CN102093981A - Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells - Google Patents
Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells Download PDFInfo
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Abstract
The invention provides a method for efficiently inducing reprogramming of human body cells into pluripotent stem cells, belonging to the field of biomedicine. The method provided by the invention comprises the following step: before carrying out nuclear reprogramming on body cells, culturing the body cells for 2-4 days in a culture medium containing thyroid hormone T3. The method provided by the invention can increase the iPSC (induced pluripotent stem cell) induction efficiency by 20-100 times to about 1.2%, and shorten the induction cycle to 12-16 days; and meanwhile, different clones of the iPSC obtained by induction have uniform cellular morphology, multiplication capacity and pluripotent gene expression, and can be used for researching human diseases.
Description
Technical field
The present invention relates to biomedical sector, relating in particular to a kind of reprogramming of somatic cells of efficiently inducing is the method for pluripotent stem cell.
Background technology
Stem cell is subjected to accurate regulation and control to adult cells whose development and differentiation at gene level, is reprogrammed and be reversed into somatic differentiation state to the stem cell state of pluripotency.Scientist (the Takahashi et al. 2007 of Japan in 2007, the U.S.; Yu et al. 2007; Park et al. 2008) reports in succession, in human somatocyte, express four transcription factor gene (Oct4 excessively by retrovirus (or slow virus), Sox2, Klf4 and c-Myc, perhaps Oct4, Sox2, Nanog and Lin28), reverse the differentiation state of human adult cell, obtain multipotency potential stem cell (iPSC, induced pluripotent stem cell).Having report to confirm, import Oct3/4 and two kinds of transcription factor genes of Sox2 by virus vector, can be pluripotent stem cell (Danwei Huangfu, 2008) with reprogramming of somatic cells.On March 5th, 2009, the patient Parkinson iPSC that Cell report Rudolf Jaenisch study group utilizes the Cre-recombinase system to make and induces success does not have exogenous factor, the iPSC clinical application is pushed away again go a step further.On March 26th, 2009, Science report Yu Jun English etc. by a kind of nonconformity plasmid Episomal Vector successfully induce no foreign gene also carrier free be incorporated into people such as iPSC(Yu Jun English on the genome, carrier free does not have the people's of exogenous gene sequence induced multi-potent stem cells yet, science, 2009.324:7797-801).Human reprogramming of somatic cells is that pluripotent stem cell (iPSC) is the important milestone incident in stem-cell research field over the past two years.(Kennedy 2007 to be chosen as ten big sciences progress by the science magazine in continuous 2 years at 2007-2008; Vogel 2008).
By the regulation and control reprogramming of somatic cells, make into somatocyte and obtain and the similar multipotency of embryonic stem cell (ESC), have very important research and potential cell replacement therapeutic value.Human iPSC and embryonic stem cell can specific expression SSEA-3, SSEA-4, Tra-1-60, surface antigens such as Tra-1-81, and nuclear transcription factor-2 genes such as Nanog, Oct4.By suspension culture, after iPSC and embryonic stem cell can form embryoid (EB), can be differentiated to form the cell of human embryonic three germinal layers, express three peculiar genes of germinal layer.These characteristics are to judge that iPSC has similar undifferentiated hESC's standard.Because pluripotent stem cell (iPSC) has multidirectional differentiation potential, more and more researchers is being explored external evoked iPSC differentiation, is used for the replacement therapy of non-renewable cell.In addition, because the iPSC in patient source carries corresponding Disease-causing gene and special cell pathology process, the researchist induces acquisition iPSC on one's body from the patient of type i diabetes, Parkinson's disease and other at least tens kinds of diseases, the special iPSC of these diseases can be used as the cell model of disease, and is significant for the research human diseases.
Although iPSC attracts the various countries investigator to carry out big quantity research, yet the existing mankind of inducing or primates somatocyte are that the technical system of iPSC faces following problem at present: 1. its efficient is extremely low, and is about about 1/10000, and 2. induction time is very long, about about 24 days.3. its cell phenotype of the iPSC that each research group obtained, genetic expression and function thereof are all variant.Above-mentioned technical problem has limited the research of iPSC to a great extent and has used.
