CN102994447B - A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells - Google Patents
A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells Download PDFInfo
- Publication number
- CN102994447B CN102994447B CN201110268905.3A CN201110268905A CN102994447B CN 102994447 B CN102994447 B CN 102994447B CN 201110268905 A CN201110268905 A CN 201110268905A CN 102994447 B CN102994447 B CN 102994447B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- hair follicle
- hair
- follicle 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.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 55
- 210000003780 hair follicle Anatomy 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 22
- 210000002894 multi-fate stem cell Anatomy 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 50
- 210000004209 hair Anatomy 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 241001430294 unidentified retrovirus Species 0.000 claims abstract description 5
- 230000000249 desinfective effect Effects 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims abstract description 3
- 230000006698 induction Effects 0.000 claims abstract description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
- 230000001177 retroviral effect Effects 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 210000004709 eyebrow Anatomy 0.000 claims description 5
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 claims description 4
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 4
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 4
- 230000003203 everyday effect Effects 0.000 claims description 4
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 claims description 3
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 claims description 3
- 206010038997 Retroviral infections Diseases 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 6
- 230000003321 amplification Effects 0.000 abstract description 3
- 230000029087 digestion Effects 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 208000028990 Skin injury Diseases 0.000 abstract description 2
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 238000012797 qualification Methods 0.000 abstract description 2
- 102000018317 Keratin-19 Human genes 0.000 abstract 1
- 108010066302 Keratin-19 Proteins 0.000 abstract 1
- 108091005804 Peptidases Proteins 0.000 abstract 1
- 239000004365 Protease Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 12
- 240000001624 Espostoa lanata Species 0.000 description 7
- 235000009161 Espostoa lanata Nutrition 0.000 description 7
- 210000001671 embryonic stem cell Anatomy 0.000 description 7
- 235000015097 nutrients Nutrition 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 4
- 210000002304 esc Anatomy 0.000 description 4
- 210000003128 head Anatomy 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000700605 Viruses Species 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 108010007093 dispase Proteins 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 210000002444 unipotent stem cell Anatomy 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000003995 blood forming stem cell Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 210000000472 morula Anatomy 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000009256 replacement therapy Methods 0.000 description 2
- 230000008672 reprogramming Effects 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 2
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 1
- 241000772415 Neovison vison Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000754835 Sapovirus isolates Species 0.000 description 1
- 206010042566 Superinfection Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000009668 clonal growth Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 210000002337 follicle stem cell Anatomy 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002660 stem cell treatment Methods 0.000 description 1
- 239000003206 sterilizing agent Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 210000003014 totipotent stem cell Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to and a kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells, its concrete steps are: root is pasted at the position of the hair for preparing to choose and cuts off to only staying about 0.2-0.3cm hair root, 75% alcohol disinfecting 10min; Rinsing; Single hair kind is entered in culture dish, adopts hair follicle stem cells culture medium culturing; Often within 3-4 days, liquid is changed once after 1 week; E, retrovirus are packed; Induction hair follicle stem cells reprogrammed is iPS cell; The iPS cell maintain in hair follicle stem cells source and qualification.Process of the present invention is simple; Become unicellular without the need to protease digestion; In the hair follicle stem cells of amplification, β-integrin and cytokeratin-19 positive rate reach more than 90%; For the molecular mechanism research of the quick reparation of skin injury, the growth of hair follicle stem cells and generation, epigenetics research and reprogrammed for induced multi-potent stem cells provides sufficient seed cell to originate.
Description
Technical field
The present invention relates to stem cell field, particularly a kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells.
