Patent Application: US-201113990722-A

Abstract:
a method for preparing induced pluripotent stem cells , which comprises steps as follows : step 1 , introducing one or more stem cell pluripotency factors into somatic cells ; step 2 , culturing the somatic cells , into which the stem cell pluripotency factor has been introduced in the step 1 , by using medium supplemented with lithium salt ; and step 3 , identifying and characterizing the induced pluripotent stem cells . furthermore , there provided a medium for preparing induced pluripotent stem cells , which comprising lithium salt . the medium supplemented with lithium salt is used for efficiently inducing pluripotent stem cells . lithium salt is able to increase the production efficiency of mouse ips cells by 5 - 60 times . the present method for inducing ips cells is in favor of improving the safety of ips technique and application of ips cells in regenerative medicine .

Description:
the traditional technologies of molecular biology , microbiology , cell biology , immunology and dna recombination used in the present experiments fall into the scope of technologies in the art , unless otherwise specified . references includes but not limited to : sambrook , fritsch and maniatis , molecular cloning : a laboratory manual ( third edition ), 2002 ; current protocols in molecular biology ( f . m . ausubel et al . ( 1987 )); series of books such as methods in enzymology ( academic press , inc . ); pcr2 : a practical approach ( m . j . macpherson , b . d . hames and g . r . taylor , ( 1995 )); antibodies , a laboratory manual and animal cell culture ( r . i . freshney , ( 1987 )); handbook of stem cells , vol . 2 ( w . french et al .). unless otherwise stated , all the terms in this text have the meanings as a person skilled in the art regularly understands . in order to assist the understanding of the present invention , some of the terms used herein are defined as follows : the term “ induced pluripotent stem cells ( ips cells )” used herein refer to those cells obtained from in vitro reprogramming of somatic cells into which stem cell pluripotency factors have been introduced . such cells are very similar to mouse es ( embryonic stem ) cells in many aspects such as cell morphology , growth characteristics , expression of specific gene , manner of dna methylation and the like , and are very similar to mouse es cells in aspects such as teratoma formation , chimeric animal formation and germline transmission , etc ., under the culture conditions of es cells . the said “ stem cell pluripotency factors for the induction of somatic cells reprogramming ” used herein are key factors in maintaining stem cell pluripotency . somatic cells can be reprogrammed to stem cells by introducing these factors into the somatic cells . many factors have been reported to have an ability to induce the reprogramming of somatic cells [ 14 - 18 ]. preferably , the pluripotency factor ( s ) is / are one or more selected from the group consisting of oct4 , sox2 ( or sox1 ), klf4 ( or klf2 or klf5 ), nanog , c - myc ( or l - myc or n - myc ), lin28 and esrrb . the aforementioned stem cell pluripotency factors may be derived from any desired species , and mouse stem cell pluripotency factors are preferable . the said “ induction of reprogramming ” ( sometimes only be simplified as “ induction ”) used herein refers to a process of de - differentiating somatic cells into pluripotency stem cells . preferably , somatic cells can be induced to de - differentiate into pluripotent stem cells by introducing cdna of pluripotency factors essential for maintaining stem cell pluripotency into the somatic cells . among them , preferably , the pluripotency factor ( s ) is / are one or more selected from the group consisting of oct4 , sox2 ( or sox1 ), klf4 ( or klf2 or klf5 ), nanog , c - myc ( or l - myc or n - myc ), lin28 and esrrb . the method for introducing cdna of stem cell pluripotency factors into somatic cells may be performed in various ways , such as viral infection , liposome transfection , electroporation , particle bombardment , transposon - mediated insertion expression , membrane permeable proteins or drug induction , etc . it is preferable that the viral vector containing the cdna is used for transfection . the viral vectors include lentivirus , retrovirus , adenovirus , and etc . it is preferable to use retroviral vectors ( pmx vector ). the said “ medium ” used herein may be dmem ( dulbcco &# 39 ; s modified eagle &# 39 ; s medium ) supplemented with 15 % fetal bovine serum , 1000 u / ml leukaemia inhibitory factor ( lif ), l - glutamine , non - essential amino acids , penicillin / streptomycin and β - mercaptoethanol ( mes medium ), and knockout dmem supplemented with 15 % serum replacement , 1000 u / ml leukaemia inhibitory factor ( lif ), l - glutamine , non - essential amino acids , penicillin / streptomycin and β - mercaptoethanol ( ksr medium ). the said “ reporter gene ” used herein is capable of indicating a state in which cells have been transformed into cells similar to the embryonic stem cells . the reporter gene comprises a fluorescent protein sequence or antibiotics resistant gene sequence introduced by using transgenic or homologous recombinant means , which is under the control of the promoter of certain embryonic stem cell - specific genes , and therefore the expression of the fluorescent protein gene or the antibiotic resistance gene is activated when cells come into the state similar to that of embryonic stem cells , so that the cells have certain detectable characteristics . in the embodiment , the used mouse embryonic fibroblasts are og2 ( oct4 - gfp +/+ ) cells isolated from 13 . 5 - day embryos obtained by mating homozygous male og2 ( oct4 - gfp +/+ ) mice with female 129 mice . according to the present invention , the method for detecting cell pluripotency is well applied by a person skilled in the art , including ap staining , cell fluorescence staining , quantitative pcr analysis of the stem cell - specific gene expression and viral gene silencing , in vivo teratoma formation analysis , chimera formation analysis and germline transmission detection . the following examples are provided to illustrate the standard laboratory practices performed by the inventors . it is to be understood that the following examples are illustrative only and the present invention is not limited thereto . each substance mentioned in the description is commercially available from invitrogen , unless otherwise specified . experimental procedures for the production of retrovirus and the generation of ips cells are as follows : retrovirus were produced by transfecting plat - e cells with pmxs retroviral vectors containing the cdnas of mouse oct4 , sox2 , klf4 and c - myc ( purchased from addgene ) using fugene hd ( roche ) according to the manufacturer &# 39 ; s protocol . after 48 hours , virus - containing supernatants were collected and filtered . the filtered supernatant was supplemented with 8 mg / l of polybrene , and then was used to infect mouse embryonic fibroblasts . the day when adding virus containing supernatants is counted as “ day 0 ”. the fibroblasts infected with the virus were cultured in the mes medium , and ips colonies were picked from day 14 to the day 18 after infection ( four - factor infection experiment ) or from day 20 to day 24 after infection ( three - factor infection experiment ), based on the expression of oct - gfp and the typical morphology of stem cells . experimental procedure for the quantitation of reprogramming efficiency is as follows : the main method for calculating the reprogramming efficiency is the counting of the oct - gfp + cells , including : 1 . using flow cytometry to determine the percentage of oct - gfp + cells in the cell culture , which were infected with four factors for 10 to 12 days or with three factors for 14 to 16 days , or 2 . directly counting the oct - gfp + colonies in the wells by using fluorescence microscope . experimental procedure for the characterization of ips cells is as follows : alkaline phosphatase staining , fluorescence staining of stem cell marker proteins , identification of expression of pluripotency genes , silencing of viral genes , teratoma formation , chimera formation and germline transmission are performed [ 19 , 20 ]. the stem cell medium supplemented with lithium salt can promote the formation of four - factor induced ips cells a . same volumes of the viruses carrying oct4 , sox2 , klf4 or c - myc were mixed and added to a total of 180 , 000 og2 mouse embryonic fibroblasts in one well of a 6 - well plate and cells were cultured in dmem medium supplemented with 10 % fetal bovine serum at 37 ° c . and 5 % co 2 . the day of viral infection was counted as “ day 0 ”. on day 2 , cells were trypsinized and resuspended in the mes medium , and then seeded at a density of 5 , 000 cells per well onto a 96 - well plate pre - seeded with irradiated mouse embryonic fibroblast feeders . the mes medium supplemented with different concentrations of licl ( 0 . 6 mm , 1 . 2 mm , 2 . 5 mm , 5 mm , 10 mm , 20 mm and 40 mm ) were used from day 3 . on day 6 , the medium was replaced with ksr medium supplemented with different concentrations of licl ( 0 . 6 mm , 1 . 2 mm , 2 . 