Therefore, human iPSC is used widely in regenerative medicine and disease are studied, must explores a kind of strategy of efficiently inducing, shorten the reprogrammed required time, simplify the process of inducing.
Summary of the invention
The purpose of this invention is to provide that can efficiently to induce reprogramming of somatic cells be pluripotent stem cell, shorten the means of reprogrammed time simultaneously, another object of the present invention provides the method for these means of use.
The inventor to be realizing that above-mentioned purpose is that target has been carried out extensive studies, and find with the nuclear reprogramming factor with before somatocyte contacts, with containing Triiodothyronine T3 or T4 culture medium culturing somatocyte, can increase iPSC formation efficient significantly.
A kind of reprogramming of somatic cells of inducing is the method for pluripotent stem cell: may further comprise the steps:
A. with culture medium culturing somatocyte 2-4 days that contains Triiodothyronine T3 or T4;
B. the nuclear reprogramming factor is joined in the somatic cell culture liquid and contact with somatocyte, continue to cultivate, the clone who selects the similar embryonic stem cell of the form cultivation of going down to posterity meets the cell clone of embryonic stem cell characteristic by screening, obtains pluripotent stem cell.
Described Triiodothyronine T3 or T4 concentration preferred 10
-10-10
-7Mol/L.The Triiodothyronine T3 or the T4 that add can promote cell proliferation and cellular energy metabolism, improve reprogramming efficiency and shorten induction duration.
The described nuclear reprogramming factor is the factor of pluripotent stem cell for inducing reprogramming of somatic cells, and this nuclear reprogramming factor contacts with somatocyte with DNA, mRNA or protein form.
The described nuclear reprogramming factor can derive from different species, and preferably with the biology in the identical source of use somatocyte, when being the human fibroblasts as somatocyte, the described nuclear reprogramming factor preferably is people's the nuclear reprogramming factor and a variant thereof.
Preferably, the described nuclear reprogramming factor contacts with somatocyte with dna form, and described DNA is for having the carrier of corresponding nuclear reprogramming factor encoding sequence respectively.
Preferred, described carrier is virus vector or plasmid.
Described virus vector and plasmid all have sophisticated supply of commodities, can buy from the market.
Preferred, described virus vector is retroviral vector, lentiviral vectors or adenovirus carrier.
Preferred in above-mentioned arbitrary scheme, the described nuclear reprogramming factor comprises a kind of among Oct3/4 and Sox2, the Sox1.
Preferred, the described nuclear reprogramming factor also comprises Klf4, c-Myc, Nanog, at least a among the Lin28.
Described Klf4 is replaceable to be Klf5 or Klf2.
Described c-Myc is replaceable to be L-Myc or N-Myc.
Preferred, the described nuclear reprogramming factor is Oct3/4, Sox2, Klf4, c-Myc or Oct3/4, Sox2, Klf4, Nanog.
Above-mentioned each nuclear reprogramming factor encoding sequence all can be searched on NCBI and obtain.
Preferred in above-mentioned arbitrary scheme, described somatocyte can derive from different kinds, includes but not limited to the mankind, gorilla, monkey etc.
Preferred, described somatocyte is skin flbroblast, mucomembranous epithelial cell, lymphocyte, hair follicle cell, the fat mesenchymal stem cell of primate, umbilical cord mesenchymal stem cells, mesenchymal stem cells MSCs can separate obtaining or buy from relevant cell bank from primate species.
Preferred, skin flbroblast, mucomembranous epithelial cell, lymphocyte, hair follicle cell, fat mesenchymal stem cell that described somatocyte is behaved, umbilical cord mesenchymal stem cells, mesenchymal stem cells MSCs.
Preferred in above-mentioned arbitrary scheme, the nuclear reprogramming factor is being joined in the somatic cell culture liquid with after somatocyte contacts, need to continue culture medium culturing 5-8 days with containing Triiodothyronine T3 or T4, described Triiodothyronine T3 or T4 concentration are 10
-10-10
-7Mol/L can further increase iPSC by above-mentioned processing and form efficient.
