CN107022522A - Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell - Google Patents

Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell Download PDF

Info

Publication number
CN107022522A
CN107022522A CN201610067377.8A CN201610067377A CN107022522A CN 107022522 A CN107022522 A CN 107022522A CN 201610067377 A CN201610067377 A CN 201610067377A CN 107022522 A CN107022522 A CN 107022522A
Authority
CN
China
Prior art keywords
cell
cord blood
mscs
transdifferentiation
stem cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610067377.8A
Other languages
Chinese (zh)
Inventor
袁卫平
张孝兵
程涛
庞雅坤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Traceability Life Polytron Technologies Inc
Original Assignee
Traceability Life Polytron Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Traceability Life Polytron Technologies Inc filed Critical Traceability Life Polytron Technologies Inc
Priority to CN201610067377.8A priority Critical patent/CN107022522A/en
Publication of CN107022522A publication Critical patent/CN107022522A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/605—Nanog
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Abstract

The present invention relates to a kind of induction of cord blood CD34 positive cells to the direct transdifferentiation technology of Derived from Mesenchymal Stem Cells, belong to technical field of bioengineering.The invention provides a kind of technique for gene engineering that cord blood CD 34 positive cell can be made directly to obtain iMSCs from the efficient transdifferentiation of candidate stem cell, candidate stem cell and certain composition are turned into MSCs by the co-cultivation transdifferentiation of enough time, above-mentioned composition includes at least one of OCT4, NANOG, MYCL1, SNAI1, SNAI2, TWIST1 transcription factor, can also include one or more cell factors disclosed by the invention and other compounds.The technical method provided by the present invention, the method for using virus to infect causes associated transcription factor transient expression in cord blood CD 34 positive cell, coordinates induction MSCs culture medium, can be in the time of 2-3 weeks, from 1 × 106Cord blood CD 34 positive cell transdifferentiation can obtain 1 × 109MSCs, transdifferentiation and come iMSCs cells shows for MSCs form and express MSCs cell surface markers, such as CD29, CD44, CD73, CD90.

