF of 20ng/mL；
2%Neural additive 2mL；
Rapamycin is added into culture medium again to final concentration of 100nmol/L.
The composition and proportion of Neural differentiation culture medium described in step (3) are as follows:
1 ×Culture medium 97mL；
2%Serum-free additive 2mL；
The GlutaMAX of 2mMTM- I additive 1mL；
Rapamycin is added into culture medium again to final concentration of 10nmol/L.
Autophagy is present in the induction atomization of ips, and body cell has more organelle, and iPS organelle is less, because
This by somatic induction be iPS during need autophagy to participate in.Autophagy is a phagocytosis own cells matter albumen or organelle
And so that its coating is entered vesica, and form autophagy lysosome with lysosome fusion, the process for the content that it is wrapped up of degrading, by
This realizes that the metabolism of cell itself needs the update with certain organelles.And iPS is divided into neural stem cell and neuron also has
There are different organelle and eucaryotic cell structure, autophagy is promoting the conversion of organelle to work, therefore can accelerate iPS and be divided into nerve
Stem cell and neuron.
The present invention has the following advantages and effects with respect to the prior art:
1, the method for the present invention can accelerate being induced to differentiate to form for neuron.In normal iPS cell Differentiating Into Neurons mistake
Cheng Zhong, being added to after rapamycin promotes autophagy can make cell the form of neuron occurred at 14 days or so.
2, the method for the present invention improves the neure growth state of three type iPS of spinocerebellar ataxia.Spinocerebellum is total
Ji imbalance three types (SCA3) iPS is divided into the extension after neuron with incubation time, and state can gradually be deteriorated, and it is mould that thunder pa is added
After element, the state of neuron is not than adding the neuron state of rapamycin good.
Detailed description of the invention
Fig. 1 be differentiation culture by the 14th day when culture medium in neuronal cell protrusion aspect graph；Wherein, A- comparative example
(×4)；B- comparative example (× 10)；C- embodiment (× 4)；D- embodiment (× 10).
Fig. 2 is the aspect graph for inducing multi-potent stem cell the neuronal cell protrusion in differentiation incubation of patient SCA3；
Wherein, A- is not added with (× 4) of rapamycin；(× 4) of B- addition rapamycin.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment
A method of promote people to induce multi-potent stem cell and break up to Neuronal induction, comprising the following steps:
(1) people cultivated under the conditions of non-trophoblast induces multi-potent stem cell (iPS), when degrees of fusion is 70-80%,
Preheating is addedIt is incubated for 5 minutes for 37 DEG C after cell dissociation buffer, it is then several with pipette pressure-vaccum
It is secondary, so that cell is dispersed into individual cells suspension, then by cell point kind in culture hole；Dividing kind of density is each culture hole inoculation
2.5×105–3.0×105A cell；When dividing kind, 10 μM of ROCK inhibitor Y27632 can be added, in culture hole to prevent
Cell death；
(2) first day after dividing kind, the supernatant for abandoning culture hole was inhaled to remove not adherent cell, preheating is addedNerve-inducing complete medium is placed in and continues to cultivate in incubator；Hereafter liquid is changed every other day, can be obtained within the 7th day after point kind
The neural stem cell (NSCs) for obtaining P0 generation continues the neural stem cell that culture obtains P1 generation for 3-4 days；
(3) poly ornithine and the coated culture dish of laminin are placed in 37 DEG C, 5%CO2Incubator in cultivate 1h,
Then Neuronal induction culture medium is added in culture dish, then the neural stem cell in the P1 generation after step (2) passage is inoculated into
In culture dish, inoculum density is 2.5 × 104–5×104A cell/cm2；Rapamycin is added in Neuronal induction culture medium
To promote the generation of autophagy；After culture 2 days, culture medium is changed to Neural differentiation culture medium, replacement in every 3-4 days is primary
Culture medium, culture can induce to 12-15 days and be divided into neuronal cell；
The composition and proportion of Neuronal induction culture medium described in step (3) are as follows:
1 × KnockOutTMDMEM/F-12 culture medium 97mL；
The GlutaMAX of 2mMTM- I additive 1mL；
The 20 μ L of bFGF of 20ng/mL；
The 20 μ L of EGF of 20ng/mL；
2%Neural additive 2mL；
Rapamycin is added into culture medium again to final concentration of 100nmol/L.
The composition and proportion of Neural differentiation culture medium described in step (3) are as follows:
1 ×Culture medium 97mL；
2%Serum-free additive 2mL；
The GlutaMAX of 2mMTM- I additive 1mL；
Rapamycin is added into culture medium again to final concentration of 10nmol/L.
Comparative example
A method of promote people to induce multi-potent stem cell and break up to Neuronal induction, in Neuronal induction culture medium and mind
Through not adding rapamycin in first cell differential medium, the same embodiment of other steps and operations.
