CN102161980A - Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast - Google Patents
Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast Download PDFInfo
- Publication number
- CN102161980A CN102161980A CN 201110036898 CN201110036898A CN102161980A CN 102161980 A CN102161980 A CN 102161980A CN 201110036898 CN201110036898 CN 201110036898 CN 201110036898 A CN201110036898 A CN 201110036898A CN 102161980 A CN102161980 A CN 102161980A
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cells
- mesenchymal stem
- ipscs
- people
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 210000004263 induced pluripotent stem cell Anatomy 0.000 title abstract description 10
- 210000002901 mesenchymal stem cell Anatomy 0.000 title abstract description 8
- 238000012258 culturing Methods 0.000 title abstract description 3
- 210000002993 trophoblast Anatomy 0.000 title abstract 4
- 210000004027 cell Anatomy 0.000 claims abstract description 51
- 230000003321 amplification Effects 0.000 claims abstract description 38
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 38
- 210000003953 foreskin Anatomy 0.000 claims abstract description 5
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims description 39
- 210000002304 esc Anatomy 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 13
- 230000009849 deactivation Effects 0.000 claims description 10
- 206010043276 Teratoma Diseases 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 8
- 238000001890 transfection Methods 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 6
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 6
- 230000003203 everyday effect Effects 0.000 claims description 6
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 238000003757 reverse transcription PCR Methods 0.000 claims description 5
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 4
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 4
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 claims description 4
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 claims description 4
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 4
- 238000001727 in vivo Methods 0.000 claims description 4
- 102000002734 Collagen Type VI Human genes 0.000 claims description 3
- 108010043741 Collagen Type VI Proteins 0.000 claims description 3
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 claims description 3
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 claims description 3
- 108091023040 Transcription factor Proteins 0.000 claims description 3
- 102000040945 Transcription factor Human genes 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 230000024835 cytogamy Effects 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000010297 mechanical methods and process Methods 0.000 claims description 3
- 230000005226 mechanical processes and functions Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 241001430294 unidentified retrovirus Species 0.000 claims description 3
- 210000001778 pluripotent stem cell Anatomy 0.000 claims 6
- 238000011156 evaluation Methods 0.000 claims 1
- 210000002950 fibroblast Anatomy 0.000 abstract description 5
- 238000007710 freezing Methods 0.000 abstract description 2
- 230000008014 freezing Effects 0.000 abstract description 2
- 238000010257 thawing Methods 0.000 abstract description 2
- 210000001185 bone marrow Anatomy 0.000 abstract 3
- 238000007796 conventional method Methods 0.000 abstract 1
- 230000001573 trophoblastic effect Effects 0.000 description 22
- 239000000243 solution Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 17
- 101100078010 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MSC7 gene Proteins 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 7
- 210000002894 multi-fate stem cell Anatomy 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 230000001464 adherent effect Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 102100029284 Hepatocyte nuclear factor 3-beta Human genes 0.000 description 2
- 102100028707 Homeobox protein MSX-1 Human genes 0.000 description 2
- 101001062347 Homo sapiens Hepatocyte nuclear factor 3-beta Proteins 0.000 description 2
- 101000985653 Homo sapiens Homeobox protein MSX-1 Proteins 0.000 description 2
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 2
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 description 2
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 2
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 2
- 238000011789 NOD SCID mouse Methods 0.000 description 2
- 108010032788 PAX6 Transcription Factor Proteins 0.000 description 2
- 102100037506 Paired box protein Pax-6 Human genes 0.000 description 2
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 2
- 210000002534 adenoid Anatomy 0.000 description 2
- 238000004115 adherent culture Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000000280 densification Methods 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 239000012997 ficoll-paque Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 101150117600 msc1 gene Proteins 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102100024810 DNA (cytosine-5)-methyltransferase 3B Human genes 0.000 description 1
- 101710123222 DNA (cytosine-5)-methyltransferase 3B Proteins 0.000 description 1
- 102100037126 Developmental pluripotency-associated protein 4 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000881868 Homo sapiens Developmental pluripotency-associated protein 4 Proteins 0.000 description 1
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 1
- 101000835745 Homo sapiens Teratocarcinoma-derived growth factor 1 Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102100026404 Teratocarcinoma-derived growth factor 1 Human genes 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000000251 trophoblastic cell Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for culturing human induced pluripotent stem cells (iPSCs) by using human bone marrow mesenchymal stem cells as a trophoblast. The method comprises the following steps of: obtaining the human iPSCs from child foreskin fibroblasts by using third to fifth generation human bone marrow mesenchymal stem cells obtained through subculture and culture after thawing from low-temperature freezing, performing amplification culture, and inoculating into trophoblast cells to obtain the human iPSCs. In the method, the human iPSCs are cultured by using the human bone marrow mesenchymal stem cells as the trophoblast cells instead of mouse embryonic fibroblasts in the conventional method, so that the pollution of heterologous cells in a culture system is reduced, that the culture system can make the human iPSCs amplified in vitro is proved, biological characteristics and multipotency of the human iPSCs are maintained for a long time, and the possibility of clinical application of the human iPSCs is provided.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of cultivating induced multi-potent stem cells with human marrow mesenchymal stem cell as trophoderm.
