WO2013004135A1 - 诱导性多能干细胞的制备方法和用于制备诱导性多能干细胞的培养基 - Google Patents

诱导性多能干细胞的制备方法和用于制备诱导性多能干细胞的培养基 Download PDF

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WO2013004135A1
WO2013004135A1 PCT/CN2012/077579 CN2012077579W WO2013004135A1 WO 2013004135 A1 WO2013004135 A1 WO 2013004135A1 CN 2012077579 W CN2012077579 W CN 2012077579W WO 2013004135 A1 WO2013004135 A1 WO 2013004135A1
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medium
osmotic pressure
cells
oct4
factor
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谢欣
许新秀
张儒
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中国科学院上海药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a method for preparing induced pluripotent stem cells and a medium for preparing induced pluripotent stem cells.
  • Embryonic stem cells are known as "universal cells". In theory, they can be differentiated into any of the 220 cells of the human body, thus forming various complex tissues and organs of the body, along with mouse embryonic stem cells in 1981 and humans in 1998.
  • the application prospect of embryonic stem cells is mainly for transplantation therapy. With embryonic stem cells as the starting cells, a large amount of tissue and organ transplantation materials can be provided for clinical treatment by in vitro culture and directed differentiation.
  • many specific cell types such as nerve cells, cardiomyocytes, etc.
  • iPS Cells Induced Pluripotent Stem Cells (iPS Cells) [3].
  • iPS Cells Induced Pluripotent Stem Cells
  • the Yamanaka group and the Thomson group reprogrammed human somatic cells into iPS cells [4-5].
  • researchers first used iPS cells to obtain surviving and fertile mice through tetraploid blastocyst injection, demonstrating the true versatility of iPS cells [6].
  • iPS As a technique for inducing pluripotent stem cells, iPS is of great value in the treatment of clinical diseases because it does not require human eggs or human embryos as in the case of traditional nuclear transfer or cell fusion. Stem cells, thus circumventing many obstacles such as ethics and law, and it avoids immune rejection by reprogramming adult cells into stem cells, making autologous transplantation more feasible; on the other hand, iPS studies stem cell self-renewal mechanisms, Research on stem cell signaling regulation and exploring the pathogenesis of many diseases and finding treatments are of great significance. However, the application of iPS cells still has two major problems to be solved: biosafety issues and induced efficiency problems.
  • the exogenous gene overexpressed in the original experimental system contained two proto-oncogenes (c-Myc, Klf-4), and the foreign gene was introduced by retrovirus, and the virus was in the genome. Insertion with multiple copies can also result in genetic mutations and cancerous changes. Therefore, everyone is working to optimize iPS technology to improve the safety of its applications, for example, efficient preparation of virus-free iPS cells by transposon-mediated transgenic methods [7-8]; The previously introduced transcription factor gene is removed from iPS cells [9]; iPS cells can be efficiently obtained with the introduction of fewer exogenous genes in the induction of iPS [10-17] And reprogramming directly by adding a transmembrane transcription factor protein [18].
  • the induction efficiency is also a rate-limiting step that restricts the development and application of iPS cell technology.
  • the initial iPS induction efficiency is generally less than 1%, and the use of small molecule compounds can greatly improve the reprogramming efficiency and reduce the number of transcription factors used.
  • small molecules are rich in resources and flexible in combination application.
  • stem cell differentiation can be inhibited and self-renewal can be promoted (for example, adding LIF to the culture medium (for mouse stem cells) and bFGF (for human stem cells). Etc. [19-20]);
  • LIF to the culture medium
  • bFGF for human stem cells
  • Etc. [19-20] The optimization of the microenvironment during somatic cell reprogramming can also significantly increase iPS cell induction efficiency.
  • changes in the physical environment of hypoxia can also improve the reprogramming efficiency of human and mouse somatic cells or promote ES cell self-renewal, etc. [21-23] Therefore, the change of the physical environment is also a worthwhile direction to improve the efficiency of induced reprogramming, and it also plays an important role in understanding the reprogramming mechanism.
  • Hyperosmosis acts as an environmental stimulating stress cell, which activates p38 MAPK signaling pathway [24-26], changes the expression of related transcription factors and induces stem cells. Differentiation [27-28]. Therefore, the medium currently used for stem cell culture, Knockout DMEM, is an optimized hypotonic medium having an osmotic pressure of 270 mOSM/kg. Traditionally, the optimized medium osmotic pressure and mouse embryonic tissue osmotic pressure are close, which is more suitable for the growth of undifferentiated embryonic stem cells and induced pluripotent stem cells [29-30].
  • the present invention provides a method for suppressing the induction of pluripotent stem cells by changing the medium, the method comprising the steps of:
  • Step 1 introducing one or more stem cell pluripotency factors into the somatic cells
  • Step 2 culturing the stem cells into which the stem cell pluripotency factor was introduced in the step 1 is cultured using a stem cell culture medium, wherein, in the process, further including culturing the cells using a high osmotic pressure medium;
  • Step 3 Identify induced pluripotent stem cell clones.
  • the method further comprises introducing a reporter gene into the somatic cell to thereby indicate the production of the induced pluripotent stem cell and the efficiency of its production by the reporter gene.
  • the reporter gene is Oct4-GFP or Nanog-GFP.
  • the reporter gene is Oct4-GFP.
  • the cDNA of the stem cell pluripotency factor is introduced into mouse embryonic fibroblasts in a viral infection manner.
  • the stem cell pluripotency factor may be selected from the group consisting of Oct4, Sox2, Sox Klf4, K1Q, Klf5, Nanog, c-Myc, L-Myc, N-Myc, Lin28 and Esrrb. And more preferably, the stem cell pluripotency factor may be a four factor including Oct4, Sox2, Klf4, and c-Myc; or, the stem cell pluripotency factor may be a three factor including Oct4, Sox2, and Klf4; The stem cell pluripotency factor may be a two factor including Oct4 and Sox2.
  • the cells are cultured for 1 to 9 days using high osmotic pressure medium from the 3rd to 6th day after introduction of the factor.
  • the cells are cultured using high osmotic pressure medium on days 3 to 12 after introduction of the factors, or on days 6 to 9 after introduction of the factors.
  • the high osmotic medium is osmotic or non-ionic by adding NaCl, sucrose, mannitol, Na 2 SO 4 , meglumine or the like in a common isotonic medium.
  • the substance for increasing the osmotic pressure is selected from the group consisting of NaCl and sucrose. Most preferably, in the high osmotic pressure medium, the substance used to increase the osmotic pressure is NaCl.
  • the osmotic pressure of the high osmotic pressure medium is 380-480 mOSM/kg.
  • the osmotic pressure is 480 mOSM/kg (ie plus 100 in isotonic medium) mM NaCl or 200 mM sucrose). Or more preferably, the osmotic pressure is 380 mOSM/kg under conditions of two-factor (Oct4 and Sox2)-infected MEF-induced iPS (g is added to the isotonic medium plus NaCl 50 mM).
  • Oct4 and Sox2 two-factor
  • the high osmotic pressure medium may be an osmotic pressure increasing mES medium and/or an osmotic pressure increasing KSR medium.
  • the hyperosmotic medium further comprises a small molecule compound selected from the group consisting of VPA, LiCl, CHI 9902 epsox, Parnate, and the like.
  • the mES medium is DMEM (Dulbcco, supplemented with 15% fetal calf serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin/streptomycin, and ⁇ -mercaptoethanol.
  • the KSR medium is supplemented with 15% replacement serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin/streptomycin, and ⁇ -mercapto Knockout DMEM for ethanol.
  • LIF leukemia inhibitory factor
  • L-glutamine L-glutamine
  • non-essential amino acids penicillin/streptomycin
  • ⁇ -mercapto Knockout DMEM for ethanol.
  • the cells are cultured using a high osmotic pressure medium to culture the cells for 0 to 3 days using an osmotic pressure-enhancing mES medium, and then the osmotic pressure is increased.
  • the cells were cultured for 0 to 6 days in KSR medium.
  • the present invention provides a method for preparing induced pluripotent stem cells, comprising: Step 1, introducing Oct4, Sox2, Klf4 and c-Myc four factors or Oct4, Sox2, Klf4 three factors or Oct4, Sox2 two factors Mouse embryonic fibroblasts, wherein, further comprising introducing a reporter gene Oct4-GFP into the somatic cell;
  • Step 2 The mouse embryonic fibroblasts introduced in step 1 into which the Oct4, Sox2, Klf4 and c-Myc four factors or Oct4, Sox2, Klf4 three factors or Oct4, Sox2 factors are introduced are digested and inoculated the next day.
  • the osmotic pressure was 380 ⁇ 480 mOSM/kg in high osmotic pressure mES medium, and on the sixth day, it was changed to high osmotic pressure KSR medium with osmotic pressure of 380 ⁇ 480 mOSM/kg, and changed to iso-osmosis on the eighth day.
  • the KSR medium was further cultured, and the introduction of Oct4, Sox2, Klf4 and c-Myc four factors or Oct4, Sox2, Klf4 three factors or Oct4, Sox2 into mouse embryonic fibroblasts was recorded as the first step. 0 days; and
  • Step 3 Select clones with good stem cell morphology or Oct4-GFP positive for passage.
  • “Good stem cell-like morphology” refers to a clone that is similar in morphology to mouse stem cells.
  • “Oct4-GFP positive” refers to a clone of the transgenic Oct4-GFP reporter gene. Oct4 is a stem cell-specific gene whose expression is more realistic in characterizing that mouse embryonic fibroblasts have been reprogrammed into stem cells.
  • step 3 the identified pluripotent stem cell clones were identified by AP staining, endogenous Oct4 expression detection, fluorescent quantitative PCR for detection of pluripotency marker expression, silencing of exogenous viral genes, and formation of teratomas.
  • the somatic cells are derived from a somatic cell of a mammal.
  • the mammal is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a sheep, a cow, a monkey or a human.
  • the present invention provides a medium for preparing induced pluripotent stem cells, which is further used to increase the osmotic pressure medium, and further to provide hyperosmotic conditions in the early stage of induction, which is more conducive to iPS formation, and later to hyperosmotic conditions. No significant effect.
  • the medium for preparing the induced pluripotent stem cells may be an osmotic pressure increasing medium, preferably, an osmotic pressure increasing mES medium and/or an osmotic pressure increasing KSR medium.
  • the high osmotic pressure medium further comprises a small molecule compound selected from the group consisting of VPA, LiCl, CHI 99021, epsox, Parnate, and the like.
  • the mES medium is DMEM (Dulbcco, supplemented with 15% fetal bovine serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin/streptomycin, and ⁇ -mercaptoethanol.
  • the KSR medium is supplemented with 15% replacement serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin I streptomycin and ⁇ -mercapto Knockout DMEM for ethanol.
  • LIF leukemia inhibitory factor
  • L-glutamine L-glutamine
  • non-essential amino acids penicillin I streptomycin
  • ⁇ -mercapto Knockout DMEM for ethanol.
  • the osmotic pressure medium has an osmotic pressure of 380-480 mOSM/kg.