At the end of experiment, whether there is neuronal cell protrusion using common inverted microscope observation culture medium.As a result
As shown in Figure 1, when differentiation culture was by the 14th day in culture medium neuronal cell protrusion form, be added to the cell of rapamycin
Faster there is neuron morphology.It can be seen that the differentiation of neuron can be accelerated by being added to rapamycin.
Induce multi-potent stem cell (iPS) using the people in SCA3 patient skin source simultaneously and test, using embodiment and
Whether the method for comparative example there is neuronal cell protrusion using common inverted microscope observation culture medium at the end of experiment.
As a result as shown in Fig. 2, patient's SCA3 induces multi-potent stem cell in differentiation incubation, neuronal cell state is preferable.It can
See, the neure growth state of three type iPS of spinocerebellar ataxia is improved after addition rapamycin.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (3)
1. a kind of promote people to induce multi-potent stem cell the method broken up to Neuronal induction, it is characterised in that the following steps are included:
(1) people of degrees of fusion 70-80% induces multi-potent stem cell, and is added after the cell dissociation buffer of preheating and is incubated for 5 minutes for 37 DEG C, connects
With pipette pressure-vaccum several times, so that cell is dispersed into individual cells suspension, then by cell point kind in culture hole；Every hole adds
37 DEG C, 5%CO are put into after entering ROCK inhibitor2Incubator in cultivate；
(2) first day after dividing kind, the supernatant for abandoning culture hole was inhaled to remove not adherent cell, preheating is added
Nerve-inducing complete medium is placed in and continues to cultivate in incubator；Hereafter liquid is changed every other day, the 7th day available P0 generation after point kind
Neural stem cell continues the neural stem cell that culture obtains P1 generation for 3-4 days；
(3) poly ornithine and the coated culture dish of laminin are placed in 37 DEG C, 5%CO2Incubator in cultivate 1h, then
Neuronal induction culture medium is added in culture dish, then the neural stem cell in the P1 generation after step (2) passage is inoculated into culture
In ware, inoculum density is 2.5 × 104–5×104A cell/cm2；Rapamycin is added in Neuronal induction culture medium to promote
Into the generation of autophagy；After culture 2 days, culture medium is changed to Neural differentiation culture medium, the primary culture of replacement in every 3-4 days
Base, culture can induce to 12-15 days and be divided into neuronal cell；
People described in step (1), which induces multi-potent stem cell, refers to that the people in SCA3 patient skin source induces multi-potent stem cell；
People described in step (1) induce multi-potent stem cell be under the conditions of non-trophoblast culture obtain it is few without breaking up or containing only
Measure the clone of differentiation；
ROCK inhibitor described in step (1) is ROCK inhibitor Y27632；
The composition and proportion of Neuronal induction culture medium described in step (3) are as follows:
1 × KnockOutTMDMEM/F-12 culture medium 97mL；
The GlutaMAX of 2mMTM- I additive 1mL；
The 20 μ L of bFGF of 20ng/mL；
The 20 μ L of EGF of 20ng/mL；
2%Neural additive 2mL；
Rapamycin is added into culture medium again to final concentration of 100nmol/L；
The composition and proportion of Neural differentiation culture medium described in step (3) are as follows:
1 ×Culture medium 97mL；
2%Serum-free additive 2mL；
The GlutaMAX of 2mMTM- I additive 1mL；
Rapamycin is added into culture medium again to final concentration of 10nmol/L.
2. according to claim 1 promote people to induce multi-potent stem cell the method broken up to Neuronal induction, feature exists
In: cell dissociation buffer described in step (1) isCell dissociation buffer.
3. according to claim 1 promote people to induce multi-potent stem cell the method broken up to Neuronal induction, feature exists
In: point kind of the density in step (1), for each culture hole inoculation 2.5 × 105–3.0×105A cell.
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| CN111849899B (en) * | 2017-07-28 | 2022-06-14 | 杨涛 | Induction medium for directionally inducing differentiation of hipscs into neural cell system |
| CN109868258B (en) * | 2017-12-27 | 2023-12-05 | 华南师范大学 | Compositions and methods for inducing astrocytes into functional neurons |
| CN109385404B (en) * | 2018-10-19 | 2021-07-06 | 国典(北京)医药科技有限公司 | Method for inducing stem cells to differentiate into neurons, neurons and application |
| CN110699321B (en) * | 2019-11-14 | 2021-04-09 | 信阳师范学院 | Method for in vitro inducing neural stem cells to directionally differentiate into neurons |
| CN112522198A (en) * | 2020-12-17 | 2021-03-19 | 深圳先进技术研究院 | Engineered nerve cell and preparation method and application thereof |
| CN113416709A (en) * | 2021-06-21 | 2021-09-21 | 香港再生医学有限公司 | Method, culture medium and system for promoting iPSC to differentiate into peripheral neuron cells |
| CN113712995A (en) * | 2021-07-20 | 2021-11-30 | 河北医科大学 | Application of neural stem cell and umbilical cord mesenchymal stem cell in spinal cord injury |
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