Background technology
(Induced Pluripotent Stem Cells iPSCs) is the breakthrough that has milestone significance in the stem-cell research field in recent years to induced multi-potent stem cells.It is by ectopic expression several with keep embryonic stem cell (Embryonic Stem Cells, ESCs) the relevant key nuclear factor of multipotency, make somatocyte generation reprogrammed, the multipotential cell of a kind of similar ESCs that obtains can be divided into tridermic all histocytes in vivo and in vitro.The iPSCs that is come by reprogramming of somatic cells obtains the multipotential stem cell consistent with patient self genetic background to have increased a new way, and no longer use human body early embryo and ovocyte, so ethical arguement will be calmed down thereupon, nuclear transfer technology lacks ovocyte and the numerous and diverse awkward situation of technology has also obtained rational solution.Be the desirable seed cell of organizational project and regenerative medicine, disease model, drug screening, therefore have wide clinical application background.At present, the investigator induces differentiation with people iPSCs to various histocytes external, as hematopoietic stem, red corpuscle, T cell, B cell, myocardial cell, beta Cell of islet, liver cell etc.; Set up multiple heredopathia people iPSCs disease model, be used for Study on Pathogenesis, and on animal model, attempted by the iPSCs that hereditary defect is arranged being carried out genetic modification, the treatment heredopathia; Set up kinds of tumors iPSCs disease model, be used for Study on Pathogenesis and drug screening.
The vitro culture of people iPSCs needs the interaction of cell and cell, various cytokine, matrix to wait to keep its self and multipotency.Its classical training mode is that (Mouse Embryonic fibroblasts MEF) is trophoderm, with containing bFGF, serum substitute (serum replacement, nutrient solution cultivation SR) with mouse embryo fibroblasts.But before people iPSCs entered clinical application, heterologous protein, cell contamination were problems that must solve in the removal culture system.Therefore, after people iPSCs occurred, the investigator just began to attempt improving its culture system, when keeping its self and multipotency, reduces the pollution of heterologous protein, cell.As 2010, Christian etc. used the human skin fibroblast of immortalization as trophoderm, and cultivator iPSCs finds that this humanized trophoderm can produce and keep the cultivation of people iPSCs, but its incubation time is lacked (the cultivation algebraically of report was 7 generations); The same year, Kristiina etc. are with a kind of RegES nutrient solution cultivator iPSCs of no heterologous protein of definite ingredients, find its for a long time (＞80 generation) keep self and the multipotency of iPSCs, but proof is not in the reprogrammed process, and it is iPSCs that this nutrient solution can make the human body cell reprogrammed.Ruth etc. are with the mTeSRTM nutrient solution suspension culture people iPSCs of another kind of definite ingredients, after finding to cultivate for 17 generations, still can keep its self and multipotency, but this culture system costs an arm and a leg, and same proof is not in the reprogrammed process, and it is iPSCs that this nutrient solution can make the human body cell reprogrammed.Therefore, further the humanized culture system of development research people iPSCs comprises the reprogrammed process and keeps cultivation, and to the influence of iPSCs biological characteristics, is that people iPSCs moves towards premise for clinical application.
