CN109679918A - A kind of preparation method of convenient and fast people's inductive pluripotent stem cells - Google Patents
A kind of preparation method of convenient and fast people's inductive pluripotent stem cells Download PDFInfo
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- CN109679918A CN109679918A CN201811586070.4A CN201811586070A CN109679918A CN 109679918 A CN109679918 A CN 109679918A CN 201811586070 A CN201811586070 A CN 201811586070A CN 109679918 A CN109679918 A CN 109679918A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention discloses a kind of preparation methods of convenient and fast people's inductive pluripotent stem cells, carrier will be reprogrammed to import in human somatic cell by the method for liposome transfection, human somatic cell after being transfected, human somatic cell after transfection is subjected to Fiber differentiation in human embryonic stem cell medium, it clones and generates to human embryo stem cell sample, picking human embryo stem cell sample clone carries out amplification cultivation, obtains people's inductive pluripotent stem cells after the versatility of identification of cell.The preparation method provided through the invention can obtain people's inductive pluripotent stem cells of no exogenous origin gene integrator, and compared with prior art, and the low and requirement to experimental facilities of the preparation method experimental cost in the present invention is low, simple and easy to operate.
Description
Technical field
The present invention relates to field of biomedicine more particularly to a kind of preparation sides of convenient and fast people's inductive pluripotent stem cells
Method.
Background technique
Multipotential stem cell (pluripotent stem cell) is that one kind has self-renewal capacity and divides to triploblastica
The stem cell of change has broad application prospects in regenerative medicine field.The preparation side of people's inductive pluripotent stem cells (iPSC)
The imagination by somatic conversion for inductive pluripotent stem cells may be implemented in method, comes self so as to obtain abundance, patient
The iPSC in source.IPSC can be applied not only to the regenerative medicine treatment of disease, additionally it is possible to be effectively prevented from immune rejection problems.Mesh
Preceding most common iPSC preparation method includes that retrovirus or slow virus induction, outer satellite electricity turn induction, sendai virus induction.
However, the preparation method of currently used iPSC still has much room for improvement.Wherein induced by reverse transcription or slow virus
The iPSC arrived, due to there is the integration of foreign gene, the safety in utilization of iPSC can be reduced.Pass through outer satellite electricity turn and sendai virus
Obtained iPSC is induced, although safety in utilization with higher, outer satellite electricity turns induction to reality without exogenous origin gene integrator
Equipment requirement height is tested, and sendai virus Induction experiments are at high cost, and common lab can not be prepared, it is necessary to it buys, limits
The research and application of iPSC.
Summary of the invention
Technical problems based on background technology, the invention proposes a kind of convenient and fast people's inductive pluripotent stem cells
Preparation method uses the method, it is only necessary to have basic cell culture condition, can easily obtain no exogenous origin gene integrator
People's inductive pluripotent stem cells.
A kind of preparation method of convenient and fast people's inductive pluripotent stem cells proposed by the present invention, comprising the following steps:
A, carrier will be reprogrammed to import in human somatic cell by the method for liposome transfection, mankind's body after being transfected
Cell；
B, the human somatic cell after transfection is subjected to Fiber differentiation in human embryonic stem cell medium, until Human embryo is dry thin
Born of the same parents' sample clone generates, and picking human embryo stem cell sample clone carries out amplification cultivation, and it is dry that people's inductive pluripotent is obtained after identification
Cell.
Preferably, the human somatic cell is urine cell.
Preferably, the reprogramming carrier includes:
First reprogramming carrier, the first reprogramming carrier includes OriP replication site, EF1 α promoter, EBNA1 table
Up to sequence, miR-302-367 cluster；
Second reprogramming carrier, the second reprogramming carrier includes OriP replication site, EF1 α promoter, transcription because
Son.
Preferably, the quantity of the first reprogramming carrier is 1, and single second reprogramming carrier includes 1 or 2 and turns
Record the factor.