Description

Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell
Technical field
The present invention relates to direct transdifferentiation from a kind of induction of cord blood CD34 positive cells to Derived from Mesenchymal Stem Cells Technology, belongs to technical field of bioengineering.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is a kind of originating from mesoblastic more competent Cell, can be separated, such as marrow and adipose tissue from the Various Tissues of people.One of the characteristics of MSCs is that they have There is the ability for being divided into a variety of interstitial tissues, including bone, cartilage, tendon, muscle, bone marrow interstital and adipose tissue Deng this biological characteristics causes MSCs cells to be with a wide range of applications in clinical treatment and regenerative medicine.
MSCs can be obtained easily from a variety of adult tissues, including adipose tissue and myeloid tissue, and its point From the method with culture in field it is very ripe (for example, United States Patent (USP) 5,486,359).However, by external The MSCs et al. Ke rates of culture are low and differentiation capability is extremely limited, and this turns into the MSCs using adult origin Carry out the significant obstacle of high efficiency cell treatment.The MSCs of embryonic origin has a longer telomere, more preferable proliferation rate, And with higher et al. Ke rate and outstanding differentiation capability.The MSCs of derived from embryonic stem cells acquisition methods It is known, for example, in the method for U.S. Patent Publication No. US2008/0219957 descriptions, being done by breaing up embryo Cell clone simultaneously results in embryonic origin under conditions of without stroma cell using the serum free medium containing FGF2 Mescenchymal stem cell.Disputed on however, the use of embryonic stem cell inherently has height.
Cord blood (Umbilical Cord Blood, UCB) is extremely readily available after neonate is born, and is separated and is cultivated UCB MSCs method has built up (for example, United States Patent (USP) 7704739)；However, obtained at present in UCB MSCs quantity is considerably less.Cord blood convenient material drawing, between the induction come in the absence of Medical Ethics dispute, its transdifferentiation Mesenchymal stem cells (induced Mesenchymal Stem Cells, iMSCs) multiplication capacity is strong and differentiation capability comprehensively, Transplanting efficiency is higher than any MSCs for separating and obtaining from adult tissue in vivo.Cord blood cell induction MSCs's Method has been reported, but typically has low-down efficiency.Peters etc. reports a kind of using independent of nourishing The dual cultivating system of confluent monolayer cells induces MSCs by cord blood cell.In addition, from other embryonic tissues, such as placenta, The method that MSCs is obtained in Whartons jelly and amniotic fluid is also widely described.But, in order that MSCs allogeneics are moved Plant can be widely used for clinical treatment and benefit more patients, and the MSCs warehouses for setting up enough scale are very must Want.Therefore a method for being capable of the high-quality MSCs of high efficiency acquisition is set up, with extensive clinical practice valency Value.
The content of the invention
The problem of to solve above-mentioned Cord blood induction MSCs inefficiencies, it is an object of the invention to provide one kind induction Cord blood CD 34 positive cell is divided into MSCs technique for gene engineering, and the iMSCs obtained using the method The clinical treatment of progress.
To reach above-mentioned purpose, it can make cord blood CD 34 positive cell directly from Hematopoietic Stem the invention provides one kind Cell high-efficient transdifferentiation obtain iMSCs technique for gene engineering, CD34 molecules be optionally expressed in the mankind and other Mammalian hematopoietic stem cell surface, and be at present using than wide as the ripe of cell gradually weakens to disappearing Human hematopoietic stem cell surface marker.The technical method provided by the present invention, the method for using virus to infect causes Associated transcription factor transient expression in cord blood CD 34 positive cell, coordinates induction MSCs culture medium, can In the time of 2-3 weeks, from 1 × 106Cord blood CD 34 positive cell transdifferentiation can obtain 1 × 109MSCs, Transdifferentiation and come iMSCs cells shows for MSCs form and express MSCs cell surface markers, such as CD29、CD44、CD73、CD90。
Transcription factor used in umbilical cord blood hematopoietic stem cell transdifferentiation involved in the present invention include OCT4, In the core former times acid sequence combination of this 6 genes of NANOG, MYCL1, SNAI1, SNAI2, TWIST1 at least It is a kind of.OCT4(POU5F1；Accession no.NM_001173531；amino acid sequence provided in SEQ ID NO：1) it is a kind of well-known reprogramming transcription factor, candidate stem cell can be changed into mesoderm Stem cell, and the propagation of mesodermal stem cell can be promoted.NANOG(Accession no.AAP49529；amino acid sequence provided in SEQ ID NO：2) it is another stem cell transcription factor, cell can be promoted Propagation and MSCs into clone ability.The gene of Myc families, such as MYCL1 (Accession Nos. NP_001028253 (isoform 1), NP_005367 (isoform 2), and NP_001028254 (isoform 3)；amino acid sequences provided in SEQ ID