Summary of the invention
The purpose of this invention is to provide a kind of with human marrow mesenchymal stem cell (Mesenchymal Stem Cells MSCs) as the method for trophoderm cultivator induced multi-potent stem cells (iPSCs), is achieved through the following technical solutions:
(1) trophocyte's preparation: the human marrow mesenchymal stem cell of using the 3rd ~ the 5th generation of cultivating behind go down to posterity cultivation, the low temperature cryopreservation resuscitation is as the trophocyte.Human marrow mesenchymal stem cell adopts Ficoll-paque density gradient centrifugation commonly used at present to cultivate in conjunction with adherent screening and separating and obtains.During as trophoderm, on six orifice plates of cell inoculation after handle with 0.1%gelatin, the cytogamy degree reaches 80%-90%(about 2 * 10
4/ cm
2), and handle deactivation in 2 hours with the ametycin of 10ug/ml, the cell after the deactivation used in 24 hours;
(2) acquisition of people iPSCs: get 2-3 year children foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor.Second day vitaminize C 25ug/ml in culture system added VPA2 μ M(and used altogether 5 days on the 5th day), the inoblast after the transfection was inoculated on the trophocyte in the 6th day, the 7th day, use the people ESCs nutrient solution that contains bFGF, alternative serum instead.Change liquid subsequently every day, the clone up to class ESCs occurring selects, amplification cultivation;
(3) amplification cultivation of people iPSCs: microscopically is selected the clone of class ESCs, after 5-10 minute, dispels into little agglomerate with the enzymic digestion of IV Collagen Type VI gently, is inoculated on the ready trophocyte.Change liquid every day.Went down to posterity once in every 7-8 days.Cell after the amplification goes down to posterity with mechanical process, and promptly the glass dropper with 5ml scrapes cell from trophoderm gently, dispels into little agglomerate subsequently gently, is inoculated on the ready trophocyte;
(4) keeping of the biological characteristics of people iPSCs and multipotency: be that amplification cultivation is identified the ESCs specific gene expression of amplification cultivation descendant iPSCs after 14 generations on the trophoderm with the RT-PCR method with the human marrow mesenchymal stem cell; Whether detect it with EB forming method and immunodeficient mouse teratoma forming method simultaneously can break up to triploblastica in vivo and in vitro.
The invention provides a kind of is the method for human marrow mesenchymal stem cell as trophoderm acquisition cultivator iPSCs with the humanization cell, but biological property and the multipotency of this humanized trophocyte's long term maintenance people iPSCs; Thus obtained people iPSCs does not have the pollution of allos cell, for its clinical application provides possibility.End user's mesenchymal stem cells MSCs of the present invention replaces using in the traditional method mouse embryo fibroblasts as the trophocyte, the cultivator induced multi-potent stem cells, reduced the pollution of allos cell in the culture system, and proved that this culture system can make people's induced multi-potent stem cells at amplification in vitro (seeing embodiment 4), and kept its biological characteristics and multipotency (seeing embodiment 5), provide possibility for people's induced multi-potent stem cells enters clinical application.
Description of drawings
Fig. 1 is technological line figure.
Fig. 2 is the 4th generation human marrow mesenchyme stem cell.Before A is deactivation; After B is the ametycin deactivation.
The people iPSCs of Fig. 3 for obtaining as trophoderm with human marrow mesenchymal stem cell.A is inoblast transfection after 21-25 days, the granuloma sample of appearance clone (scale is 100 μ m), and B is the iPSCs clone (scale is 100 μ m) who forms after the amplification cultivation, C is clone's local cells form (scale is 20 μ m).
To be 8-11 with the 4th generation human marrow mesenchyme stem cell be growth curve in the trophoblastic culture system for people iPSCs to Fig. 4.