Preferably, the size of the reprogramming carrier is no more than 10000bp.
Preferably, the transcription factor be OCT4, SOX2, SOX1, KLF4, KLF5, l-MYC, n-MYC, c-MYC,
One of ESRRB, LRH, NANOG, LIN28, SV40LT or a variety of.
Preferably, the transcription factor is OCT4, SOX2, KLF4, SV40LT.
Preferably, the transcription factor OCT4 is connected with SOX2 by IRES sequence or 2A sequence.
A kind of preparation method of convenient and fast people's inductive pluripotent stem cells proposed by the present invention, OriP replication site exist
In the presence of EBNA1 expressed sequence, reprogramming carrier can be made to be replicated independently of cellular genome, and with cell
It passes on and gradually loses, people's inductive pluripotent stem cells that utility preparation method provided by the invention obtains are without foreign gene
Integration, improves the safety in utilization of people's inductive pluripotent stem cells.In the reprogramming carrier that the present invention uses, each rearrange
Cheng Zaiti only includes 1-2 transcription factor, controls reprogramming carrier size within 10000bp, significantly improves lipid
The efficiency of body transfection.In addition, the EF1 α promoter, miR-302-367 cluster in reprogramming carrier can be improved to urine cell
Inducing effect, so that compared with prior art, the present invention the experimental cost for obtaining people's inductive pluripotent stem cells is low and to experiment
The requirement of equipment is low, simple and easy to operate.
Detailed description of the invention
Fig. 1 is the microscope photo that urine cell carries out before liposome transfection in embodiment 1；
Fig. 2 is the microscope photo that urine cell carries out 6h after liposome transfection in embodiment 1；
Fig. 3 is the 4th day microscope photo of Fiber differentiation after urine cell transfecting carrier in embodiment 1；
Fig. 4 is the 14th day microscope photo of Fiber differentiation after urine cell transfecting carrier in embodiment 1；
Fig. 5 is the 25th day microscope photo of Fiber differentiation after urine cell transfecting carrier in embodiment 1；
Fig. 6 is the human embryo stem cell sample clone that urine cell is formed by Fiber differentiation in embodiment 1；
Fig. 7 is the expression that people's inductive pluripotent stem cells marker OCT4 is detected in embodiment 1；
Fig. 8 is the expression that people's inductive pluripotent stem cells surface marker SSEA4 is detected in embodiment 1；
Fig. 9 is the expression that people's inductive pluripotent stem cells surface marker TRA-1-81 is detected in embodiment 1；
Figure 10 is that the people's inductive pluripotent stem cells obtained in embodiment 1 have the ability for being divided into entoderm；
Figure 11 is that people's inductive pluripotent stem cells for obtaining have and are divided into mesoblastic ability in embodiment 1；
Figure 12 is that people's inductive pluripotent stem cells for obtaining have and are divided into ectodermic ability in embodiment 1.
Specific embodiment
In the following, technical solution of the present invention is described in detail by specific embodiment, it is not specified in embodiment specific
Technology or conditions, it the technology or conditions that describe according to the literature in the art or is carried out according to product description.
Embodiment 1
A kind of preparation method of convenient and fast people's inductive pluripotent stem cells proposed by the present invention:
(1) building of carrier is reprogrammed
It include hygromycin and CMV starting in endonuclease RruI and KpnI excision pCEP4 (Invitrogen) plasmid
Nucleic acid sequence including son:
5 μ l10 × FastDigest buffer (Thermo Scientific), 1 μ g are added in 1.5mLEP pipe
PCEP4 plasmid, 1 μ lFastDigest RruI (Thermo Scientific), 1 μ l FastDigest KpnI (Thermo
Scientific), ddH is added2O complements to 50 μ l, and digestion 2h under the conditions of 37 DEG C is recycled by agarose gel electrophoresis
The segment of 7500bp or so obtains the digestion post-fragment of pCEP4 plasmid.