Background technology
Stem cell can be divided into adult stem cell (adultstemcells) and embryonic stem cell (embryonicstemcells) according to cell development process, according to features such as the differentiation potentials of stem cell, stem cell can be able to be divided into myeloid-lymphoid stem cell (totipotentstemcells), multipotential stem cell (pluripotent/multipotentstemcells), specially can stem cell (unipotentstemcells) and terminally differentiated cells.Myeloid-lymphoid stem cell can be differentiated to form whole cell that human body has or grow and form new individuality, refers to that zygote in embryo development procedure in early days and zygote divide formation 2, the cell in 4 or 8 blastomere periods further.Multipotential stem cell (English is expressed as: pluripotentstemcells) can be differentiated to form whole cell that human body has but can not grow and form new individuality, is mainly present among embryo.In early days in embryo development procedure, zygote divides the morula of formation further, and morula is differentiated to form blastocyst more further; The one-tenth idiosome cell of blastocyst, and the cell before this stage, as long as extract a cell, can be differentiated to form whole cells that human body has.Another notional multipotential stem cell (English is expressed as: multipotentstemcells) has been growth more late period of above-mentioned cell, as cell and the hemopoietic stem cell of each germinal layer.These cells can be divided into a variety of cell, as hemopoietic stem cell can be divided into various white corpuscle, red corpuscle, thrombocyte etc.Unipotent stem cell or specially energy stem cell, can only be divided into a kind of cell, or be divided into the close two kinds of cells (also transdifferentiationof can occur under special conditions) of Relationship Comparison.Unipotent stem cell exists widely, has existence at most tissues organ.
Embryonic stem cell (embryonicstemcells, EScells) a kind ofly derives from body early embryo, can cultivate, maintain normal karyotype, have the multipotential cell that self and differentiation and development are 3 embryonic tissue cell potential by Long Term Passages in vitro.The people such as Britain Evans in 1981 set up the biochemical embryonic stem cell strain of use of first mouse.After this, scientist establishes the ES clones such as pig, ox, sheep, goat, rabbit, mink, hamster, rhesus monkey, marmoset again in succession.For research cytodifferentiation, animal development, set up correlative study model, illustrate gene function etc. and open wide research space.1998, Univ Wisconsin-Madison USA Thomson taught, and after obtaining embryo owner agreement, isolates embryonic stem cell, and cultivate in vitro successfully from being the remaining embryo of tube baby.In the same year, the people such as professor Gearhart of John-John Hopkins University of the U.S. isolate primordial germ stem cell from the ovary and testis tissue of early stage (about 6 weeks) Aborted fetus, and establish embryonic germ cell system.To modify the variation day by day of directed differentiation means and ripe in vitro of the widespread use in animal model, the foundation of mammal body-cell neucleus transplanting technology and day by day perfect, ES cell at the mammalian genetic such as mouse along with homologous recombination technique, make ES cell in multiple field, as the aspect such as cell therapy, organizational project illustrates great potential, be considered to the hope in the future of Transplanted cells replacement therapy.
2006,4 transcription factor: Oct4 are found when the systematic study mouse such as Yamanaka and people ESCs multipotency correlation factor, Sox2, Klf4, c-Myc, after these four factors are imported mouse fibroblast cell, inoblast reprogrammed forms the similar multipotential stem cell of ESCs, be called as induced multi-potent stem cells (mouseinducedPluripotentStemcells, miPSCs).Yamanaka and Yu in 2007 etc. independently establish people's iPS cell line (humaniPSCs, hiPSCs).This achievement in research is described as the new milestone of life science, the regulatory mechanism that it is not only research versatility brings notional innovation and provides new research model, more solve maximum difficult point in current cellular replacement therapy it-transplant after immune rejection problems-open new approach.IPSCs cell is consistent with ESCs on form, versatility maintenance and differentiation potential, and overcomes the problems such as the inevitable ethics dispute of ESCs and variant cell transplant rejection.Therefore, hiPSCs replaces hESCs to become the important cell derived of future clinical translational medicine gradually.