Fig. 5 is the 9th, 10 generation people iPSCs at different donors the 4th generation human marrow mesenchyme stem cell of originating is amplification times (hMSC1 and hMSC2 are women's donor source, and hMSC3 originates for male sex's donor) in the trophoblastic culture system.
Fig. 6 for RT-PCR detect people iPS/MSC7 human marrow mesenchymal stem cell be in the trophoblastic culture system 14 generations of amplification cultivation with the expression of descendant ESCs specific gene.
Fig. 7 iPS/MSC7 that behaves is that amplification cultivation is broken up situation at external formation EB to triploblastica after 14 generations in the trophoblastic culture system at human marrow mesenchymal stem cell.A is that the suspension EB(scale that the EB cultivation formed after 8 days is 50 μ m); B-D cultivates the adherent all kinds cell that is divided into of EB (scale is 100 μ m) after 14 days; E is that PT-PCT detects EB differentiation back pluripotency gene (SOX2 and OCT4) and tridermic marker gene (FOXA2 and AFP are the entoderm marker gene; MSX1 and BRACHYURY are the mesoderm marker gene; MAP2 and PAX6 are the ectoderm marker gene) expression (U represents not break up, and D represents the differentiation back).
Fig. 8 iPS/MSC7 that behaves is that amplification cultivation forms the teratoma situation in the trophoblastic culture system in NOD-SCID mouse body after 14 generations at human marrow mesenchymal stem cell.A is for growing teratomatous mouse; B is the adenoid tissue (HE dyeing, scale is 20 μ m) that forms in the teratoma; C is the muscle sample tissue (HE dyeing, scale is 20 μ m) that forms in the teratoma.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1: human marrow mesenchymal stem cell separation and Culture, cryopreservation resuscitation
(1) human marrow mesenchymal stem cell separation and Culture
Aseptic condition is gathered the marrow of 3 parts of healthy donors down, and anticoagulant heparin is with Ficoll-paque(proportion 1.077) density gradient centrifugation collection mononuclearcell, with 5 * 10
5Individual cell/cm
2Density inoculation, nutrient solution is for containing 10%(V/V) the low sugar DMEM(LG-DMEM of foetal calf serum and 1% glutamine) nutrient solution, place 37
0C, 5%CO2 incubator are cultivated.Change nutrient solution after 24 hours, discard non-adherent cell, as seen the fusiformis attached cell is arranged.Changed liquid 1 time in per afterwards 3 days, the adherent fusion of left and right sides primary cell in 14 days reaches 90%-95%, is arranged with obvious directivity, becomes whirlpool shape, netted, radial (seeing Fig. 2 A).With 0.25% trypsinase-1 mmol EDTA digestion, divide the bottle cultivation of going down to posterity in the 1:3 ratio, and be labeled as the P1 cell.Changed liquid once in per 3 days in the culturing process that goes down to posterity, cell reaches 90% and merges the back had digestive transfer culture, and is labeled as P2, by that analogy.
(2) the frozen and recovery of human marrow mesenchymal stem cell
When frozen, will be in the mescenchymal stem cell digestion of logarithmic phase after, with frozen protection liquid (60% LG-DMEM, 30% foetal calf serum, 10% DMSO) re-suspended cell, final concentration of cells is 5 * 10
6/ ml divides to change in the frozen pipe cell title, algebraically, frozen time and frozen person on the mark over to.After immediately frozen pipe being put into the Virahol freezing storing box, put into-80
0C, the back of spending the night moves into-196
0The C liquid nitrogen container is preserved.During cell recovery, from liquid nitrogen, take out cell and put into 37
0C constant water bath box quick-thawing treats that frozen storing liquid thaws to little ice crystal, moves into super clean bench.Under the aseptic condition, cell suspension is moved into the 15ml centrifuge tube, dropwise add the nutrient solution 10ml of precooling, behind the centrifugal 5min of 200g, supernatant discarded.With cell mass with the resuspended cultivation of an amount of preheating nutrient solution.Adherent fully behind the recovery survivaling cell inoculation 6h, cellular form becomes fusiformis gradually.Cell is bred rapidly through beginning after 1-2 days resting stages.Cellular form after the recovery and frozen preceding basically identical.
