Method for establishing induced pluripotent stem cells from human amniotic epithelial cells
阅读说明：本技术 一种从人羊膜上皮细胞建立诱导性多能干细胞的方法 (Method for establishing induced pluripotent stem cells from human amniotic epithelial cells ) 是由 陆建峰 张传宇 蒙都 曹立宁 于 2020-11-27 设计创作，主要内容包括：本发明提供一种将人羊膜上皮细胞重编程为诱导性多能干细胞的新方法：利用非脂阳离子转染试剂Neofect~(TM)将携带OCT4、SOX2、KLF4、LIN28、L-MYC等5个转基因和1个P53基因shRNA共6个重编程因子的多个oriP/EBNA1游离型质粒载体导入hAECs。该方法过程简单、操作方便、费用低廉,且游离型质粒载体是非基因整合型的重编程方法,减少致瘤性；整个重编程过程耗时较短、效率较高；获得的hAE-iPSCs克隆在无动物源性的TeSR~(TM)-E8~(TM)-人重组玻连蛋白体系中扩大培养,为临床应用奠定基础。(The invention provides a novel method for reprogramming human amniotic epithelial cells into induced pluripotent stem cells, which comprises the following steps: neofect using non-lipid cation transfection reagent TM A plurality of oriP/EBNA1 episomal plasmid vectors carrying 6 reprogramming factors in total, such as 5 transgenes including OCT4, SOX2, KLF4, LIN28 and L-MYC, and 1 shRNA of the P53 gene, were introduced into hAECs. The method has simple process, convenient operation and low cost, and the free plasmid vector is a non-gene integration type reprogramming method, thereby reducing the tumorigenicity; the whole reprogramming process is short in time consumption and high in efficiency; the obtained hAE-iPSCs are cloned in animal origin-free TeSR TM ‑E8 TM And the expanded culture in a human recombinant vitronectin system lays a foundation for clinical application.)
1. A method for establishing induced pluripotent stem cells from human amniotic epithelial cells by transfecting more than 2 oriP/EBNA1 free-form plasmids containing 6 reprogramming factors of OCT4, SOX2, KLF4, LIN28, cDNA of L-MYC and shRNA of p53 with a non-lipid cationic transfection reagent NeofectTMAnd (3) introducing the human amniotic epithelial cells, and reprogramming the human amniotic epithelial cells into induced pluripotent stem cells.
2. The method of claim 1, wherein the human amniotic epithelial cells are human amniotic epithelial cells cultured in vitro within two generations.
3. The method according to claim 1, wherein 2 or more oriP/EBNA 1-episomal plasmids are simultaneously introduced into human amniotic epithelial cells.
4. The method of claim 3, wherein the oriP/EBNA 1-episomal plasmid is three plasmids, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL.
5. The method according to any one of claims 1 to 4, wherein the transfected human amniotic epithelial cells are cultured on a primary culture plate until the induced pluripotent stem cell clone grows and is picked out.
6. The method of claim 5, further comprising the step of seeding the human recombinant glass-linked egg after the clones of induced pluripotent stem cells obtained are pickedWhite-coated plates, in TeSRTM-E8TMAnd carrying out amplification culture in a culture medium.
7. Method according to claim 1, characterized in that it comprises the following steps:
(1) inoculating the human amniotic epithelial cells into a culture plate, and culturing for 2 days by using a human amniotic epithelial cell culture medium;
(2) after the culture medium of the human amniotic epithelial cells is replaced, a non-lipid cationic transfection reagent Neofect is addedTMAnd a mixture containing the episomal plasmids pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL, and then adding the mixture into the culture medium of the human amniotic epithelial cells for transfection;
(3) after 24 hours of transfection, TeSR was changedTM-E8TMThe stem cell culture medium is replaced by fresh culture medium every other day;
(4) selecting out induced pluripotent stem cell clone, seeding on a culture plate coated with human recombinant vitronectin, and adopting TeSRTM-E8TMAnd performing amplification culture on the culture medium to obtain the induced pluripotent stem cells.