NO：3-5；Respectively), promotion that can be long-term MSC breeds.C-MYC is one kind of proto-oncogene, and continuous expression c-MYC may trigger tumour, but instantaneously Expression c-MYC be also be capable of safety be applied to transdifferentiation, not being found MYCL1 and tumour at present has phase Guan Xing.SNAI1(Accession no.NM_005985；amino acid sequence provided in SEQ ID NO： 3), SNAI2 (Accession no.NM_003068XR_017857；amino acid sequence provided in SEQ ID NO：4).SNAI is a transcription factor family, by suppressing adhesion molecule in embryo development procedure E-cadherin regulation Epithelial and stromal conversions (Epithelial.Mesenchymal Transition, EMT).TWIST1 (Accession no.NM_000474；amino acid sequence provided in SEQ ID NO：3) it is alkaline spiral shell One of the spiral of a ring one (basic Helix-Loop-Helix, bHLH) transcription factor family member is revolved, it is non-in evolutionary process Often conservative, its nucleotides and amino acid sequence are highly conserved between different genera, are played in embryo is developed Important function.In addition TWIST1 is considered as oncogene, influences apoptosis of tumor cells, and be used as EMT processes In key regulator, invasion and attack and transfer of the TWIST1 to tumour have a major impact.TWIST1 may participate in a variety of The differentiation of cell, including myocyte, cartilage cell, osteogenic cell, and it is relevant with the development of heart valve.
The expression vector for being used to convert umbilical cord blood hematopoietic stem cell involved in the present invention includes, but are not limited to slow virus Carrier, non-integration slow virus vector, gland relevant viral vector, adenovirus vector and circles episomal vector.This A little carriers can also add appropriate control element and ripe control technique, such as TET-OnTM systems (Clontech Labs., Mountain View, CA；Baron＆Bujard, Methods Enzymol.2000, 327：1-21).The promoter of expression vector involved in the present invention can be SFFV, MSCV, EF1- α and ubiquitin Any one of promoter.
The cell factor added during transdifferentiation involved in the present invention or compound can be following at least one Kind, involved cell factor includes but is not limited to EGF (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (such as FGF-2), transforminggrowthfactor-β1 (TGF-β 1), and Bones morphology hair Raw albumen 3 (BMP-3).Involved compound includes but is not limited to be capable of enhanced MSC s multiplication capacities WNT3A or WNT5A or GSK-3 inhibitor and the VEGF and/or IGF-1 for promoting MSCs growths.
It can make cord blood CD 34 positive cell directly from the efficient transdifferentiation of candidate stem cell the invention provides one kind IMSCs technique for gene engineering is obtained, candidate stem cell and certain composition are turned by the co-cultivation of enough time Differentiation turns into MSCs, and above-mentioned composition includes OCT4, NANOG, MYCL1, SNAI1, SNAI2, TWIST1 At least one of transcription factor, one or more cell factors disclosed above and other compounds can also be included.
The technical method provided using the present invention can be obtained uniformly from cord blood CD 34 positive cell Induction Transformation MSCs, this MSCs is substantially similar or identical, with close gene expression profile and cellular morphology.For example make The cell surface marker CD73 for the identification of M SCs that MSCs expression prepared by the technical method provided with the present invention is generally acknowledged, At least one of CD105, CD166, CD90, CD271, CD29, CD44.
Technical method disclosed by the invention available for from CD34 positive cells except preparing MSCs, it can also be used to uses CD133 positive cells or the cord blood cell of non-purifying prepare MSCs.
The disclosure of invention is except utilizing the technical method that CD34 positive cells transdifferentiation is MSCs, in addition to root The MSCs and pharmaceutically acceptable carrier obtained according to disclosed technical method.
Technical method provided by the present invention can be changed into directly by already present cord blood stem cell storehouse is convenient and safe For mesenchyma stem cell, it is worth with more far-reaching medical applications.
Brief description of the drawings
Fig. 1 builds schematic diagram for the slow virus carrier used in embodiment 1.
Fig. 2 is the circles expression vector schematic diagram that uses in embodiment 2.
Fig. 3 is the karyotyping figure after the iMSCs long-term cultivations from embodiment 2 in embodiment 3.
Fig. 4 be in embodiment 4 iMSCs from embodiment 2 to lipoblast, cartilage cell and Gegenbaur's cell Differentiation figure.
The cell phenotype analysis chart that Fig. 5 is the iMSCs from embodiment 2 in embodiment 5.
Embodiment
In order to which technical characteristic, purpose and beneficial effect to the present invention are more clearly understood from, now to the skill of the present invention Art scheme carry out it is described further below, but it is not intended that to the present invention can practical range restriction.
Embodiment 1
Present embodiments provide a kind of using the acquisition of slow-virus infection Cord Blood-Derived CD34 Positive Hematopoietic Stem Cells MSCs method, it comprises the following steps：
The enrichment and storage of 1.CD34 positive cells：
1) Cord blood is fully mixed with HES 5: 1, is stored at room temperature 40 minutes precipitation red blood cells.
