WO2012079278A1 - 诱导性多能干细胞的制备方法以及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以及用于制备诱导性多能干细胞的培养基 Download PDF

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WO2012079278A1
WO2012079278A1 PCT/CN2011/001875 CN2011001875W WO2012079278A1 WO 2012079278 A1 WO2012079278 A1 WO 2012079278A1 CN 2011001875 W CN2011001875 W CN 2011001875W WO 2012079278 A1 WO2012079278 A1 WO 2012079278A1
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lithium
cells
medium
lithium salt
oct4
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PCT/CN2011/001875
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French (fr)
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谢欣
王荃
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中国科学院上海药物研究所
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Priority to CA2823464A priority Critical patent/CA2823464A1/en
Priority to SG2013044862A priority patent/SG191101A1/en
Priority to KR1020137018543A priority patent/KR20130101135A/ko
Priority to JP2013543496A priority patent/JP2013545480A/ja
Priority to EP11849759.3A priority patent/EP2653535A1/en
Priority to US13/990,722 priority patent/US20130295579A1/en
Priority to AU2011345137A priority patent/AU2011345137A1/en
Publication of WO2012079278A1 publication Critical patent/WO2012079278A1/zh

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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. Background technique
  • Embryonic stem cells are derived from the inner cell mass of early embryos and are capable of self-renewing, maintaining pluripotency and having the ability to differentiate into three germ layer cells in vitro. With the success of mouse embryonic stem cells in 1981 and human embryonic stem cells in 1998 [1, 2], the study of regenerative medicine really opened the curtain. The application prospect of embryonic stem cells is mainly for transplantation therapy. Embryonic stem cells are used as the starting cells. By in vitro culture and directed differentiation, a large number of tissues and organs can be provided for clinical treatment. Through the study of the mechanisms of self-renewal and directed differentiation of embryonic stem cells, many specific cell types (eg, nerve cells, cardiomyocytes, etc.) have been tested for mature differentiation methods.
  • specific cell types eg, nerve cells, cardiomyocytes, etc.
  • embryonic stem cells face many technical and ethical issues, from in vitro studies to clinical applications. For example, how to obtain embryonic stem cells without immune rejection? How to get the patient's own embryonic stem cells? and many more.
  • mouse fibroblasts can be reversely differentiated into cells with embryonic stem cell characteristics by overexpressing four transcription factors Oct4, Sox2, Klf4 and c-Myc, and named as more inducible.
  • iPS Cells Induced Pluripotent Stem Cells
  • the researchers initially selected 24 genes related to embryonic stem cell signaling regulation as candidate genes, retroviruses as gene transfection vectors, and mixed in many different ways.
  • the virus of the gene infects mouse fibroblasts, and finally uses the morphological characteristics of cells, the expression of specific genes of embryonic stem cells, and the formation of teratomas as iPS Identification index of cellular pluripotency.
  • 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, and its reprogramming from adult cells to stem cells avoids immune rejection and makes autologous transplantation more feasible. On the other hand, iPS has research on stem cell self-renewal mechanism, stem cell signal regulation, and many diseases. Great meaning.
  • iPS cells still has two major problems, safety issues and induced efficiency problems.
  • the exogenous gene overexpressed in the original experimental system contained two oncogenes (c-Myc and Klf-4), and the introduction of the foreign gene was retrovirus, and the virus was more in the genome. Insertion of a copy can also cause mutations and canceration.
  • researchers are working to optimize iPS technology to increase the safety of their applications, for example, by reducing the number of transcription factors [10, 11], or by using gene-transfection methods that are not integrated into the genome [12], as well as adding directly.
  • the form of the transmembrane transcription factor protein is reprogrammed [13].
  • the present invention provides a method for preparing induced pluripotent stem cells, the method comprising the following steps:
  • Step 1 introducing one or more stem cell pluripotency factors into the somatic cells
  • Step 2 culturing a somatic cell into which a stem cell pluripotency factor is introduced in a medium in which a lithium salt is added;
  • Step 3 Identification of 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 a reporter gene.
  • the reporter gene is Oct4-GFP or Nanog-GFP, and more preferably 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, Soxl, Klf4, Klf2, Klf5, Nanog, c-Myc, L-Myc, N-Myc, Lin28 and Esrrb. And more preferably, the stem cell pluripotency factor may comprise Oct4, Sox2, Klf4 and c-Myc. And more preferably, the stem cell pluripotency factor may comprise Oct4, Sox2 and Klf4.
  • the lithium salt comprises lithium chloride, lithium carbonate, lithium acetate, lithium bromide, lithium protonate, lithium gamma linolenate, lithium heparin, lithium sulfate, lithium nitrate, etc. all containing lithium ions Chemicalization
  • the lithium salt is lithium chloride.
  • the lithium salt has a working concentration of from 0.1 to 40 mM. More preferably, the work of the lithium salt The concentration is 0.5-20 mM. And more preferably, the lithium salt has a working concentration of 1 - 10 mM. And more preferably, the lithium salt has a working concentration of 5 to 10 mM. Most preferably, the lithium salt has a working concentration of 10 mM.
  • the medium used in the step 2 may be DMEM supplemented with 15% fetal calf serum, 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids, penicillin/streptomycin and ⁇ -mercaptoethanol.