  • the high osmotic pressure medium refers to a medium to which a ionic or nonionic osmotic pressure adjusting substance such as NaCl, sucrose, Na 2 SO 4 or meglumine is added to a common isotonic medium. More preferably, In the high osmotic pressure medium, the substance for increasing the osmotic pressure is selected from the group consisting of NaCl and sucrose. Most preferably, in the high osmotic pressure medium, the substance used to increase the osmotic pressure is NaCl.
  • the present invention efficiently produces iPS cells by increasing the osmotic pressure of the medium.
  • the clone counting experiment showed that the hypertonic experimental group increased the iPS induction efficiency by 15-20 times (transduction four-factor experiment) and 6-fold (transduction two-factor experiment) compared with the control group.
  • Increasing the osmotic pressure of the medium during iPS induction can also speed up the process of reprogramming.
  • the hyperosmolar-induced experimental group can detect Oct4-GFP-positive clones on the 6th day after infection, while the control group usually takes 8 days. Oct4-GFP positive clones can only be detected later.
  • the iPS cells induced by hyperosmosis have good pluripotency, and the iPS clones transduced with four factors or two factors can form teratomas and differentiate into three germ layers.
  • the present invention proposes for the first time that a hypertonic osmotic culture environment is conducive to reprogramming, and it is proved that the physical microenvironment change and the somatic cell reprogramming are closely related in the cell culture process. Hypertonic environments are more straightforward and easier than other methods of increasing reprogramming efficiency.
  • the method for efficiently inducing iPS cells provided by the present invention has important significance for promoting basic research and clinical application of iPS.
  • Figure 1 is a flow chart showing the experimental procedure for promoting the formation of iPS cells using a stem cell medium which increases osmotic pressure.
  • Figure 2 is a graph showing that high osmotic pressure stem cell media enhances four-factor induced iPS efficiency and accelerates iPS induction.
  • A is a photograph showing a representative image of three pairs of wells in a 96-well plate on the 14th day after infection, in which more Oct-GFP-positive clones appeared in the wells treated with 50 mM and 100 mM NaCl;
  • B is shown to Oct- A graph of the statistics of GFP-positive clones, 10-15 or so of Oct-GFP-positive clones were observed in the wells treated with 50 mM and 100 mM NaCl hypertonic medium on the 8th day after infection. The well has no clones.
  • Figure 3 is a graph showing the improvement of the four-factor induced iPS efficiency by flow cytometry for detecting hypertonic environment.
  • A is a graph showing the percentage of GFP-positive cells analyzed by flow cytometry after cell digestion on day 14 after infection.
  • B is a graph showing statistical data of 3 independent experiments. ***, p ⁇ 0.001 o
  • Figure 4 is a graph showing that the addition of 100 mM NaCl or 200 mM sucrose to a stem cell culture medium can increase the efficiency of four-factor induced iPS.
  • A is a photograph showing a representative image of three pairs of wells in a 96-well plate on the 14th day after infection.
  • B is a graph showing three independent experimental statistics on Oct-GFP positive clones, wherein an experimental group in which NaCl or sucrose was added to increase the osmotic pressure of the medium obtained nearly 30 clones on the 14th day after infection, while the control group only There are 3-5 clones. Compared with the control group, **, p ⁇ 0.01; ***, ⁇ 0 ⁇ 001.
  • Figure 5 is a graph showing that increasing the osmotic pressure of the medium in the early stage of reprogramming is more advantageous for the formation of iPS.
  • A is a schematic diagram of the time period of hypertonic treatment.
  • B shows representative images of 96-well plates in hypertonic treatment on days 3-6, 6-9 or 9-12 after infection with four-factor virus, respectively, on day 14 post-infection.
  • C is a graph showing three independent experimental statistics for Oct-GFP positive clones in panel B.
  • *, , D showed hypertonic treatment on the first day, 3-6 days, 3-8 days, 3-10 days or 3-12 days after infection, on the 14th day after infection 96
  • Figure 6 is a graph showing the interaction of high osmotic medium with other chemical small molecules such as valproic acid (VPA) or LiCl to increase the efficiency of four-factor induced reprogramming.
  • A is a statistical plot of the number of GFP positive clones in 96-well plates on day 14 post infection.
  • the figure shows that both NaCl and VPA can increase the induction efficiency of iPS, and the synergistic effect of the two at low concentrations (VPA 0.5 mM + NaC1 50 mM).
  • the figure is a statistical analysis of 3 independent replicates, **, p ⁇ 0.01; ***, p B is the number of GFP-positive clones in 96-well plates on day 14 post-infection.
  • the figure shows that both NaCl and LiCl can increase the induction efficiency of iPS, and the synergistic effect of the two at low concentrations (LiCl 5 mM + NaCl 50 mM).
  • the figure is the statistical data of 3 independent replicate experiments, **, p ⁇ 0.01; ***, ⁇ 0 ⁇ 001 relative to the control group.
  • Figure 7 shows that when induced by two factors (Oct4, Sox2), high osmotic pressure conditions can be combined with small molecule compounds (Repsox 1 ⁇ , CHIR 3 ⁇ and Parnet 5 ⁇ ) to significantly increase the number of iPS clone formation.
  • A is a picture showing that the iPS clone induced to form under high osmotic pressure on the 18th day after the two-factor infection has an embryonic stem cell-like morphology and expresses Oct4-GFP.
  • B is a statistical plot of the number of GFP positive clones on day 18 post infection.
  • the control group was a two-factor induction, and the number of clones obtained when a small molecule compound combination (Repsox 1 ⁇ , CHIR 3 ⁇ and Parnet 5 ⁇ ) was added to the isotonic medium. Adding 50 mM NaCl under the above conditions to increase the osmotic pressure can greatly improve The number of clones with high iPS.
  • C is a diagram showing the identification of viral DNA in a two-factor iPS clone. Among them, the iPS clone induced by four factors was used as a positive control, and water as a negative control. It can be seen that all the two-factor iPS clones have only the integration of Oct4 and Sox2 genes, and no Kif4 and c-Myc gene insertion.
  • Figure 8 is a graph showing staining of pluripotency markers of iPS clones induced under high osmotic pressure conditions.
  • A is a graph showing alkaline phosphatase staining and immunofluorescence staining of iPS clones induced by four factors under high osmotic pressure conditions.
  • the first line of the picture shows that the iPS cell line induced under hypertonic conditions has a morphology similar to that of embryonic stem cells, strongly expressing Oct4-GFP, alkaline phosphatase (AP) staining is positive and uniform; second and third rows Immunofluorescence staining for stem cell-specific proteins Nanog and SSEA-1, iPS cell lines expressed these two stem cell-specific proteins, and Hoechst showed nuclear DNA staining.
  • B is an alkaline phosphatase staining and immunofluorescence staining of iPS clones induced by two factors (Oct4 and Sox2) under high osmotic pressure.
  • the first line of the picture shows that the iPS cell line induced under hypertonic conditions has a morphology similar to that of embryonic stem cells, strongly expressing Oct4-GFP, and the alkaline phosphatase staining is positive and uniform; the second and third lines are stem cells respectively.
  • Immunofluorescence staining of specific proteins Nanog and SSEA-1, iPS cell lines expressed these two stem cell-specific proteins, and Hoechst showed nuclear DNA staining.
  • Figure 9 is a graph showing the expression of stem cell-specific genes and the silencing of foreign viral genes by real-time PCR.
  • A Mouse embryonic fibroblast (MEF) was used as a negative control, and mouse embryonic stem cell line E14 (ES) was used as a positive control to detect the expression of endogenous specific genes in stem cells of iPS clones. All four-factor iPS clones induced under high osmotic pressure highly expressed stem cell-specific genes, including endogenous Oct4, endogenous Sox2, Nanog and Rexl; B: positive for mouse embryonic fibroblasts 4 days after virus infection In contrast, ES cells were negative controls and the silence of the exogenous viral gene was detected. All four-factor iPS clones induced under high osmotic pressure showed good exogenous viral gene silencing.
  • Figure 10 is a graph showing the results of a four-factor iPS-forming teratoma experiment induced under hypertonic conditions.
  • the HE staining and judging by the tissue structure, the iPS cell line can differentiate into the unique tissues of the three germ layers, where a is the neural tube-like structure of the ectoderm, b is the cartilage-like structure of the mesoderm, and c is the epidermal-like structure of the ectoderm. , d is the gastrointestinal lumen epithelial-like structure of the endoderm.
  • experiments of the present invention will use molecular biology, microbiology, cell biology Learning, immunology, and recombinant DNA conventional techniques are within the skill of the art. See for example,
  • inducible pluripotent stem cells is a cell derived from somatic cells reprogrammed in vitro by introduction of stem cell pluripotency factors. Such cells are cultured under embryonic stem cell (ES) conditions, The properties of cell morphology, growth characteristics, specific gene expression, and DNA methylation are very similar to those of mouse ES cells, and also in mouse ES in terms of teratoma formation, chimeric animal formation, and germline transmission. The cells are very similar.
  • the "stem cell pluripotency factor that induces somatic cell reprogramming" as described herein is a factor that is critical for stem cell pluripotency, and can be induced to reprogram into stem cells by introducing these factors into somatic cells. A number of such reports have been reported on factors that can induce reprogramming [31-35].
  • the pluripotency factors include Oct4, Sox2 (or Soxl), Klf4 (or Klf2 or Klf5), Nanog, c-Myc (or L-Myc or N-Myc), Lin28 and Esrrb.
  • the above stem cell pluripotency factor can be derived from any species, preferably a mouse stem cell pluripotency factor, as needed.
  • induced reprogramming refers to the process of dedifferentiating somatic cells into pluripotent stem cells.
  • the somatic cells are dedifferentiated into pluripotent stem cells by introducing a pluripotency factor cDNA required to maintain stem cell pluripotency into somatic cells.
  • the pluripotency factor comprises one of Oct4, Sox2 (or Soxl), Klf4 (or Klf2 or 5), Nanog, c-Myc (or L- or N-Myc), Lin28 and Esrrb Or multiple.
  • the method of introducing the stem cell pluripotency factor cDNA into a somatic cell can be carried out by various methods including viral infection, lipofection, electroporation, particle bombardment, transposon-mediated insertional expression, transmembrane protein, drug Various methods of inducing the transfer of DNA into cells.
  • the vector is transfected with a viral vector comprising a cDNA, including a lentivirus, a retrovirus, an adenovirus, and the like. a viral vector.
  • a retroviral vector (PMX vector) is preferred.
  • the medium described herein may be DMEM supplemented with 15% fetal calf serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin I streptomycin and ⁇ -mercaptoethanol.
  • LIF leukemia inhibitory factor
  • L-glutamine L-glutamine
  • non-essential amino acids penicillin I streptomycin and ⁇ -mercaptoethanol.
  • Knockout DMEM (KS medium) of streptomycin and ⁇ -mercaptoethanol.
  • the substances used to increase the osmotic pressure and the osmotic pressure of the medium shown in this paper were determined by the FM-8P automatic freezing point osmometer (Shanghai Medical University Instrument Factory). Specific values: 100 mM NaCl osmotic pressure is 190 mOSM/kg, 200 mM sucrose osmotic pressure is 190 mOSM/kg, 50 mM NaCl osmotic pressure is 90 mOSM/kg, 100 mM sucrose osmotic pressure is 90 mOSM/kgo DMEM medium ( GIBCO) The osmotic pressure is 290 mOSM/kg and is considered to be isotonic.