2) packing 15ml lymphocyte separation mediums are placed in equilibrium at room temperature in 50ml centrifuge tubes.
3) supernatant in drawing 1), is slowly added to equilibrate in the lymphocyte separation medium of room temperature, notes not The liquid level of separating liquid is disturbed, cumulative volume can reach 50ml.Centrifuge, which is set, removes lock, and 400G is centrifuged 25 minutes.
4) the 5 times of volume PBS of tunica albuginea layer addition drawn between two liquid phases are fully mixed.500G is centrifuged 10 minutes. Obtain mononuclearcell.
5) the specification enrichment single core of Cord blood according to U.S.'s day Ni people CD34 positive cells enrichment magnetic bead kit is thin CD34 positive cells (Miltenyi Biotec, Auburn, CA) in born of the same parents.
2.CD34 positive cell pre-stimulations：Fresh separated or the cord blood CD 34 positive cell of recovery, in hematopoiesis Cultivated under stem cell cultivation conditions：Iscove ' s modified Dulbecco ' s medium (IMDM)+10%FBS +100ng/ml TPO+100ng/ml SCF+100ng/ml FL+100ng/ml G-CSF+10ng/ml IL3.Cell The factor is purchased from ProSpec (East Brunswick, NJ).37 DEG C, 5%CO2 is cultivated 2 days.
3. slow-virus infection：
1) 24 orifice plates use fibronectin (RetroNectin) (CH-296；Takara Bio, Inc., Shiga, Japan) Pre-processed according to product description.Room temperature is placed 2 hours or 4 spent after night, and PBS washes away residual fibers connection egg In vain, it can use.
2) candidate stem cell is per hole 1x104It is inoculated into 24 orifice plates being coated with.Then the band that MOI is 10 is added There is 37 DEG C of the slow virus of the related transcription factor of transdifferentiation, 5%CO2 is cultivated 4-5 hours, and slow virus carrier is built Flow is as shown in Figure 1.
3) change containing virulent culture medium, add fresh candidate stem cell culture medium, and carry out after 24 hours Superinfection.
4. transdifferentiation culture：By the cell culture after second of gene transfer in transdifferentiation culture medium, transdifferentiation training Support base composition as follows：α MEM+10%FBS+10-20ng/mlFGF2+10~20ng/ml EGF2+10-20ng/ml PDGFB.37 DEG C, after 5%CO2 is cultivated 4 to 5 days, most cells transdifferentiation is MSCs samples iMSCs.
Embodiment 2
Present embodiments provide one kind makes Cord Blood-Derived CD34 Positive Hematopoietic Stem Cells turn to divide using non-integrated vector Change the method for obtaining iMSCs, it comprises the following steps：
1. the structure of non-integrated vector
1) in order to preferably meet the standard that clinical cytology is treated, we have developed carried out using non-integrating vectors MSCs transdifferentiation method.We test the episomal vector based on EBNA1 first.Consideration convey contaminates bleeding of the umbilicus After CD34 positive cells one week, we have obtained MSC like cells.
2) from Invitrogen companies (Carlsbad, CA).Hygromycin gene element and CMV promoter are logical Two restriction enzyme sites of NruI and BamHI are crossed to be removed and by OCT4, NANOG, MYCL1, SNAI1, At least one of nucleotide sequence combination of this 6 genes of SNAI2, TWIST1 cuts and inserted from slow virus carrier Enter pCEP4 episomal vectors.Fig. 2 is that the circles that CD34 positive cells are used to iMSCs transdifferentiations are expressed The schematic diagram of carrier.
3) reprogramming gene is cloned on pCEP4 carriers backbone；The expression of gene is SFFV (Spleen Focus-forming virus U3 promoter, spleen focus forms viral U3 promoters) driving.2A is one and come from The autologous cracking site sequence of horse Coryzavirus.Wpre, posttranscriptional regulatory element；SV40polyA, SV40 disease Malicious polyadenylation signal；The reproduction element of OriP, Epstein-barr virus, EBV viral origin；EBNA1, Ai Baisitan-Ba Er antigen 1s, it is for the duplication during infection cell and maintains the persistence of free plasmid to play Vital effect.
2. episomal vector consideration convey contaminates
1) the fresh or culture of bleeding of the umbilicus CD34 positive cells recovered after freezing in candidate stem cell cultivating system, Both TPO, SCF, and FL are added in the IMDM containing 10% serum, cell factor final concentration is 100 millimicros Grams per milliliter.
2) after three days, harvesting uses CD34 cellsKit (Lonza) and The program u-008 of Nucleofector II consideration convey instrument transfects 12 μ g pCEP plasmids.
3) after transfecting, cell is planted in RetroNectin pre-processed boards so that candidate stem cell recovers immediately.
4) the cell culture system culture using foregoing MSCs in second day.
IMSCs and the slow virus transdifferentiation come using non-integrated vector transdifferentiation and the iMSCs cell biologies come Characteristic is identical.Transdifferentiation after 3 weeks 99% cell detection less than OCT4 express.
The iMSCs of embodiment 3 long-term cultivation and the identification of Genome stability
The present embodiment is to carry out long-term cultivation to the iMSCs obtained in embodiment 2, and carries out Genome stability Identification, is followed the steps below：
1. marrow iMSCs is cultivated 40 more than generation by us under MSCs condition of culture, population doubling time is 18-36 Hour.Cell culture and passage specific method：