Preferred in above-mentioned arbitrary scheme, the nuclear reprogramming factor is joined in the somatic cell culture liquid with after somatocyte contacts, continue to cultivate 18-30 hour, change then and contain the 20%FBS-DMEM cultivation, be passaged to feeder layer cells after 3-5 days and continue to cultivate 3-5 days, change the ESC culture medium culturing at last and can obtain pluripotent stem cell in 3-5 days.
Described 20%FBS-DMEM is high sugared DMEM or the DMEM/F12 substratum that contains 20% foetal calf serum, adding final concentration is 2mmol/L L-glutamine, final concentration is a 0.1mmol/L b-mercaptoethanol, final concentration is 5-100ng/mL bFGF, and final concentration is 0.1mmol/L non-essential amino acid and microbiotic.
The preferred final concentration of this microbiotic is 50 units/mL penicillin and 50mg/mL Streptomycin sulphate.
Described feeder layer cells can be the l cell of 13.5 days embryonic stages, embryonic stage the human skin cell, the adult skin inoblast, human umbilical cord mesenchymal stem cells etc. are preferably with mitomycin and handle the l cell that makes 13.5 days its embryonic stages that loses multiplication capacity.
Preferred, the nuclear reprogramming factor joined in the somatic cell culture liquid to replenish when contacting with somatocyte add and fresh medium, keep cell proliferation institute energy requirement, this fresh medium is to add final concentration in the DMEM/F12 substratum to be respectively the 0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L the b-mercaptoethanol, the bFGF of 10ng/mL, 20%(vol/vol) FBS and microbiotic.
The preferred final concentration of this microbiotic is 50 units/mL penicillin and 50mg/mL Streptomycin sulphate.
The substratum preferred, that described ESC substratum is made up of knockout DMEM or DMEM/F12,5-100ng/L Prostatropin, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20% serum substitute, 5-25ug/mL anti-mycoplasma drug plasmocin and microbiotic.
The preferred final concentration of this microbiotic is 50 units/mL penicillin and 50mg/mL Streptomycin sulphate.
The pluripotent stem cell that obtains by the inventive method has and the similar characteristic of embryonic stem cell (ES), and these characteristics comprise: multiplication capacity, express the embryonic stem cell mark, form comprise interior, in, the tissue of outer three germinal layers and the teratoma of cell.
Compared with prior art, the invention has the beneficial effects as follows: the reprogramming of somatic cells of efficiently inducing provided by the invention is the method for pluripotent stem cell, can improve and induce iPSC efficient 20-100 doubly, reach about 1.2%, and shorten induction duration to 12-16 days, induce cellular form, multiplication capacity, pluripotency genetic expression homogeneous between the different clones of iPSC of acquisition simultaneously, can be used in the cell replacement treatment.
Description of drawings
Fig. 1 is the expression that 3 groups of cells are induced TRA-1-60 after 13 days respectively.
Fig. 2 is that 3 groups of cells are induced TRA-1-60 positive expression rate after 13 days respectively.
Fig. 3 is embodiment 5 gained iPSC form pictures.
Fig. 4 is that embodiment 5 gained iPSC express human ESC pluripotency relevant cell signature.
Fig. 5 is embryoid body and the differentiation figure thereof that embodiment 5 gained iPSC form.
Embodiment
The present invention is further described below in conjunction with description of drawings and embodiment.
One. efficiently inducing reprogramming of somatic cells is pluripotent stem cell.
Embodiment 1. contains the amplification of the retroviral vector of nuclear reprogramming factor encoding sequence.
By prior art, buy the retroviral vector that comprises people Oct3/4, Sox2, Klf4, c-Myc respectively from Addgene company.After the amplification, purifying obtains containing the retroviral vector of corresponding gene in intestinal bacteria.
Embodiment 2. packing retrovirus.