  • LIF leukemia inhibitory factor
  • L-glutamine L-glutamine
  • ⁇ -mercaptoethanol DMEM
  • the step 2 specifically includes the following steps:
  • Mouse embryonic fibroblasts introduced in step 1 into which four factors (Oct4, Sox2, Klf4, and c-Myc) or three factors (Oct4, Sox2, Klf4) were introduced were digested and inoculated into feeder cells on the next day.
  • the medium was cultured using mES medium, and cultured on the third day using a mES medium supplemented with a lithium salt, and on the sixth day, the medium was cultured in a KSR medium supplemented with a lithium salt, and on the eighth day, the medium was changed to a KSR medium; as well as
  • Clones with good stem cell morphology or Oct4-GFP positive were selected for passage.
  • “Good stem cell-like morphology” refers to a clone that is similar in morphology to mouse stem cells.
  • “Oc4-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 identification of induced pluripotent stem cell clones including AP staining, detection of endogenous Oct4 expression, detection of pluripotency marker expression by fluorescent quantitative PCR, silencing of exogenous viral genes, formation of teratoma, chimerism Form, and whether there is germ line transmission.
  • 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 further comprises a lithium salt.
  • the lithium salt comprises all lithium ion-containing chemicals such as lithium chloride, lithium carbonate, lithium acetate, lithium bromide, lithium protonate, lithium gamma linolenate, lithium heparin, lithium sulfate, lithium nitrate, and the like.
  • the lithium salt is lithium chloride.
  • the lithium salt has a working concentration of 0.1-40 mM. More preferably, the lithium salt has a working concentration of from 0.5 to 20 mM. And more preferably, the lithium salt has a working concentration of from 1 to 10 mM. And more preferably, the lithium salt has a working concentration of 5-10 mM. Most preferably, the lithium salt has an operating concentration of 10 mM.
  • lithium salt concentration exceeds 40 mM, long-term culture will lead to cell death; when the lithium salt concentration is less than 0.1 mM, the induction efficiency of iPS cells cannot be significantly increased.
  • the medium is a mES medium supplemented with a lithium salt and a KSR medium supplemented with a lithium salt.
  • the lithium salt of the present invention can efficiently produce iPS cells.
  • the computational efficiency of the flow cytometry experiment showed that the experimental group to which lithium salt was added was about 5 times higher than that of the control group (transduced four-factor experiment) and 60 times (transduced 3-factor experiment:).
  • the addition of lithium salt during the iPS induction process can also speed up the process of reprogramming.
  • the experimental group added with lithium salt can detect Oct4-GFP positive clones on the 8th day after infection, while the control group usually does not detect until 10 days later. Oct4-GFP positive clone.
  • iPS cells induced by lithium salt have good pluripotency, and iPS clones transduced with four factors or three factors can form chimeric mice. Four-factor iPS chimeric mice achieve germline transmission and produce offspring. .
  • the method for efficiently inducing iPS cells provided by the present invention has important significance for promoting basic research and clinical application of iPS.
  • Fig. 1 is a flow chart showing the experimental procedure for promoting the formation of iPS cells using a stem cell medium supplemented with lithium salt.
  • Figure 2 shows that the addition of lithium salt to stem cell media increases the induction of iPS efficiency by four factors (Oct4, Sox2, Klf4, c-Myc) and accelerates iPS induction.
  • A is a 96-well plate showing the 10th day after infection. Photograph of a representative image of the inner well, in which more Oct-GFP positive clones appeared in the wells treated with 10 mM LiCl; B is a graph showing statistical data on Oct-GFP positive clones, on the 8th day after infection In the LiCl-treated wells, about 10 Oct-GFP-positive clones were observed, while the control wells were not.
  • C is a graph showing statistical data of Oct-GFP positive clones with different concentrations of LiCl, and a significant dose effect can be observed, with LiCl concentration being 10 mM being most effective. *, p ⁇ 0.05
  • Figure 3 shows the flow cytometry detection of lithium salts to improve the four-factor induced iPS efficiency.
  • A is a graph showing the percentage of GFP-positive cells analyzed by flow cytometry after cell digestion on the 12th day after infection;
  • B is a graph showing statistical data of 3 independent experiments. ***, p ⁇ 0.001 o
  • Figure 4 shows that the addition of lithium salt to the stem cell medium increased the three-factor (Oct4, Sox2, Klf4)-induced iPS efficiency.
  • A is a graph showing statistical data on Oct-GFP positive clones. On day 14 after infection, 5 mM or 10 mM LiCl can significantly increase iPS induction efficiency; B is shown after cell digestion on day 16 after infection.
  • Figure 5 shows alkaline phosphatase staining and immunofluorescence staining.
  • the upper part shows that the iPS cell line has a morphology similar to that of embryonic stem cells, strongly expressing Oct4-GFP, and the alkaline phosphatase staining is positive and uniform.
  • the lower half is immunofluorescent staining of the stem cell-specific proteins Nanog and SSEA-1, and the iPS cell line expresses these two stem cell-specific proteins.
  • Figure 6 shows the detection of stem cell-specific gene expression and silencing of foreign viral genes by real-time PCR.
  • A Mouse embryonic fibroblasts were used as a negative control, and mouse embryonic stem cell line E14 cells were used as a positive control to detect the expression of stem cell-specific genes. All four-factor and three-factor plus lithium salt-induced iPS clones highly expressed stem cell-specific genes, including endogenous Oct4, endogenous Sox2, Nanog, and Rexl; B, positive for mouse embryonic fibroblasts 4 days after viral infection In contrast, ES cells were negative controls and the silencing of exogenous viral genes was detected. All four factors and three factors plus lithium salt induced iPS Clones all showed good exogenous viral gene silencing.