1) 24 orifice plates use fibronectin (RetroNectin) (CH-296；Takara Bio, Inc., Shiga, Japan) Pre-processed according to product description.Room temperature is placed 2 hours or 4 spent after night, and PBS washes away residual fibers connection egg In vain, it can use.
2) MSCs cultures were in the pretreated culture plates of RetroNectin, per 2-3 days, using Accutase or Five minutes digestion iMSCs of trypsin treatment are passed on into individual cells, and by 4-6 times.
2. passing through long-term in vitro culture, cell keeps typical MSCs forms, flow cytometry cell surface The expression of molecular labeling is identical with the MSCs cells of early stage transdifferentiation.
3. chromosome karyotype analysis result is found as shown in figure 3, not having in the iMSCs after long-term in vitro culture Chromosome abnormality.Chromosomal G-banding method：It is complete using DNA standard spectrum karyotyping programs and HiSKY Cytogenetics system (Applied Spectral Imaging, Inc.Vista, CA).Each clone selects 10 and is in The cell of interphase cell, carries out chromosome karyotype analysis.
The iMSCs vitro differentiations ability of embodiment 4 is detected
The present embodiment is to carry out long-term cultivation to the iMSCs obtained in embodiment 2, and carries out vitro differentiation ability Identification, bone marrow-drived mesenchymal stem can be divided into Gegenbaur's cell, cartilage cell and fat cell.Obtained using the above method The iMSC obtained is cultivated about 3 weeks in differential medium, it has been found that mesenchymal stem cells MSCs can be very good ground Be divided into the pedigree of multiple skeletal tissues, it was demonstrated that transdifferentiation and the MSCs differentiation capabilities that come are complete.It is specific according to Following steps are carried out：
1. the differentiation of fat cell：
1)iMSCs(4×103cells/cm2) inoculation.Cell growth adds adipogenic induction differential medium to converging： - MEM+10%/FBS+1 μM of μ g/ml insulin of dexamethasone+10 of α, 0.5mM isobutyl group xanthine+10ng/ml FGF2。
2) culture medium is changed once for every three days, changes liquid after three times every 2 days, 37 DEG C, 5%CO2 is cultivated 2 weeks.
3) particle in oil red decoration method colour developing oil and fat cell：Nutrient solution is sucked, is washed with PBS 2 times.Add 3.7% Formaldehyde, room temperature fixes 30min, then is washed 2 times with distillation.Oil red-O the dye liquors of addition 0.2%, in 37 DEG C of dyeing 30min, sucks dye liquor, with distillation washing 2 times.1ml distilled water is eventually adding, observes and takes pictures under the microscope.
2. Chondrocyte Differentiation：
1)iMSCs(4×103cells/cm2) inoculation.Cell growth adds into chondrocyte induction differential medium to converging： + 0.1 μM of dexamethasone+10ng/ml TGF-βs 3+10ng/ml of α-MEM+10% hyclones 1+1%ITS+200 μM of ascorbic acid -2- phosphoric acid of TGF-β.
2) culture medium is changed once for every three days, changes liquid after three times every 2 days, 37 DEG C, 5%CO2 is cultivated 3 weeks.
3) nutrient solution is sucked, the paraformaldehyde for gently adding 4% is fixed, and then takes out cell mass and does paraffin section. The expression of II Collagen Type VIs is dyed or detected with SABC with alcian blue.
4) alcian blue colouring method：Paraffin section is immersed in first in 1% acid alcian blue dye liquor and dyed 30min, then with 0.1M salt acid elution 5min.Observe and take a picture under the microscope.
3. Osteoblast Differentiation：
1)iMSCs(4×103cells/cm2) inoculation.Cell growth adds Osteoinductive differentiation culture medium to converging： α-MEM culture mediums+10%FBS+10mM β -+0.1 μm of phosphoglycerol dexamethasone+10ng/ml BMP2+10ng/ml BMP4+10ng/ml FGF2+200 μM ascorbic acid.
2) culture medium is changed once for every three days, changes liquid after three times every 2 days, 37 DEG C, 5%CO2 is cultivated 3 weeks.
3) Alizarin red staining：The nutrient solution in 6 orifice plates is sucked first, is washed with PBS 2 times, is then added methanol third Ketone mixed liquor (the two mixed proportion is 1: 1), room temperature fixes 10min, then is washed 3 times with PBS.It is eventually adding 0.5% Alizarin red dye liquor, dyes 30min at 37 DEG C, sucks alizarin red dye liquor, washed with PBS after 3 times, in microscope Lower observation is simultaneously taken a picture.
Three be differentiated result as shown in figure 4, iMSCs can break up lipoblast, cartilage cell and Gegenbaur's cell.
The flow cytomery cell phenotype of embodiment 5
The present embodiment is to carry out cell phenotype detection to the iMSCs obtained in embodiment 2, is followed the steps below：
1. collecting iMSCs cells growns to logarithmic phase, individual cells are digested to using pancreatin, 400G centrifuges 5 points Clock removes supernatant.Cell mass is resuspended with 100 μ l PBS+2%EDTA.
2. the antibody for marking MSC surface markers related, 4 degree of incubations 30min, 1ml PBS+2%EDTA wash first Secondary, cell mass is resuspended with 300 μ l PBS+2%EDTA, the expression of Flow cytometry related antigen.
3. streaming antibody is purchased from EBioscience companies (San Diego, CA, USA)
CD29：Anti-Human CD29(Integrin beta 1)
CD44：Anti-Human/Mouse CD44(IM7)
CD90：Anti-Human CD90(Thy-1)
CD166：Anti-Human CD166(ALCAM)
CD73：Anti-Human CD73(AD2)
CD105：Anti-Human CD105(Endoglin)
As shown in figure 5, after cultivating 3 weeks, all iMSC expression MSC marks：CD29、CD44、CD90 And CD166, most iMSCs express CD73 and CD105 simultaneously.