The EF1 α promoter sequence containing endonuclease KpnI restriction enzyme site is obtained by PCR:
Specific primer used in 1 PCR of table
| Primer sequence | |
| Upstream primer 1 | GGCTCCGGTGCCCGTCAGT |
| Downstream primer 1 | AACGGTACCTCACGACACCTGAAATGGAAGAAA |
2 PCR system of table
| 10×KOD Plus buffer(TOYOBO) | 5μl |
| 2.5mM dNTP | 5μl |
| Upstream primer (5 μM) | 2.5μl |
| Downstream primer (5 μM) | 2.5μl |
| KOD Plus(TOYOBO) | 1μl |
| MgSO4(25mM) | 2μl |
| PCEP4 plasmid | 100ng |
| ddH2O | Complement to 50 μ l |
It is expanded according to following PCR program:
The purpose product that PCR product is carried out agarose gel electrophoresis and recycles 1200bp or so, with nuclease restriction endonuclease
KpnI carries out single endonuclease digestion to purpose product:
5 μ l10 × FastDigest buffer (Thermo Scientific), 20 μ l purposes are added in 1.5mLEP pipe
DdH is added in product, 1 μ lFastDigest KpnI (Thermo Scientific)2O complements to 50 μ l, under conditions of 37 DEG C
Digestion 2h is directly recycled to obtain junction fragment.
The digestion post-fragment of pCEP4 plasmid is connect with junction fragment by ligase:
PCR pipe be added 5 μ lSolution I, the digestion post-fragment of 0.5 μ lpCEP4 plasmid, 4.5 μ l junction fragments,
30min is connected under conditions of 16 DEG C and obtains connection product, and connection product is transformed into bacillus coli DH 5 alpha competent cell, is applied
It is distributed on LB plate with ampicillin, 37 DEG C of overnight incubations.Picking monoclonal expands culture, extracting plasmid, reflects through sequencing
Obtained after fixed correct containing OriP replication site, EF1 α promoter, EBNA1 expressed sequence pEP4M plasmid.
EBNA1 expressed sequence in pEP4M plasmid is cut off with endonuclease NsiI and ClaI:
5 μ l10 × FastDigest buffer (Thermo Scientific), 1 μ g are added in 1.5mLEP pipe
PEP4M plasmid, 1 μ lFastdigest Bsu15I (i.e. ClaI) (Thermo Scientific), 1 μ lFastDigest
DdH is added in Mph1103I (i.e. NsiI) (Thermo Scientific)2O complements to 50 μ l, digestion 2h under conditions of 37 DEG C,
By the segment of agarose gel electrophoresis recycling 5000bp or so, the digestion post-fragment of pEP4M plasmid is obtained.
Gone out in pEP4M plasmid by PCR amplification by the OriP replication site of endonuclease NsiI and ClaI part excision
Sequence:
Specific primer used in 3 PCR of table
| Primer sequence | |
| Upstream primer 2 | GCTATCCTAATCTGTATCCGGGTAGCA |
| Downstream primer 2 | AATTCCATCGATTAAATTCACCTAAGAATGGGAGCAACC |
4 PCR system of table
| 10×KOD Plus buffer(TOYOBO) | 5μl |
| 2.5mM dNTP | 5μl |
| Upstream primer (5 μM) | 2.5μl |
| Downstream primer (5 μM) | 2.5μl |
| KOD Plus(TOYOBO) | 1μl |
| MgSO4(25mM) | 2μl |
| PEP4M plasmid | 100ng |
| ddH2O | Complement to 50 μ l |
Expansion sign is carried out according to following PCR program:
The purpose product that PCR product is carried out agarose gel electrophoresis and recycles 500bp or so, by purpose product nucleic acid
Restriction endonuclease NsiI, ClaI carry out double digestion:
5 μ l10 × FastDigest buffer (Thermo Scientific), 20 μ l purposes are added in 1.5mLEP pipe
Product, 1 μ lFastdigest Bsu15I (i.e. ClaI) (Thermo Scientific), 1 μ lFastDigest Mph1103I
DdH is added in (i.e. NsiI) (Thermo Scientific)2O complements to 50 μ l, digestion 2h under conditions of 37 DEG C, directly recycles
Obtain junction fragment.