  • Knockout DMEM medium has an osmotic pressure of 270 mOSM/kg and is also considered to be isotonic.
  • DMEM plus 100 mM NaCl had an osmotic pressure of 480 mOSM/kg and was considered hypertonic.
  • the osmotic pressure of DMEM plus 200 mM sucrose was 480 mOSM/kg, which was considered as hypertonic condition.
  • the osmotic pressure of DMEM plus 50 mM NaCl was 380 mOSM/kg, which was considered as medium hypertonic condition.
  • the osmotic pressure of DMEM plus 100 mM sucrose was 380 mOSM/kg, which was considered as medium hypertonic condition.
  • the reporter gene of the present invention refers to a stage capable of indicating that a cell has been transformed into a similar embryonic stem cell, and includes a fluorescent protein sequence added by a transgene or homologous recombination or a resistance gene sequence directed against an antibiotic, and the sequence is in an embryonic stem cell. Under the control of the promoter of some genes specifically expressed, the expression of this fluorescent protein or resistance gene can be activated when the cell reaches a state similar to the embryonic stem cell, so that the cell has certain characteristics that can be detected.
  • the mouse embryonic fibroblasts used in this example were OG2 (Oc4-GFP + A ) cells isolated from the 13.5 day embryos produced by the mating of homozygous OG2 (Oct4-GFP + + ) males and 129 female rats.
  • the method for detecting cell pluripotency according to the present invention is well known to those skilled in the art, including AP staining, cell fluorescence staining, PCR to identify stem cell-specific gene expression, real-time PCR analysis of viral gene silencing, and analysis of in vivo differentiation into teratogenicity. The ability of the tumor.
  • Retroviral vectors containing cDNAs of mouse Oct4, Sox2, Klf4, c-Myc were purchased from Addgene. Transfection was performed to Plat-E cells using Fugene HD according to the manufacturer's instructions to generate virus. After 48 hours, the virus supernatant was collected and filtered to supplement 1,5-dimethyl-1,5-diaza eleven Mouse embryonic fibroblasts were infected with methylene poly-methane bromide (8 mg/L). The day when the virus supernatant was added was defined as day 0.
  • the virus-infected fibroblasts were cultured in mES medium and replaced with KSR medium on the 6th day after infection, on days 14-18 after infection (four-factor infection test) or 20-24 days (two factors) Infection experiments) Pick up iPS colonies, which are based on Oct-GFP expression and typical stem cell morphology.
  • the main method for calculating the efficiency of reprogramming is to count Oct-GFP positive clones: 1. Count the number of Oct-GFP positive clones directly in the original wells by fluorescence microscopy on the 14th day after the four-factor or two-factor infection; The percentage of Oct-GFP positive cells was determined by flow cytometry on cells on day 14 after four factor infection or on day 18 after infection with two factors.
  • the identification process of iPS cells is as follows:
  • Example 1 ⁇ osmotic dry cell culture medium supplemented with NaCl osmotic pressure can promote the formation of four-factor induced iPS
  • a four-factor (Oct4, Sox2, Klf4, c-Myc) virus was mixed in equal volumes and added to a hole in a 6-well plate for a total of 180,000 OG2 mouse embryonic fibroblasts at 37 ° C, The culture was carried out in a 5% CO 2 environment in DMEM medium supplemented with 10% fetal bovine serum. On the day of the addition of the virus, day 0, the cells were digested and resuspended in mES medium on day 2, and inoculated into 96-well plates pre-planted with feeder cells (radiation-treated mouse embryonic fibroblasts). 5000 cells per well.
  • the mES medium with different osmotic pressure values was used from the 3rd day (that is, the isotonic medium was added to the hypertonic medium by adding 50 mM or 100 mM NaCl, and the osmotic pressure of the hypertonic medium was 380 mOSM/kg and 480 mOSM/kg) culture, starting on day 6 and changing to high osmolality KSR medium (ie adding 50 mM or 100 mM NaCl to make isotonic medium hyperosmotic
  • the culture medium and the osmotic pressure of the hypertonic medium were cultured at 380 mOSM/kg and 480 mOSM/kg, respectively. From the 8th day, the culture was carried out by changing to KSR medium (isotonic medium condition), and the whole process is shown in Fig. 1.
  • the control group was cells cultured under isotonic medium conditions (290 mOSM/kg).
  • FIG. 2A is a representative picture of a 96-well plate at day 14, and it can be seen that the wells treated with the medium supplemented with 100 mM NaCl had more Oct-GFP positive clones than the untreated control wells.
  • Figure 2B is a statistical data of Oct-GFP positive clones.
  • FIG. 3A is a representative flow data plot
  • Figure 3B is a statistical data of three experiments showing that treatment with medium supplemented with 100 mM NaCl increased the iPS induction efficiency by 28-fold under four-factor induction conditions.
  • Example 2 Stem cell medium supplemented with sucrose also promotes the formation of four-factor induced iPS
  • a four-factor (Oct4, Sox2, Klf4, c-Myc) virus mixed in equal volumes, infected into a hole in a 6-well plate, a total of 180,000 OG2 mouse embryonic fibroblasts, at 37 ° C,
  • the culture was carried out in a 5% CO 2 environment in DMEM medium supplemented with 10% fetal bovine serum.
  • the cells were digested and resuspended in mES medium on the 2nd day, and seeded in a 96-well plate pre-planted with feeder cells (radiation-treated mouse embryonic fibroblasts). 5000 cells in the well.
  • the high osmolality mES medium was used from the third day (that is, the isotonic medium was added to the hypertonic medium by adding 100 mM NaCl and 200 mM sucrose respectively, and the osmotic pressure of the hypertonic medium was 480 mOSM/ Kg) cultured, starting on day 6 and switching to high osmolality KSR medium (ie adding 100 mM NaCl and 200 mM sucrose respectively to make isotonic medium into hypertonic medium, osmotic pressure of hypertonic medium) Both were cultured at 480 mOSM/kg). On the 8th day, the culture was further changed to KSR medium (isotonic medium condition).
  • the control group was cells cultured under isotonic medium conditions (290 mOSM/kg). The whole process is shown in Figure 1.
  • b Using the method described in a, from day 12, the number of Oct-GFP positive clones was observed and counted under an inverted fluorescence microscope every day while photographing using a fluorescence microscope.
  • Figure 4A is a representative picture of a 96-well plate at day 14.
  • Example 3 The osmotic pressure of the medium was more favorable for the formation of iPS cells in the early stage of reprogramming.
  • a four-factor (Oct4, Sox2, Klf4, c-Myc) virus mixed in equal volumes, infected into a well of a 6-well plate, a total of 180,000 OG2 mouse embryonic fibroblasts, at 37 ° C,
  • the culture was carried out in a 5% CO 2 environment in DMEM medium supplemented with 10% fetal bovine serum.
  • the cells were digested and resuspended in mES medium on the 2nd day, and seeded in a 96-well plate pre-planted with feeder cells (radiation-treated mouse embryonic fibroblasts). 5000 cells in the well.
  • the medium was incubated with high osmotic pressure mES medium (ie, medium supplemented with 100 mM NaCl) for 1 to 3 days, and on the sixth day, it was changed to high osmotic pressure KSR medium (ie, culture with 100 mM NaCl). Base) The culture was further cultured for 0 to 6 days, and then cultured in a normal osmotic pressure KSR medium to continue cloning. The time at which the high osmotic pressure starts to be processed and the length of the treatment time are as shown in Fig. 5A.
  • the control group was cells cultured under isotonic medium conditions.
  • Figure 5D shows hypertonic treatment on days 3-4, 3-6, 3-8, 3-10 or 3-12 days after infection, respectively, in the 96-well plate on day 14 post-infection.
  • Example 4 The osmotic medium interacts with other chemical small molecules to extract 4 factor-induced reprogramming efficiency
  • the control group was cells that were transferred to four factors, cultured under isotonic medium conditions, and no small molecule compounds were added.
  • Example 5 Lifting osmotic pressure combined with other small molecule compounds can promote the formation of two-factor induced iPS
  • a two-factor (Oct4 and Sox2) virus mixed in equal volumes, infected into a hole in a 6-well plate, a total of 180,000 OG2 mouse embryonic fibroblasts, at 37 ° C, 5% CO 2 environment
  • the medium was cultured in DMEM medium supplemented with 10% fetal calf serum.
  • the experimental group was replaced with a small molecule compound combination (CHIR99021 3 ⁇ , Repsox 1 M, Parnet 5 ⁇ ), and 50 mM NaCl KSR medium (medium hypertonic medium conditions).
  • the control group was changed to a KSR (isotonic medium condition) medium in which only a small molecule compound combination (CHIR99021 3 ⁇ , Repsox 1 ⁇ , Parnet 5 ⁇ ) was added.
  • KSR isotonic medium condition
  • the experimental group and the control group were changed to KSR with only the combination of small molecule compounds (CHI 99021 3 ⁇ , Repsox 1 ⁇ , Parnet 5 ⁇ ).
  • Isotonic medium conditions Medium, on day 10, replaced with KSR medium without compound addition (isotonic medium conditions).
  • FIG. 7A the two-factor iPS clone iPS cell line on the 18th day after infection had a morphology similar to that of embryonic stem cells.
  • Figure 7B is a statistical data of Oct-GFP positive clones. On the 18th day after infection, there were only 2 clones in the control wells, and 13 Oct-GFP positive clones were observed in wells treated with medium hypertonic medium supplemented with 50 mM NaCl.
  • iPS clone pick the iPS clone on day 20, subculture the digested cells to obtain a two-factor iPS cell line, and extract the DNA to detect the integration of the viral gene by PCR, confirming that the iPS clone is indeed two factors. (Oct4 and Sox2) induced production. Before extracting DNA, the trophoblast cells were removed by differential adherence, and the total DNA of iPS cells was extracted using Trizol (Invitrogen) reagent according to the manufacturer's instructions, and TaKaRa TaqTM kit (Takara), Bio-ad was applied. PCR was performed on the DNA Engine PC instrument. The above PCRs were performed using conventional PCR conditions according to the kit manufacturer's instructions.
  • IPS cell line induced under osmotic culture conditions is pluripotent a.
  • OG2 is infected with four factors (Oct4, Sox2, Klf4, c-Myc) or two factors (Oct4 and Sox2).
  • Mouse embryonic fibroblasts were cultured in hypertonic medium, and representative clones were picked according to clonal morphology and fluorescence expression 14 days after infection (four factors) or 18 days (two factors), and uniform iPS was formed after passage. Cell line.
  • iPS cell lines Morphological observation of selected iPS cell lines and staining of stem cell-specific proteins. As shown in Figure 8, the four-factor and two-factor iPS cell lines obtained by hypertonic conditions have characteristic morphology similar to embryonic stem cells, strongly expressing Oct-GFP, and alkaline phosphatase (AP) staining is positive. Immunofluorescence staining of iPS cells using antibodies against stem cell signature proteins revealed that iPS cell lines express stem cell-specific proteins Nanog and SSEA-1.