By prior art, the retroviral vector transfection 293T cell of Oct3/4, Sox2, Klf4, c-Myc gene will be contained, the packing retrovirus respectively.And with by 30-100KD ultrafiltration pipe, 5000 * g, 4 ℃, centrifugal 30 minutes, collect viral liquid, make it concentrate about 10 times respectively, final concentration is about 5 * 10
6-5 * 10
7The single nuclear reprogramming factor of transducing units/mL/.The retrovirus equal-volume that contains Oct3/4, Sox2, Klf4, c-Myc gene respectively after concentrating is mixed, get 0.6-1.0 ml virus solution infectosome cell, the virus titer MOI (multiplicity of infection) of this moment is about the single nuclear reprogramming factor of 5-50/.
Embodiment 3. contains the amplification of the lentiviral vectors of nuclear reprogramming factor encoding sequence.
By prior art, from the slow virus plasmid of the purchaser Oct3/4 of Addgene company, Sox2, Nanog and Lin28.After the amplification, purifying obtains containing the slow virus plasmid of corresponding gene in intestinal bacteria.
Embodiment 4. packing slow viruss.
By prior art, the lentiviral vectors transfection 293T cell of Oct3/4, Sox2, Nanog, Lin28 gene will be contained, the packing slow virus respectively.And with by 30-100KD ultrafiltration pipe, 5000 * g, 4 ℃, centrifugal 30 minutes, collect viral liquid, make it concentrate about 10 times respectively.Final concentration is about 5 * 10
6-5 * 10
7The single nuclear reprogramming factor of transducing units/mL/.The quality of packing cell is very crucial to efficiency of infection, and the slow virus after concentrating is mixed, and gets 0.6-1.0 ml virus solution infectosome cell, and the virus titer MOI (multiplicity of infection) of this moment is about the single nuclear reprogramming factor of 5-50/.
The preparation of embodiment 5. pluripotent stem cells.
The human skin inoblast is passaged to 12 orifice plates, the about 2.5-3 in every hole * 10
4Individual.(adding final concentration in the DMEM/F12 substratum is the 0.1mmol/L non-essential amino acid at substratum, final concentration is 2mmol/L L-glutamine, final concentration is a 0.1mmol/L b-mercaptoethanol, final concentration is the bFGF of 10ng/mL, 20% (vol/vol) KSR (knockout serum replacement), final concentration is that 50 units/mL penicillin and final concentration are the 50mg/mL Streptomycin sulphate) the middle Triiodothyronine T3 that adds, making its final concentration is 5 * 10
-9Mol/L, cultivate after 3 days, adding embodiment 2 gained reverse transcription disease venom 0.6-1.0mL infects, and (the interpolation final concentration is respectively the 0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol in the DMEM/F12 substratum to add the 0.5mL fresh medium, the bFGF of 10ng/mL, 20% (vol/vol) FBS, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate), keep cell proliferation institute energy requirement.Change behind the 24h and add final concentration in high sugared DMEM of 20%FBS-DMEM(or the DMEM/F12 substratum and be respectively 20%FBS, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 30ng/mL bFGF, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) to cultivate, the cell proliferation behind the virus infection is slowed down, and the form that occurred changing to epithelial cell in metainfective about the 3rd day changes.Change 20%FBS-DMEM cultivate back the 5th day with cell with 4-10 * 10
2The density of/cm2 is passaged to feeder layer cells and cultivates.Be passaged to after the feeder layer cells the 3rd day and begin to use instead people ESC culture medium culturing, the iPSC of reprogrammed is cloned in and uses appearance in about the 4th day behind the people ESC substratum instead, expresses pluripotency gene SSEA4, TRA-1-60, TRA-1-81, E-cadherin, NANOG and endogenous Oct3/4, Sox2 etc.Above-mentioned ESC substratum is respectively the substratum that 30ng/L Prostatropin, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20% serum substitute, 5-25ug/mL anti-mycoplasma drug plasmocin and 50 units/mL penicillin and 50mg/mL Streptomycin sulphate are formed by adding final concentration in the knockout DMEM substratum.Through further screening, obtain meeting the cell clone of embryonic stem cell characteristic, i.e. pluripotent stem cell.In 6 days after adding retroviral infection human skin inoblast, need to add Triiodothyronine T3 in substratum, making its final concentration is 5 * 10
-9Mol/L.