In recent years, investigators successively from various kinds of cell type reprogrammed to obtain hiPSCs thin, as skin flbroblast, mesenchymal stem cells MSCs, liver cell and gastric epithelial cell, neural precursor, islet cells, human skin and hair follicle keratinocyte, fat stem cell, amniotic fluid-derived cell, Blood precursor cells, neural stem cell, cord blood stem cell, umbilical cord Wharton jelly and amnion-derived cell, periphery blood T lymphocyte etc.Thisly obtain by surgical means the ethnics Problem that iPS that seed cell prepares patient-specific is expected to overcome immunological rejection and the deep layer brought in embryonic stem cell treatment by biopsy or other.Above-mentioned different somatocyte has different features in hiPSCs reprogrammed process, and aforesaid method is in the middle of the seed cell process required for obtaining, because will by means of the means such as biopsy or blood drawing, damage to a certain extent will be caused to patient, so need a kind of new Wicresoft or noninvasive method to obtain seed cell, and this seed cell can be iPS cell by reprogramming expeditiously.
Summary of the invention
For the problem in background technology, the invention provides following technical scheme:
Cultivate amplifying human hair follicle stem cells and reprogrammed is a method for induced multi-potent stem cells, its concrete steps are:
A, by prepare choose hair position paste root cut off to only staying about 0.2-0.3cm hair root, 75% alcohol disinfecting 10min;
B, fast root of hair to be extracted with clean eyebrow tweezer, and insert rinsing in aseptic PBS fast;
C, single hair kind is entered in culture dish, adopt hair follicle stem cells culture medium culturing;
D, observe the growing state of hair every day, after 1 week, often within 3-4 days, change liquid once;
E, retrovirus are packed: adopt reagent Fugene6 and PLATA cell to carry out retroviral packaging, utilize pMX-hOct4, pMX-hSox2, pMX-hKlf4 and pMX-hc-Myc to pack out four kinds of retrovirus respectively;
F, by retroviral infection hair follicle stem cells, impel it to express the mankind Oct4, Sox2, Klf4 and c-Myc gene of external source, induction hair follicle stem cells reprogrammed is iPS cell;
The iPS cell maintain in G, hair follicle stem cells source and qualification.
As the preferred technical solution of the present invention, in described steps A: the sterilizing before use of little scissors, tweezers and cotton balls, preferably to prepare between one aseptic, or through the room of ultraviolet sterilization, or (spray whole room with the ethanol of 70%) in patients home temporarily; As collection head originator, cut off the long hair of 2cmx2cm area after ear, only stay the hair of 2-3 millimeters long, cut hair part and peripheral part 10X10cm area with the ethanol cotton balls erasing of 70%, after five min again with 70% the wiping of ethanol cotton balls once.
As the preferred technical solution of the present invention, the hair follicle stem cells substratum cultivated in described step C is: containing the DMEM/F12 of 1%B27,10ng/mLbFGF and 50ng/mLEGF.
As the preferred technical solution of the present invention, in described step e: first 2 days of transfection, prepare 4 100mm culture dish, respectively inoculate 2 × 106 PLATA cells; Transfection same day, the PLAT-A cell confluency degree of 4 100mm culture dish reaches 70%, meets cell quantity requirement needed for transfection; Get 4 EP pipe, eachly add 300ulOPTI-MEM and 27ulFugene6, play even gently, incubated at room 5min; The each 9 μ g of pMX-hOct4, pMX-hSox2, pMX-hKlf4 and pMX-hc-Myc add in above-mentioned 4 EP pipes respectively, fully after mixing, and incubated at room 15min; Mixed solution in above-mentioned 4 EP pipe is added to 4 100mm culture dish respectively, and after shaking up culture dish gently, be placed on 37 DEG C, the incubator of 5%CO2 is cultivated; After transfection 24h, 4 culture dish carry out changing liquid, respectively add 10mL not containing dual anti-PLATA minimum medium; After transfection 48h, collect 4 containing transcription factor viral supernatants to 50mL centrifuge tube, mixing, about 36mL; For infecting hair follicle stem cells after collecting above-mentioned virus.