8. The method of claim 7, further comprising the steps of:
(1) step (4), freezing the cultured induced pluripotent stem cells by adopting a serum-free cell freezing medium;
(2) and (4) identifying a small number of cells in the step (5) to express stem cell markers NANOG, OCT4, SOX2 and TRA-1-60, namely the induced pluripotent stem cells.
9. The method of claim 7, further comprising the steps of:
(7) and (4) screening the cells after the expanded culture in the step (4) to obtain induced pluripotent stem cells expressing stem cell markers NANOG, OCT4, SOX2 and TRA-1-60.
10. The method according to any one of claims 7 to 9, characterized in thatCharacterized in that in the step (2), the episomal plasmid is firstly added into DMEM to be diluted until the concentration of each plasmid is 0.05-0.01 mu g/mu L, and then the episomal plasmid is added into the DMEM to be transfected by a non-lipid cationic transfection reagent NeofectTMMixing, standing for 20 min, and adding into human amniotic epithelial cell culture medium.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for reprogramming human amniotic epithelial cells into induced pluripotent stem cells.
Background
Induced Pluripotent Stem Cell (iPSCs) technology is a technology that obtains self-renewal capacity and multi-differentiation potential by dedifferentiating somatic cells by introducing exogenous genes or gene products. The iPSCs have great application prospect in cell replacement therapy, gene therapy, disease models, development models and drug screening. Patient-specific or disease-specific iPSCs can help us to better understand disease and treat disease. Currently iPSCs have been used in the treatment of several diseases, including genetic diseases, degenerative diseases, and the like. The advantages of iPSCs are that they can be produced using adult somatic cells, there are no ethical problems, and patient-specific iPSCs have low immunogenicity for autologous transplantation. However, the reprogramming steps are complicated, the used culture system contains animal-derived components, and the cost is high, which is a difficult problem existing in the clinical application of the iPSCs technology.
Methods have been used to date to reprogram a variety of somatic cells (e.g., fibroblasts, peripheral blood mononuclear cells, etc.) into human iPSCs. In order to obtain high-quality human iPSCs suitable for clinical application more efficiently, consideration should be given to selecting which type of somatic cells as seed cells to reprogram to iPSCs. Human amniotic epithelial cells (hAECs) are derived from the amniotic membrane of a fetus, are fetal appendages and medical wastes, do not involve ethical problems, and are abundant in source. hAECs are similar to fetal fibroblasts, and are less aged and less environmentally affected than adult human cells, and accumulate less DNA damage or mutations due to environmental stimuli and age, and have less apparent epigenetic memory than adult human cells. Therefore, the iPSCs obtained by reprogramming hAECs have higher application value.
Research results published by Easley et al in 2011 indicate that shorter time is required for hAACs to Reprogram iPSCs than dermal fibroblasts of adults and newborns, the number of obtained iPSCs is more, and iPSCs obtained by reprogramming hAACs are closer to Embryonic Stem Cells (ESCs) than iPSCs obtained by reprogramming fibroblasts, and are closer to ESCs in gene expression level, function and metabolism (Easley et al Cell Reprogram, Vol.14 of 2012, No. 3 of 2012, page 193-203). However, the specific reason why hAECs reprogram to iPSCs is more efficient is not clear. According to their experiments and previous studiesThis is probably because hAECs have basal expression of OCT4(OCT4 gene also known as POU5F1 or OCT3/4), SOX2, KLF4, c-MYC, and these 4 genes are closely related to reprogramming. The relative expression of KLF4 and c-MYC genes of hAECs is not much different from that of human embryonic stem cells, while the expression of OCT4 and SOX2 are very low, which indicates that hAECs are possibly in a preparation state for reprogramming. The basal expression of an endogenous gene is generally more stable than the ectopic expression of an exogenous gene in a cell. In the non-gene-integrated iPSCs technology, exogenous reprogramming factors introduced into somatic cells are gradually lost with the passage of time, and the expression of endogenous reprogramming factors of hAECs is favorable for maintaining the reprogramming state of the iPSCs. In addition, hAECs have the potential of differentiation of three germ layers, and express stem cell markers such as OCT4, NANOG, REX-1, SSEA-3, SSEA4, TDGF1, DNMT3B, GBRB3, GDF3, TRA-1-60 and TRA-1-81, wherein some genes are transcription factors which are important for maintaining self-renewal capacity and pluripotency. These secondary cofactors, except for the most major reprogramming quadractors (OCT4, NANOG, c-MYC, KLF4), may be one of the reasons for the greater efficiency of hAECs reprogramming. The initial phase of reprogramming is the development of mesenchymal-epithelial transformation (MET), using hAECs as seed cells, perhaps skipping the MET process, and therefore faster obtaining of iPSCs clones than fibroblasts. Amniotic fluid cells (amniotic fluid cells) also have advantages similar to hAECs in reprogramming to iPSCs, but the amniotic fluid cells are generally obtained by amniocentesis and are invasive operation, the cell amount is far lower than that of hAECs, the placenta can be obtained by non-invasive operation, and 1-10 × 10 of placenta can be harvested from one human placenta7A personal amniotic epithelial cell. In conclusion, hAECs are ideal cell types for establishing iPSCs.