  • Figure 7 shows a four-factor plus lithium salt-induced iPS-forming teratoma experiment. Judging from the organizational structure, the iPS cell line is capable of differentiating into the unique tissues of the three germ layers.
  • the left side is the epidermal-like structure belonging to the ectoderm
  • the middle is the cartilage-like and muscle-like structure belonging to the mesoderm
  • the right side is the digestion belonging to the endoderm.
  • the lumen structure is the left side.
  • Figure 8 shows the four-factor plus lithium salt-induced iPS cell formation chimera and germline transmission experiments.
  • the iPS cell line treated with 4 factors plus lithium salt on the left has germline transmission ability
  • the iPS cell line treated with 3 factors plus lithium salt on the right has chimeric formation ability.
  • the experiments of the present invention use molecular biology, microbiology, cell biology, immunology, and recombinant DNA conventional techniques, which are within the skill of the art. See, for example, Sambrook, Fritsch and Maniatis, Guide to Molecular Cloning, Third Edition (2002); Current Protocols in Molecular Biology (FM Ausubel et al., ed. (1987); Series Methods in Enzymology (Academic Method) Inc. ); PCR2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, ed., (1995)); Antibodies, A Laboratory Manual and Animal Cell Culture (RI) Edited by Freshney, (1987)); Handbook of Stem Cells, Volume 2 (edited by W. French et al.).
  • iPS cells inducible pluripotent stem cells
  • iPS cells are cells derived from somatic cells which are reprogrammed in vitro by introduction of stem cell pluripotency factors.
  • Such cells under embryonic stem cell (ES) culture conditions, and ES cells in cell morphology, growth characteristics, specific gene expression,
  • ES embryonic stem cell
  • the properties of DNA methylation are very similar to those of mouse ES cells, and are also very similar to mouse ES cells in terms of teratoma formation, chimeric animal formation, and germline transmission.
  • the pluripotency factor that induces somatic cell reprogramming is a key factor in maintaining the pluripotency of stem cells, and the introduction of these factors into somatic cells can induce somatic cell reprogramming into stem cells.
  • the pluripotency factor comprises a group selected from the group consisting of Oct4, Sox2 (or Soxl), Klf4 (or Klf2 or Klf5), Nanog, c-Myc (or L-Myc or N-Myc), Lin28 and Esrrb. one or more.
  • the above stem cell pluripotency factor may be derived from any species as desired, and is preferably a mouse stem cell pluripotency factor.
  • 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 cDNA of a pluripotency factor required to maintain stem cell pluripotency into somatic cells.
  • the pluripotency factor comprises a group selected from the group consisting of Oct4, Sox2 (or Soxl), Klf4 (or Klf2 or Klf5), Nanog, c-Myc (or L-Myc or N-Myc), Lin28 and Esrrb. One or more of them.
  • the method of introducing the cDNA of the stem cell pluripotency factor into a somatic cell can be carried out by various methods including viral infection, lipofection, electroporation, particle bombardment, transposon-mediated insertion expression, transmembrane protein, Various methods of transferring DNA into cells, such as drug induction.
  • transfection is carried out using a viral vector comprising cDNA.
  • the viral vector includes a plurality of viral vectors such as a lentivirus, a retrovirus, and an adenovirus.
  • a retroviral vector is used.
  • 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/streptomycin and ⁇ -mercaptoethanol.
  • LIF leukemia inhibitory factor
  • mES medium fetal calf serum
  • replacement serum 1000 U/mL leukemia inhibitory factor (LIF), L-glutamine, non-essential amino acids
  • KSR medium Knockout DMEM
  • the "reporter gene” as used in the present invention means that the cell has been transformed into a similar embryonic fine The stage of the cell, including the use of transgene or homologous recombination to join a fluorescent protein sequence or a resistance gene sequence against an antibiotic, which is under the control of a promoter of some genes specifically expressed by embryonic stem cells, and thus can reach a similar embryo in the cell.
  • the expression of this fluorescent protein or resistance gene is activated in the stem cell state, thereby giving this cell certain features that can be detected.
  • the mouse embryonic fibroblasts used in this example were OG2 (Oct4-GFP +/- ) cells isolated from the 13.5-day embryos produced by the mating of homozygous OG2 (Oct4-GFP +/+ ) males and 129 females. .
  • 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, real-time PCR analysis of stem cell-specific gene expression and viral gene silencing, and analysis of differentiation into teratomas in vivo.
  • Ability formation of chimeric mice and transmission of the reproductive system.
  • Retroviral vectors containing cDNAs of mouse Oct4, Sox2, Klf4, c-Myc were purchased from Addgene. Transfected into PlatE 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 with 1,5-dimethyl-1,5-diaza eleven Mouse embryonic fibroblasts were infected after polymethyl 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 iPS colonies were picked 14-18 days after infection (4 factor infection experiments) or 20-24 days (3 factor infection experiments), which was based on Oct-GFP. The expression and typical stem cell morphology are picked.
  • the quantitative experimental process of reprogramming efficiency is as follows: The primary method used to calculate reprogramming efficiency is to count Oct-GFP positive clones: 1. Oct-GFP positive for iPS cells after 10-12 days of four-factor infection or 14-16 days after three-factor infection using flow cytometry The proportion of cells; 2. Count the number of Oct-GFP positive clones directly in the original wells by fluorescence microscopy.