The preparation of embodiment 6. pluripotent stem cells.
The human mucosa epithelial cell is passaged to 12 orifice plates, the about 2.5-3 in every hole * 10
4Individual.In substratum, (contain DMEM/F12,0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, the bFGF of 10 ng/mL, 20%KSR (knockout serum replacement), 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) add Triiodothyronine T3, making its final concentration is 5 * 10
-8Mol/L, cultivate after 2 days, the slow virus liquid 0.6-1.0mL that adds embodiment 4 gained infects, and (the interpolation final concentration is respectively the 0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L beta-mercaptoethanol in the DMEM/F12 substratum to add the 0.5mL fresh medium, the bFGF of 10ng/mL, 20%FBS, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate), keep cell proliferation institute energy requirement.Using 20%FBS-DMEM behind the 18h instead (adds final concentration and is respectively 20%FBS in high sugared DMEM or the DMEM/F12 substratum, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 5-100ng/mL bFGF, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) cultivate.Cell proliferation behind the virus infection is slowed down, and the form that occurred changing to epithelial cell in metainfective about the 3rd day changes.Change 20%FBS-DMEM cultivate back the 4th day with cell with 4-10 * 10
2/ cm
2Density be passaged to feeder layer cells and cultivate.Generation began to use instead people ESC substratum on the 4th day to the feeder layer cells, and (adding final concentration in the DMEM/F12 substratum is 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 60ng/mL bFGF, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) cultivate, the iPSC of reprogrammed is cloned in and uses appearance in about the 4th day behind the people ESC substratum instead, express pluripotency gene SSEA4, TRA-1-60, TRA-1-81, E-cadherin, NANOG and endogenous Oct3/4, Sox2 etc.Through further screening, obtain meeting the cell clone of embryonic stem cell characteristic, i.e. pluripotent stem cell.In 7 days after adding retroviral infection human skin inoblast, need to add Triiodothyronine T3 in substratum, making its final concentration is 5 * 10
-8Mol/L.
The preparation of embodiment 7. pluripotent stem cells.
Human umbilical cord mesenchymal stem cells is passaged to 12 orifice plates, the about 2.5-3 in every hole * 10
4Individual.(adding final concentration in the DMEM/F12 substratum is the 0.1mmol/L non-essential amino acid at substratum, final concentration is 2mmol/L L-glutamine, final concentration is a 0.1mmol/L b-mercaptoethanol, final concentration is the bFGF of 60ng/mL, 20% (vol/vol) KSR (knockout serum replacement), final concentration is that 50 units/mL penicillin and final concentration are the 50mg/mL Streptomycin sulphate) the middle Triiodothyronine T4 that adds, making its final concentration is 10
-7Mol/L, cultivate after 3 days, adding embodiment 2 gained reverse transcription disease venom 0.6-1.0mL infects, and (the interpolation final concentration is respectively the 0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol in the DMEM/F12 substratum to add the 0.5mL fresh medium, the bFGF of 10ng/mL, 20% (vol/vol) FBS, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate), keep cell proliferation institute energy requirement.Change behind the 30h and add final concentration in high sugared DMEM of 20%FBS-DMEM(or the DMEM/F12 substratum and be respectively 20%FBS, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 40ng/mL bFGF, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) to cultivate, the cell proliferation behind the virus infection is slowed down, and the form that occurred changing to epithelial cell in metainfective about the 3rd day changes.Change 20%FBS-DMEM cultivate back the 3rd day with cell with 4-10 * 10
2The density of/cm2 is passaged to feeder layer cells and cultivates.Be passaged to after the feeder layer cells the 5th day and begin to use instead people ESC culture medium culturing, the iPSC of reprogrammed is cloned in and uses appearance in about the 5th day behind the people ESC substratum instead, expresses pluripotency gene SSEA4, TRA-1-60, TRA-1-81, E-cadherin, NANOG and endogenous Oct3/4, Sox2 etc.Above-mentioned ESC substratum is respectively the substratum that 5-100ng/L Prostatropin, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20% serum substitute, 5-25ug/mL anti-mycoplasma drug plasmocin and 50 units/mL penicillin and 50mg/mL Streptomycin sulphate are formed by adding final concentration in the knockout DMEM substratum.Through further screening, obtain meeting the cell clone of embryonic stem cell characteristic, i.e. pluripotent stem cell.In 8 days after adding retroviral infection human skin inoblast, need to add Triiodothyronine T4 in substratum, making its final concentration is 10
-7Mol/L.