The digestion post-fragment of pEP4M plasmid is attached with junction fragment:
It is added 5 μ l Solution I in PCR pipe, the digestion post-fragment of 0.5 μ lpEP4M plasmid, 4.5 μ l junction fragments,
30min is connected under conditions of 16 DEG C, is transformed into bacillus coli DH 5 alpha competence, is coated on LB plate with ampicillin
On, 37 DEG C of overnight incubations.Picking monoclonal expands culture, extracting plasmid, obtains replicating comprising OriP after sequencing identification is correct
Site, EF1 α promoter and the pEP4MM plasmid for not carrying EBNA1 expressed sequence.
MiR-302-367 cluster is cloned into the multiple cloning sites in pEP4M plasmid after EF1 α promoter, first is obtained and rearranges
Cheng Zaiti；By transcription factor OCT4 and SOX2 by 2A sequence connect rear clone enter it is more after EF1 α promoter in pEP4MM plasmid
Cloning site obtains the second reprogramming carrier 1；Transcription factor Klf4, SV40LT is cloned into EF1 in another pEP4MM plasmid
Multiple cloning sites after α promoter obtain the second reprogramming carrier 2.Three kinds of reprogramming carriers are extracted through endotoxin-free respectively
Kits will be reprogrammed by transfection reagent specification requirement after mixing using liposome (lipofectamine 3000)
It is intracellular that carrier is transfected into urine.
(2) Fiber differentiation of urine cell
The preparation of induced medium:
Four kinds of promotion cells are added in human embryonic stem cell medium mTeSR1 (Stem Cell Technologies)
Small molecule compound PD0325901 (Selleck), the CHIR99021 (Selleck), A-83-01 (sigma- of reprogramming
Aldrich) and Thiazovivin (Selleck), make four kinds of small molecule compounds working concentration be respectively 0.5 μM/L, 3 μM/
L、0.5μM/L、0.5μM/L。
Posterior segment urine is collected with sterile receipts urine cup, the urine being collected into is transferred in centrifuge tube, 400g is centrifuged 10min,
It sucks supernatant to every pipe and leaves about 1-5ml liquid, be added and contain dual anti-(penicillin 500U/ml, 500 μ g/ml of streptomysin)
(GIBCO) PBS (Genom), 400g are centrifuged 10min.Supernatant is sopped up to remaining 0.5-1ml liquid, remaining liquid is resuspended
It is added in 6 well culture plates of coating 0.1% gelatin (Millipore) afterwards, and 3ml is added and contains 0.2% (v/v) Primocin
(InvivoGen) REGM culture medium (LONZA).
6 well culture plates are placed in 37 DEG C of carbon dioxide incubators stationary culture 3 days, were observed under the microscope in the 3rd day
Whether cell pollutes, if pollution, is collected again；If pollution-free, continue about 2 week of stationary culture, during which every 3 days
A subculture is replaced, first generation urine cell is obtained.
Culture medium is sucked, after obtained first generation urine cell is cleaned twice with PBS, 500 μ l0.25% pancreatin-are added
EDTA (GIBCO), room temperature, which is digested to tapping culture plate cell, to be hiked up, and FBS (GIBCO) is added and terminates digestion.
Piping and druming dispersion urine cell is simultaneously collected in centrifuge tube, and 200g is centrifuged 5min, sucks supernatant, and REGM culture medium is added
(LONZA), it is passed to and has been coated in the 6 orifice plates of Matrigel (BD) after cell dispels, cultivated in 37 DEG C of carbon dioxide incubators,
When cell grows to 70% degrees of fusion, is operated, will be weighed using lipofectamine3000 (ThermoFisher) by specification
Programming vector is transfected into urine cell.Transfection 6h after change liquid be induced medium, Fiber differentiation 10 days or so, then more
Human embryonic stem cell medium mTeSR1 (Stem Cell Technologies) is changed to be cultivated for 15 days or so, it is during which every
Its one subculture of replacement.Picking human embryo stem cell sample clone carries out amplification cultivation, and amplification cultivation to the 10th generation or so carries out
People's inductive pluripotent stem cells identification experiment.