  • the identification process of iPS cells is as follows:
  • Example 1 The stem cell medium supplemented with lithium salt promotes the formation of four-factor-induced iPS a.
  • the four-factor (Oct4, Sox2, Klf4, c-Myc) viruses are mixed in equal volumes and infected into a 6-well plate.
  • a total of 180,000 OG2 mouse embryonic fibroblasts in one well were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37 ° C in a 5% CO 2 environment.
  • 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 per well, starting on day 3, using 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), starting on the 6th day KSR medium with different concentrations of LiCl (0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM and 40 mM) was added. On the 8th day, it was replaced with KSR medium. The whole process is shown in Figure 1. Show.
  • FIG. 2A is a representative picture of a 96-well plate at day 10, and it can be seen that 10 mM LiCl-treated wells have more Oct-GFP positive clones relative to untreated wells.
  • Figure 2B is a statistical data of Oct-GFP positive clones. On day 8, almost no Oct-GFP positive clones were seen in the control wells, and about 10 clones were observed in the 10 mM LiCl treated wells.
  • FIG. 2C shows the number of positive clones that occurred after treatment with different concentrations of LiCl (0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, and 40 mM). A significant dose-response curve was found, where LiCl concentration was The effect is most pronounced at 10 mM.
  • FIG. 3A is a representative flow data plot
  • Figure 3B is a statistical data from three experiments showing that 10 mM LiCl treatment can increase iPS induction efficiency by 5-6 fold under four factor induction conditions.
  • Example 2 The dry cell culture medium supplemented with lithium salt promoted the formation of three-factor-induced iPS a.
  • the three-factor (Oct4, Sox2, Klf4) viruses were mixed in equal volumes and infected into one well of a 6-well plate.
  • mice embryonic fibroblasts were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37 ° C in a 5% CO 2 environment. On the 0th day of the day when the virus was added, 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:).
  • iPS cell line has a characteristic morphology similar to that of embryonic stem cells, strongly expressing Oct-GFP green fluorescence, and alkaline phosphatase (AP) 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 sample extract total RNA from iPS cells according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScriptTM kit (Takara), and Real-Time PCR analysis was performed using a JumpStartTM Taq ReadyMixTM kit (Sigma), Mx 3000P fluorescence quantitative PCR instrument (ABI). All of the above PCR conditions were performed using conventional PCR conditions, following the manufacturer's instructions. A list of primers used to identify iPS-specific genes is shown in Table 1.
  • the iPS cell line obtained using the method of the present invention expresses higher levels of pluripotency factors, including endogenous Oct4, endogenous Sox2, Nanog, and Rexl, expression levels and mouse embryonic stem cell lines. E14 is quite. These factors have no detectable expression in the original mouse embryonic fibroblasts.
  • the iPS cell line obtained by the method of the present invention can better silence the exogenous viral gene, and also indicates that the cell has completed the reprogramming to the stem cell state.
  • mice 4-factor iPS clone obtained by trypsinization with lithium salt were added, and 1 million iPS cells were resuspended in 200 mES medium and injected into the inner thigh muscle of NOD-SCID mice. In the flesh. 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. 7, tissues derived from three germ layers can be found in teratoma slices formed by iPS cells, including epidermal-like tissues representing ectoderm, cartilage and muscle tissues representing mesoderm, and digestion representing endoderm. Tumor-like tissue. It is indicated that the mouse 4-factor iPS clone obtained by adding a lithium salt can have pluripotency of different types of cells differentiated into three germ layers like mouse embryonic stem cells.
  • mice 4-factor iPS clone obtained by adding lithium salt is injected into the ICR mouse blastocyst, so that it is mixed with the embryonic stem cells in the injected mouse blastocyst, and the blastocyst is transplanted into the surrogate mother. Growth in the uterus. After the mice were born, the skin color of the mice was observed to determine the fit. When chimerism was detected, chimeric mice were mated with white ICR mice, and the hair color of the offspring mice was observed to determine the presence or absence of germline transmission.
  • both the 3-factor and 4-factor iPS cell lines were able to produce chimeric mice, and the 4-factor iPS cell line produced chimeric mice with black mice in the progeny mice, demonstrating the addition of lithium.
  • the salt-derived iPS cell line does have a germline transmission capacity, indicating that the cell does have pluripotency similar to that of embryonic stem cells.
  • the present invention enables the efficient production of iPS cells using a medium supplemented with a lithium salt.
  • the computational efficiency of the flow cell count experiment showed that the experimental group with lithium salt was increased by about 5-6 times (transduction of four-factor experiment:) and 60 times (transduction of 3-factor experiment:) compared with the control group.
  • the addition of lithium salt during the iPS induction process can also speed up the process of reprogramming.
  • the experimental group added with lithium salt can detect Oct4-GFP positive clones on the 8th day after infection, while the control group usually does not detect until 10 days later. Oct4-GFP positive clone.
  • iPS cells induced by lithium salt have good pluripotency, and iPS clones transduced with four factors or three factors can form chimeric mice.
  • Four-factor iPS chimeric mice achieve germline transmission and produce offspring. .
  • the method for efficiently inducing iPS cells of the present invention has important significance for promoting basic research and clinical application of iPS.