The preparation of embodiment 8. pluripotent stem cells.
Human marrow mesenchymal stem cell is passaged to 12 orifice plates, the about 2.5-3 in every hole * 10
4Individual.(adding final concentration in the DMEM/F12 substratum is the 0.1mmol/L non-essential amino acid at substratum, final concentration is 2mmol/L L-glutamine, final concentration is a 0.1mmol/L b-mercaptoethanol, final concentration is the bFGF of 40ng/mL, 20% (vol/vol) KSR (knockout serum replacement), final concentration is that 50 units/mL penicillin and final concentration are the 50mg/mL Streptomycin sulphate) the middle Triiodothyronine T4 that adds, making its final concentration is 5 * 10
-8Mol/L, cultivate after 4 days, adding embodiment 2 gained reverse transcription disease venom 0.6-1.0mL infects, and (the interpolation final concentration is respectively the 0.1mmol/L non-essential amino acid, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol in the DMEM/F12 substratum to add the 0.5mL fresh medium, the bFGF of 10ng/mL, 20% (vol/vol) FBS, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate), keep cell proliferation institute energy requirement.Change behind the 24h and add final concentration in high sugared DMEM of 20%FBS-DMEM(or the DMEM/F12 substratum and be respectively 20%FBS, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20ng/mL bFGF, 50 units/mL penicillin and 50mg/mL Streptomycin sulphate) to cultivate, the cell proliferation behind the virus infection is slowed down, and the form that occurred changing to epithelial cell in metainfective about the 3rd day changes.Change 20%FBS-DMEM cultivate back the 4th day with cell with 4-10 * 10
2The density of/cm2 is passaged to feeder layer cells and cultivates.Be passaged to after the feeder layer cells the 4th day and begin to use instead people ESC culture medium culturing, the iPSC of reprogrammed is cloned in and uses appearance in about the 4th day behind the people ESC substratum instead, expresses pluripotency gene SSEA4, TRA-1-60, TRA-1-81, E-cadherin, NANOG and endogenous Oct3/4, Sox2 etc.Above-mentioned ESC substratum is respectively the substratum that 5-100ng/L Prostatropin, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20% serum substitute, 5-25ug/mL anti-mycoplasma drug plasmocin and 50 units/mL penicillin and 50mg/mL Streptomycin sulphate are formed by adding final concentration in the knockout DMEM substratum.Through further screening, obtain meeting the cell clone of embryonic stem cell characteristic, i.e. pluripotent stem cell.In 5 days after adding retroviral infection human skin inoblast, need to add Triiodothyronine T4 in substratum, making its final concentration is 5 * 10
-8Mol/L.
Except that special statement, in the foregoing description the reagent that uses all can buy from Invitrogen company, cell strain is type strain.
Utilization contains the adenovirus carrier of nuclear reprogramming factor encoding sequence or method that plasmid is induced iPSC can not repeat them here with reference to the foregoing description.BFGF among the present invention in each substratum all can well promote the growth of cell in its corresponding content range.
Nuclear reprogramming combinations of factors used in the present invention can be made corresponding adjustment according to prior art, does not repeat them here.
Two. the evaluation of the pluripotent stem cell of acquisition.