(3) people's inductive pluripotent stem cells identification experiment
People's inductive pluripotent stem cells pluripotency marker test experience:
Primary antibody: Mouse anti-OCT4 antibody (Santa Cruz Biotechnology sc-5279)
Mouse anti-SSEA4 antibody(Abcam ab16287)
TRA-1-81antibody(Millipore MAB4381)
Secondary antibody: Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody,Alexa Fluor Plus 488(Invitrogen)
Cell is cleaned one time with PBS, addition 0.25% pancreas enzyme -EDTA (GIBCO) room temperature digests thin to tapping culture plate
Born of the same parents hike up, and FBS (GIBCO) is added and terminates digestion, gently blows and beats and disperses cell, collect 200g in centrifuge tube and are centrifuged 5min.
Supernatant is sucked, after being washed once with PBS, 200g is centrifuged 5min and abandons supernatant.200 μ l Cytofix Fixation Buffer are added
(BD) the fixed 20min of room temperature, is cleaned 2 times with PBS.SSEA4 and TRA-1-81 antibody is carried out according to the ratio of 1:50 with PBS dilute
It is added in cell after releasing, is incubated for 30min under conditions of 37 DEG C；It is added before OCT4 antibody, 200 μ l is added into cell and spend
Ionized water is centrifuged on abandoning by Perm/Wash Buffer I (BD), penetrating 15min under the conditions of 4 DEG C of the dilution proportion of 1:10
It is cleaned 2 times, is added in cell after OCT4 antibody is diluted with PBS according to the ratio of 1:50, in 37 DEG C of item with PBS after clear
30min is incubated under part.It is cleaned 2 times after SSEA4, TRA-1-81, OCT4 antibody are discarded with PBS.Secondary antibody PBS is pressed into 1:100
It is added in cell after dilution proportion, is protected from light under the conditions of 37 DEG C and is incubated for 30min, PBS is lured after washing 2 times with flow cytomery people
The expression of the property led multipotential stem cell marker OCT4, people's inductive pluripotent stem cells surface marker SSEA4, TRA-1-81.
(2) differentiation capability of people's inductive pluripotent stem cells is detected
Enter immunodeficient mouse groin position by the cell infusion that Fiber differentiation obtains for about 5,000,000, passes through
It 1-2 months, takes out the intracorporal teratoma of mouse and is fabricated to histotomy and carries out hematoxylin eosin staining, microscopically observation
By the differentiation capability for the cell that Fiber differentiation obtains.
Cell state of the urine cell before and after transfection is as shown in Figure 1 and Figure 2 in embodiment 1, from the comparison of Fig. 1, Fig. 2
As can be seen that the method for reprogramming carrier by liposome transfection in the present invention is smaller to cellular damage.Fig. 3, Fig. 4, it Fig. 5, shows
The variation of urine cell cellular morphology during Fiber differentiation in embodiment 1 is shown, Fig. 6 is that urine cell passes through Fiber differentiation
The monoclonal of formation shows that preparation method provided by the invention can form human embryo stem cell sample clone with inducing somatic.It is logical
Overflow-type cell instrument detects people's inductive pluripotent stem cells marker OCT4, people's inductive pluripotent stem cells surface marker
SSEA4, TRA-1-81, which pass through in embodiment 1 in the cell of Fiber differentiation, to express, as a result respectively as shown in Fig. 7, Fig. 8, Fig. 9,
Show that people's inductive pluripotent stem cells pluripotency marker is the positive.It can be observed to exist in teratoma by entoderm under microscope
The body of gland of development, as shown in Figure 10；Cartilage from mesoderm development, as shown in figure 11；And it is developed by ectoderm
Nerve fiber show that cell that the preparation method Fiber differentiation in through the invention obtains has as shown in figure 12 and be divided into
It is interior, in, ectodermic ability, there is versatility, it was demonstrated that it is dry that the method in the present invention can easily obtain people's inductive pluripotent
Cell.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
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Claims (8)
1. a kind of preparation method of convenient and fast people's inductive pluripotent stem cells, which comprises the following steps:
A, carrier will be reprogrammed to import in human somatic cell by the method for liposome transfection, mankind's body after being transfected is thin
Born of the same parents；
B, the human somatic cell after transfection is subjected to Fiber differentiation in human embryonic stem cell medium, until human embryo stem cell sample
Clone generates, and picking human embryo stem cell sample clone carries out amplification cultivation, obtains people's inductive pluripotent stem cells after identification.