Claims (8)

1. it is a kind of efficiently by cord blood CD 34 positive cell from the method that candidate stem cell transdifferentiation is iMSCs, By candidate stem cell and certain composition by co-culturing transdifferentiation for MSCs.
2. according to the method described in claim 1, it is characterised in that the composition includes：
Transcription factor needed for umbilical cord blood hematopoietic stem cell transdifferentiation, the expression for converting umbilical cord blood hematopoietic stem cell are carried Body and the cell factor or compound added during transdifferentiation.
3. method according to claim 1 or 2, it is characterised in that the umbilical cord blood hematopoietic stem cell turns to divide Transcription factor needed for changing is in OCT4, NANOG, MYCL1, SNAI1, SNAI2 and TWIST1 It is at least one.
4. the method according to claim any one of 1-3, it is characterised in that described to be made for converting Cord blood The expression vector of hemocytoblast is selected from slow virus carrier, non-integration slow virus vector, gland relevant viral vector, adenovirus At least one of carrier and circles episomal vector.
5. the method according to claim any one of 1-3, it is characterised in that described to be made for converting Cord blood The promoter of the expression vector of hemocytoblast is selected from any of SFFV, MSCV, EF1- α and ubiquitin promoter.
6. the method according to claim any one of 1-5, it is characterised in that described to add during transdifferentiation Plus cell factor be selected from EGF, platelet-derived growth factor, fibroblast growth factor, turn Change at least one of grouth factor beta 1 and Bone morphogenetic protein 3 or more than one.
7. the method according to claim any one of 1-6, it is characterised in that described to add during transdifferentiation Plus compound be selected from WNT3A, WNT5A and GSK-3 any of inhibitor, and, VEGF and IGF-1 At least one of.
8. application of the method described in claim any one of 1-7 in MSCs is prepared from CD34 positive cells with And from the cord blood cell of CD133+ cells or non-purifying prepare MSCs in application.
CN201610067377.8A 2016-02-01 2016-02-01 Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell Pending CN107022522A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610067377.8A CN107022522A (en) 2016-02-01 2016-02-01 Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610067377.8A CN107022522A (en) 2016-02-01 2016-02-01 Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell

Publications (1)

Publication Number Publication Date
CN107022522A true CN107022522A (en) 2017-08-08

Family

ID=59524005

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610067377.8A Pending CN107022522A (en) 2016-02-01 2016-02-01 Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell

Country Status (1)

Country Link
CN (1) CN107022522A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250502A (en) * 2008-04-01 2008-08-27 中国科学院上海生命科学研究院 Method for preparing evoked pluripotent stem cell
CN101792776A (en) * 2009-02-01 2010-08-04 中国人民解放军第二军医大学东方肝胆外科医院 Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector
CN102459575A (en) * 2009-06-05 2012-05-16 细胞动力国际有限公司 Reprogramming t cells and hematophietic cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250502A (en) * 2008-04-01 2008-08-27 中国科学院上海生命科学研究院 Method for preparing evoked pluripotent stem cell
CN101792776A (en) * 2009-02-01 2010-08-04 中国人民解放军第二军医大学东方肝胆外科医院 Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector
CN102459575A (en) * 2009-06-05 2012-05-16 细胞动力国际有限公司 Reprogramming t cells and hematophietic cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MENG XIANMEI, ET AL.: "Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor", 《CELL RESEARCH》 *
张峰等主编: "《细胞工程》", 31 July 2014, 中国农业大学出版社 *

Similar Documents

Publication Publication Date Title
Qiao et al. Human mesenchymal stem cells isolated from the umbilical cord
Steens et al. Current strategies to generate human mesenchymal stem cells in vitro
Bonnamain et al. Human dental pulp stem cells cultured in serum-free supplemented medium
Quattrocelli et al. Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles
CN109234229B (en) Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same
CN106085952A (en) A kind of placental villi plate mescenchymal stem cell and extracting method thereof
CN101857854A (en) Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof
CN102827805A (en) HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method
CN1778905B (en) Separating culture and use for fatty mesenchymal dry cell
CN107287156A (en) A kind of isolated culture method of fat mesenchymal stem cell and its application
CN109897815B (en) Efficient separation and culture method of adipose endothelial progenitor cells without coating
CN106119191A (en) A kind of placental villi plate mescenchymal stem cell and clinicization preparation method
CN107267452B (en) Dental pulp stem cell recovery liquid and recovery method of dental pulp stem cells
CN109628388B (en) Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition
Ma et al. Location, isolation, and identification of mesenchymal stem cells from adult human sweat glands
KR101592401B1 (en) Method for Preparing patient-specific Induced Pluripotency Stem Cell from adipose-derived Mesenchymal Stem Cell and Production thereof
CN109294994A (en) Effectively repair the method and application of thalassemia Westmead mutation
CN107119020A (en) A kind of hepatic injury targeting mescenchymal stem cell based on miR 9 and preparation method and application
CN115820540A (en) Endothelial differentiation inducer for mesenchymal stem cells
CN103146755A (en) Method for inducing synovium mesenchymal stem cells to be differentiated to chondrocytes by in-vitro lentivirus mediated BMP-2 (Bone Morphogenetic Protein) genes
JP6711756B2 (en) Method for differentiating into adipocytes using pluripotent stem cells derived from mesenchymal stem cells
Zhang et al. Effects of RPE‑conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration
CN107022522A (en) Direct transdifferentiation technology from cord blood CD 34 positive cell to mescenchymal stem cell
CN106318979A (en) Method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells
CN105624115B (en) Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into nerve-like cells and induction method thereof

Legal Events

Date Code Title Description
DD01 Delivery of document by public notice

Addressee: Zhao Kan

Document name: Notification of Passing Examination on Formalities

PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170808

RJ01 Rejection of invention patent application after publication