The in-induced pluripotent stem cells (iPS) technology not only solves the ethical problems associated with the source of embryonic stem cells, but also avoids the immune rejection caused by transplantation of allogeneic embryonic stem cells. Currently, several transcription factors related to the induction of iPS cells from adult cells, as well as the induction method, how to improve the efficiency of induction of iPS cells, and the clinical application of iPS cells are all improvements on the induction protocol reported by Yamanaka.
Principle
The principle of the Yamanaka method of inducing pluripotent stem cells is to clone the genes of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4, into a viral vector and introduce them into mouse fibroblasts, and find that they can be induced to transform, and the resulting iPS cells are similar to the embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cellular multiplication, embryoid and teratomatoid tumor generation, and differentiation. The resulting iPS cells were similar to embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cell multiplication capacity, embryoid and teratoma generation capacity, and differentiation capacity.
Operation method
Induction of multipotent stem cells
Principle
The principle of the Yamanaka method of inducing pluripotent stem cells is to clone the genes of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4, into a viral vector and introduce them into mouse fibroblasts, and find that they can be induced to transform, and the resulting iPS cells are similar to the embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cellular multiplication, embryoid and teratomatoid tumor generation, and differentiation. The resulting iPS cells were similar to embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cell multiplication capacity, embryoid and teratoma generation capacity, and differentiation capacity.
Materials and Instruments
Equipment: Move The basic process of induction of multipotent stem cells can be divided into the following steps: (2) Preparation of embryonic stem cell culture medium: DF12 medium, 200 ml; serum substitute, 50 ml; 200 mmol/L-glutamine + 2-mercaptoethanol solution, 1.25 ml; non-essential amino acid 100x solution, 2.5 ml; b-FGF solution, 5 ml All the components were added into 250 ml culture flasks and filtered through 0.22 μm filters to remove bacteria. All components were added to 250 ml culture bottles and filtered through a 0.22 μm filter to remove bacteria. The medium should preferably be used within 2 weeks. (3) Discard the culture supernatant of Fbx15 βgeo/βgco-derived MEF cells, add the virus solution prepared in the previous step, and add 2.5 ml of virus solution to each well of a 6-well culture plate. Incubate at 37 ℃ for 4 h until overnight. (2) Growth characteristics. Caveat (1) Many combinations of transcription factors have been reported, based on the Yamanaka four factors, combinations of OCT4, SOX2, NANOG and LIN28; OCT4, SOX2 and KLF4; Sox2, c-Myc and Klf4 have also been reported; freeze before use. Dispense in 50 ml portions and store frozen; thaw before use.(2) Currently, there are many means of overexpressing the above core transcription factors, including retroviral vectors, adenoviral vectors, transfection plasmids, recombinant protein induction methods, and chemical small molecules [e.g., the small molecule inhibitor of G9a histone methyltransferase, BIX-01294 (BIX)].(3) In terms of improving induction efficiency, MSC as inducer cells have a higher rate of induction into iPS than skin-derived fibroblasts: the histone deacetylase inhibitor Valproic Acid (VPA) and the recently reported Vitamin C can significantly increase the rate of induction of iPS.(4) Screening methods for positive clones include reporter gene screening and morphological criteria screening. The reporter gene screening method refers to the use of genetic recombination technology to establish that resistance of mouse fibroblasts with drug resistance genes is regulated by endogenous nanog or ()ct4 expression, and that clones expressing the drug resistance genes grow predominantly by stress screening. For more product details, please visit Aladdin Scientific website.
① 37 ℃ water bath
② Disposable sterile petri dishes
③ Disposable sterile pipette
④ T75 culture flask
⑤ Gelatin-coated 6-well plate ⑥ 15 ml centrifuge tube, syringe and needle
⑤ Gelatin-coated 6-well plate ⑥ 15 ml centrifuge tube, syringe and needle
Reagents:
① Material: pre-prepared feeder layer cells
② DMEM medium
③ Fetal bovine serum
③ Fetal bovine serum ④ Glutamine
⑤ Non-essential amino acids
⑥ DF12 medium
⑦ Serum substitutes
⑧ Glutamine + 2-mercaptoethanol solution
⑨ b-FGF solution
⑩ β-mercaptoethanol
(I) Preparation of reagents
(1) Preparation of MEF medium DMEM medium, 450 ml; fetal bovine serum (heat inactivated at 56 ℃ for 30 min), 50 ml; 200 mmol/L-glutamine, 5 ml; and non-essential amino acids, 5 ml. After mixing all the components, filter the medium through 0.22 μm filter to remove the bacteria. After mixing all the components, filter with a 0.22 μm filter to remove bacteria, and store at 4 ℃ to maintain sterility.
(4) Preparation of b-FGF: b-FGF, 10 μg; 0.1% BSA, sterile, 5 ml; dissolve in 0.5 ml portions and store frozen.
(II) Preparation before starting
(1) Acquisition of mouse embryonic fibroblasts A double-selective marker system, such as βgeo (neo for G418 screening and βGal for development) gene cassette, was homologously recombined into the mouse Nanog gene locus by homologous recombination, so that βgeo was under the control of endogenous Nanog/O)ct promoter. since Nanog is expressed only in ESCs. Since Nanog is only expressed in ESCs, somatic cells derived from Fbx 158geo / 3gco mice cannot be grown in G418 due to the lack of ES properties. Fbx15βgeo/βgco mice were isolated and cultured from Fbx15βgea/βgeo-derived MEFs according to the MEF preparation method.
(2) Classical "Yamanaka" method OCT4, SOX2, KLF4, and C-MYC viruses were prepared.
(3) Preparation of MEF feeder layer cells.
(C) Cell induction
(1) One day before infection, Fbx15 Pgco/βgeo-derived MEF cells in the logarithmic growth phase were spread in 6-well culture plates to achieve 80% confluence of cells at the time of infection.
(2) Add the appropriate amount of concentrated viral supernatant at 10 pfμ/cell according to the measured viral titer, top up with MEF medium to a volume of 2.5 ml per well, and then add polybrene at a final concentration of 8 μg/ml and mix well.
(5) Add G418 at a final concentration of 0.3 mg/ml to the culture medium 3 d after infection.
(6) Change the culture medium of fresh embryonic stem cells every 2-3 days, add G418 at a final concentration of 0.3 mg/ml, and carry out clone screening for 2-3 weeks.
(7) Observe the clone formation during the culture process. The clones with similar shape to ES cells were amplified and passed on according to the ESC culture method and identified.
(D) Experimental results and analysis
Successfully induced iPS should be identified for pluripotency, which includes the following aspects.
(1) Morphological criteria.
(4) Expression of marker molecules.
(5) Epigenetic characterization.