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Description

诱导性多能干细胞的制备方法以及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以及用 于制备诱导性多能干细胞的培养基。 背景技术
胚胎干细胞来源于早期胚胎的内细胞团， 在体外培养中能够自我更新、 保持多能性并具有向三个胚层细胞分化的能力。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地拉开了序 幕。 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的检 测标准， 这些研究为帕金森氏病、 老年痴呆症、 心肌损伤、 糖尿病、 肝硬化 等疾病的治疗带来了希望。 然而， 胚胎干细胞真正从体外研究到进入临床应 用还面临许多技术和伦理问题， 比如， 如何获得无免疫排斥的胚胎干细胞？ 如何获得病人自身的胚胎干细胞？ 等等。
2006年， Yamanaka实验室首先报道可以利用过表达 Oct4、 Sox2、 Klf4 和 c-Myc四种转录因子使小鼠的成纤维细胞逆分化成具有胚胎干细胞特性的 细胞， 并将其命名为诱导性多能干细胞 (Induced Pluripotent Stem Cells, iPS Cells)[3]。在这篇发表在《Cell》杂志上的文章中， 研究人员最初是选取了 24 个与胚胎干细胞信号调控相关的基因作为候选基因， 以逆转录病毒为基因转 染载体， 用混合有多种不同基因的病毒感染小鼠成纤维细胞， 最终以细胞形 态学特征、 胚胎干细胞特定基因的表达以及畸胎瘤的形成等指标作为 iPS细 胞多能性的鉴定指标。他们在实验中最终将 24个候选基因减少到 Oct4、Sox2、 Klf4和 c-Myc这四个转录因子。 虽然在这篇文章中， 研究人员最终没有得到 嵌合体小鼠， 但是直接从已分化的细胞得到干细胞为胚胎干细胞领域甚至生 命科学领域都带来了概念性的革新。 iPS 细胞诱导技术的出现也引起了人们 对其生物医学应用的极大关注， 因为我们可以病人的体细胞为来源得到病人 特异的 iPS细胞， 这种 iPS细胞又可以分化为具有功能的细胞、 组织和器官， 用于疾病治疗。 这样的应用方式可以避免免疫相容性和伦理问题。
2007年，三个不同的实验室均发表文章报道获得了具有种系嵌合能力的 小鼠 iPS细胞 [4-6]。在此之后，也陆续有实验室报道获得了人的 iPS细胞 [7, 8]。 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 [9]。 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， 并且它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很多疾病的研究都有很大意义。
但是， iPS 细胞的应用仍然有两大问题， 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癌基因 (c-Myc和 Klf-4)， 并且外源基因的导入方式是逆转录病毒， 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， 研究人员正在致力于优 化 iPS技术使其应用的安全性增加， 例如， 减少转录因子的数目 [10, 11]， 或 者使用非整合到基因组的基因转染方法 [12]， 还有直接添加透膜的转录因子 蛋白的形式进行重编程 [13]。 但是， 这些方法会导致 iPS 细胞的诱导效率更 加低下， 目前 iPS细胞的形成效率一般都低于 1 %， 这对 iPS细胞的发展和 应用造成了巨大障碍。 因此， 寻找能够提高 iPS细胞诱导效率的培养条件、 发明内容
因此，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， 本发明提供了一种诱导性多能干细胞的 制备方法， 该方法包括以下歩骤：
歩骤 1， 将一个或多个干细胞多能性因子导入体细胞；
歩骤 2， 使用添加了锂盐的培养基培养歩骤 1中导入了干细胞多能性因 子的体细胞； 以及
歩骤 3， 鉴定诱导性多能干细胞克隆。
优选地， 所述方法进一歩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。 且更优选地， 所述报告 基因为 Oct4-GFP或 Nanog-GFP， 且更优选为 Oct4-GFP。
优选地， 在歩骤 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，在歩骤 1中，所述干细胞多能性因子可选自 Oct4、 Sox2、 Soxl、 Klf4、 Klf2、 Klf5、 Nanog, c-Myc、 L-Myc、 N-Myc、 Lin28和 Esrrb中。 且 更优选地， 所述干细胞多能性因子可包括 Oct4、 Sox2、 Klf4和 c-Myc。 且更 优选地， 所述干细胞多能性因子可包括 Oct4、 Sox2和 Klf4。
优选地， 在歩骤 2中， 所述锂盐包括氯化锂、 碳酸锂、 醋酸锂、 溴化锂、 门东氨酸锂、 γ亚麻酸锂、 肝素锂、 硫酸锂、 硝酸锂等所有包含锂离子的化
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更优选地， 在歩骤 2中， 所述锂盐为氯化锂。
优选地， 所述锂盐的工作浓度为 0.1-40mM。更优选地， 所述锂盐的工作 浓度为 0.5-20 mM。 且更优选地， 所述锂盐的工作浓度为 1 -10 mM。 且更优 选地， 所述锂盐的工作浓度为 5- 10 mM。 且最优选地， 所述锂盐的工作浓度 为 10 mM。
歩骤 2中使用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 /链霉素和 β-巯基乙醇的 DMEM(Dulbcco's Modified Eagle's Medium) ( mES培养基） 以及添加有 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 ( KSR培养基）。
优选地， 所述歩骤 2具体包括如下歩骤：
将歩骤 1中制备的导入了四因子 (Oct4、 Sox2、 Klf4和 c-Myc)或三因子 (Oct4, Sox2、 Klf4)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， 并在第三天使用添加了锂盐的 mES培养基培养， 在第六天换为添加锂盐的 KSR培养基培养， 在第八天换为 KSR培养基继续 培养； 以及
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。