  • RNA was extracted Before the RNA was extracted, the trophoblast cells were removed by differential adherence, and the total RNA of the iPS cells was extracted using Trizol (Invitrogen) reagent according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScriptTM kit (Takara), and Real-Time PCR analysis of exogenous gene silencing and endogenous using the JumpStartTM Taq ReadyMixTM kit (Sigma), Mx 3000P real-time PCR instrument (Stratagene) Gene expression, PCR were performed using conventional PCR conditions, following the kit manufacturer's instructions. A list of primers used to identify iPS-specific genes is shown in Table 2.
  • the iPS cell lines obtained using the methods of the present invention all expressed higher levels of pluripotency factors, including endogenous Oct4, endogenous Sox2, Nanog and exl, expression levels of mouse embryonic stem cells.
  • the line E14 is equivalent. These factors have no detectable expression in the original mouse embryonic fibroblasts.
  • the iPS cell lines obtained by the method of the present invention are capable of better silencing the exogenous viral gene, and also indicate that the cells are reprogrammed to stem cells.
  • iPS cells were resuspended in 200 ⁇ L mES medium and injected into NOD- SCID mouse in the inner thigh muscle. After 4-5 weeks, the mice were sacrificed, teratoma was embedded in paraffin, sectioned, and histological analysis was performed using hematoxylin/eosin staining. As shown in Fig. 10, tissues derived from three germ layers can be found in teratoma slices formed by iPS cells, including neural tube-like tissues representing ectoderm, epidermal-like structures, cartilage tissues representing mesoderm, and representative tissues. Digestive lumen-like tissue of the germ layer.
  • the mouse four-factor iPS clone obtained by hyperosmosis can have the pluripotency of different types of cells differentiated into three germ layers like mouse embryonic stem cells.
  • the hypertonic medium environment of the present invention is capable of efficiently producing iPS cells.
  • the computational efficiency of the clone counting experiment showed that the hypertonic experimental group increased about 10-25 times (transduced four-factor experiment) and 6-fold (transduced 2-factor experiment) compared with the control group, and the iPS cells induced by hyperosmosis had good versatility. Sex.
  • the method for efficiently inducing iPS cells discovered by the present invention is a basic research for promoting iPS and Clinical application has important implications.
  • Zhao, X.Y., et al, iPS cells produce viable mice through tetraploid comp lemen tation .
  • Esteban, MA, et al Vitamin C enhances the generation of mouse and human induced pluripotent stem cells.
  • Ichida, JK, et al A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell, 2009. 5(5): p. 491-503.
  • Shi, Y., et al Induction of Puluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.

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Abstract

提供了一种诱导性多能干细胞的制备方法，包括以下步骤：步骤1，将一个或多个干细胞多能性因子导入体细胞；步骤2，使用干细胞培养基对步骤1中制备的导入了干细胞多能性因子的体细胞进行培养，其中，在该过程中，进一步包括使用高渗透压培养基对所述细胞进行培养；和步骤3，鉴定诱导性多能干细胞克隆。还提供了一种用于制备诱导性多能干细胞的培养基，将高渗透压的培养基应用于制备iPS细胞中。高渗透压环境能够使小鼠iPS细胞的产生效率提高10-25倍。该iPS细胞诱导方法有利于iPS技术安全性的提高和iPS细胞在再生医学领域的应用。

Description

诱导性多能干细胞的制备方法和用于制备诱导性多能干细胞的培养基 技术领域
本发明涉及生物医药领域，具体涉及诱导性多能干细胞的制备方法和用于 制备诱导性多能干细胞的培养基。
背景技术
胚胎干细胞被誉为"万能细胞"， 从理论上讲， 可分化为人体 220种细胞中 的任何一种， 从而构成机体各种复杂的组织器官， 随着 1981年小鼠胚胎干细 胞和 1998年人的胚胎干细胞的建系成功 [1-2]， 再生医学的研究真正开始。 胚 胎干细胞的应用前景主要是用于移植治疗， 以胚胎干细胞作为起始细胞， 通 过体外培养及定向分化， 可以为临床治疗中提供大量的组织和器官的移植材 料。 通过对于胚胎干细胞自我更新和定向分化机制的研究， 许多特异性的细 胞类型 (例如神经细胞、 心肌细胞等)已经具备了成熟的分化方法和相应的检 测标准。 它可以用于治疗帕金森氏症、 脊髓损伤和糖尿病等组织器官缺损或 功能障碍等多种疾病。 但一直以来， 为了获取人体 ES细胞， 必须摧毁胚胎， 这一点使胚胎干细胞治疗研究一直面临的伦理和法律等诸多障碍。 于是科学 家们开始寻求不通过胚胎而将分化细胞重编程直接逆转为干细胞的新方法。
2006年 8月， Yamanaka小组将 24种转录因子排列组合导入小鼠成纤维 细胞， 最终确定最少有 4种转录因子组合一 Oct4、 Sox2、 c-Myc和 Klf4即可 将小鼠成纤维细胞重编程为诱导性多能干细胞 （Induced Pluripotent Stem Cells, iPS Cells) [3]。 2007年底， Yamanaka小组和 Thomson小组先后将人 的体细胞重编程为 iPS细胞 [4-5]。 2009年， 研究人员首次利用 iPS细胞通过 四倍体囊胚注射得到存活并具有繁殖能力的小鼠， 证明了 iPS细胞具有真正 的全能性 [6]。 iPS作为诱导产生多能性干细胞的一种技术， 在临床疾病治疗 方面的应用价值是巨大的， 因为它不像传统的核移植或者细胞融合等获取干 细胞的方式那样需要人的卵子或人的胚胎干细胞， 从而绕开了伦理和法律等 诸多障碍， 并且它利用由成体细胞重编程为干细胞的特性避免了免疫排斥使 得自体移植变得更加可行； 另一方面， iPS对于干细胞自我更新机制的研究、 干细胞信号调控的研究以及探索很多疾病的发病机制并寻找治疗方法都有很 大意义。 但是， iPS细胞的应用仍然有两大难题亟待解决： 生物安全问题和诱导 效率问题。 在生物安全问题方面， 最初实验体系中过表达的外源基因中含有 两个原癌基因 (c-Myc， Klf-4), 并且外源基因的导入方式是通过逆转录病毒， 病毒在基因组中有多个拷贝的插入也会导致基因突变及癌变。 因此， 大家致 力于优化 iPS技术以提高其应用的安全性， 例如， 借助转座子介导的转基因方 法高效制备了无病毒 (virus-free) iPS细胞 [7-8]; 成功地从所获得的 iPS细胞中 移除先前导入的转录因子基因 [9]; iPS的诱导过程中使用特定小分子化合物， 在更少的外源基因导入的情况下即可高效率获得 iPS细胞 [10-17];以及直接通 过添加透膜的转录因子蛋白的形式进行重编程 [18]。 这些发现使人们向制备 无遗传修饰的 iPS细胞迈出了一大步。 另外诱导效率也是制约 iPS细胞技术发 展和应用的一个限速步骤， 最初 iPS的诱导效率一般都低于 1%， 而选用小分 子化合物可大幅度提高重编程效率并且减少转录因子使用个数。 同时， 小分 子资源丰富而且组合应用灵活多样， 通过加入小分子改变细胞培养环境， 从 而激活细胞重编程相关信号通路， 提高体细胞重编程效率， 这一技术领域正 逐步为各国科学家所重视。 细胞的培养环境对干细胞自我更新和分化尤为重 要， 通过添加利于干细胞生长的生长因子可以抑制干细胞分化， 促进自我更 新（例如在培养基中加入 LIF (适合鼠干细胞)， 加入 bFGF (适合人干细胞）等 等 [19-20] ) ; 同样在体细胞重编程过程中微环境的优化也可以显著提高 iPS细 胞诱导效率。 除了添加相应生长因子及小分子化合物改变细胞生长微环境之 外， 低氧这一物理环境的改变也可以提高人和小鼠体细胞重编程效率或者促 进 ES细胞自我更新等等 [21-23]， 因此， 物理环境的改变也是提高诱导重编程 效率的一个值得探索的方向， 对理解重编程机制也有着十分重要的作用。
在以往的应用研究中， 高渗环境对于 ES细胞有着促进分化的作用， 高渗 作为一种环境剌激胁迫细胞， 通过激活 p38 MAPK 信号途径 [24-26]， 改变相 关转录因子表达进而诱导干细胞分化 [27-28]。 因此， 目前用于干细胞培养的 培养基 Knockout DMEM是经过优化后的低渗培养基， 经测定其渗透压为 270 mOSM/kg。 传统上认为， 经过优化的培养基渗透压和小鼠胚胎组织渗透压接 近， 更适于未分化的胚胎干细胞和诱导性多能干细胞的生长 [29-30]。 但是， 在小鼠体细胞重编程过程中应用高渗环境， 研究高渗透压对于小鼠体细胞重 编程过程的影响， 以及高渗环境如何导致相关转录因子表达的改变， 目前尚 无报道。 因此， 研究渗透压在体细胞重编程过程的作用， 寻找能够提高 iPS 细胞诱导效率的培养条件，对推动 iPS细胞的基础研究和临床应用将有巨大帮 助。
发明内容
针对现有技术中存在的问题， 本发明人进行了广泛和深入的研究， 最终 完成本发明。
为了提高 iPS细胞的诱导效率， 本发明提供了通过改变培养基渗透压制 备诱导性多能干细胞的方法， 该方法包括以下步骤：
步骤 1， 将一个或多个干细胞多能性因子导入体细胞；
步骤 2， 使用干细胞培养基对步骤 1 中制备的导入了干细胞多能性因子 的体细胞进行培养， 其中， 在该过程中， 进一步包括使用高渗透压培养基对 所述细胞进行培养； 以及
步骤 3， 鉴定诱导性多能干细胞克隆。
优选地， 所述方法进一步包括将报告基因导入体细胞以此通过报告基因 来指示所述诱导性多能干细胞的产生及其产生效率。 优选地， 所述报告基因 为 Oct4-GFP或 Nanog-GFP。 且更优选地， 所述报告基因为 Oct4-GFP。
优选地， 在步骤 1中， 将所述干细胞多能性因子的 cDNA以病毒感染方 式导入小鼠胚胎成纤维细胞。
优选地，在步骤 1中，所述干细胞多能性因子可选自 Oct4、 Sox2、 Sox Klf4、 K1Q、 Klf5、 Nanog、 c-Myc、 L-Myc、 N-Myc、 Lin28禾卩 Esrrb中。 且 更优选地， 所述干细胞多能性因子可为包括 Oct4、 Sox2、 Klf4和 c-Myc的四 因子； 或者， 所述干细胞多能性因子可为包括 Oct4、 Sox2和 Klf4的三因子； 或者， 所述干细胞多能性因子可为包括 Oct4和 Sox2的二因子。
优选地， 在步骤 2中， 从导入所述因子后第 3至 6天开始使用高渗透压 培养基对所述细胞进行培养 1至 9天。
更优选地， 在步骤 2中， 在导入所述因子后第 3至第 6天， 或者在导入 所述因子后第 3至第 8天，或者在导入所述因子后第 3至第 10天，或者在导 入所述因子后第 3至第 12天，或者在导入所述因子后第 6至第 9天，使用高 渗透压培养基对所述细胞进行培养。 优选地， 在步骤 2中， 所述高渗透压培养基是通过在普通等渗培养基中 外加 NaCl、 蔗糖 (sucrose)、 甘露醇、 Na2SO4、 葡甲胺等离子型或非离子型渗 透压调节物质而制备的培养基。 更优选地， 在所述高渗透压培养基中， 用来 提高渗透压的物质选自 NaCl和蔗糖中。 最优选地， 在所述高渗透压培养基 中， 用来提高渗透压的物质为 NaCl。 优选地， 在步骤 2中， 所述高渗透压培 养基的渗透压为 380-480 mOSM/kg。更优选地，在四因子 (Oct4、 Sox2、 c-Myc 和 Klf4)感染小鼠胚胎成纤维细胞 (MEF)诱导 iPS 条件下， 渗透压为 480 mOSM/kg (即在等渗培养基中外加 100 mM NaCl或 200 mM蔗糖）。 或更优 选地， 在二因子 (Oct4 和 Sox2)感染 MEF 诱导 iPS 条件下， 渗透压为 380 mOSM/kg(g卩在等渗培养基中外加 NaCl 50 mM)。 渗透压超过 480 mOSM/kg 时， 长期培养将导致细胞死亡； 渗透压低于 380 mOSM/kg时， 不能显著增加 iPS细胞的诱导效率。
步骤 2中， 所述高渗透压培养基可为提高渗透压的 mES培养基和 /或提 高渗透压的 KSR培养基。优选地，所述高渗透压培养基进一步包含选自 VPA、 LiCl、 CHI 9902 epsox, Parnate等中的小分子化合物。 所述 mES培养基 为添加有 15%胎牛血清、 1000 U/mL白血病抑制因子 (LIF)、 L-谷氨酰胺、 非 必需氨基酸、 青霉素 /链霉素和 β-巯基乙醇的 DMEM(Dulbcco，s Modified Eagle's Medium); 以及所述 KSR培养基为添加有 15%替代血清、 1000 U/mL 白血病抑制因子 (LIF)、 L-谷氨酰胺、 非必需氨基酸、 青霉素 /链霉素和 β- 巯基乙醇的 Knockout DMEM。
优选地， 在步骤 2中， 所述使用高渗透压培养基对所述细胞进行培养为 先使用提高渗透压的 mES培养基对所述细胞进行培养 0至 3天，再使用所述 提高渗透压的 KSR培养基对所述细胞进行培养 0至 6天。
优选地， 本发明提供了一种诱导性多能干细胞的制备方法， 包括： 步骤 1， 将 Oct4、 Sox2、 Klf4和 c-Myc四因子或 Oct4、 Sox2、 Klf4三 因子或 Oct4、 Sox2两因子导入小鼠胚胎成纤维细胞， 其中， 进一步包括将报 告基因 Oct4-GFP导入体细胞；
步骤 2， 将步骤 1中制备的导入了 Oct4、 Sox2、 Klf4和 c-Myc四因子或 Oct4、 Sox2、 Klf4三因子或 Oct4、 Sox2两因子的小鼠胚胎成纤维细胞在第 二天消化并接种到饲养层细胞中，使用 mES培养基培养， 并在第三天使用渗 透压为 380〜480 mOSM/kg的高渗透压 mES培养基培养，在第六天换为渗透 压为 380〜480 mOSM/kg的高渗透压 KSR培养基培养，在第八天换为等渗透 压 KSR培养基继续培养， 其中， 以步骤 1 的将 Oct4、 Sox2、 Klf4和 c-Myc 四因子或 Oct4、 Sox2、 Klf4三因子或 Oct4、 Sox2两因子导入小鼠胚胎成纤 维细胞当天记为第 0天； 以及
步骤 3， 挑选具有良好干细胞形态或 Oct4-GFP阳性的克隆进行传代。
"良好干细胞样形态 "是指与小鼠干细胞形态相似的克隆。 "Oct4-GFP 阳 性"是指转基因 Oct4-GFP报告基因阳性的克隆。 Oct4是干细胞特异性基因， 其表达比较真实地表征小鼠胚胎成纤维细胞已经重编程为干细胞。
步骤 3中， 鉴定诱导多能性干细胞克隆包括 AP染色， 内源 Oct4表达 检测， 荧光定量 PCR检测多能性标志物表达水平， 外源病毒基因的沉默， 以 及畸胎瘤的形成。
本发明的方法中， 优选地， 所述体细胞来自哺乳动物的体细胞。 且更优 选地， 所述哺乳动物选自小鼠、 大鼠、 兔、 猪、 羊、 牛、 猴或人。
此外， 本发明提供了一种用于制备诱导性多能干细胞的培养基， 其进一 步为提高渗透压的培养基， 更进一步为诱导前期给予高渗条件更利于 iPS形 成， 后期给与高渗条件并无明显效果。
所述用于制备诱导性多能干细胞的培养基可为提高渗透压培养基， 优选 地，可为提高渗透压的 mES培养基和 /或提高渗透压的 KSR培养基。优选地， 所述高渗透压培养基进一步包含选自 VPA、 LiCl、 CHI 99021、 epsox, Parnate 等中的小分子化合物。 所述 mES培养基为添加有 15%胎牛血清、 1000 U/mL 白血病抑制因子 (LIF)、 L-谷氨酰胺、 非必需氨基酸、 青霉素 /链霉素和 β- 巯基乙醇的 DMEM(Dulbcco，s Modified Eagle's Medium);以及所述 KSR培养 基为添加有 15%替代血清、 1000 U/mL白血病抑制因子 (LIF)、 L-谷氨酰胺、 非必需氨基酸、 青霉素 I链霉素和 β-巯基乙醇的 Knockout DMEM。
优选地，所述提高渗透压培养基的渗透压为 380-480 mOSM/kg。优选地， 所述高渗透压培养基是指在普通等渗培养基中外加 NaCl、 蔗糖 (sucrose )、 Na2SO4、 葡甲胺等离子型或非离子型渗透压调节物质的培养基。 更优选地， 在所述高渗透压培养基中， 用来提高渗透压的物质选自 NaCl和蔗糖中。 最 优选地， 在所述高渗透压的培养基中， 用来提高渗透压的物质为 NaCl。
本发明通过提高培养基渗透压能高效产生 iPS细胞。 克隆计数实验显示 高渗的实验组比对照组 iPS诱导效率提高 15-20倍 （转导四因子实验） 和 6 倍（转导二因子实验）。在 iPS诱导过程中提高培养基的渗透压也可以加快重 编程的过程， 高渗诱导的实验组在感染后第 6天即可检测到 Oct4-GFP阳性 的克隆， 而对照组一般要到 8天以后才能检测到 Oct4-GFP阳性的克隆。 通 过高渗诱导的 iPS细胞具有良好的全能性， 转导四因子或二因子的 iPS克隆 均能形成畸胎瘤， 向三个胚层分化。 与现有技术不同， 本发明首次提出高渗 透压培养环境有利于重编程的进行， 证明细胞培养过程中物理微环境的改变 和体细胞重编程有着紧密联系。 高渗环境比其他提高重编程效率的方法更直 接， 更容易。 本发明提供的高效诱导 iPS细胞的方法对推动 iPS的基础研究 和临床应用有着重要的意义。
附图说明
图 1为利用提高渗透压的干细胞培养基促进 iPS细胞的形成的实验操作 流程图。
图 2为显示高渗透压的干细胞培养基提高四因子诱导 iPS效率并加快 iPS 诱导进程的图。 A为显示感染后第 14天 96孔板内三副孔的代表性图像的照 片， 其中， 50 mM和 100 mM NaCl处理的孔中出现较多的 Oct-GFP阳性克 隆； B为显示对 Oct-GFP阳性克隆的统计数据的图， 在感染后第 8天， 添加 了 50 mM和 100 mM NaCl高渗培养基处理的孔中即能分别观察到 10-15个 左右 Oct-GFP阳性克隆， 而对照孔无克隆。 在感染后第 11 天， 高渗处理的 孔中即能观察到 20-30个左右 Oct-GFP阳性克隆， 而对照孔为 3-5个左右。 在感染后第 14天，高渗处理的孔中即能观察到 30-40个左右 Oct-GFP阳性克 隆， 而对照孔仅有 5个左右。 与对照组相比， *， p<0.05 ; **， p<0.01 ; ***， ρ<0·001。
图 3 为显示流式细胞仪检测高渗环境提高四因子诱导 iPS效率的图。 A 为显示感染后第 14天细胞消化后经流式细胞仪分析的 GFP阳性细胞百分比 的图。 B为显示 3次独立实验的统计数据的图。 ***， p<0.001 o 图 4为显示向干细胞培养基中添加 100 mM NaCl或 200 mM蔗糖均可提 高四因子诱导 iPS效率的图。 A为显示感染后第 14天 96孔板内三副孔的代 表性图像的照片。 B为显示对 Oct-GFP阳性克隆的 3次独立实验统计数据的 图， 其中， 加入 NaCl或蔗糖提高培养基渗透压的实验组可以在感染后第 14 天获得近 30个克隆， 而对照组仅有 3-5个克隆。 与对照组相比， **， p<0.01 ; ***， ρ<0·001。
图 5为显示在重编程的早期提高培养基渗透压对 iPS的形成更为有利的 图。 A为高渗处理的时间段示意图。 B显示了分别在四因子病毒感染后第 3-6 天、 6-9天或 9-12天予以高渗处理， 在感染后第 14天 96孔板内的代表性图 像。 C为显示对图 B中 Oct-GFP阳性克隆的 3次独立实验统计数据的图。 与 对照组相比， *， ， D显示了分别在感染后第 天、 3-6天、 3-8天、 3-10天或 3-12天予以高渗处理， 在感染后第 14天 96孔板 内 Oct-GFP阳性克隆的 3次独立实验统计数据。 与对照组相比， *， p<0.05; **， ρ<0·01。
图 6为显示了高渗透压培养基与其他化学小分子如丙戊酸 (VPA)或 LiCl 共同作用提高四因子诱导重编程效率的图。 A为感染后第 14天 96孔板内 GFP 阳性克隆数的统计图。 该图显示了 NaCl和 VPA均能增加 iPS的诱导效率， 而两者在低浓度时(VPA 0.5 mM + NaC1 50 mM)的效果有协同作用。 该图为 3 次独立重复实验统计数据， 与对照组相比， **， p<0.01 ; ***， p B 为感染后第 14天 96孔板内 GFP阳性克隆数的统计。该图显示了 NaCl和 LiCl 均能增加 iPS的诱导效率， 而两者在低浓度时 （LiCl 5 mM + NaCl 50 mM) 的效果有协同作用。该图为 3次独立重复实验统计数据， 相对于对照组， **， p<0.01 ; ***， ρ<0·001。
图 7显示了二因子 (Oct4， Sox2)诱导时， 高渗透压条件可与小分子化合物 组合 （Repsox 1 μΜ， CHIR 3 μΜ和 Parnet 5 μΜ) 共同作用， 显著提高 iPS 克隆形成数目。 A为显示二因子感染后第 18天在高渗透压条件下诱导形成的 iPS克隆具有类似胚胎干细胞的形态且表达 Oct4-GFP的图片。 B为感染后第 18天 GFP阳性克隆数的统计图。 其中， 对照组为二因子诱导， 在等渗培养 基中加入小分子化合物组合 （Repsox 1 μΜ， CHIR 3 μΜ和 Parnet 5 μΜ) 时 获得的克隆数。 在上述条件下额外加入 50 mM NaCl提高渗透压可以大大提 高 iPS的克隆数目。 C为显示二因子 iPS克隆中病毒 DNA的鉴定的图。其中， 以四因子诱导得到的 iPS克隆作为阳性对照， 以水作为阴性对照， 可以看到 所有二因子 iPS克隆均只有 Oct4和 Sox2的基因整合，而无 Kif4和 c-Myc的 基因插入。
图 8为显示在高渗透压条件下诱导的 iPS克隆的多能性标记物染色的图。 A为显示四因子在高渗透压条件下诱导的 iPS克隆的碱性磷酸酶染色及免疫 荧光染色的图。 其中， 第一行图片显示在高渗条件下诱导的 iPS细胞系具有 类似胚胎干细胞的形态， 强烈表达 Oct4-GFP， 碱性磷酸酶 (AP)染色呈阳性并 且均一； 第二行和第三行分别为干细胞特异性蛋白 Nanog和 SSEA-1的免疫 荧光染色， iPS 细胞系表达这两种干细胞特异性蛋白， Hoechst 显示细胞核 DNA染色。 B为显示了二因子 (Oct4和 Sox2)在高渗透压条件下诱导的 iPS克 隆的碱性磷酸酶染色及免疫荧光染色。 其中， 第一行图片显示在高渗条件下 诱导的 iPS细胞系具有类似胚胎干细胞的形态， 强烈表达 Oct4-GFP， 碱性磷 酸酶染色呈阳性并且均一； 第二行和第三行分别为干细胞特异性蛋白 Nanog 和 SSEA-1 的免疫荧光染色， iPS 细胞系表达这两个干细胞特异性蛋白， Hoechst显示细胞核 DNA染色。
图 9为荧光定量 PCR检测干细胞特异性基因的表达以及外源病毒基因的 沉默的图。 A: 以小鼠胚胎成纤维细胞 (MEF)为阴性对照， 小鼠胚胎干细胞系 E14细胞 (ES)为阳性对照， 检测 iPS克隆中干细胞内源特异基因的表达。 所 有在高渗透压条件下诱导的四因子 iPS克隆均高表达干细胞特异基因， 包括 内源 Oct4， 内源 Sox2， Nanog和 Rexl ; B: 以病毒感染后 4天的小鼠胚胎成 纤维细胞为阳性对照， ES细胞为阴性对照， 检测外源病毒基因的沉默。所有 在高渗透压条件下诱导的四因子 iPS克隆均显示良好的外源病毒基因沉默。
图 10为显示高渗条件下诱导的四因子 iPS形成畸胎瘤实验的结果的图。 经 HE染色并由组织结构判断， iPS细胞系能够分化形成三个胚层的特有组 织， 其中， a为外胚层的神经管样结构， b为中胚层的软骨样结构， c为外胚 层表皮样结构， d为内胚层的消化道管腔上皮样结构。
具体实施方式
定义和技术
除非另外指明， 本发明的实验将使用分子生物学、 微生物学、 细胞生物 学、 免疫学和重组 DNA 传统技术， 其属于本领域技术范围。 参见例如，
Sambrook, Fritsch和 Maniatis， 分子克隆实验指南， 第三版（2002) ; Current Protocols in Molecular Biology ( F. M. Ausubel等人编著（ 1987))；丛书 Methods in Enzymology (酶学方法 ) (Academic Press, Inc. )； PC 2: A Practical Approach ( PC 实验方法） （M. J. MacPherson, B. D. Hames和 G. . Taylor编著， ( 1995 ) )； Antibodies, A Laboratory Manual and Animal Cell Culture (抗体实 验手册及动物细胞培养） ( R. L Freshney编著， ( 1987) )； Handbood of Stem Cells (干细胞手册）， 卷 2 (W. French等人编著)。
除非另外说明， 本文中所用的术语均具有本领域技术人员常规理解的含 义， 为了便于理解本发明， 将本文中使用的一些术语进行了下述定义。
本文所述的"诱导性多能干细胞 (ipsy '是这样的细胞， 其来源是体细胞通 过导入干细胞多能性因子在体外重编程而成。 这样的细胞在胚胎干细胞 (ES) 培养条件下， 在细胞形态、 生长特性、 特异性基因表达、 DNA甲基化方式等 性质均与小鼠 ES细胞非常相似， 而且在畸胎瘤形成、 嵌合体动物形成和生 殖系传递等方面也与小鼠 ES细胞非常相似。
本文所述的"诱导体细胞重编程的干细胞多能性因子"为对于干细胞多能 性维持关键的因子， 通过向体细胞导入这些因子可以诱导体细胞重编程为干 细胞。 已有多篇文献报导了多个这样的可以诱导重编程的因子 [31-35]。 优选 地，所述的多能性因子包括 Oct4， Sox2(或 Soxl)，Klf4(或 Klf2或 Klf5)，Nanog， c-Myc (或 L-Myc或 N-Myc)， Lin28及 Esrrb。 上述干细胞多能性因子可以根 据需要来源于任何物种， 优选为小鼠的干细胞多能性因子。
本文所述的"诱导重编程" （有时也仅被简化为 "诱导"） 是指将体细胞去 分化为多能性干细胞的过程。 优选地， 通过将维持干细胞多能性所需的多能 性因子 cDNA导入体细胞可以诱导体细胞去分化为多能干细胞。 其中， 优选 地， 所述的多能性因子包括 Oct4， Sox2 (或 Soxl )， Klf4 (或 Klf2或 5 )， Nanog, c-Myc (或 L-或 N-Myc)， Lin28及 Esrrb中的一个或多个。
将所述干细胞多能性因子 cDNA导入体细胞的方法可采用多种方法， 包 括病毒感染、 脂质体转染、 电穿孔、 粒子轰击、 转座子介导的插入表达、 穿 膜蛋白、药物诱导等各种将 DNA转入细胞的方法。优选地， 使用包含 cDNA 的病毒载体进行转染， 所述病毒载体包括慢病毒、 逆转录病毒、 腺病毒等多 种病毒载体。 优选地逆转录病毒载体 （PMX载体）。
本文所述的培养基可为添加有 15%胎牛血清、 1000U/mL白血病抑制因 子 (LIF)、L-谷氨酰胺、非必需氨基酸、青霉素 I链霉素和 β-巯基乙醇的 DMEM
(Dulbcco's Modified Eagle's Medium) (mES培养基） 和添加有 15%替代血 清、 1000U/mL 白血病抑制因子 (LIF)、 L-谷氨酰胺、 非必需氨基酸、 青霉素
I链霉素和 β-巯基乙醇的 Knockout DMEM (KS 培养基）。
本文所示用来提高渗透压的物质以及培养基的渗透压由 FM-8P全自动 冰点渗透压计测定 （上海医大仪器厂）。 具体数值： 100 mM NaCl渗透压为 190 mOSM/kg, 200 mM蔗糖渗透压为 190 mOSM/kg, 50 mM NaCl渗透压为 90 mOSM/kg, 100 mM蔗糖渗透压为 90 mOSM/kgo DMEM培养基 (GIBCO) 渗透压为 290 mOSM/kg, 视为等渗条件。 Knockout DMEM培养基 (GIBCO) 渗透压为 270 mOSM/kg, 亦视为等渗条件。 DMEM外加 100 mM NaCl后渗 透压值为 480 mOSM/kg,视为高渗条件。 DMEM外加 200 mM蔗糖后渗透压 值为 480 mOSM/kg, 视为高渗条件。 DMEM外加 50 mM NaCl后渗透压为 380 mOSM/kg,视为中等高渗条件。 DMEM外加 100 mM蔗糖后渗透压为 380 mOSM/kg, 视为中等高渗条件。
本发明所述的报告基因是指能够指示细胞已经转变到类似胚胎干细胞的 阶段， 包括利用转基因或同源重组手段加入的一段荧光蛋白序列或针对抗生 素的抗性基因序列， 这段序列处于胚胎干细胞特异表达的一些基因的启动子 控制下， 故而可以在细胞到达类似胚胎干细胞状态时激活这段荧光蛋白或抗 性基因的表达， 从而使这个细胞具有某些可以被检测的特征。 本实施例中使 用的小鼠胚胎成纤维细胞为 OG2 ( Oct4-GFP+A ) 细胞， 分离自纯合 OG2 (Oct4-GFP+ +) 雄鼠与 129母鼠交配产生的 13.5天的胚胎。
本发明所述的检测细胞多能性的方法是本领域技术人员熟知的，包括 AP 染色， 细胞荧光染色， PCR鉴定干细胞特异基因表达， 荧光定量 PCR分析 病毒基因的沉默， 分析体内分化为畸胎瘤的能力。
下列实施例举例说明了发明人的标准实验室实践， 用于示范本发明的模 式， 而不应将本发明理解为限定于这些实施例的范围。
本发明中所用技术概述： 除了特别说明， 以下实施例中提及的各种物质均购自 Invitrogen。
反转录病毒生产以及产生 iPS细胞的实验过程如下：
从 Addgene公司购置包含小鼠 Oct4， Sox2， Klf4， c-Myc的 cDNA的反 转录病毒载体 （pMXs)。 使用 Fugene HD (罗氏） 按其产品说明书进行转染 至 Plat-E细胞以产生病毒， 48小时后收集病毒上清液并且过滤， 补充 1，5-二 甲基 -1，5-二氮十一亚甲基聚甲溴化物（8 mg/L)后感染小鼠胚胎成纤维细胞。 添加病毒上清液的当天被定义为第 0天。 将病毒感染后的成纤维细胞培养在 mES培养基中，在感染后第 6天更换为 KSR培养基，在感染后第 14-18天（四 因子感染实验） 或第 20-24天 （二因子感染实验） 挑取 iPS集落， 这是基于 Oct-GFP的表达和典型的干细胞形态来挑取的。
重编程效率的定量的实验过程如下：
用于计算重编程效率的主要方法是计数 Oct-GFP 阳性克隆： 1、 对四因 子或二因子感染后第 14 天的细胞在原孔中直接用荧光显微镜计数 Oct-GFP 阳性克隆数； 2、 利用流式细胞仪对四因子感染后第 14天的或二因子感染后 第 18天的细胞测定 Oct-GFP阳性的细胞比例。
iPS细胞的鉴定实验过程如下：
进行碱性磷酸酶染色、 干细胞标记蛋白荧光染色、 鉴定多能性基因的表 达、 病毒基因的沉默、 畸胎瘤的形成 [36-37]。
实施例 1: 加入 NaCl提髙渗透压的髙渗干细胞培养基能促进四因子诱导的 iPS的形成
a. 将四因子 （Oct4， Sox2, Klf4， c-Myc) 的病毒以等体积混合， 加入 到 6孔板的一孔中共计 18万个 OG2小鼠胚胎成纤维细胞中，在 37°C、 5% CO2 的环境中培养在添加了 10%胎牛血清的 DMEM培养基中。 以加入病毒当天 记为第 0天，第 2天将细胞消化并重悬于 mES培养基中， 并接种到预先种满 饲养层细胞 （放射线处理的小鼠胚胎成纤维细胞） 的 96孔板中， 每孔 5000 个细胞。从第 3天开始使用具有不同渗透压值的 mES培养基（即添加 50 mM 或 100 mM的 NaCl使等渗培养基成为高渗培养基， 高渗培养基的渗透压分 别为 380 mOSM/kg和 480 mOSM/kg) 进行培养， 第 6天开始换为高渗透压 值的 KSR培养基（即添加 50 mM或 100 mM的 NaCl使等渗培养基成为高渗 培养基， 高渗培养基的渗透压分别为 380 mOSM/kg和 480 mOSM/kg)进行培 养。 从第 8天开始再换为 KSR培养基（等渗培养基条件）进行培养， 整个过 程如图 1所示。 对照组为等渗培养基条件 （290 mOSM/kg) 培养的细胞。
b . 使用如 a所述的方法， 从第 8天开始， 每天在倒置荧光显微镜下观察 并计数 Oct-GFP阳性克隆数， 同时使用荧光显微镜拍照。 图 2A为第 14天时 96孔板中代表性的图片， 可以看到添加了 100 mM NaCl的培养基处理的孔 相对于不处理的对照孔， 有更多 Oct-GFP阳性的克隆。 图 2B是对 Oct-GFP 阳性克隆的统计数据。 在感染后第 8天， 添加了 50 mM和 100 mM NaCl高 渗培养基处理的孔中即能分别观察到 10-15个左右 Oct-GFP阳性克隆， 而对 照孔无克隆。 在感染后第 1 1 天， 高渗处理的孔中即能观察到 20-30个左右 Oct-GFP阳性克隆， 而对照孔为 3-5个左右。 在感染后第 14天， 高渗处理的 孔中即能观察到 30-40个左右 Oct-GFP阳性克隆， 而对照孔仅有 5个左右。 统计数据可以看到高渗环境可以提高重编程效率达到对照组的 10倍左右。
c. 使用如 a所述的方法， 在第 14 天消化细胞， 利用流式细胞仪检测 Oct-GFP阳性的细胞比例。 图 3A为代表性的流式数据图； 图 3B是对 3次实 验的统计数据， 显示在四因子诱导条件下， 添加 100 mM NaCl的培养基处理 可提高 iPS诱导效率 28倍。
实施例 2: 加入蔗糖的干细胞培养基也能促进四因子诱导的 iPS的形成
a. 将四因子 （Oct4， Sox2, Klf4， c-Myc) 的病毒以等体积混合， 感染 到 6孔板的一孔中共计 18万个 OG2小鼠胚胎成纤维细胞中，在 37°C、 5% CO2 的环境中培养在添加了 10%胎牛血清的 DMEM培养基中。 以加入病毒当天 为第 0天，第 2天将细胞消化并重悬于 mES培养基中， 并种在预先种满饲养 层细胞 （放射线处理的小鼠胚胎成纤维细胞） 的 96孔板中， 每孔 5000个细 胞。 从第 3天开始分别使用高渗透压值的 mES培养基（即分别添加 100 mM NaCl和 200 mM蔗糖使等渗培养基成为高渗培养基，高渗培养基的渗透压值 均为 480 mOSM/kg) 进行培养， 第 6天开始分别换为高渗透压值的 KSR培 养基（即分别添加 100 mM NaCl和 200 mM蔗糖使等渗培养基成为高渗培养 基， 高渗培养基的渗透压值均为 480 mOSM/kg) 进行培养。 第 8天开始再换 为 KSR培养基（等渗培养基条件）进行培养。对照组为等渗培养基条件（290 mOSM/kg) 培养的细胞。 整个过程如图 1所示。 b . 使用如 a所述的方法， 从第 12天开始， 每天在倒置荧光显微镜下观 察并计数 Oct-GFP阳性克隆数， 同时使用荧光显微镜拍照。 如图 4A为第 14 天时 96孔板代表性图片。
c 使用如 a所述的方法， 在第 14天固定细胞， 如图 4B对 Oct-GFP阳 性克隆的统计数据所示， 与对照组相比， 添加 lOO mM NaCl和 200 mM蔗糖 的培养基均能够显著提高 iPS效率， 且二者效率并无显著差异。
实施例 3： 在重编程前期提髙培养基渗透压更有利于 iPS细胞的形成。
a. 将四因子 （Oct4， Sox2, Klf4， c-Myc ) 的病毒以等体积混合， 感染 到 6孔板的一孔中，共计 18万个 OG2小鼠胚胎成纤维细胞，在 37°C、5% CO2 的环境中培养在添加了 10%胎牛血清的 DMEM培养基中。 以加入病毒当天 为第 0天，第 2天将细胞消化并重悬于 mES培养基中， 并种在预先种满饲养 层细胞 （放射线处理的小鼠胚胎成纤维细胞） 的 96孔板中， 每孔 5000个细 胞。 从第 3天开始使用高渗透压的 mES培养基 （即添加 100 mM NaCl的培 养基）培养 1至 3天，第 6天开始换为高渗透压的 KSR培养基（即添 100 mM NaCl的培养基） 再培养 0至 6天， 以后换为正常渗透压的 KSR培养基继续 培养至克隆计数。高渗透压开始处理的时间及处理时间的长短如图 5A所示。 对照组为等渗培养基条件培养的细胞。
b . 使用如 a所述的方法， 从第 8天开始， 每天在倒置荧光显微镜下观察 并计数 Oct-GFP阳性克隆数， 同时使用荧光显微镜拍照。 图 5B所示为感染 后第 14天 96孔板内的代表性图像。 图 5C为感染后第 14天对 96孔板内 Oct-GFP阳性克隆的统计数据。 可以看出在第 3-6天使用高渗培养基所诱导 的 iPS数量最多， 第 6-9天使用高渗培养基也有显著效果， 而在 9-12天使用 高渗培养基则无明显效果。 这说明高渗培养基发挥作用只在重编程的前期。 图 5D显示了分别在感染后第 3-4天、 3-6天、 3-8天、 3-10天或 3-12天予以 高渗处理， 在感染后第 14天 96孔板内 Oct-GFP阳性克隆的 3次独立实验统 计数据。 进一步说明高渗培养基发挥作用只在重编程前期， 而且过长时间用 高渗培养基处理对 iPS细胞的生长不利。
实施例 4: 髙渗培养基与其他化学小分子共同作用提髙 4因子诱导的重编程 效率
a. 将四因子 （Oct4， Sox2, Klf4， c-Myc ) 的病毒以等体积混合， 加入 到 6孔板的一孔中共计 18万个 OG2小鼠胚胎成纤维细胞中，在 37°C、 5% CO2 的环境中培养在添加了 10%胎牛血清的 DMEM培养基中。 以加入病毒当天 记为第 0天，第 2天将细胞消化并重悬于 mES培养基中， 并接种到预先种满 饲养层细胞 （放射线处理的小鼠胚胎成纤维细胞） 的 96孔板中， 每孔 5000 个细胞。 从第 3天开始使用不同渗透压值的 mES培养基 （即添加 50 mM或 100 mM NaCl的高渗培养基，渗透压分别为 380 mOSM/kg和 480 mOSM/kg) , 并与不同浓度的小分子化合物 （VPA和 LiCl) 联用； 第 6天开始换为高渗透 压值的 KSR培养基（即添加 50 mM或 100 mM NaCl的高渗培养基， 渗透压 分别为 380 mOSM/kg和 480 mOSM/kg), 同时添加不同浓度的小分子化合物 (VPA和 LiCl) (培养基和小分子化合物的具体情况如图 6所示）。 第 8天开 始再换为 KSR培养基培养 （等渗培养基条件）， 整个过程如图 1所示。 对照 组为转入四因子， 等渗培养基条件培养的细胞， 并且不加入任何小分子化合 物。
b . 使用如 a所述的方法， 从第 8天开始， 每天在倒置荧光显微镜下观察 并计数 Oct-GFP阳性克隆数， 同时使用荧光显微镜拍照。如图 6A显示， NaCl 和 VPA均能增加 iPS的诱导效率， 而两者在低浓度合用时 （VPA 0.5 mM + NaCl 50 mM) 的效果有协同作用。 图 6B显示了 NaCl和 LiCl均能增加 iPS 的诱导效率， 而两者在低浓度合用时 （LiC1 5 mM + NaC1 50 mM) 的效果有 协同作用。 显示了高渗透压培养基与其他化学小分子（VPA或 LiCl)共同作 用提高四因子诱导重编程效率。
实施例 5: 提髙渗透压并结合其他小分子化合物能促进二因子诱导的 iPS的 形成
a. 将两因子 (Oct4和 Sox2)的病毒以等体积混合， 感染到 6孔板的一孔中 共计 18万个 OG2小鼠胚胎成纤维细胞中， 在 37°C、 5% CO2的环境中培养 在添加了 10%胎牛血清的 DMEM培养基中。 以加入病毒当天为第 0天，第 2 天实验组换成加入小分子化合物组合（CHIR99021 3 μΜ, Repsox 1 M，Parnet 5 μΜ)， 以及 50 mM NaCl的 KSR培养基（中等高渗培养基条件）； 对照组换 成仅加入小分子化合物组合（CHIR99021 3 μΜ， Repsox 1 μΜ， Parnet 5 μΜ) 的 KSR (等渗培养基条件） 培养基。 第 6天实验组和对照组均换液为仅加入 小分子化合物组合 ( CHI 99021 3 μΜ， Repsox 1 μΜ， Parnet 5 μΜ) 的 KSR (等渗培养基条件） 培养基， 第 10天换为无化合物添加的 KSR培养基 （等 渗培养基条件） 继续培养。
b . 使用如 a所述的方法， 从第 12天开始， 每天在倒置荧光显微镜下观察 并计数 Oct-GFP阳性克隆数， 同时使用荧光显微镜拍照。 如图 7A所示， 感 染后第 18天二因子 iPS克隆 iPS细胞系具有类似胚胎干细胞的形态。 图 7B 是对 Oct-GFP阳性克隆的统计数据。 在感染后第 18天， 对照孔仅有 2个克 隆， 而添加了 50 mM NaCl 的中等高渗培养基处理的孔中能观察到 13 个 Oct-GFP阳性克隆。
c 使用如 a所述的方法， 在第 20天挑取 iPS克隆团， 消化细胞传代培养 后得到两因子 iPS细胞系，并提取 DNA通过 PCR检测病毒基因的整合情况， 证实 iPS克隆确实为两因子 (Oct4和 Sox2)诱导生成。 在提取 DNA之前， 利 用差速贴壁法去除滋养层细胞， 使用 Trizol (Invitrogen公司） 试剂， 按照制 造商说明提取 iPS细胞的总 DNA， 同时应用 TaKaRa Taq™试剂盒 （Takara 公司）， Bio- ad DNA Engine PC 仪进行 PCR鉴定。 上述 PCR均使用常规 PCR条件， 按照试剂盒制造商说明进行。 其中鉴定病毒基因插入使用的引物 列表如表 1所示。如图 7C所示，所有两因子 iPS克隆均只有病毒 Oct4和 Sox2 的基因插入， 未见 Kif4和 c-Myc的插入。 表 1
Figure imgf000017_0001
实施例 6： 髙渗培养条件下诱导获得的 iPS细胞系具有多能性 a. 如上所述， 用四因子 （Oct4， Sox2， Klf4， c-Myc) 或两因子 （Oct4 和 Sox2) 感染 OG2小鼠胚胎成纤维细胞， 在高渗培养基中培养， 感染后 14 天（四因子）或 18天（两因子）根据克隆形态及荧光表达挑取具有代表性的 克隆， 经过传代后形成均匀的 iPS细胞系。
b . 对于挑选出来的 iPS细胞系进行形态观察， 并进行干细胞特异性蛋白 的染色。 如图 8所示， 利用高渗条件获得的四因子和两因子 iPS细胞系均具 有类似胚胎干细胞的特征形态， 强烈表达 Oct-GFP， 并且碱性磷酸酶 （AP) 染色呈阳性。使用针对干细胞特征蛋白的抗体对 iPS细胞进行免疫荧光染色， 结果表明 iPS细胞系均表达干细胞特异性蛋白 Nanog和 SSEA-1。
c 在提取 RNA 之前， 利用差速贴壁法去除滋养层细胞， 使用 Trizol ( Invitrogen 公司） 试剂， 按照制造商说明提取 iPS 细胞的总 RNA。 用 PrimeScript™试剂盒 （Takara 公司） 进行逆转录， 并使用 JumpStart™ Taq ReadyMix™试剂盒 （Sigma公司）， Mx 3000P荧光定量 PCR仪 （Stratagene 公司） 进行 Real-Time PCR分析外源基因沉默以及内源基因表达， PCR均使 用常规 PCR条件， 按照试剂盒制造商说明进行。其中鉴定 iPS特异基因使用 的引物列表如表 2所示。 如图 9A所示， 使用本发明方法获得的 iPS细胞系 均表达较高水平的多能性因子， 包括内源性 Oct4， 内源性 Sox2， Nanog和 exl , 其表达水平与小鼠的胚胎干细胞系 E14相当。 而这些因子在原始的小 鼠胚胎成纤维细胞中均没有可检测到的表达。 同时如图 9B所示， 利用本发 明方法获得的 iPS细胞系均能够较好地沉默外源的病毒基因， 也说明细胞完 成了重编程到干细胞的状态。
表 2
Figure imgf000018_0001
Nanog TGAAACCTGTCCTTGAGTGC 内源 -Sox2 F AGGGCTGGGAGAAAGAAGAG 内源 -Sox2 R CCGCGATTGTTGTGATTAGT exl F CAGCCAGACCACCATCTGTC exl GTCTCCGATTTGCATATCTCCTG 外源 -Sox2 F GGGTGGACCATCCTCTAGAC 外源 -Sox2 GGGCTGTTCTTCTGGTTG 外源 -Klf4 F GGGTGGACCATCCTCTAGAC 外源 -Klf4 GCTGGACGCAGTGTCTTCTC 外源 -cMyc F GGGTGGACCATCCTCTAGAC 外源 -cMyc CCTCGTCGCAGATGAAATAG 外源 -Oct4 F GCTTGGATACACGCCGC 外源 -Oct4 TTCATGTCCTGGGACTCCTC d. 使用胰酶消化高渗诱导得到的小鼠四因子 iPS克隆， 将 100万个 iPS 细胞重悬在 200 μL mES培养基中注射入 NOD-SCID小鼠大腿内侧肌肉中。 4-5周后， 处死小鼠， 畸胎瘤用石蜡包埋、 切片， 使用苏木精 /伊红染色进行 组织结构上的分析。如图 10所示， 在 iPS细胞形成的畸胎瘤切片中能够发现 源于 3个胚层的组织， 包括代表外胚层的神经管样组织、 表皮样结构， 代表 中胚层的软骨组织， 以及代表内胚层的消化管腔样组织。 说明高渗诱导得到 的小鼠四因子 iPS克隆能够像小鼠胚胎干细胞那样具有分化为 3个胚层的不 同类型细胞的多能性。总之， 本发明的高渗培养基环境能高效产生 iPS细胞。 克隆计数实验计算效率显示高渗的实验组比对照组提高约 10-25倍 （转导四 因子实验）和 6倍（转导 2因子实验）， 且利用高渗诱导的 iPS细胞具有良好 的全能性。 本发明发现的高效诱导 iPS细胞的方法对推动 iPS的基础研究和 临床应用有着重要的意义。
以上所示仅为本发明较佳的实施例而已， 当然不能以此来限定本发明的 权利范围， 因此依本发明权利要求所作的等同变化， 仍属本发明所涵盖的范 围。
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Claims

权 利 要 求
1、 一种诱导性多能干细胞的制备方法， 该方法包括以下步骤： 步骤 1， 将一个或多个干细胞多能性因子导入体细胞；
步骤 2，使用干细胞培养基对步骤 1中制备的导入了干细胞多能性因子 的体细胞进行培养， 其中， 在该过程中， 进一步包括使用高渗透压培养基 对所述细胞进行培养； 和
步骤 3， 鉴定诱导性多能干细胞克隆。
2、 根据权利要求 1所述的方法， 其中， 进一步包括将报告基因导入体 细胞， 以此报告基因来指示所述诱导性多能干细胞的产生及其产生效率。
3、 根据权利要求 1所述的方法， 其中， 所述报告基因为 Oct4-GFP或 Nanog-GFP, 优选为 Oct4-GFP。
4、 根据权利要求 1所述的方法， 其中， 在步骤 1中， 将所述干细胞多 能性因子的 cDNA以病毒感染方式导入小鼠胚胎成纤维细胞。
5、 根据权利要求 1所述的方法， 其中， 在步骤 1中， 所述干细胞多能 性因子选自 Oct4、 Sox2、 Soxl、 Klf4、 Klf2、 Klf5、 Nanog、 c-Myc, L-Myc、 N-Myc、 Lin28禾卩 Esrrb中。
6、 根据权利要求 5所述的方法， 其中， 在步骤 1中， 所述干细胞多能 性因子为包括 Oct4、 Sox2、 Klf4和 c-Myc的四因子，或者为包括 Oct4、 Sox2 和 Klf4的三因子， 或者为包括 Oct4和 Sox2的二因子。
7、 根据权利要求 1所述的方法， 其中， 在步骤 2中， 从导入所述因子 后第 3至 6天开始使用高渗透压培养基对所述细胞进行培养 1至 9天。
8、 根据权利要求 1所述的方法， 其中， 在步骤 2中， 在导入所述因子 后第 3至第 6天， 或者在导入所述因子后第 3至第 8天， 或者在导入所述 因子后第 3至第 10天， 或者在导入所述因子后第 3至第 12天， 或者在导 入所述因子后第 6至第 9天， 使用高渗透压培养基对所述细胞进行培养。
9、 根据权利要求 1所述的方法， 其中， 在步骤 2中， 所述高渗透压培 养基是通过在普通等渗培养基中外加离子型或非离子型渗透压调节物质 (优选为 NaCl、 蔗糖、 甘露醇、 Na2SO4和葡甲胺)而制备的培养基， 更优选 地， 所述渗透压调节物质包括 NaCl和蔗糖， 最优选地， 所述渗透压调 节物质为 NaCl。
10、 根据权利要求 1所述的方法， 其中， 在步骤 2中， 所述高渗透压培 养基的渗透压为 380〜480 mOSM/kg。
11、 根据权利要求 10所述的方法， 其中， 在步骤 2中， 所述高渗透压培 养基为提高渗透压的 mES培养基和 /或提高渗透压的 KSR培养基， 优选地， 所述高渗透压培养基进一步包含选自 VPA、 LiCl、 CHI 9902 epsox 和 Parnate中的小分子化合物， 其中， 所述 mES培养基为添加有 15%胎牛血清、 1000 U/mL白血病抑制因子、 L-谷氨酰胺、 非必需氨基酸、 青霉素 /链霉素 和 β-巯基乙醇的 DMEM;以及所述 KSR培养基为添加有 15%替代血清、 1000 U/mL 白血病抑制因子、 L-谷氨酰胺、 非必需氨基酸、 青霉素 /链霉素和 β- 巯基乙醇的 Knockout DMEM。
12、 如权利要求 1所述的方法， 其中， 在步骤 2中， 所述使用高渗透压 培养基对所述细胞进行培养为先使用提高渗透压的 mES 培养基对所述细胞 进行培养 0至 3天， 再使用提高渗透压的 KSR培养基对所述细胞进行培养 0 至 6天。
13、 根据权利要求 1所述的方法， 包括： 步骤 1， 将 Oct4、 Sox2、 Klf4和 c-Myc四因子或 Oct4、 Sox2、 Klf4三 因子或 Oct4、 Sox2两因子导入小鼠胚胎成纤维细胞， 其中， 进一步包括将报 告基因 Oct4-GFP导入小鼠胚胎成纤维细胞；
步骤 2， 将步骤 1中制备的导入了 Oct4、 Sox2、 Klf4和 c-Myc四因子或 Oct4、 Sox2、 Klf4三因子或 Oct4、 Sox2两因子的小鼠胚胎成纤维细胞在第 二天消化并接种到饲养层细胞中，使用 mES培养基培养， 并在第三天使用渗 透压为 380〜480 mOSM/kg的高渗透压 mES培养基培养，在第六天换为渗透 压为 380〜480 mOSM/kg的高渗透压 KSR培养基培养，在第八天换为等渗透 压 KSR培养基继续培养， 其中， 以步骤 1 的将 Oct4、 Sox2、 Klf4和 c-Myc 四因子或 Oct4、 Sox2、 Klf4三因子或 Oct4、 Sox2两因子导入小鼠胚胎成纤 维细胞当天记为第 0天； 以及
步骤 3， 挑选具有良好干细胞形态或 Oct4-GFP阳性的克隆进行传代。
14、 根据前述权利要求中任一项所述的方法， 其中， 所述体细胞是来自 哺乳动物的体细胞。
15、根据权利要求 14所述的方法，其中，所述哺乳动物选自小鼠、大鼠、 兔、 猪、 羊、 牛、 猴或人。
16、 一种用于制备诱导性多能干细胞的培养基， 其特征在于， 所述培养 基为提高渗透压的培养基， 优选地， 所述高渗透压培养基进一步包含选自 VPA、 LiCl、 CHI 9902K Repsox和 Parnate中的小分子化合物。
17、根据权利要求 16所述的培养基，其中，所述培养基的渗透压为 380〜 480 mOSM/kg。
18、根据权利要求 17所述的培养基， 其中， 所述培养基为提高渗透压的 mES培养基和 /或提高渗透压的 KSR培养基， 其中， 所述 mES培养基为添加 有 15%胎牛血清、 1000 U/mL白血病抑制因子、 L-谷氨酰胺、 非必需氨基酸、 青霉素 I链霉素和 β-巯基乙醇的 DMEM;以及所述 KSR培养基为添加有 15% 替代血清、 1000 U/mL白血病抑制因子、 L-谷氨酰胺、 非必需氨基酸、 青霉 素 I链霉素和 β-巯基乙醇的 Knockout DMEM。
PCT/CN2012/077579 2011-07-01 2012-06-27 诱导性多能干细胞的制备方法和用于制备诱导性多能干细胞的培养基 WO2013004135A1 (zh)

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