Embodiment 2: human marrow mesenchymal stem cell is as trophocyte's preparation
Get the 3-5 generation human marrow mesenchyme stem cell as trophoblastic cell.Use 0.1%gelatin 37 in advance 6 orifice plates
0C is hatched 1h to spending the night.Human marrow mesenchymal stem cell is with 2 * 10
4/ cm
2Density is inoculated on six orifice plates after gelatin handles, makes second day cytogamy degree reach 80%-90%.Ametycin with 10ug/ml is handled deactivation in 2 hours, basically identical (see figure 2) before cellular form after the deactivation and the deactivation.Cell after the deactivation used in 24 hours.
Embodiment 3: with human marrow mesenchymal stem cell is inducing of trophoblastic people iPSCs
Get third generation children (2-3 year) foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor.Transfection finish the back in culture system vitaminize C 25ug/ml up to selected clone, adding VPA2 μ M(on the 5th day used 5 days altogether), inoblast after the transfection was inoculated into on the human marrow mesenchymal stem cell trophocyte in the 6th day, used the people ESCs nutrient solution of the bFGF, 1% glutamine, 1% non-essential amino acid, 100 μ M and 20% alternative serum that contain 8ng/ml on the 7th day instead.Change liquid subsequently every day, up to the clone who class ESCs occurs (seeing Fig. 3 A), clear border, cell densification.Microscopically is selected one by one, amplification cultivation.Technological line is seen Fig. 1
Embodiment 4: be the amplification cultivation of trophoblastic people iPSCs with human marrow mesenchymal stem cell
(1) people iPSCs is being the cellular form after the amplification cultivation in the trophoblastic culture system with human marrow mesenchymal stem cell
Microscopically is selected the clone of class ESCs, and the IV Collagen Type VI enzymic digestion of using 1mg/ml dispels into little agglomerate after 5-10 minute gently, is inoculated on the ready trophocyte, and personnel selection ESCs nutrient solution is 37
0C, 5%CO
2The middle cultivation.Change liquid every day.Went down to posterity once in the every 7-8 of iPSCs days.Cell after the amplification goes down to posterity with mechanical process, and promptly the glass dropper with 5ml scrapes cell from trophoderm gently, dispels into little agglomerate subsequently gently, is inoculated on the ready trophocyte with 1:2-1:3.People iPSCs is being that the amplification back forms typical people ESCs sample clone in the trophoblastic culture system with human marrow mesenchymal stem cell: clear border, and the cell densification, most of cell is circular, and kernel is obvious, and the border is class fibroblast-like cells (seeing Fig. 3 B, C).
(2) people iPSCs with human marrow mesenchymal stem cell is being growth kinetics in the trophoblastic culture system
Three strain people iPSCs(iPS/MSC1, iPS/MSC7 and iPS/MSC10 with the 8th generation) with 5 * 10
4Cells/well inoculation (with little agglomerate inoculation) is in trophoblastic 12 orifice plates to being covered with human marrow mesenchymal stem cell, establishes three multiple holes.Cultivate after 7 days, people iPSCs is scraped from trophoderm, get the part counting, calculate amplification times; Getting simultaneously and wherein partly being inoculated into the new human marrow mesenchymal stem cell that is covered with is in trophoblastic 12 orifice plates, establishes three multiple holes, continues to cultivate 7 days, and counting is the calculating amplification times also.By that analogy, cultivate the three generations altogether, calculate amplification times.People iPSCs is being sustainable amplification in the trophoblastic culture system with human marrow mesenchymal stem cell, 2.32 ± 0.05 times of (see figure 4)s of average per generation amplification.