The earliest reprogramming of hAECs into iPSCs was Easley et al, who introduced 4 reprogramming factors OCT4, SOX2, KLF4, c-MYC into hAECs via lentiviral vectors, repeated the virus transduction process 24 hours later, transferred hAECs to matrigel-coated 6-well plates 24 hours later, and changed mTeSR-1 stem cell media the following day. iPSCs clones appeared at day 14. Finally, an average of 3 clones of iPSCs were obtained per well of 6-well plates, and these iPSCs were propagated in a trophoblast matrigel-mTeSR-1 system (Easley et al Cell Reprogram, vol 14, 7/2012, p 3, 193-. Zhao et al also successfully reprogrammed hAECs to iPSCs in 2017. In their method, only 3 reprogramming factors OCT4, SOX2 and YAP were used, and the advantage was that YAP was used to activate the Hippo-YAP pathway, thereby replacing protooncogenes c-MYC and KLF4(Zhao et al Exp Ther Med, vol.14, No. 1, p.199-. However, their means of introducing reprogramming factors into cells is still a genetically integrated lentiviral vector.
Since the Yamanaka team obtained iPSCs by introducing four genes, Oct4, Sox2, c-Myc and Klf4, into mouse fibroblasts by viral vectors in 2006, the iPSCs technology developed rapidly, resulting in many methods for establishing iPSCs. Methods for introducing reprogramming factors into lentiviral vectors are widely applied, but the lentiviral vectors enable exogenous genes to be randomly integrated into host cell genomes, so that the risk of tumorigenesis is increased. Therefore, non-gene integration methods have been developed, including adenovirus, Sendai virus, episomal plasmid, piggyBac transposon system, direct introduction of reprogramming proteins or mRNAs, induction using chemicals, and the like. Although the non-gene integration method is safer, the efficiency is generally lower than that of the conventional gene integration method, wherein Sendai virus and direct introduction of protein are expensive, and the introduction of protein or mRNA requires repeated introduction of reprogramming factors, which results in a large increase in workload. A safer, efficient, economical, simple and fast iPSC technology is still under development.
The Thomson team found that the use of lentiviral vectors containing the four factors OCT4, SOX2, NANOG and LIN28 was sufficient to induce reprogramming of adult fibroblasts to iPSCs, but with low efficiency. If the lentiviral vector is replaced with a non-gene-integrated episomal plasmid vector, reprogramming efficiency will be lower. Therefore, they developed a non-gene integration method of oriP/EBNA1 episomal plasmid vector, which was based on four factors of OCT4, SOX2, NANOG and LIN28, introduced c-MYC, KLF4 to improve reprogramming efficiency, and in addition SV40TL counteracted cytotoxicity of c-MYC, resulting in a three-plasmid method containing 7 factors (Yu et al Science, 5 months 2009, 324, 5928, 797-page 801).