Be raw material with human skin fibroblast (HDF), human umbilical cord mesenchymal stem cells (UCMSC) respectively, prepare iPSC by embodiment 5 methods, establish an athyreosis hormone T3 simultaneously and induce control group (HDF), after inducing 13 days, as shown in Figure 1, the human skin fibroblast (HDF) of adding the T3 group iPSC clone occurs with human umbilical cord mesenchymal stem cells (UCMSC), and TRA-1-60 expresses the positive and the most clones of control group show as TRA-1-60 feminine gender or false positive; As shown in Figure 2, in human skin fibroblast (HDF) and human umbilical cord mesenchymal stem cells (UCMSC), add T3 the positive rate of TRA-1-60 positive colony is all improved about 20-100 times than un-added control group, wherein OSKM is OCT3/4, SOX2, the abbreviation of Klf4 and c-Myc.In addition, as shown in Figure 3, the iPSC that obtains by present embodiment 5 on form to human ESC(H9) similar, and form homogeneous between the different iPSC clones that obtain) under condition of suspension culture, can form embryoid body (EB), energy neuralward cytodifferentiation is expressed neuroglial acidic protein (GFAP) in the N2 substratum.
Gained iPSC is carried out the molecular biology aspect to be identified, the result shows the iPSC cell expressing pluripotency gene SSEA-3 of formation, SSEA-4(and do not express mouse ES specific mark SSEA-1), TRA-1-60, TRA-1-81, E-cadherin, NANOG (seeing accompanying drawing 4) and endogenous OCT3/4, SOX2 etc.By RT-PCR endogenous OCT3/4 and NANOG gene expression analysis are shown endogenous OCT3/4 and the expression of NANOG gene promoter in the people iPSC cell that obtains.Demethylation is finished in the corresponding site with the NANOG gene promoter area of dna methylation analytical results demonstration OCT3/4.Gained iPSC can form embryoid body (EB) by suspension culture, inwardly China and foreign countries' triploblastica differentiation (seeing accompanying drawing 5) under differentiation condition.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
- One kind to induce reprogramming of somatic cells be the method for pluripotent stem cell: may further comprise the steps:A. with culture medium culturing somatocyte 2-4 days that contains Triiodothyronine T3 or T4;B. the nuclear reprogramming factor is joined in the somatic cell culture liquid and contact with somatocyte, continue to cultivate, the clone who selects the similar embryonic stem cell of the form cultivation of going down to posterity meets the cell clone of embryonic stem cell characteristic by screening, obtains pluripotent stem cell; The described nuclear reprogramming factor contacts with somatocyte with DNA, mRNA or protein form.
- 2. method according to claim 1 is characterized in that: described Triiodothyronine T3 of steps A or T4 concentration are 10 -10-10 -7Mol/L; The described somatocyte of steps A is skin flbroblast, mucomembranous epithelial cell, lymphocyte, hair follicle cell, the fat mesenchymal stem cell of primate, umbilical cord mesenchymal stem cells, mesenchymal stem cells MSCs.
- 3. method according to claim 2 is characterized in that: step B is described to join in the somatic cell culture liquid nuclear reprogramming factor with after somatocyte contacts, and needing to continue with final concentration be 10 -10-10 -7The Triiodothyronine T3 of mol/L or T4 culture medium culturing 5-8 days.
- 4. method according to claim 1 is characterized in that: the described nuclear reprogramming factor of step B comprises a kind of among Oct3/4 and Sox2, the Sox1.
- 5. method according to claim 4 is characterized in that: the described nuclear reprogramming factor also comprises Klf4, c-Myc, Nanog, at least a among the Lin28.
- 6. method according to claim 5 is characterized in that: the described nuclear reprogramming factor of step B contacts with somatocyte with dna form; Described DNA is the carrier that has corresponding nuclear reprogramming factor encoding sequence; Described carrier is virus vector or plasmid.
- 7. method according to claim 6 is characterized in that: described virus vector is retroviral vector, lentiviral vectors or adenovirus carrier.