As the preferred technical solution of the present invention, in described step F: the total cellular score that the above-mentioned four kinds of mixing retroviral infections with 1: 1: 1: 1 shift to an earlier date 24h preparation is the inoblast of 0.5-1x105, and selects 10ng/mLPolybrene to increase efficiency of infection; Change fresh medium into after 8 hours, repeat once after 24 hours.Last repetition shifted infected cell to MEFs after 24 hours, iPS nutrient solution is changed into after 24 hours, change training liquid every day afterwards, observation of cell metamorphosis also records first clone's time of occurrence, until choose clone's enlarged culturing when Ahau clone is enough large.
As the preferred technical solution of the present invention, in described step G: primary iPS clone, enough large or iPS clonal growth went down to posterity before converging, and within general 5 ~ 7 days, went down to posterity once; Two kinds of propagating methods can be adopted: mechanical process, be mainly used in early stage iPS clone and select and go down to posterity: under stereoscopic microscope, with syringe needle, iPS clone is peeled off with feeder layer cells, and be cut into several piece; With 200uL rifle head gently by its reverse shovel at the bottom of feeder layer and culture dish, and transferred species is on new feeder layer, moves in incubator and cultivates; Next day, major part iPS clone was adherent, changed liquid every day subsequently and observed; Digestion method: the nutrient solution added containing 1mg/mLCollageIV and 1mg/mLDispase is placed in incubator and digests 10-15min; Collection iPS clone is also centrifugal, the resuspended rear recentrifuge of precipitation iPS nutrient solution; The precipitation iPS nutrient solution of iPS clone is resuspended, and pressure-vaccum becomes the little agglomerate of 50 ~ 100 cells gently, by 1: 3 ~ 1: 5 points of kinds on new feeder layer, continues to cultivate in incubator; Timing is observed and changes liquid.
The beneficial effect that detection method of the present invention and detection kit are brought is:
1, the hair follicle stem cells amplifying preparation process in a kind of human hair, recklessly moustache, armpit hair and pubes source is provided.
2, the hair follicle stem cells substratum in hair, recklessly moustache, armpit hair and pubes source is provided;
3, the method that hair follicle stem cells reprogrammed is induced multi-potent stem cells is provided;
4, method provided by the invention only needs collection to be low to moderate single hair, without the need to carrying out biopsy to individuality or extracting blood, greatly reduces the misery of people;
Only need directly to choose with after sterilizing agent process five min when 5, gathering hair follicle, process is simple;
6, single hair follicle can obtain 0.5-1x10 through the cultivation of two weeks
5hair follicle stem cells number, cell concentration is enough attached most importance to and is programmed for the cell derived that generate induced pluripotent stem cells provides sufficient, simultaneously for the molecular mechanism research of the quick reparation of skin injury, the growth of hair follicle stem cells and generation, epigenetics research and induced multi-potent stem cells provide the seed cell of abundance to originate.
Accompanying drawing is sketched
Fig. 1 shows the hair follicle stem cells ball (magnification 20x) grown out from people's hair of suspension culture.
Fig. 2 shows induced multi-potent stem cells clone (magnification 10x) come from hair follicle stem cells reprogramming.
Embodiment
Below preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
In embodiment, the meaning that following shortenings is expressed as follows, the shortenings not adding definition uses its usual acceptable definition:
PBS=phosphoric acid buffer; DMEM/F12=DMEM and F12 is 1: 1 mixed cell culture base; Cm=centimetre; BFGF=Prostatropin; EGF=epithelical cell growth factor; HBSS=Hank ' s balanced salt solution; DEG C=degree Celsius; Min=min; ML=milliliter; μ L=microlitre.
First part: hair follicle stem cells collection
1, reagent and instrument
1. 70% ethanol;
②PBS；
③DMEM/F12；
4. 100x green grass or young crops/Streptomycin sulphate storing solution;
5. cotton balls;
6. 500 milliliters of Plastic Bottles with nozzle;
7. 24 well culture plates;
8. one, little scissors;
9. eyebrow tweezers one.