Okita et al successfully established iPSCs of human dermal fibroblasts and dental pulp cells by using oriP/EBNA1 episomal plasmid vectors carrying 6 factors including shRNA of OCT4, SOX2, KLF4, LIN28, L-MYC and p 53. Wherein, the shRNA of p53 is used for inhibiting p53 and improving reprogramming efficiency; and the C-MYC is replaced by L-MYC, so that the efficiency is higher. The 6 factors are contained in 3 episomal plasmids and are introduced into human dermal fibroblasts and dental pulp cells by electroporation (Okita et al Nature Methods, vol.8, No. 5, p.409-412, 5.2011). Cells need to be digested from the culture plate during electrotransformation, the cells are resuspended in electrotransformation buffer, and the cells are seeded on a new culture plate after electrotransformation is finished. Seven days later cells were transferred to trophoblasts (mouse embryonic fibroblasts) by trypsinization. Clones of iPSCs appeared after two weeks of electroporation. Although the method can obtain the iPSCs quickly, the process is somewhat complicated, and animal-derived substances are introduced.
Therefore, there is a pressing need in the art to develop a simpler method that meets the criteria for clinical use to reprogram hAECs to iPSCs.
Disclosure of Invention
The invention aims to solve the technical problem of providing a simple, quick, economic and efficient method for establishing iPSCs, which is in accordance with clinical application standards, aiming at the existing problem of establishing iPSCs.
The invention provides a method for establishing induced pluripotent stem cells from human amniotic epithelial cells, which is characterized in that more than 2 oriP/EBNA1 free plasmids containing 6 reprogramming factors of cDNA of OCT4, SOX2, KLF4, LIN28 and L-MYC and shRNA of p53 are transfected by a non-lipid cation transfection reagent NeofectTMAnd (3) introducing the human amniotic epithelial cells, and reprogramming the human amniotic epithelial cells into induced pluripotent stem cells.
The human amniotic epithelial cells are human amniotic epithelial cells cultured in vitro within two generations.
The above-mentioned 2 or more oriP/EBNA 1-free plasmids were simultaneously introduced into human amniotic epithelial cells.
The oriP/EBNA 1-free plasmid is three plasmids, namely pCXLE-hOCT3/4-shp53-F (Addgene,27077), pCXLE-hSK (Addgene,27078) and pCXLE-hUL (Addgene, 27078).
And continuously culturing the transfected human amniotic epithelial cells on an original culture plate until the induced pluripotent stem cells are cloned and grown and are picked out.
The method also comprises the step that after the obtained induced pluripotent stem cells are cloned and picked out, the cells are planted on a culture plate coated by the human recombinant vitronectin and are planted on TeSRTM-E8TMAnd carrying out amplification culture in a culture medium.
The method comprises the following steps:
(1) inoculating the human amniotic epithelial cells into a culture plate, and culturing for 2 days by using a human amniotic epithelial cell culture medium;
(2) after the culture medium of the human amniotic epithelial cells is replaced, a non-lipid cationic transfection reagent Neofect is addedTMAnd a mixture containing the episomal plasmid pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL, and then adding the mixture into the culture medium of the human amniotic epithelial cells;
(3) after 24 hours of transfection, TeSR was changedTM-E8TMStem cell culture medium, changing liquid every other day;
(4) selecting out induced pluripotent stem cell clone, seeding on a culture plate coated with human recombinant vitronectin, and adopting TeSRTM-E8TMAnd performing amplification culture on the culture medium to obtain the induced pluripotent stem cells.
The above method further comprises the steps of:
(5) step (4), freezing the cultured induced pluripotent stem cells by adopting a serum-free cell freezing medium;
(6) and (4) identifying a small number of cells in the step (5) to express stem cell markers NANOG, OCT4, SOX2 and TRA-1-60, namely the induced pluripotent stem cells.
The above method further comprises the steps of:
(7) and (4) screening the cells after the expanded culture in the step (4) to obtain induced pluripotent stem cells expressing stem cell markers NANOG, OCT4, SOX2 and TRA-1-60. The stem cells obtained by screening can be directly applied to clinic.
In the step (2), the episomal plasmids are firstly added into DMEM together to be diluted until the concentration of each plasmid is 0.05-0.01 mu g/mu L,then non-lipid cation transfection reagent NeofectTMMixing, standing for 20 min, and adding into human amniotic epithelial cell culture medium.