歩骤 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， 畸胎 瘤的形成， 嵌合体的形成， 以及是否有生殖系传递 (germ line transmission)。
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 歩包含锂盐。 优选地， 所述锂盐包括氯化锂、 碳酸锂、 醋酸锂、 溴化锂、 门东氨酸锂、 γ亚麻酸锂、 肝素锂、 硫酸锂、 硝酸锂等所有包含锂离子的化学品。
且更优选地， 所述锂盐为氯化锂。
优选地， 所述锂盐的工作浓度为 0.1-40 mM。 更优选地， 所述锂盐的工 作浓度为 0.5-20 mM。 且更优选地， 所述锂盐的工作浓度为 1-10 mM。 且更 优选地， 所述锂盐的工作浓度为 5-10 mM。 且最优选地， 所述锂盐的工作浓 度为 10 mM。
锂盐浓度超过 40 mM时， 长期培养将导致细胞死亡； 锂盐浓度低于 0.1 mM时， 不能显著增加 iPS细胞的诱导效率。
优选地， 所述培养基为添加了锂盐的 mES培养基和添加了锂盐的 KSR 培养基。
本发明的锂盐能高效产生 iPS细胞。 流式细胞计数实验计算效率显示添 加锂盐的实验组比对照组提高约 5倍 (转导四因子实验)和 60倍 (转导 3因子实 验：)。在 iPS诱导过程中添加锂盐也可以加快重编程的过程， 添加锂盐的实验 组在感染第 8天即可检测到 Oct4-GFP阳性的克隆， 而对照组一般要到 10天 以后才能检测到 Oct4-GFP阳性的克隆。利用锂盐诱导的 iPS细胞具有良好的 全能性， 转导四因子或三因子的 iPS克隆均能形成嵌合体小鼠， 其中四因子 iPS 的嵌合体小鼠实现了生殖系传递， 并产下后代。 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为利用添加锂盐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 显示了添加锂盐的干细胞培养基提高四因子 (Oct4、 Sox2、 Klf4、 c-Myc)诱导 iPS效率并加快 iPS诱导进程。 A为显示感染后第 10天 96孔板 内一孔的代表性图像的照片， 其中， 10 mM LiCl 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， LiCl处理的孔中即能观察到 10个左右 Oct-GFP阳性克隆， 而对 照孔则没有； 第 10天， LiCl处理的孔中的 Oct-GFP阳性克隆数能达到 20个 左右。 *， p<0.05; C为显示添加不同浓度 LiCl的 Oct-GFP阳性克隆的统计数 据的图， 可以观察到明显的剂量效应， 其中 LiCl浓度为 10 mM时效果最为 显著。 *， p<0.05
图 3显示了流式细胞仪检测锂盐提高四因子诱导 iPS效率。 A为显示了 感染后第 12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 为显示了 3次独立实验统计数据的图。 ***， p<0.001 o
图 4显示了添加锂盐的干细胞培养基提高三因子 (Oct4、 Sox2、 Klf4)诱导 iPS效率。 A为显示了对 Oct-GFP阳性克隆的统计数据的图， 在感染后第 14 天， 5 mM或 10 mM的 LiCl均能显著提高 iPS诱导效率； B为显示了感染后 第 16天细胞消化后流式细胞仪检测 GFP阳性细胞百分比的图。 *， p<0.05， **， ρ<0·01。
图 5显示了碱性磷酸酶染色及免疫荧光染色。 上半部分显示在 iPS细胞 系具有类似胚胎干细胞的形态， 强烈的表达 Oct4-GFP， 碱性磷酸酶染色呈阳 性并且均一。 下半部分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。
图 6显示了荧光定量 PCR检测干细胞特异基因的表达和外源病毒基因的 沉默。 A、 以小鼠胚胎成纤维细胞为阴性对照， 小鼠胚胎干细胞系 E14细胞 为阳性对照， 检测干细胞特异基因的表达。 所有四因子及三因子加锂盐诱导 的 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。所有四因子及三因子加锂盐诱导的 iPS 克隆均显示良好的外源病毒基因沉默。
图 7显示了四因子加锂盐诱导的 iPS形成畸胎瘤实验。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， 左侧为属于外胚层的表皮样 结构， 中间为属于中胚层的软骨样及肌肉样结构， 右侧为属于内胚层的消化 道管腔结构。
图 8显示了四因子加锂盐诱导的 iPS细胞形成嵌合体及生殖系传递实验。 左侧为 4因子加锂盐处理得到的 iPS细胞系具有生殖系传递能力， 右侧为 3 因子加锂盐处理得到的 iPS细胞系具有嵌合体形成能力。 具体实施方式
定义和技术
除非另外指明， 本发明的实验使用分子生物学、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. )； PCR2： A Practical Approach (PCR实验方法） （M. J. MacPherson, B. D. Hames和 G. R. 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. I. Freshney编著， ( 1987))； Handbook 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"（iPS细胞:)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， 与 ES细胞在细胞形态、 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"是维持干细胞多 能性的关键因子， 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 [14-18]。优选地， 所述的多能性因子包括选自 Oct4、 Sox2(或 Soxl)、 Klf4(或 Klf2或 Klf5)、 Nanog, c-Myc (或 L-Myc或 N-Myc)、 Lin28及 Esrrb中的一个或多个。 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 能性因子的 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选自 Oct4、 Sox2(或 Soxl)、 Klf4(或 Klf2或 Klf5)、 Nanog, c-Myc (或 L-Myc或 N-Myc)、 Lin28及 Esrrb中的一个或多个。