2, the collection of hair follicle:
1. little scissors, eyebrow tweezers and cotton balls sterilizing before use, under aseptic condition, the 24 every holes of well culture plate add 200uLDMEM/F12 and 2uL100x green grass or young crops/Streptomycin sulphate storing solution;
2. aseptic is prepared between one, or through the room of ultraviolet sterilization, or (spray whole room with the ethanol of 70%) in patients home temporarily;
3. the ethanol that collected hair follicle person changes aseptic clothes or clothes spray 70% enters aseptic and room through ultraviolet sterilization;
4. collection head originator: the long hair cutting off 2cmx2cm area after ear, only stays the hair of 2-3 millimeters long;
5. hair part and peripheral part 10X10cm area is cut with the ethanol cotton balls erasing of 70%;
6. the ethanol cotton balls again with 70% after five min is wiped once;
7. hold eyebrow tweezers after ten min under operator's aseptic condition and gather hair root 5 to 10, under aseptic condition, put into 24 well culture plates;
8. send back in laboratory in one hour, unconditional person 4 degree of stored refrigerated can send laboratory back in 24 hours.
Second section: hair follicle stem cells is cultivated and gone down to posterity
1, the cultivation of hair follicle stem cells
Reagent and instrument
1. DMEM/F12 liquid nutrient medium (Invitrogen, Cat.no.11330-32);
2. B27 additive;
3. bFGF cytokine;
4. EGF Urogastron;
5. 100X is dual anti-;
⑥PBS；
⑦HBSS(Invitrogen，Cat.no.14170-088)；
8. Dispase enzyme (bectonDickinson, S.A., cat.no.354235);
⑨TrypLEExpressStableTrypsinReplacement(Invitrogen，Cat.no.12604-039)；
10. CO2 incubator;
bechtop;
culturing bottle.
2, hair follicle stem cells is cultivated
1. the configuration of hair follicle stem cells substratum: DMEM/F12,2%B27,20ng/mlbFGF, 20ng/mlEGF, 1% is dual anti-;
2. every hole adds 1mlPBS rinsing hair once;
3. every hole adds 2ml hair follicle stem cells substratum;
4. put 37 DEG C, cultivate in 5%CO2 incubator, within every 3 days, change liquid once, totally two weeks.
3, hair follicle stem cells digests and goes down to posterity
1. by hair follicle stem cells with PBS rinsing once, add 0.5mlHBSS configuration Dispase Digestive system (500U/ml), depending on hair follicle stem cells group size, 4 degree digest 1--5 hour;
2. Dispase Digestive system is exhausted, add the TrypLEExpressStableTrypsinReplacement of 0.5ml, 37 degree of digestion 20min;
3. neutralize with the serum of 10%, more extremely unicellular with the rifle head piping and druming of 200ul;
4. be transferred in the Ep pipe of 1.5ml sterilizing, the centrifugal 5min of 300xg;
5. single hair follicle stem cells is again planted in or goes down to posterity without in the 6 hole culture dish (first embedding at the bottom of ware with Matrigel) of MEF.
Part III: hair follicle stem cells reprogrammed is generate induced pluripotent stem cells (iPS)
1, reagent prepares
1. pMSCV retroviral plasmid (containing Oct-4, Sox-2, Klf4, c-Myc and GFP expressed sequence, Addgene);
2. FuGENE6 transfection reagent (RocheAppliedScience, cat.no.1181509001);
③EpiLife。
2, the retroviral amplification of pMSCV
1. thaw pipe 293 cell, to the fusion of 80% in amplification cultivation to the culture dish of 10cm diameter, adds the pMSCV retroviral plasmid DNA (extremely slow adds) in nutrient solution of the FuGENE6/9ug of 27ul;
2. put 37 DEG C, cultivate 24 hours in 5%CO2 incubator;
3. add fresh substratum 10ml, put 32 DEG C, cultivate 24 hours again in 5%CO2 incubator;
4. gather vial supernatant, and add the new DMEM substratum of people;
5. vial supernatant 0.45ulPVDF membrane filtration, and add 1ulpolybrene (10mg/ml)/ml and help and turn agent.