The invention provides a novel method for reprogramming hAECs into iPSCs, namely, a novel method for reprogramming iPSCs by using a non-lipid cation transfection reagent NeofectTM3 oriP/EBNA 1-free plasmids pCXLE-hOCT3/4-shp53-F, pCXLE-hSK reported by Okita et al, pCXLE-hUL (six factors including cDNA of OCT4, SOX2, KLF4, L-MYC and LIN28 and shRNA of p 53) were introduced into hECs, reprogrammed to iPSCs by one-step method, and cultured in TeSRTM-E8TMHuman recombinant vitronectin animality system.
The invention makes the following 4 point improvements on the Okita method (Okita, K et al. A more effective method to generation integration-free human iPS cells, Nature Methods 2011 May; 8(5): 409-:
1. hAECs are selected as seed cells to be reprogrammed to iPSCs instead of human dermal fibroblasts or dental pulp cells, and the advantages of hAECs are fully utilized; hAECs are selected as seed cells to be reprogrammed to iPSCs instead of human dermal fibroblasts or dental pulp cells, and the advantages of hAECs are fully utilized;
2. the transfection method abandons the electricity and adopts a non-lipid cation transfection reagent Neofect which is simple to operateTMWhen in transfection, only a transfection reagent and the free plasmid are mixed and dripped into a culture, and a fresh culture medium is replaced the next day without trypsinization and cell resuspension;
3. human amniotic epithelial cell medium was changed to TeSR on day 2 post transfectionTM-E8TMThe stem cell culture medium is changed every other day until iPSCs clone appears, and compared with the method of Okita, the method omits the step of transferring cells to a trophoblast and is a one-step method;
4. cloning the iPSCs obtained by reprogramming and selecting the iPSCs on a culture plate coated by the human recombinant vitronectin, and culturing the iPSCs in animal origin-free TeSRTM-E8TMHuman recombinant vitronectin system, which is more in accordance with the clinical application standard.
In addition, episomal plasmid vectors are a reprogramming method of a non-gene integration type, reducing tumorigenicity.
The method of the invention can rapidly reprogram hAECs into iPSCs. hAE-iPSCs clones appeared at day 10 and 23 after transfection and could be picked at TeSRTM-E8TMProliferation passages in the human recombinant vitronectin system. The nucleus-to-cytoplasm ratio of the harvested hAE-iPSCs is large, the edges of cell clusters are sharp, the shapes of the cell clusters are similar to those of ESCs, and the karyotype is normal. In vitro experiments show that hAE-iPSCs express stem cell markers NANOG, OCT4, SOX2 and TRA-1-60. hAE-iPSCs form teratomas in immunodeficient mice, demonstrating their multipotentiality. However, reprogramming human dermal fibroblasts with the method of the present invention, although the iPSCs clone was initially formed, since this method does not comprise a step of transferring the cells to a trophoblast after transfection, human dermal fibroblasts were reprogrammed at TeSRTM-E8TMThe iPSCs can not grow up because the stem cell culture medium does not die and is not separated from the culture plate, and finally iPSCs clone derived from adult dermal fibroblasts can not be obtained. Therefore, the reprogramming of human amniotic epithelial cells into iPSCs is necessary in the method of the invention and is also the creative place of the invention, the reprogramming of human dermal fibroblasts into iPSCs cannot achieve the effects of 'one-step method' and 'no animal origin', while the reprogramming of human amniotic epithelial cells into hAE-iPSCs by the method of the invention has the effects of 'one-step method' and 'no animal origin', and can be directly applied to clinic.
Drawings
FIG. 1 is a schematic representation of the basic operational flow of reprogramming hAECs to iPSCs.
FIG. 2 illustrates the formation process of 2 hAE-iPSCs.
a) hAECs form normal before transfection, and the fusion degree is about 60%; b) hAE-iPSCs clones appeared at day 10 post-transfection (indicated by arrows), and cells that were not successfully reprogrammed to iPSCs were in the TeSRTM-E8TMGradually apoptosis in the stem cell culture medium and detachment from the culture plate; c) the clone morphology of hAE-iPSCs is obvious at 16 days after transfection; d) hAE-iPSCs clone grows up at 23 days after transfection, and the edges of cell groups are sharp; e) hAE-iPSCs cells are closely connected, and the nucleus-to-cytoplasm ratio is large; f) hAE-iPSCs can survive and proliferate after being transferred to a culture plate coated by human recombinant vitronectin, and are clonedMorphology and cell morphology maintain the morphology of similar embryonic stem cells.
FIG. 3 is a graphical representation of the effect of reprogramming human dermal fibroblasts to iPSCs using the present method.
a) Human dermal fibroblasts are normal in morphology before transfection, and the fusion degree is about 60%; b) iPSCs were cloned in the plates at day 16 post-transfection (indicated by arrows), and cells that were not successfully reprogrammed to iPSCs were in the TeSRTM-E8TMThe stem cells are not separated from the culture plate in the culture medium; c) iPSCs clone still does not grow up after 33 days of transfection, and most of human dermal fibroblasts also do not separate from the culture plate; d) on day 45 after transfection, human dermal fibroblasts were morphologically abnormal, but most were attached to the culture plate, while iPSCs clones were detached from the culture plate.
FIG. 4hAE-iPSCs karyotype was identified as a normal human karyotype.
FIG. 5 shows that hAE-iPSCs express stem cell markers NANOG, OCT4, SOX2, TRA-1-60.
FIG. 6 is a graphic representation of teratoma formation from 6 hAE-iPSCs.
The hAE-iPSCs obtained by the method are injected to the back of a SCID-beige immunodeficient mouse subcutaneously on both sides, and a tumor body is formed after 45 days. Tumor body HE staining showed 3 germ layer cells.
Detailed Description
All cells below were at 5% CO2And culturing in a cell culture box at constant temperature of 37 ℃.
Example 1 reprogramming procedure and Effect of human amniotic epithelial cells
The steps (figure 1) and effects of reprogramming the human amniotic epithelial cells into hAE-iPSCs are as follows: day-2: seed cells
At 5X 104hAECs within two in vitro culture generations were plated at a density of cells per square centimeter in 6-well plates, i.e., 4.8X 10 cells per well of 6-well plates5And one hAECs.
Day 0: transfection
Taking a well of a 6-well plate as an example
1) Replacing the cells with fresh human amniotic epithelial cell culture medium hAECM two hours before transfection;
2) plasmid dilution: 1 mu g of each plasmid pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL is uniformly mixed with 100 mu L of DMEM;
3) add 3. mu.L Neofect directly to the DNA dilutionTMThe transfection reagent is mixed gently and evenly, and is kept stand for 20 minutes at room temperature, and the preparation of the transfection compound is finished;
4) the transfection complex was added to the cells and gently mixed.
Day 1:
transfected cells were replaced with fresh hAACM.
Day 2:
aspirate hAMCs and replace with TeSRTM-E8TMStem cell culture medium, change the liquid every other day.
Day 23 and later: selecting, cloning and expanding culture
An average of 8 hAE-iPSCs clones can be obtained in one hole of a 6-hole culture plate, and the yield is 0.0017%;
1) individual stem cell clones were picked up with a 10. mu.L pipette tip to human recombinant vitronectin (0.5. mu.g/cm)2) In one well of a 24-well coated plate and in TeSRTM-E8TMAdding ROCK inhibitor Y-27632(10 mu M) into the culture medium;
2) the picked clones were grown and passaged to one well of a 12-well plate using ReLeSR (FIG. 2f), also human recombinant vitronectin (0.5. mu.g/cm)2) Coated and incorporated in TeSRTM-E8TMAdding Y-27632 into the culture medium;
3) passing hAE-iPSCs to one well of 6-well culture plate by the same method about one week;
4) the hAE-iPSCs can be subjected to expanded culture, frozen storage and identification;
5) in order to ensure that hAE-iPSCs do not contain xenogeneic animal components, the frozen stock is used for freezing and storing cellsSerum-free cell cryopreservation solution.
hAECs were normal in morphology and viability in culture plates before transfection (FIG. 2a), and hAE-iPSCs were cloned 10 days after transfection without successful reprogramming to iPSCsCells in TeSRTM-E8TMApoptosis progressed in stem cell culture medium and detached from the plate (fig. 2 b). The hAE-iPSCs clone morphology was evident at day 16 post-transfection (FIG. 2 c). hAE-iPSCs were cloned and grown up 23 days after transfection, with sharp cell population edges (FIG. 2d), hAE-iPSCs with tight junctions between cells and large nuclear-cytoplasmic ratios (FIG. 2 e). hAE-iPSCs were viable and proliferated by transferring to human recombinant vitronectin-coated plates, and the clonal morphology and cell morphology maintained the morphology similar to that of embryonic stem cells (FIG. 2 f). hAE-iPSCs were karyotyped as normal human karyotypes (FIG. 4). Cell immunofluorescence staining experiments showed that hAE-iPSCs expressed stem cell markers NANOG, OCT4, SOX2, TRA-1-60 (FIG. 5). hAE-iPSCs were injected bilaterally subcutaneously into the back of SCID-beige immunodeficient mice, and tumors formed 45 days later. HE staining of tumor body revealed 3 tissues of neuroepithelium, striated muscle and gland, which are derived from ectoderm, mesoderm and endoderm, respectively (fig. 6).
Example 2 reprogramming procedure and Effect of human dermal fibroblasts
The reprogramming steps and effects of the method on the human dermal fibroblasts are as follows:
day-2: seed cells
At 5X 104The human dermal fibroblasts cultured in vitro within four generations were plated in 6-well plates at a density of cells per square centimeter, i.e., 4.8X 10 cells per well of 6-well plates5Human dermal fibroblasts.
Normal morphology and viability of human dermal fibroblasts in culture plates was observed prior to transfection (fig. 3 a).
Day 0: transfection
Taking a well of a 6-well plate as an example
1) Two hours before transfection, cells were replaced with fresh human fibroblast culture medium;
2) plasmid dilution: 1 mu g of each plasmid pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL is uniformly mixed with 100 mu L of DMEM;
3) add 3. mu.L Neofect directly to the DNA dilutionTMThe transfection reagent is mixed gently and evenly, and is kept stand for 20 minutes at room temperature, and the preparation of the transfection compound is finished;
4) the transfection complex was added to the cells and gently mixed.
Day 1:
the transfected cells were replaced with fresh human fibroblast culture medium.
Day 2:
the human fibroblast culture medium is aspirated and replaced by TeSRTM-E8TMStem cell culture medium, change the liquid every other day. Day 16: iPSCs clone exists in the culture plate, and human fibroblast which is not successfully reprogrammed to iPSCs is cultured in TeSRTM-E8TMThe stem cells did not leave the plate in culture medium (FIG. 3 b).
Day 33: human fibroblast-derived iPSCs clones remained unexplored, and most of the human fibroblasts did not detach from the culture plate (fig. 3 c).
Day 45: human fibroblasts were morphologically abnormal, but most remained attached to the plate, while human fibroblast-derived iPSCs clones fell off the plate (fig. 3 d).
Required reagent
1. Amniotic epithelial cell culture medium hAECM [ F12/DMEM, 10% KSR (knock out Serum replacement),2mmol/L L-glutamine, 1% nonnessial amino acid, 55. mu. mol/L2-mercaptoethanol, 1mmol/L sodium pyroltate, 1% antistic-antimycotic (all from Gibco) and 10ng/mL EGF (Peprotech) ]
2.3 episomal plasmids pCXLE-hOCT3/4-shp53-F (Addgene,27077), pCXLE-hSK (Addgene,27078), pCXLE-hUL (Addgene,27080)
3.NeofectTMDNA transfection reagent (zero guests create intelligence, TF201201)
4.DMEM(HyClone,SH30243.01)
5. Human fibroblast culture medium: 90% DMEM (HyClone, SH30243.01), 10% FBS (Thermo Fisher Scientific,10100147)
6.TeSRTM-E8TMStem cell culture medium (Stemcell Technologies, #05990)
7. Human recombinant vitronectin (Thermo Fisher Scientific, A14700)
8.ReLeSR(Stemcell Technologies,#05872)
9.Y-27632(Topscience,129830-38-2)
10.Serum-free cell freezing medium (Yikesai biology, VUC00-N011)
11.pCXLE-hOCT3/4-shp53-F(Addgene,27077)、pCXLE-hSK(Addgene,27078)、pCXLE-hUL(Addgene,27078)