5 mm , 5 mm , 10 mm , 20 mm and 40 mm ). on day 8 , the medium was replaced with ksr medium again . the entire process was shown in fig1 . b . using the method described in step a , from day 8 , oct - gfp + colonies were observed and counted under an inverted fluorescence microscope every day , and pictures were also taken with a fluorescence microscope . fig2 a was representative images of wells in the 96 - well plate on day 10 , in which there were more gfp + colonies in 10 mm licl treated well than in untreated well . fig2 b showed statistical data of oct - gfp ± colonies . on day 8 , gfp + colony could barely be observed in control wells , while approximately 10 gfp + colonies could be observed in 10 mm licl treated wells . on day 10 , approximately 20 gfp + colonies could be observed in 10 mm licl treated wells , with induction efficiency 5 to 6 times of that of control wells &# 39 ;. fig2 c showed the number of positive colonies generated after the treatment with different concentrations of licl ( 0 . 6 mm , 1 . 2 mm , 2 . 5 mm , 5 mm , 10 mm , 20 mm and 40 mm ). significant dose effect was observed , wherein the most effective dose of licl was 10 mm . c . using the method described in step a , on day 12 , cells were trypsinized and percentage of oct - gfp + cells was determined by using flow cytometry . fig3 a was representative facs plots . fig3 b showed the statistical data of three independent experiments , demonstrating that the induction efficiency of wells treated with 10 mm licl was increased by 5 to 6 times than that of wells without treatment , when using four factors to induce reprogramming . the stem cell medium supplemented with lithium salt can promote the formation of three - factor induced ips cells a . same volumes of the viruses carrying oct4 , sox2 or klf4 were mixed and added to a total of 180 , 000 og2 mouse embryonic fibroblasts in one well of a 6 - well plate and cells were cultured in dmem medium supplemented with 10 % fetal bovine serum at 37 ° c . and 5 % co 2 . the day of viral infection was counted as “ day 0 ”. on day 2 , cells were trypsinized and resuspended in the mes medium , and seeded at a density of 5 , 000 cells per well onto a 96 - well plate pre - seeded with irradiated mouse embryonic fibroblast feeders . the mes medium supplemented with different concentrations of licl ( 0 . 6 mm , 1 . 2 mm , 2 . 5 mm , 5 mm , 10 mm , 20 mm and 40 mm ) were used from day 3 . on day 6 , medium was replaced with ksr medium supplemented with licl having different concentrations ( 0 . 6 mm , 1 . 2 mm , 2 . 5 mm , 5 mm , 10 mm , 20 mm and 40 mm ). at day 8 , medium was replaced with ksr medium again . the entire process was shown in fig1 . b . using the method described in step a , from day 12 , oct - gfp + colonies were observed and counted under an inverted fluorescence microscope every day , and pictures were also taken with a fluorescence microscope . on day 14 , as compared with control groups , the induction efficiency of ips cells in wells treated with 5 mm or 10 mm licl was significantly improved , wherein the induction efficiency of ips cells in wells treated with 10 mm licl could be increased by approximately 7 times , as shown in fig4 a . c . using the method described in step a , on day 16 , cells were trypsinized and the percentage of oct - gfp + cells was determined by using flow cytometry . the percentage of oct - gfp + cells in control groups was only 0 . 24 %, while the percentage of oct - gfp + cells treated with 10 mm of licl in experimental groups reached 14 . 5 %, with an increase of approximately 60 times , as shown in fig4 b . ips cell lines obtained by addition of lithium salt are pluripotent a . as described above , mouse embryonic fibroblasts were infected with four factors of oct4 , sox2 , klf4 and c - myc or three factors of oct4 , sox2 and klf4 , and cultured in the stem cell medium supplemented with licl . on day 14 after infection ( four factors ) or on day 20 after infection ( three factors ), representative colonies were picked out based on morphology and fluorescence expression , and were used to generate homogeneous ips cell lines through passages . b . the established ips cell lines were observed in terms of their morphology and stained to detect stem cell - specific proteins . as shown in fig5 , ips cells had characteristic morphology similar to that of embryonic stem cells , strongly expressed oct - gfp , and were positive in alkaline phosphatase ( ap ) staining . immunofluorescent staining of the ips cells was carried out by using antibodies against stem cell - specific proteins , and results demonstrated that ips cells expressed stem cell - specific proteins nanog and ssea - 1 . c . feeder cells were removed by using differential adhesion method prior to extraction of rna from ips cells . total rna was extracted using trizol ( invitrogen ) reagent according to the manufacturer &# 39 ; s protocol . reverse transcription was performed using primescript ™ kit ( takara co .). real - time pcr was performed by using jumpstart ™ taqready mix ™ kit ( sigma co .) and mx 3000p thermal cycler ( abi co .). common pcr conditions were used according to the manufacturer &# 39 ; s protocol . primers for identifying ips specific genes were listed in table 1 . as shown in fig6 a , ips cells obtained by using the method of the invention expressed high - level of pluripotency factors including endogenous oct4 , endogenous sox2 , nanog and rex1 , and their expression levels were comparable to those of embryonic stem cell line e14 . on the contrary , expression of these factors was not detectable in the original mouse embryonic fibroblasts . meanwhile , as shown in fig6 b , ips cells obtained by using the method of the invention could silence the viral genes fairly well , demonstrating that cells were completely reprogrammed into stem cells . d . mouse four - factor induced ips cells obtained by adding lithium salt were trypsinized , and approximately 1 × 10 6 ips cells were resuspended in 200 μl of mes medium and injected into the muscle of femoribus internus in nod - scid mice . 4 - 5 weeks later , mice were sacrificed and teratomas were embedded in paraffin , sectioned , he stained and analyzed . as shown in fig7 , tissues originating from three germ layers were found in sections of teratomas formed from ips cells , which including epidermis - like tissue representing ectoderm , muscle - and cartilage - like tissues representing mesoderm , and lumen - like tissues of alimentary tract representing endoderm . thus , similar to mouse embryonic stem cells , mouse four - factor induced ips cells obtained by adding lithium salt were pluripotent and could contribute to different types of cells of the three germ layers . e . mouse four - factor induced ips cells obtained by adding lithium salt were injected into the blastocysts of icr mouse and thus mixed with the mouse embryonic stem cells in the blastocyst . the blastocysts were then transplanted into the uterus of the pseudopregnant female icr mice for normal develop . the coat color of the pups was observed to judge whether a chimera was formed . if chimeras were detected , they were mated with white icr mice and the coat color of the offspring animals was observed to judge whether the germline transmission occurred . as shown in fig8 , both three - factor and four - factor ips cells could produce chimera mice . and black mice appeared in the offsprings of the chimera mice generated by four - factor ips cells . these results confirmed that the ips cells obtained by adding lithium salt did contribute to the germline , demonstrating that these cells are pluripotent and very similar to embryonic stem cells . in short , ips cells can be efficiently generated by using the medium supplemented with lithium salt in the present invention . it is demonstrated by the results from flow cytometry analysis that the induction efficiency of experimental groups treated with lithium salt can be increased by 5 to 6 times ( four - factor induction assay ) and approximately 60 times ( three - factor induction assay ) than that of the control groups . the reprogramming time can also be shortened by adding lithium salt during ips induction . oct4 - gfp + colonies can be detected on day 8 after infection in experimental groups treated with lithium salt , while oct4 - gfp + colonies have not been found until day 10 in the control groups . ips cells induced by using lithium salt have excellent pluripotency . ips clones transduced with four factors or three factors can form chimera mice , wherein germline transmission and birth to offsprings are observed in chimera mice generated with four - factor ips cells . the present invention provides efficient method for inducing ips cells , and the method might substantially promote the basic research and clinical application of ips cells . the contents shown above is only the preferred examples of the present invention , obviously , they can not define the scope of the rights of the present invention , therefore equivalent changes made according to claims claimed in the present invention still fall into the scope of the present invention . 1 . evans , m . j . and m . h . kaufman , establishment in culture of pluripotential cells from mouse embryos . nature , 1981 . 292 ( 5819 ): p . 154 - 6 . 2 . thomson , j . a ., et al ., embryonic stem cell lines derived from human blastocysts . science , 1998 . 282 ( 5391 ): p . 1145 - 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