将所述干细胞多能性因子的 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、 药物诱导等的各种将 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， 使用逆转录病毒载体 （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 /链霉素和 β-巯基乙醇的 DMEM(Dulbcco's Modified Eagle's Medium) (mES培养基） 以及添加有 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 (KSR培养基）。
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 +/—)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， 荧光定量 PCR分析干细胞特异基因表达及病毒基因的 沉默， 分析体内分化为畸胎瘤的能力， 嵌合鼠的形成及生殖系的传递。 实施例
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， 本说明书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 PlatE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 14-18天（4因子感染实验）或 20-24天（3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. 利用流式 细胞仪对四因子感染后 10-12 天或三因子感染后 14-16 天的 iPS 细胞测定 Oct-GFP阳性的细胞比例； 2. 在原孔中直接用荧光显微镜计数 Oct-GFP阳性 克隆数。
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、 嵌合体的形成及生殖系传递 [19, 20]。 实施例 1 : 添加了锂盐的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， 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% C02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， 第 3天开始使用添加了不同浓度 LiCl(0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM禾口 40 mM)的 mES培养基，第 6 天开始换为添加了不同浓度 LiCl(0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM和 40 mM)的 KSR培养基， 第 8天开始再换为 KSR培养 基培养， 整个过程如图 1所示。
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 10天时 96孔板中代表性的图片，可以看到 10 mM LiCl处理的孔相 对于不处理的孔， 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。 第 8天时， 对照孔几乎看不到 Oct-GFP阳性克 隆，而 lO mM LiCl处理的孔可以观察到 10个左右克隆。而到第 10天， 10 mM LiCl处理的孔可以观察到 20个左右克隆， 其效率是对照组的 5-6倍。图 2C是用不同浓度的 LiCl (0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM和 40 mM)处理之后出现的阳性克隆数， 可以发现明显的 剂量效应曲线， 其中 LiCl浓度为 10 mM时效果最为显著。
c 使用如 a所述的方法， 在第 12 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， 10 mM LiCl处理可 提高 iPS诱导效率 5-6倍。 实施例 2: 添加锂盐的干细胞培养基能促进三因子诱导的 iPS的形成 a. 将三因子 (Oct4、 Sox2、 Klf4)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% C02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， 第 3天开始使用添加了不同浓度 LiCl(0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM和 40 mM)的 mES培养基， 第 6天开始 换为添加了不同浓度 LiCl(0.6 mM, 1.2 mM, 2.5 mM, 5 mM, 10 mM, 20 mM和 40 mM)的 KSR培养基， 第 8天开始再换为 KSR培养基培养， 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 所示， 第 14天时， 与对照组相比， 添加 5 mM或 10 mM LiCl均能够 显著提高 iPS效率， 其中添加 10 mM LiCl的效率可增加 7倍左右。 c 使用如 a所述的方法， 在第 16 天消化细胞， 利用流式细胞仪检测 Oct-GFP阳性的细胞比例。如图 4B所示，对照组的 Oct-GFP阳性的细 胞比例仅为 0.24%。而添加 10 mM LiCl的实验组的 Oct-GFP阳性细胞 比例可达到 14.5%， 增加了近 60倍。 实施例 3 : 添加锂盐得到的 iPS细胞系具有多能性
a. 如上所述，用 4因子 (Oct4、 Sox2、 Klf4、 c-Myc)或 3因子 (Oct4、 Sox2、 Klf4)感染小鼠胚胎成纤维细胞， 在添加 LiCl的干细胞培养基中培养， 感染后 14天 (4因子)或 20天 (3因子：)， 根据克隆形态及荧光表达挑取 具有代表意义的克隆团， 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 5所示， iPS细胞系具有类似胚胎干细胞的特征形态， 强烈表达 Oct-GFP的绿色荧光， 并且碱性磷酸酶 (AP)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 PrimeScriptTM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（ ABI 公司）进行 Real-Time PCR分析。所有上述 PCR条件均使用常规 PCR 条件， 按照制造商说明进行。 其中鉴定 iPS特异基因使用的引物列表 如表 1所示。
表 1]
Figure imgf000014_0001
内源 -Oct4 F TAGGTGAGCCGTCTTTCCAC
内源 -Oct4 R GCTTAGCCAGGTTCGAGGAT
Nanog F CTCAAGTCCTGAGGCTGACA