(3) human marrow mesenchymal stem cell of people iPSCs in different donors source is the growing state in the trophoblastic culture system
Three strain people iPSCs(iPS/MSC1, iPS/MSC7 and iPS/MSC10 with the 9th, 10 generations) with 5 * 10
4Cells/well inoculation (with little agglomerate inoculation) is to the human marrow mesenchymal stem cell (hMSC1, hMSC2, the hMSC3 that are covered with different donors source, wherein hMSC1 and hMSC2 are women's donor, hMSC3 is male sex's donor) in trophoblastic 12 orifice plates, establish three multiple holes.Cultivate after 7 days, people iPSCs is scraped from trophoderm, get the part counting, calculate amplification times.The mesenchymal stem cells MSCs in different donors source is trophoblastic culture system does not have the significant difference (see figure 5) to the amplification of people iPSCs cell.
Embodiment 5: people iPSCs is the situation of keeping of amplification cultivation biological characteristics and multipotency after 14 generations in the trophoblastic culture system at human marrow mesenchymal stem cell
(1) people iPSCs expressing human ESCs specific gene situation
Being collected in human marrow mesenchymal stem cell is the people iPSC/MSC7 cell mass of amplification cultivation after 14 generations in the trophoblastic culture system, extract total RNA with TRIZOL, carry out RT-PCR subsequently, detect the expression of people ESCs specific gene: comprise endogenous KLF4, SOX2, c-Myc, OCT4 and NANOG, DNMT3B, DPPA4, hTERT, NODAL, REX13, TDGF1 etc.Be contrast with H1 people ESCs and children's foreskin inoblast simultaneously.Detected result shows that the expression level of these genes in people iPSCs is similar to H1 people ESCs.
(2) people iPSCs breaks up situation at external formation EB to triploblastica
At human marrow mesenchymal stem cell is that the people iPSC/MSC7 of amplification cultivation after 14 generations scrapes from trophoderm in the trophoblastic culture system, is seeded in ultralow adhesion 6 orifice plates with cell mass, and nutrient solution is for not containing the people ESCs nutrient solution of bFGF, 37
0C, 5%CO
2The middle cultivation changed liquid every other day; Cultivate after 8 days, be inoculated in 6 orifice plates after 0.1%gelatin handles, continue with the people ESCs nutrient solution that does not contain bFGF, 37
0C, 5%CO
2Middle adherent culture 8 days.Experimental result shows that the people iPSCs after the amplification can see Fig. 7 A at external formation EB(), after these EB adherent culture, can be divided into various types of cells (seeing Fig. 7 B-D).Collect the cell of these differentiation, detect triploblastica mark expression of gene situation with RT-PCR, the result shows that (FOXA2 and AFP are the entoderm marker gene for cell triploblastica marker gene after the differentiation; MSX1 and BRACHYURY are the mesoderm marker gene; MAP2 and PAX6 are the ectoderm marker gene) obviously increase of expression.Show at human marrow mesenchymal stem cell to be that the people iPSCs of amplification cultivation after 14 generations still kept its multidirectional differentiation potential in the trophoblastic culture system.
(3) people iPSCs forms teratoma in vivo and breaks up situation to triploblastica
To be that the people iPSC/MSC7 of amplification cultivation after 14 generations scrapes from trophoderm in the trophoblastic culture system at human marrow mesenchymal stem cell, with twice of the PBS buffer solution for cleaning that does not contain calcium ions and magnesium ions, with PBS that people iPSCs is resuspended, get in the back leg root muscle that 200ul is expelled to the NOD-SCID mouse, the cell quantity of each injection point is 2-3 * 10
6Observe into the knurl situation after the injection.After treating that teratoma forms, take out teratoma row paraffin section and HE dyeing, observe it and break up situation to triploblastica.Experimental result shows that the people iPSCs after the amplification can form teratoma (seeing Fig. 8 A) in the immunodeficient mouse body, see after the HE dyeing muscle sample is arranged in the knurl, adenoid tissue forms (seeing Fig. 8 B, C).Being illustrated in human marrow mesenchymal stem cell is that the people iPSCs of amplification cultivation after 14 generations still kept its multidirectional differentiation potential in the trophoblastic culture system.