2. the preparation method of convenient and fast people's inductive pluripotent stem cells according to claim 1, which is characterized in that the mankind
Body cell is urine cell.
3. the preparation method of convenient and fast people's inductive pluripotent stem cells according to claim 1 or claim 2, which is characterized in that described
Reprogramming carrier includes:
First reprogramming carrier, the first reprogramming carrier include OriP replication site, EF1 α promoter, EBNA1 expression sequence
Column, miR-302-367 cluster；
Second reprogramming carrier, the second reprogramming carrier includes OriP replication site, EF1 α promoter, transcription factor.
4. the preparation method of convenient and fast people's inductive pluripotent stem cells according to claim 3, which is characterized in that described first
The quantity for reprogramming carrier is 1, and single second reprogramming carrier includes 1 or 2 transcription factors.
5. the preparation method of any one of -4 convenient and fast people's inductive pluripotent stem cells according to claim 1, which is characterized in that
The size of the reprogramming carrier is no more than 10000bp.
6. according to the preparation method of the convenient and fast people's inductive pluripotent stem cells of claim 3 or 4, which is characterized in that described
Transcription factor be OCT4, SOX2, SOX1, KLF4, KLF5, l-MYC, n-MYC, c-MYC, ESRRB, LRH, NANOG, LIN28,
One of SV40LT or a variety of.
7. the preparation method of convenient and fast people's inductive pluripotent stem cells according to claim 6, which is characterized in that the transcription
The factor is OCT4, SOX2, KLF4, SV40LT.
8. the preparation method of convenient and fast people's inductive pluripotent stem cells according to claim 7, which is characterized in that the transcription
Factor OCT4 is connected with SOX2 by IRES sequence or 2A sequence.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122505A (en) * | 2021-04-13 | 2021-07-16 | 四川大学华西医院 | Method for obtaining urine source induced pluripotent stem cells through retrovirus |
| WO2023273882A1 (en) * | 2021-06-30 | 2023-01-05 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Efficient and non-genetically modified ipsc-induced, industrialized single clone selection platform, and use |
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| CHENXIA HU ET AL.: ""Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells"", 《REGEN. MED.》 * |
| DONGSHENG GUO ET AL.: ""Generation of non-integrated induced pluripotent stem cells from a 23-year-old male with multiple endocrine neoplasia type 1 syndrome"", 《STEM CELL RESEARCH》 * |
| DONGSHENG GUO ET AL.: ""Generation of non-integrated induced pluripotent stem cells from a 59-year-old female with multiple endocrine neoplasia type 1 syndrome"", 《STEM CELL RESEARCH》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122505A (en) * | 2021-04-13 | 2021-07-16 | 四川大学华西医院 | Method for obtaining urine source induced pluripotent stem cells through retrovirus |
| WO2023273882A1 (en) * | 2021-06-30 | 2023-01-05 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Efficient and non-genetically modified ipsc-induced, industrialized single clone selection platform, and use |
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