Nanog R TGAAACCTGTCCTTGAGTGC 内源 -Sox2 F AGGGCTGGGAGAAAGAAGAG 内源 -Sox2 R CCGCGATTGTTGTGATTAGT
Rexl F GACGAAGCAAGAGAAGAG
Rexl R CGATAAGACACCACAGTAC 外源 -Sox2 F GGGTGGACCATCCTCTAGAC
外源 -Sox2 R GGGCTGTTCTTCTGGTTG
外源 -Klf4 F GGGTGGACCATCCTCTAGAC
外源 -Klf4 R GCTGGACGCAGTGTCTTCTC
夕卜源 -cMyc F GGGTGGACCATCCTCTAGAC
夕卜源 -cMyc R CCTCGTCGCAGATGAAATAG
外源 -Oct4 F GCTTGGATACACGCCGC
外源 -Oct4 R TTCATGTCCTGGGACTCCTC
如图 6A所示， 使用本发明方法获得的 iPS细胞系表达较高水平 的多能性因子， 包括内源性 Oct4、 内源性 Sox2、 Nanog和 Rexl , 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 6B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。
使用胰酶消化添加锂盐得到的小鼠 4因子 iPS克隆，将 100万个 iPS 细胞重悬在 200 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 7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， 包括代表外胚层的表皮样 组织， 代表中胚层的软骨和肌肉组织， 以及代表内胚层的消化管腔样 组织。 说明添加锂盐得到的小鼠 4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。
e. 将添加锂盐得到的小鼠 4因子 iPS克隆注射入 ICR小鼠囊胚中， 使得 其与所注射的小鼠囊胚中的胚胎干细胞混合， 再将此囊胚移植入代孕 母鼠的子宫内生长发育。 待小鼠出生后， 观察小鼠的皮肤颜色判断嵌 合。 若检测到嵌合， 再将嵌合体小鼠与白色的 ICR小鼠交配， 观察子 代小鼠的毛色判断有无生殖系传递。 如图 8所示， 3因子及 4因子的 iPS细胞系均能产生嵌合体小鼠，且 4因子 iPS细胞系产生的嵌合体小 鼠的子代小鼠中有黑色小鼠， 证明此添加锂盐得到的 iPS细胞系确实 有生殖系的传递能力， 说明此细胞确实具有与胚胎干细胞相似的全能 性。
总之， 本发明使用添加了锂盐的培养基能够高效产生 iPS细胞。 流式细 胞计数实验计算效率显示， 添加锂盐的实验组比对照组提高约 5— 6倍 (转导 四因子实验:)和 60倍 (转导 3因子实验：)。在 iPS诱导过程中添加锂盐也可以加 快重编程的过程， 添加锂盐的实验组在感染第 8天即可检测到 Oct4-GFP阳 性的克隆， 而对照组一般要到 10天以后才能检测到 Oct4-GFP阳性的克隆。 利用锂盐诱导的 iPS细胞具有良好的全能性， 转导四因子或三因子的 iPS克 隆均能形成嵌合体小鼠， 其中四因子 iPS的嵌合体小鼠实现了生殖系传递， 并产下后代。 本发明的高效诱导 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， 该方法包括以下歩骤： 歩骤 1， 将一个或多个干细胞多能性因子导入体细胞；
歩骤 2， 使用添加了锂盐的培养基培养歩骤 1中导入了干细胞多能性因 子的体细胞； 以及
歩骤 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， 进一歩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 2所述的方法， 其中， 所述报告基因为 Oct4-GFP或 Nanog-GFP, 且优选为 Oct4-GFP。
4、 根据权利要求 1所述的方法， 其中， 在歩骤 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禾口 Esrrb中。
5、 根据权利要求 4所述的方法， 其中， 在歩骤 1中， 所述干细胞多能性 因子包括 Oct4、 Sox2、 Klf4和 c-Myc， 或者包括 Oct4、 Sox2和 Klf4。
6、 根据权利要求 1所述的方法， 其中， 在歩骤 2中， 所述锂盐包括氯化 锂、 碳酸锂、 醋酸锂、 溴化锂、 门东氨酸锂、 γ亚麻酸锂、 肝素锂、 硫酸锂、 硝酸锂及其它包含锂离子的化学品。
7、根据权利要求 1所述的方法，其中，在歩骤 2中，所述锂盐为氯化锂。
8、 根据权利要求 1所述的方法， 其中， 在歩骤 2中， 所述锂盐的工作浓 度为 0.1-40 mM，且优选为 0.5-20 mM，更优选为 5-10 mM，最优选为 10 mM。
9、根据权利要求 1所述的方法， 其中， 在歩骤 2中， 所述培养基为 mES 培养基和 KSR培养基， 所述 mES培养基为添加有 15%胎牛血清、 1000U/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, 且所述 KSR培养基为添加有 15%替代血清、 1000U/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。
10、 根据权利要求 1所述的方法， 其中， 所述歩骤 2进一歩包括： 将歩 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， 并在第三天使用添加了锂盐的 mES培养基培养， 在第六 天换为添加锂盐的 KSR培养基培养， 在第八天换为 KSR培养基继续培养； 以及挑选具有良好干细胞形态或 Oct4-GFP阳性的克隆进行传代。
11、 根据前述权利要求中任一项所述的方法， 其中， 所述体细胞来自哺 乳动物的体细胞。
12、根据权利要求 11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。
13、 一种用于制备诱导性多能干细胞的培养基， 其进一歩包含锂盐。
14、 根据权利要求 13所述的培养基， 其中， 所述锂盐包括氯化锂、 碳酸 锂、 醋酸锂、 溴化锂、 门东氨酸锂、 γ 亚麻酸锂、 肝素锂、 硫酸锂、 硝酸锂 及其它包含锂离子的化学品。
15、 根据权利要求 13所述的培养基， 其中， 所述锂盐为氯化锂。
16、根据权利要求 13所述的培养基，其中，所述锂盐的工作浓度为 0.1-40 mM， 且优选为 0.5-20 mM， 更优选为 5-10 mM， 最优选为 10 mM。
17、根据权利要求 12所述的培养基， 其中， 所述培养基为添加了锂盐的 mES培养基和添加了锂盐的 KSR培养基。
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