Claims (2)
1. one kind with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, is achieved through the following technical solutions:
(1) trophocyte's preparation: the human marrow mesenchymal stem cell of using the 3rd ~ the 5th generation of cultivating behind go down to posterity cultivation, the low temperature cryopreservation resuscitation is as the trophocyte, on six orifice plates of cell inoculation after handle with 0.1%gelatin, cytogamy degree 80%-90%, and handle deactivation in 2 hours with the ametycin of 10ug/ml, the cell after the deactivation used in 24 hours;
(2) acquisition of people's inductive pluripotent stem cells: get 2-3 year children's foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor, second day vitaminize C 25ug/ml in culture system, added VPA2 μ M on the 5th day, inoblast after the transfection was inoculated on the trophocyte in the 6th day, the 7th day, use the people ESCs nutrient solution that contains bFGF, alternative serum instead, change liquid subsequently every day, clone up to class ESCs occurring selects, amplification cultivation;
(3) amplification cultivation of people's inductive pluripotent stem cells: microscopically is selected the clone of class ESCs, with the enzymic digestion of IV Collagen Type VI after 5-10 minute, dispel into little agglomerate gently, be inoculated on the ready trophocyte, change liquid every day, went down to posterity once in every 7-8 days, the cell after the amplification goes down to posterity with mechanical process;
(4) keeping of the biological characteristics of people's inductive pluripotent stem cells and multipotency: with the human marrow mesenchymal stem cell be amplification cultivation after 14 generations on the trophoderm, whether the ESCs specific gene expression with RT-PCR method evaluation amplification cultivation descendant inductive pluripotent stem cells detects it with EB forming method and immunodeficient mouse teratoma forming method simultaneously and can break up to triploblastica in vivo and in vitro.
2. according to claim 1 a kind of with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, it is characterized in that, the described amplification cultivation of step (3) is that the glass dropper with 5ml scrapes cell from trophoderm gently, dispel into little agglomerate subsequently gently, be inoculated on the ready trophocyte.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110036898 CN102161980B (en) | 2011-02-12 | 2011-02-12 | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110036898 CN102161980B (en) | 2011-02-12 | 2011-02-12 | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102161980A true CN102161980A (en) | 2011-08-24 |
| CN102161980B CN102161980B (en) | 2013-07-31 |
Family
ID=44463434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110036898 Expired - Fee Related CN102161980B (en) | 2011-02-12 | 2011-02-12 | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102161980B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286532A (en) * | 2011-09-05 | 2011-12-21 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102965393A (en) * | 2012-11-06 | 2013-03-13 | 上海交通大学 | Method for preparing human induced pluripotent stem cells and use of human induced pluripotent stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104140951A (en) * | 2014-08-06 | 2014-11-12 | 深圳市科晖瑞生物医药有限公司 | Method for developing and cultivating human induced pluripotent stem cells |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN107236703A (en) * | 2017-05-25 | 2017-10-10 | 佛山科学技术学院 | A kind of feeder cells, cultural method and application |
| CN109975264A (en) * | 2019-04-18 | 2019-07-05 | 安徽农业大学 | A kind of method that slow-virus transfection blastaea trophocyte studies its gene function |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649305A (en) * | 2009-09-17 | 2010-02-17 | 中国医学科学院血液学研究所 | Method for amplifying megakaryocyte progenitor cell from human cord blood CD34<+> cell |
| WO2010099539A1 (en) * | 2009-02-27 | 2010-09-02 | Cellular Dynamics International, Inc. | Differentiation of pluripotent cells |
-
2011
- 2011-02-12 CN CN 201110036898 patent/CN102161980B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010099539A1 (en) * | 2009-02-27 | 2010-09-02 | Cellular Dynamics International, Inc. | Differentiation of pluripotent cells |
| CN101649305A (en) * | 2009-09-17 | 2010-02-17 | 中国医学科学院血液学研究所 | Method for amplifying megakaryocyte progenitor cell from human cord blood CD34<+> cell |
Non-Patent Citations (2)
| Title |
|---|
| 《Cell Reprogram》 20100630 Park Y.et al. The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture 315-328 1、2 第12卷, 第3期 * |
| 《Hum Mol Genet》 20101214 Raymond M.Anchan et al. Amniocytes can serve a dual function as a source of iPS cells and feeder layers 962-974 1、2 , * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286532B (en) * | 2011-09-05 | 2012-10-17 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102286532A (en) * | 2011-09-05 | 2011-12-21 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102965393B (en) * | 2012-11-06 | 2015-08-26 | 上海交通大学 | The preparation method and its usage of people's induced multi-potent stem cells |
| CN102965393A (en) * | 2012-11-06 | 2013-03-13 | 上海交通大学 | Method for preparing human induced pluripotent stem cells and use of human induced pluripotent stem cells |
| CN103589686B (en) * | 2013-11-06 | 2015-11-18 | 四川新生命干细胞科技股份有限公司 | A kind of human umbilical cord mesenchymal stem cells that uses is as the method for feeder layer cultivator induced multi-potent stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104140951A (en) * | 2014-08-06 | 2014-11-12 | 深圳市科晖瑞生物医药有限公司 | Method for developing and cultivating human induced pluripotent stem cells |
| CN104140951B (en) * | 2014-08-06 | 2016-08-24 | 深圳市科晖瑞生物医药有限公司 | A kind of method set up and cultivate people induced multi-potent stem cell |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN106381282B (en) * | 2016-10-12 | 2020-08-07 | 广东艾时代生物科技有限责任公司 | Passage method for induced pluripotent stem cells |
| CN107236703A (en) * | 2017-05-25 | 2017-10-10 | 佛山科学技术学院 | A kind of feeder cells, cultural method and application |
| CN107236703B (en) * | 2017-05-25 | 2021-06-01 | 佛山科学技术学院 | Feeder layer cell, culture method and application |
| CN109975264A (en) * | 2019-04-18 | 2019-07-05 | 安徽农业大学 | A kind of method that slow-virus transfection blastaea trophocyte studies its gene function |
| CN109975264B (en) * | 2019-04-18 | 2022-04-08 | 安徽农业大学 | Method for researching gene function of lentivirus transfected blastocyst trophoblast cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102161980B (en) | 2013-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102161980B (en) | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast | |
| CN103396990B (en) | Method for preparing mesenchymal stem cells | |
| CN105219707B (en) | A kind of method of recovery fat mesenchymal stem cell | |
| CN105238750B (en) | A kind of method of recovery umbilical cord mesenchymal stem cells | |
| CN103275922B (en) | A kind of fine-wool sheep hair follicle stem cells isolated culture method | |
| CN104694460A (en) | Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell | |
| CN103667349B (en) | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) | |
| CN101974488B (en) | Immortalized porcine pancreatic stem cell line and construction and differentiation methods thereof | |
| CN102140438A (en) | In-vitro culturing method for porcine skeletal muscle satellite cell (SMSC) | |
| CN102168065A (en) | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof | |
| CN104232574A (en) | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte | |
| CN105238749B (en) | A kind of method of recovery mesenchymal stem cell | |
| CN103589686B (en) | A kind of human umbilical cord mesenchymal stem cells that uses is as the method for feeder layer cultivator induced multi-potent stem cells | |
| CN105200005A (en) | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer | |
| CN102286535B (en) | Method for transdifferentiation of fibroblasts into hepatic stem cells | |
| CN105420193B (en) | Differential medium and its purposes in preparation neural stem cell | |
| CN107937346A (en) | A kind of method by the use of human urine cell as feeder layer culture induced multi-potent stem cell | |
| CN101984050B (en) | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof | |
| CN102899288A (en) | Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells | |
| CN112920989A (en) | Liver organoid model, establishment method and application thereof, and pharmaceutical composition for treating hepatocyte iron death | |
| CN111344392B (en) | Cell induction method | |
| CN102618500A (en) | Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro | |
| CN105670989A (en) | Kit and method for effectively inducing somatic cell phenotype reprogramming | |
| CN100540659C (en) | A kind of method and special culture media thereof of cultivating mouse embryo stem cell | |
| CN104140951B (en) | A kind of method set up and cultivate people induced multi-potent stem cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130731 Termination date: 20200212 |