3, the retroviral transfection of pMSCV
1. when be planted in or without MEF culture dish (first embedding at the bottom of ware with Matrigel) in hair follicle stem cells when reaching the fusion of 30%, add fresh vial supernatant and (use 0.45ulPVDF membrane filtration, and add 1ulpolybrene (10mg/ml)/ml and help and turn agent) 1ml (6 hole ware), 32 DEG C of 10min;
2. by centrifugal for 6 hole wares, 650xg, 32 DEG C of 45min;
3. after centrifugal, 6 hole wares put 32 DEG C, cultivate 20min in 5%CO2 incubator;
4. remove the liquid containing virus particle, wash secondary with PBS, add new EpiLif; e
5. after 24 hours, by 1. above-mentioned ~ 4. method superinfection virus is once;
6., after cultivating 24 ~ 48 hours again, infected cell is transferred in new MEF culture dish;
7. within every two days, liquid is changed once after, should visible little cloning cluster after 4-5 days, should large cloning cluster as seen after 14 ~ 28 days;
8. choose 5-7 typical cloning cluster to carry out identifying and going down to posterity.
The above; be only the specific embodiment of the present invention; but protection scope of the present invention is not limited thereto; any those of ordinary skill in the art are in the technical scope disclosed by the present invention; the change can expected without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (1)
1. cultivate amplifying human hair follicle stem cells and reprogrammed is a method for induced multi-potent stem cells, it is characterized in that, its concrete steps are:
A, by prepare choose hair position paste root cut off to only staying about 0.2-0.3cm hair root, 75% alcohol disinfecting 10min;
B, fast root of hair to be extracted with clean eyebrow tweezer, and be placed in aseptic PBS rinsing fast;
C, enter in culture dish by single hair kind, adopt hair follicle stem cells culture medium culturing, to obtain hair follicle stem cells, described hair follicle stem cells substratum is the DMEM/F12 containing 1%B27,10ng/mlbFGF and 50ng/mlEGF;
D, observe the growing state of hair every day, after 1 week, often within 3-4 days, change liquid once;
E, retrovirus are packed: adopt reagent Fugene6 and PLATA cell to carry out retroviral packaging, utilize pMX-hOct4, pMX-hSox2, pMX-hKlf4 and pMX-hc-Myc to pack out four kinds of retrovirus respectively;
F, by retroviral infection hair follicle stem cells, impel it to express the mankind Oct4, Sox2, Klf4 and c-Myc gene of external source, induction hair follicle stem cells reprogrammed is iPS cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110268905.3A CN102994447B (en) | 2011-09-13 | 2011-09-13 | A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110268905.3A CN102994447B (en) | 2011-09-13 | 2011-09-13 | A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102994447A CN102994447A (en) | 2013-03-27 |
| CN102994447B true CN102994447B (en) | 2015-12-02 |
Family
ID=47923560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110268905.3A Active CN102994447B (en) | 2011-09-13 | 2011-09-13 | A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102994447B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2857501A1 (en) * | 2013-10-03 | 2015-04-08 | ETH Zurich | Reprogramming of pluripotent stem cells for improved control of their differentiation pathways |
| CN104789522A (en) * | 2015-01-30 | 2015-07-22 | 上海坤爱生物科技有限公司 | Construction method for human hair follicle stem cell bank |
| CN116898988A (en) * | 2023-08-15 | 2023-10-20 | 臻赫医药(杭州)有限公司 | Skin reprogramming factor preparation, biological material and application |
-
2011
- 2011-09-13 CN CN201110268905.3A patent/CN102994447B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Efficient and rapid generation of induced pluripotentstem cells from human keratinocytes;Trond Aasen，et al;《nature biotechnology》;20081017;第26卷(第11期);1276－1284 * |
| 毛囊干细胞的分离培养与鉴定;徐红;《中国优秀硕士学位论文全文数据库.医药卫生科技辑》;20090915;第20页 * |