- 8. according to the arbitrary described method of claim 1 to 7, it is characterized in that: step B is specially the nuclear reprogramming factor is joined in the somatic cell culture liquid with after somatocyte contacts, continue to cultivate 18-30 hour, changing 20%FBS-DMEM then cultivates, be passaged to feeder layer cells after 3-5 days and continue to cultivate 3-5 days, change the ESC culture medium culturing at last and can obtain pluripotent stem cell in 3-5 days.
- 9. method according to claim 8 is characterized in that: the nuclear reprogramming factor is joined in the somatic cell culture liquid to replenish when contacting with somatocyte add fresh medium; Described feeder layer cells is for handling the l cell that makes 13.5 days its embryonic stages that loses multiplication capacity with mitomycin.
- 10. method according to claim 8 is characterized in that: the substratum that described ESC substratum is made up of knockout DMEM or DMEM/F12,5-100ng/L Prostatropin, 2mmol/L L-glutamine, 0.1mmol/L b-mercaptoethanol, 0.1mmol/L non-essential amino acid, 20% serum substitute, 5-25ug/mL anti-mycoplasma drug plasmocin and microbiotic.
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| WO2013004135A1 (en) * | 2011-07-01 | 2013-01-10 | 中国科学院上海药物研究所 | Preparation method for inductive pluripotent stem cells and culture medium for preparing inductive pluripotent stem cells |
| CN106164255A (en) * | 2013-09-20 | 2016-11-23 | 隆萨有限公司 | The method of the nuclear reprogramming of cell |
| CN106479956A (en) * | 2015-08-26 | 2017-03-08 | 中国科学院动物研究所 | A kind of culture medium for improving reprogramming efficiency of somatic cells and method |
| CN110894493A (en) * | 2019-10-28 | 2020-03-20 | 吉林大学 | Reprogramming mesenchymal stem cells and preparation method thereof |
| CN112831462A (en) * | 2021-02-09 | 2021-05-25 | 复旦大学 | Compositions, media and methods for inducing reprogramming of human cells to induced pluripotent stem cells |
| CN112941019A (en) * | 2021-03-18 | 2021-06-11 | 浙江大学 | Method for promoting reprogramming of somatic cells |
| CN112961833A (en) * | 2021-03-08 | 2021-06-15 | 浙江大学 | Method for reprogramming immortalized lymphocyte cell line into induced pluripotent stem cell |
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| CN115003795A (en) * | 2019-11-14 | 2022-09-02 | 岛崎猛夫 | Reprogramming method of cell |
| CN115948524A (en) * | 2022-11-08 | 2023-04-11 | 吉林大学 | Application of GCNA in improving reprogramming efficiency and stem cell primitive state pluripotency and promoting primordial germ cell transformation |
| CN116555169A (en) * | 2023-07-10 | 2023-08-08 | 北京北启生物医药有限公司 | Simple and rapid somatic cell chemical reprogramming method |
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| CN112831462A (en) * | 2021-02-09 | 2021-05-25 | 复旦大学 | Compositions, media and methods for inducing reprogramming of human cells to induced pluripotent stem cells |
| CN112961833A (en) * | 2021-03-08 | 2021-06-15 | 浙江大学 | Method for reprogramming immortalized lymphocyte cell line into induced pluripotent stem cell |
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| CN115948524A (en) * | 2022-11-08 | 2023-04-11 | 吉林大学 | Application of GCNA in improving reprogramming efficiency and stem cell primitive state pluripotency and promoting primordial germ cell transformation |
| CN116555169B (en) * | 2023-07-10 | 2023-10-24 | 北京北启生物医药有限公司 | Simple and rapid somatic cell chemical reprogramming method |
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Application publication date: 20110615 Assignee: Harbin Beike Health Technology Co.,Ltd. Assignor: SHENZHEN BEIKE BIO-TECHNOLOGY Co.,Ltd. Contract record no.: X2023440020002 Denomination of invention: An efficient method for inducing human somatic cells to reprogram into pluripotent stem cells Granted publication date: 20121219 License type: Common License Record date: 20230116 |