| 皮肤干细胞将成为细胞治疗神经系统疾病的新的细胞来源;杜宝玲 等;《解剖学研究》;20061231;第28卷(第3期);226－228 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102994447A (en) | 2013-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US12344861B2 (en) | Method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord | |
| CN102344906B (en) | A method for isolating and culturing hair follicle stem cells | |
| CN102827805A (en) | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method | |
| ATE517177T1 (en) | METHOD FOR CULTIVATION OF HUMAN MESENCHYMAL STEM CELLS, IN PARTICULAR FOR THE TREATMENT OF NON-HEALING BREAKS, AND BIOREACTOR FOR CARRYING OUT THIS CULTIVATION METHOD | |
| BR112020020092A2 (en) | method of inducing or improving mesenchymal stem cell wound healing properties | |
| CN104087551A (en) | Novel method for in-vitro separated culture of human epidermal cells | |
| CN102002477B (en) | Method for culturing and amplifying odontogenic epithelial cells | |
| CN103275928B (en) | Technical method for differentiating human skin stem cells in vitro into primordial germ cells | |
| CN103550828B (en) | A kind of skin renewal method based on hair follicle stem cells and Silica hydrogel dressing | |
| CN101372682B (en) | Construction method of Epinephelus fuscoguttatus fin cell line | |
| CN102994447B (en) | A kind ofly cultivate amplifying human hair follicle stem cells and reprogrammed is the method for induced multi-potent stem cells | |
| CN103396985A (en) | Method for inducing differentiation of human umbilical cord mesenchymal stem cells into hepatocytes and applications | |
| CN106754729A (en) | The method for preparing inductive pluripotent stem cells using Xist Tale inhibition transcription factors | |
| CN105695401B (en) | The preparation of all stem cells of a kind of umbilical artery and vein blood vessel and store method | |
| CN104789522A (en) | Construction method for human hair follicle stem cell bank | |
| EA017967B1 (en) | Method for producing fibroplast cells from the newborn's navel-cord | |
| CN106497863A (en) | A kind of separation of cornea of rats endothelial cell, purifying and cultural method | |
| CN103881970A (en) | Rat dermal mesenchymal stem cell culture method | |
| CN112626006B (en) | Method for separating and culturing villous goat hair follicle hair papilla cells | |
| CN201793580U (en) | Culturing device special for amniotic fluid/chorionic cell in-situ cultivation | |
| CN103382456B (en) | A kind of induction method of sexual cell | |
| CN108611316A (en) | A method of induction human fibroblasts transdifferentiation is human germ-line | |
| CN102732478A (en) | Inducer for inducing directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells and preparation and application thereof | |
| CN101892155A (en) | Culture apparatus special for in-situ culture of amniotic fluid/chorionic cells and application thereof | |
| CN106987557A (en) | Improved method for culturing human skin pluripotent stem cells (SKPs) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: ZHEJIANG XINGBO BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: CHEN BINGLAI Effective date: 20131107 Free format text: FORMER OWNER: GAO QING CENG QIAO Effective date: 20131107 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 215123 SUZHOU, JIANGSU PROVINCE TO: 315040 NINGBO, ZHEJIANG PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20131107 Address after: 315040 Zhejiang high tech District of Ningbo City Xinhui Road No. 139 Building No. 2 Applicant after: Zhejiang Cellpro Biotech Co., Ltd. Address before: Suzhou City, Jiangsu province 215123 Xinghu Street No. 218 BioBAY C15 building 401 room Applicant before: Chen Binglai Applicant before: Gao Qing Applicant before: Zeng Qiao |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 315040 Zhejiang high tech District of Ningbo City Xinhui Road No. 139 Building No. 2 Applicant after: Chen Binglai Address before: 315040 Zhejiang high tech District of Ningbo City Xinhui Road No. 139 Building No. 2 Applicant before: Zhejiang Cellpro Biotech Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |