WO2012075912A9 - 亚全能干细胞产品及其表观遗传修饰标签 - Google Patents
亚全能干细胞产品及其表观遗传修饰标签 Download PDFInfo
- Publication number
- WO2012075912A9 WO2012075912A9 PCT/CN2011/083380 CN2011083380W WO2012075912A9 WO 2012075912 A9 WO2012075912 A9 WO 2012075912A9 CN 2011083380 W CN2011083380 W CN 2011083380W WO 2012075912 A9 WO2012075912 A9 WO 2012075912A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- differentiation
- histone
- gene
- modification
- stem cells
- Prior art date
Links
Classifications
-
- 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
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
Definitions
- the present invention relates to a sub-total stem cell product, a method for inducing the production of a sub-total stem cell product, and an epigenetic modification tag for stem cell differentiation potential.
- the present invention also relates to a method for predicting the differentiation potential of stem cells and a histone modification state of a sub-total gene and/or a differentiation-related gene as an epigenetic modification label for predicting the differentiation potential of stem cells. Background technique
- iPSCs induced pluripotent stem cells
- Sex Embryonic stem cells have The omnipotence, but ethical issues, immune rejection and tumorigenicity seriously impede its clinical research and application.
- iPS has similar differentiation ability to embryonic stem cells, but it still has tumorigenicity, and from adulthood The efficiency of cell-induced iPS production is extremely low, and the induced iPS carcinogenesis rate is high. These factors greatly increase the insecurity of clinical application.
- Adult stem cells have a wide range of sources, no tumorigenicity, and no ethical issues.
- Epigenetic modifications usually include DNA methylation, histone modification, and RA modification; while histone modifications include histone methylation, acetylation, phosphorylation, and ubiquitination; the modified sites are mostly located at the N-terminus of histones. Modification can affect the state of chromatin by affecting the affinity of histones and DNA, and can also affect the binding of transcription factors to DNA sequences. It has a DNA-like genetic code for gene expression regulation, so it is called "histone.”Password”. Histone methylation refers to methylation that occurs at the N-terminal arginine or lysine residues of the H3 and H4 histones and is mediated by histone methyltransferase.
- Histone H3 4th lysine trimethylation (H3K4me3) is generally associated with promoter activation [11]
- histone H3 27th lysine trimethylation (H3K27me3) is associated with promoter expression.
- Silence related [12, 13].
- the simultaneous presence of the two promoter histone modifications, H3K4me3 and H3K27me3, is called bivalent modification. This bivalent modification keeps the gene at a relatively low level of expression and maintains a "pre-transcriptional" state. The state allows the gene to respond rapidly to transcriptional activation or inhibition according to appropriate stimuli [14-17].
- H3K4me3 modification is ubiquitous near the promoter, while H3K27me3 is only found in 10% of the gene promoter region, and that the region where H3K27me3 is modified is also modified by H3K4me3, which is subject to bivalent
- the modified gene is preferentially activated during ESC differentiation, suggesting that the presence of bivalent modification may be to maintain development-related genes in an equilibrium state, ready for later activation [15]; and those without any modification are inhibiting The state is completely silent.
- NPCs neural progenitor cells
- MEFs murine embryonic fibroblasts
- T cells primary human T cells
- Stem cell transplantation can be used to treat Parkinson's disease, cardiomyopathy, liver disease, and osteogenic treatment of bone defects, large-area burns, and much-needed skin materials.
- the adult stem cells are obtained from the autologous tissue, and the tissue induced by it does not have immunological rejection during transplantation, and has a wide range of tissue types for differentiation. It has broad application value and is expected to become the future end of stem cell transplantation for various organs. The main force of the terminal disease. However, there are still many safety problems in stem cell transplantation. If it is reported that the application of embryonic stem cells to the heart for coronary heart disease may lead to teratoma, the application of skeletal muscle stem cells may cause malignant arrhythmia.
- pluripotent stem cells depends on their differentiation potential.
- the existing methods for verifying the ability of a certain tissue-derived stem cell to differentiate are mainly through inducing differentiation and observing whether it can be differentiated into as many lineages as possible in the three germ layers. This method takes a long time and requires a lot of manpower and material resources.
- Chromatin Immunoprecipitation is the most important method for detecting histone modifications.
- Chromatin immunoprecipitation also known as binding site analysis, is a powerful tool for studying the interaction of proteins and DNA in vivo, and is commonly used in transcription factor binding sites or group-specific protein modification sites.
- Chromatin immunoprecipitation analysis is a method developed based on in vivo analysis. Its basic principle is to immobilize a protein-DNA complex in a living cell state, and randomly cut it into small fragments of chromatin within a certain length, and then pass the immunization.
- the method is to precipitate the complex, specifically enrich the DNA fragment bound by the target protein, and obtain information on the interaction between the protein and the DNA by purifying and detecting the target fragment.
- the target fragment can be detected by tiling array or high-throughput sequencing, the former being called ChlP-on-chip and the latter being called ChIP-Seq.
- the ChlP-Seq technology which combines ChIP with second-generation sequencing technology, efficiently detects DNA segments that interact with histones, transcription factors, etc., across the genome.
- ChIP chromatin immunoprecipitation
- ChlP-Seq Since the data of ChlP-Seq is the result of DNA sequencing, researchers can provide resources for further exploration of biological information. researchers can conduct research in the following aspects: (1) determining which histone modifications will occur at a particular position in the DNA strand;
- a specific separation, induction and screening system can be used to obtain stem cells with appropriate differentiation ability and to use the "histone methylation code" in the role of predicting cell differentiation and the combination of ChIP detection and bioinformatics analysis ( ChlP-seq) studies the genome-wide histone methylation profile of various grades of stem cells, finds the relationship between histone methylation and stem cell differentiation, and establishes a unified phenotype, culture conditions and identification methods for adult stem cells. Furthermore, an accurate identification and evaluation index for stem cell differentiation potential, differentiation stage and controllable specific differentiation of stem cells in vivo will be established, which will provide the most basic materials and indicators for the wide application of stem cells in clinical practice.
- Fig. 7 shows a schematic diagram of the technique of the present invention. Summary of the invention
- the object of the present invention is to induce the obtaining of a sub-total stem cell which has an epithelial-like morphology, has a Flkl-positive phenotype, has the differentiation ability of the three-embryral multi-lineage, but does not have tumorigenicity.
- the availability of such cells provides ideal seed cells for clinical regenerative repair therapy.
- a first aspect of the present invention provides a sub-total stem cell product characterized by having an epithelial-like morphology, a Flkl + phenotype, a non-angiogenicity, and the monoclonal derived cell has an induced differentiation into three germ layers in vitro.
- the ability of the tissue cells to be derived, preferably, its pluripotency genes include Oct4, Nanog c-Myc, Sall4, Sox2, Klf4; early ectodermal differentiation-related genes include Hoxal, Gbx2, Sixl, and 01ig3; mesendoderm early differentiation-associated genes T , Pgdfr a, Eomes, Tbx6 and Mixll; Mesenchymal early differentiation-related genes Kdr, Handl Gata4 and Mesp2; Definitive endoderm early differentiation-related genes Onecutl Proxl, Foxal, Foxa2, Sox7, Soxl7, Pdxl and Gsc methylation
- the modified state is mainly activated or a bivalent modification in which H3K4me3 and H3K27me3 coexist.
- a second aspect of the invention relates to a method of producing a sub-total stem cell product as described above, having the following steps:
- 1 induction medium contains l-100 ng/ml activin A+l-500 ng/ml Wnt3a+ 0.1-20% FBS+HG-DMEM, preferably 5-50 ng/ml activin A, more preferably 10-30 ng/ml activin A; preferably, 50-300 ng/ Ml Wnt3a, more preferably, 100-300 ng/ml Wnt3a ; preferably, 2-10% FBS, more preferably 5-8% FBS, said No.
- 2 induction medium contains l-100 ng/ml activin ⁇ + 1-500 ⁇ RA+ 0.1-50% FBS+HG-DMEM, 5-50 ng/ml activin oxime, more preferably 10-30 ng/ml activin oxime; preferably, 20-400 ⁇ RA, more preferably 50 -200 ⁇ RA, the obtained sub-total epithelial stem cells were subjected to RT-PCR detection, immunofluorescence staining and Western Blot detection, wherein the immunofluorescence staining indicators included Foxa2, Soxl7, Kdr, Tbx6 , Eomes, Gsc, T, Soxl, Pax6, the indicators detected by the Western Blot include Foxa2, Soxl7, T, Gsc, Epcam, Vimatin, whether the stem cells detected have the important gene phenotypic markers of the three germ layer differentiation potential, ie, limited Endoderm: Foxa2, Soxl7; mesendoderm: Gsc, T
- a third aspect of the invention relates to a method of detecting whether a stem cell product is a sub-total stem cell product, comprising the steps of:
- immunofluorescence staining detection indicators include Foxa2, Soxl7, Kdr, Tbx6, Eomes Gsc, T, Soxl, Pax6, the Western Indicators for Blot detection include Foxa2, Soxl7, T, Gsc, Epcam, Vimatin;
- neural-induced differentiation DMEM/F12 (DF12) 1 :1 basal medium was supplemented with N2/B27, 20 ng/ml EGF and 50 ng/ml IGF-1, Two weeks later, 30 ng/ml NT3 and 10 ng/ml bFGF were added. After two weeks, 30 ng/ml NT3 and 10 ng/ml BDNF were induced for 7 days.
- Adipogenic differentiation DMEM basal medium was added 10%.
- FCS 1 ⁇ dexamethasone, 0.5 mM IBMX, I mM ascorbic acid for 8 days
- osteogenic differentiation DMEM basal medium supplemented with 10% FCS, 10 mM ⁇ -glycerophosphate, 10 ⁇ dexamethasone and 0.2 mM ascorbic acid Induction for 8 days
- Hepatic epithelial-induced differentiation 20 ng/ml HGF, 10 ng/ml FGF-4, 20 ng/ml EGF and 2% FBS were added to the basal medium for 3 weeks
- Hematopoietic cells were induced to differentiate: in basal medium After induction with 150 ng/mL SCF and 200 ng/mL G-CSF for 7 days, cells were harvested and seeded in serum-free methylcellulose semi-solid medium containing 1% BSA, 50 ng/mL BMP-4.
- IL-6 50 ng/mL IL-6, 50 ng/mL SCF, 50 ng/mL Flt-3L, 10 ng/mL G-CSF, 10 ng/mL TPO; 10 g/mL EPO, 200 ⁇ glmL transferrin, 2 mM L-glutamyl Amine, Ol mM P-mercaptoethanol, 1% non-essential amino acids, induced for 9 days, collected cells, washed with methylcellulose, counted 5000 cells, re-inoculated in serum-containing methylcellulose semi-solid medium Induction for 14 days; 6) detecting the methylation status of subgenomic and tissue differentiation-related genomic proteins in the stem cells to predict their differentiation potential, as follows:
- the target gene belongs to the 4th lysine trimethylation modification of histone H3 or the lysine trimethylation modification of histone H3 position 4 and the lysine trimethylation modification of histone H3 position 27 It then indicates that the target stem cells have the ability to differentiate to the particular cell type indicated by the gene of interest.
- the gene of interest is selected from the group consisting of a sub-total gene, a three-embryonic early differentiation gene, a neural differentiation-related gene, a lipid-forming gene, an osteogenic gene, a hematopoietic-related gene, or a lineage-related gene of a liver epithelial differentiation, and a plurality of lineages.
- differentiation-related transcription factors of other lineages including totipotent genes including Oct4, Nanog, c-Myc, Sall4, Sox2, and Klf4; early ectodermal differentiation-related genes including Hoxal, Gbx2, Sixl, and 01ig3; Genes T, Pgdfr a, Eomes, Tbx6 and Mixll; mesoderm early differentiation related genes Kdr, HandK Gata4 and B Mesp2; restricted endoderm early differentiation related genes Onecutl, Proxl, Foxal, Foxa2, Sox7, Soxl7, Pdxl and Gsc , neural differentiation related genes include Tubb3, Nkx2-2, Soxl, NeurogK Ascll Bm2, MytlK Zicl, Neurog2, Hesl, Dlxl, Pax6, Tlx2, Msil, Gfral Gfra3, Mapt, Nes, 01ig2, Neurodl Neurod2, Adipogenic genes including C /EBP a,
- a fourth aspect of the invention relates to the use of a histone modification state of a sub-total gene and/or a differentiation-related gene as an epigenetic modification tag for predicting the differentiation potential of stem cells, wherein the sub-total gene and/or The histone methylation status of differentiation-related genes predicts the differentiation potential of stem cells.
- the stage of differentiation in which the cell is located is identified by detecting the histone methylation modification status of a particular lineage differentiation stage transcription factor and marker gene.
- analysis of histone modification status changes that initiate other non-target lineage differentiation-related genes identifies the specificity of cell differentiation to the target lineage.
- the histone methylation modification is a histone H3 4th lysine trimethylation modification or a histone H3 4th lysine trimethylation modification and a histone H3 27th lysine
- the acid trimethylation modification coexists.
- the sub-total gene and/or the differentiation-related gene is selected from the group consisting of a totipotent gene and an early differentiation phase of the three germ layers A gene, a lineage-related gene, a hematopoietic-related gene, or a hematopoietic-related gene or a lineage-related gene, a lineage, a plurality of lineages, or all differentiation-related transcription factors including other lineages, wherein the totipotent gene includes Oct4 , Nanog, c-Myc, Sall4, Sox2, Klf4; Early differentiation related genes of ectoderm include Hoxal, Gbx2, Ski and 01ig3; Early differentiation related genes of endoderm T, Pgdfr ⁇ , Eomes, Tbx6 and Mixl; early mesoderm Differentiation-related genes Kdr, Handl, Gata4 and Mesp2; Definitive endoderm early differentiation related genes Onecutl, ProxK Foxal, Foxa2, Sox7
- ChlP-seq or ChIP-PCR is used to detect the histone methylation modification status of the sub-total gene and/or differentiation-related gene.
- the different histone methylation status of the sub-total genes and/or differentiation-related genes indicates different differentiation potential of stem cells
- a lineage differentiation-related genomic protein methylation modification is histone H3 4th lysine Acid trimethylation modification and histone H3 4th lysine trimethylation modification and histone H3 27th lysine trimethylation modification coexist, indicating that this stem cell has differentiation to this lineage
- the potential, compared to two or more stem cells, the gene associated with the lineage is generally modified by histone H3 lysine trimethylation and histone H3 lysine trimethylation Stem cells with a high proportion of genes coexisting with the lysine trimethylation modification of histone H3 at position 27 are more likely to differentiate into this lineage.
- the target gene belongs to histidine H3 lysine trimethylation modification (H3K4me3) or histone H3 lysine trimethylation modification (H3K4me3) and histone H3 27th lysine
- H3K27me3 The coexistence of the trimethylation modification indicates that the target stem cells have the ability to differentiate to the specific cell type indicated by the gene of interest.
- the methylation modification of a lineage-associated genomic protein is the lysine trimethylation of histidine H3 and the lysine trimethylation of histidine H3 and the 27th lysine of histone H3.
- the coexistence of trimethylation modification indicates that this stem cell has the potential to differentiate into the lineage.
- the early differentiation-related genes of ectoderm include Hoxal, Gbx2, Ski and 01ig3; the early differentiation-related genes of mesendoderm T, Pgdfr a, Eomes, Tbx6 and Trim; the early differentiation-related genes of mesoderm Kdr, Handl, Gata4 and Mesp2; Restricted endoderm early differentiation related genes Onecutl, Proxl, Foxal, Foxa2, Sox7, Soxl7, Pdxl and B Gsc, neural differentiation related genes including Tubb3, Nkx2-2, Soxl, NeurogK Ascll Bm2, MytlK Zicl, Neurog2, Hesl, Dlxl , Pax6, Tlx2, Msil, Gfral, Gfra3, Mapt, Nes, 01ig2, Neurodl, Neurod2, lipid-forming genes including C/EBP ⁇ , PPAR ⁇ , ERK5, GSK3 ⁇ , GSK3 ⁇ , C/E
- Lineage or all differentiation-related transcription factors including other lineages.
- the terminally differentiated marker genes such as AP2, LPL, c-Kit, ALP, OPN, CK8, CK18, etc. should not be used as candidate genes for differentiation potential prediction
- the present application extracts mesenchymal stem cells from various tissues such as fetal/adult fat, bone marrow and umbilical cord, obtains monoclonal cells by limiting dilution method, further amplifies the monoclonal cells, and then adds an appropriate amount at an appropriate time.
- Factors such as activin A and Wnt3a induce an epithelioid-like, Flkl-positive MSC that does not form a teratoma in mice.
- RT-PCF immunofluorescence staining and Western Blot assay showed that the Flkl-positive MSCs expressed the restricted endoderm marker genes Foxa2, Soxl7, mesendoderm marker genes Gsc, T, Eomes, mesodermal marker gene Kdr, Tbx6, ectoderm marker genes Soxl, Pax6, and the like.
- Flkl + MSC revealed that it can further differentiate into three germ layer multi-lineage-derived tissues such as adipocytes, bone cells, liver epithelium, glial cells, and pancreatic stem/progenitor cells.
- sub-totipotency the ability to differentiate into intact individuals is called sub-totipotency
- sub-total-capable Flkl + MSCs are called sub-total stem cells.
- the availability of such sub-total stem cells provides ideal seed cells for research and clinical applications in regeneration and translational medicine.
- the modification status of the sub-genoplasmic H3K4 and H3K27 trimethylation of sub-total and related genes in stem cells is closely related to the differentiation ability of stem cells. It is used to predict the differentiation potential of stem cells; not only that, when stem cells differentiate into specific lineages, prior to the change in gene expression, the differentiation-related genomic protein modification state will be re-arranged, resulting in histone methylation modification status of the target lineage-related genes. It becomes more activated, and histone modifications that initiate genes related to other lineage differentiation become further suppressed or silenced.
- this histone modification state facilitates differentiation of stem cells into different lineages according to changes in microenvironment or external conditions, so differences in differentiation-related genomic modification states can be used as predictions.
- a powerful indicator for evaluating the differentiation potential of stem cells from different sources That is, it is found in the present specification that "the methylation status of the sub-total and differentiation-related genomic proteins in stem cells is closely related to the differentiation ability of the stem cells, and the stem cells can be predicted by analysis of the methylation status of the sub-total and differentiation-related genomic proteins in stem cells.
- the ability to differentiate that is, "specific sub-total and differentiation-associated genomic protein methylation-modified states can serve as a marker for predicting the ability of stem cells to differentiate from a certain source.”
- This method of predicting stem cell differentiation ability only needs to detect the histone methylation status of sub-total and differentiation-related genes in stem cells by using ChlP-seq or ChlP-PCR technology, and does not require multi-lineage differentiation of stem cells. Save time, labor and reagent supplies. Therefore, the role of histone methylation modification status in predicting stem cell differentiation has important clinical application value.
- Figure 1 shows the differentiation ability of Flkl + MSC.
- A Ammonia-derived aMSCs were obtained by limiting dilution method,
- B Flkl + MSC differentiated into adipogenic and osteogenic lineages,
- C Flkl + MSC differentiated into hematopoietic cells and identified (OC: Osteocalcin,
- BFU-E erythroid Explosive colony forming unit,
- CFU-G macrophage colony forming unit,
- CFU-MK megakaryocyte colony forming unit, HPP-CFC: high proliferative potential cell colony forming unit)
- D Flkl + MSC induced to liver epithelium Differentiation and identification
- E Flkl + MSC induced differentiation and identification in the direction of the nerve.
- Figure 2 Differential proteomic methylation modification profiles of stem cell pluripotency genes, early germline differentiation-related genes, and neural differentiation-related genes at different levels.
- A histone methylation modification of pluripotency-related genes
- B histone methylation modification of genes associated with ectodermal differentiation
- C histone methylation modification of genes associated with endoderm differentiation
- D Methylation modification of mesoderm differentiation-related genes
- E Definitive endoderm differentiation-associated genomic protein methylation modification
- F histone methylation modification of neural differentiation-related genes.
- Figure 3 shows the differential repair profile of methylation (A) and osteogenic (B) differentiation-related genes in different stem cells.
- Figure 4 shows the differential expression of methylation differences in liver epithelial (A) and hematopoietic (B) differentiation genes in different stem cells.
- Figure 5 Comparison of methylation modification of histone protein associated with adipogenic differentiation in aMSC and bMSC Comparison of differentiation ability.
- A ChalP-PCR analysis of osteogenic genomic protein methylation status in aMSC and bMSC
- B ChlP-PCR analysis of adipose genomic protein methylation status in aMSC and bMSC
- C aMSC and bMSC Comparison of osteogenic lineage and adipogenic lineage differentiation ability.
- Figure 6 shows dynamic changes in related genomic protein methylation modifications in neuronal, adipogenic and osteogenic differentiation.
- A real-time PCR to determine the expression of related genes before and after neural differentiation
- B ChlP-PCR to detect histone methylation status of related genes before and after neural differentiation
- C real-time PCR and gene chip analysis of adipogenic differentiation Pre- and post-related gene expression and histone methylation status
- D real-time PCR and gene chip analysis of related gene expression and histone methylation status before and after osteogenic differentiation
- E real-time PCR and gene chip The expression of other lineage-related genes and histone methylation status in adipogenic differentiation or osteogenic differentiation were analyzed.
- Figure 7 Schematic diagram of histone methylation status predicting stem cell differentiation potential.
- Figure 8 Morphological changes of cells before and after induction: Left: Cells are spindle-shaped before induction; Right: Cells are densely cobblestone-like after induction.
- Figure 9 Results of RT-PCR in cells before and after induction: From left to right: Upregulation of gene expression in definitive endoderm markers foxa2, Gsc, T, Eomes (P ⁇ 0.05) and ectoderm markers Soxl, Pax6 (P ⁇ 0.05) The expression of Kdr and Tbx6 genes in mesoderm markers was not significantly different (P>0.05).
- FIG 10 Western Blot assay before and after induction: After induction, the endodermal markers Foxa2, soxl7, mesendoderm markers T, Gsc expression abundance increased significantly, and the epithelial cell marker Epcam expression abundance also increased. The abundance of the interstitial cell marker Vimantin expression was not obvious.
- Figure 11 Immunofluorescence staining of cells before and after induction: Compared with uninduced cells, the cells with Foela2 and Soxl7 positive markers reached more than 90% after induction, and the cells with T-positive mesoderm markers reached more than 70%. The ectodermal marker is more than 70% positive for Soxl. detailed description
- aMSCs adult adipose-derived mesenchymal stem cells
- the cells were collected by centrifugation, and the cells were seeded at a density of 2 ⁇ 10 6 /ml in 58% DMEM/F12 + 40% MCDB-20K 5% fetal bovine serum (FCS), 10 ng/ml EGF, 10 ng/ml PDGF, lx insulin-transfer.
- FCS fetal bovine serum
- ITS Insulin-Transferrin-Selenium
- LA-BSA lx linoleic acid-bovine serum albumin
- 50 ⁇ ⁇ -mercaptoethanol 2 mM L-glutamine
- 10 ⁇ g/ml penicillin and 100 U/ml streptomycin sulfate culture medium, incubate in 37 ° C, 5% CO 2 , 95% humidity incubator.
- bMSCs bone marrow-derived mesenchymal stem cells
- the above mononuclear cells were seeded at a density of 1 ⁇ 10 6 /cm 2 in a 25 cm 2 culture flask, and the cell culture system contained 58% DMEM/F12 + 40% MCDB-201, 2% fetal bovine serum.
- FCS 10 ng/ml EGF, 10 ng/ml PDGF, l Insulin-Transferrin-Selenium (ITS), lx linoleic acid-bovine serum albumin , LA-BSA), 50 ⁇ ⁇ -mercaptoethanol, 2mM L-glutamine, 100 ⁇ ⁇ / ⁇ 1 penicillin and 100U/ml streptomycin sulfate, 37°C, 5% CO 2 , 95% humidity incubator to cultivate.
- FCS 10 ng/ml EGF
- PDGF 10 ng/ml PDGF
- ITS Insulin-Transferrin-Selenium
- LA-BSA 50 ⁇ ⁇ -mercaptoethanol
- 2mM L-glutamine 100 ⁇ ⁇ / ⁇ 1 penicillin and 100U/ml streptomycin sulfate
- 37°C 5% CO 2 , 95% humidity incubator to cultivate.
- the suspension cells were removed, the medium was supplemented, and the cells were changed once every three days.
- the cells were 70 to 80% confluent, they were digested with 0.05% trypsin-0.01% EDTA.
- the 1-2 generation mesenchymal stem cells were stored in a liquid nitrogen tank for later use.
- AMSCs and bMSCs were seeded in 96-well plates at a density of 1 cell/well by limiting dilution method. After 3 weeks, approximately 24.55% ⁇ 0.66% of the wells were observed to grow monoclonal. These monoclonals were further amplified and used as seed cells. After 4-6h cells were completely adhered, the induction medium (20ng/ml activin A + 200ng/ml wnt3a + 20% FBS + HG-DMEM) was added for 1 day. The induction medium No. 2 (20 ng/ml activin ⁇ + 100 ⁇ RA + 20% FBS + HG-DMEM) was replaced and induction culture was continued for 4 days to obtain Flkl + MSC.
- the obtained FM + MSCs with epithelial morphology were detected by RT-PCR and immunofluorescence staining (Foxa2, Soxl7, Kdr, Tbx6, Eomes, Gsc, T, Soxl, Pax6) and Western Blot (Detection of Foxa2) , Soxl7, T, Gsc, Epcam, Vimatin), the results show that the obtained Flkl + MSC has There are three major germ layer differentiation potential important gene phenotype markers (restricted endoderm: Foxa2, Soxl7; mesendoderm: Gsc, T, Eomes; mesoderm: KDR, TBX6; ectoderm: Soxl, Pax6), and have a higher Induction efficiency (Foxa2, Soxl7 positive definitive endoderm cell efficiency reached more than 90%), the results are shown in Figure 8 to Figure 11.
- the obtained Flkl + MSC was divided into 6 equal portions and induced to differentiate into hepatic epithelial, neural, hematopoietic, adipogenic and osteogenic lineages, and another cell was further expanded for control of differentiation of each lineage (Fig. 1A).
- the cytoplasm of the adipogenic induction group was filled with fat droplets under light microscope, and the positive rate of oil red 0 staining was 80%.
- Real-time quantitative PCR showed high expression of the adipogenic marker genes AP2 and LPL (Fig. 1B).
- the positive rate of ALP and alizarin red staining in osteogenic induction group was 65%.
- the cells were cultured, fixed with 1% formaldehyde, and the protein and DNA were cross-linked at room temperature for 10 minutes.
- Positive control anti-R A Polymerase II a combination of all promoter regions that activate transcription.
- Control primer promoter region of the GAPDH gene.
- Each IP requires 900 ul Dilution Buffer plus 4.5 ul PI Cocktail.
- the negative control IgG is recommended to be consistent with the species from which the protein of interest is derived.
- the product can be placed in a large tube (EP tube capable of holding 1.1 ml of solution).
- Each chromatin lOOul required product contained approximately 2 X 10 6 cells derived.
- This step is a "preclear" chromatin that removes the protein that is non-specifically bound to Protein G Agarose and adds the appropriate amount of Protein G Agarose when combined.
- each sample takes 1% of chromatin as Input.
- Positive control tube force B l .Oug anti-R A polymerase antibody.
- Negative control tube plus l.Oug normal same species IgG.
- test tube is supplemented with ⁇ -lOug antibody.
- the amount of antibody added needs to be determined based on past experience.
- the amount of Bind Reagent A added is 5 times the volume of the sample.
- step 2 Remove the spin filter, discard the liquid in the collection tube, and retain the collection tube. If precipitation is seen in step 2, this step collects sediment at the bottom of the tube, but does not affect the experiment.
- control primers including IP for positive and negative control antibodies, and Input and DNA free tubes as control tubes with or without DNA contamination.
- control primers are directed against the specific human GAPDH gene. For other substances, it is recommended that the user design specific primers based on experience.
- Hot start Taq enzymes are recommended. If the hot start Taq enzyme is not used, it is recommended to add the Taq enzyme after the initial denaturation step.
- the methylation status of the Flkl + MSC whole-genome histone K4 and K27 sites obtained after calibration according to the Human Genome Database (Hgl8).
- stem cells with different differentiation potentials such as ESC (embryonic stem cells), Flkl + MSC, HSC (hematopoietic stem cells) and HPC (hematopoietic progenitor cells) as the research object [21, 22], and analyzed the sub-totalness in these stem cells.
- the genes related to neural differentiation in the literature mainly include 22 transcription factors such as Bm2, MytlL, Zicl, Neurog2, Hesl, Dlxl, Pax6, Tlx2, Msil, Gfral, Gfra3, Mapt, Nes and 01ig2 [23-25].
- ChlP-seq data analysis showed that 17 genes in ESCs showed H3K4me3 modification or bivalent modification status; the results in Flkl + MSC were similar to those in ESC; and the analysis showed that three related to the initiation of neural differentiation
- the genes Nes, Msil and Hesl, their histone modification status in ESC and Flkl + MSC are all H3K4me3 activation state; but in HSC and HPC, some of these genes show H3K27me3 inhibitory modification, and no other modification is detected.
- Signal Figure 2F).
- H3K27me3 inhibitory or no modification signals Other lineage-related genes in HPCs are H3K27me3 inhibitory or no modification signals, and hematopoietic differentiation related genes are H3K4me3 activation modification, and their activation signals are stronger than HSC; Hematopoietic lineage differentiation differentiation related factors Gatal in ESCs, Flkl + MSCs and HSCs It is shown to be inhibited or unmodified, but is modified for H3K4me3 activation in hematopoietic progenitor cells. It is speculated that it is activated and modified in the stage of pluripotent stem cells to hematopoietic differentiation to hematopoietic progenitor cells, in order to facilitate further directed differentiation of the hematopoietic lineage.
- HSC and HPC lineage-related genes The analysis of different histone modification status of HSC and HPC lineage-related genes and the different differentiation potential of these stem cells suggest that : As pluripotency levels decline, changes in histone methylation status change, stem cells gradually lose their differentiation pluripotency (ESC) into sub-totipotency (Flkl + MSCs) or only single germ layer (HSC) Even the ability of a single lineage to differentiate (progenitor cells) cells.
- the differentiation-related genomic protein H3K4me3 and H3K27me3 modification status are closely related to the differentiation potential of stem cells, and can be used as an epigenetic modification label for predicting the differentiation potential of stem cells.
- Example 3 Analysis of lineage differentiation-related genomic proteins H3K4me3 and H3K27me3 modification states can be used to predict stem cell differentiation potential
- the differentiation of aMSC and bMSC into osteogenic and adipogenic directions showed that the differentiation ratio of aMSC and bMSC was 50% and 65%, respectively, on the 8th day of osteogenic induction, and the expression of ALP and OPN were statistically significant. Differences; on day 8 of adipogenic induction, the aMSC and bMSC differentiation ratios were 80% and 27%, respectively, and the expression of the marker genes LPL and AP2 were significantly different (Fig. 5C).
- adipogenic and osteogenic related genes of MSCs from two different sources are H3K4me3 or bivalent modification
- the proportion of adipogenic related gene H3K4me3 in aMSC is significantly higher than that of bMSC, which is more difficult than bMSC than aMSC.
- the results of differentiation into the adipogenic lineage were consistent; the methylation-activated modification of osteogenesis-associated genomic protein in bMSC was not significantly different from that of aMSC, which was consistent with the observation that bMSC and aMSC differentiated into osteogenic lineages.
- genomic protein modification analysis and differentiation ability of different sources of MSC differentiation further confirmed the feasibility of analysis of the genomic protein H3K4me3 and H3K27me3 modification status of lineage differentiation as an epigenetic modified label for predicting the differentiation potential of stem cells.
- Example 4 Dynamic analysis of the genomic proteins associated with differentiation stages H3K4me3 and H3K27me3 can be used to predict the degree of cell differentiation.
- histone methylation analysis can be used as an epigenetic modification marker to predict the differentiation potential of stem cells, and the dynamic changes of histone methylation modification of Flkl + MSCs before and after differentiation were analyzed by using ChlP-PCR. It is shown that during the differentiation of Flkl + MSCs into the neural lineage, the histone modification status of key transcription factors Pax6 and Neurog2 is changed from H3K27me3 inhibitory state to bivalent modification, Neurod2 changes from H3K27me3 inhibitory state to activated state, and Gfra2 changes from bivalent state to In the activated state, Tlx2 and Msil dominated from the bivalent K27 modification to the K4 dominant state, and Gfral changed from the unmodified state to the bivalent state; the expression of Neurog2, Pax6, Tlx2, Neurod2 and Msil was significantly up-regulated.
- the peak then expresses the dynamic process of down-regulation, which ensures that the initiation of osteogenic differentiation has further maturation using osteoblast function; the key gene of osteogenesis RUNX2 maintains the activation state of H3K4me3, and the expression level continues to increase. It further promoted the expression of its downstream target gene OSX and osteogenic marker genes ALP and OPN.
- Flkl + MSCs are involved in adipogenic differentiation, osteogenic differentiation-related transcription factors RUNX2, TAZ, MSX2, Smad5
- the histone modification of BMPR2 changed from H3K4me3 activated state to bivalent modification, and MSX2 changed from bivalent modification to inhibitory state, and the expression of these genes was down-regulated.
- the differentiation status of differentiation-related genomic proteins H3K4me3 and H3K27me3 is closely related to the differentiation ability and differentiation stage of stem cells, and can be used as an epigenetic label for predicting the differentiation potential and cell differentiation stage and maturity of different levels of stem cells from different sources.
- This finding provides a new standard for clinically better screening and identification of seed cells required for the regenerative repair of various tissues and organs.
- This histone methylation tag is relatively easy to obtain: First, use The ChlP-seq technique acquires the genome-wide histone methylation modification profile of unknown differentiation potential stem cells, and then analyzes the histone H3K4me3 and B H3K27me3 modification status of some or some lineage differentiation-related genes according to the purpose of stem cell application.
- ChlP-seq results using the ChlP-PCR technique can predict whether the stem cells have the ability to differentiate into the lineage.
- the stem cells are located on a stem cell grade pyramid with apical embryonic stem cells as the apex.
- ChlP-seq technology can quickly and easily predict whether stem cells have the potential to differentiate into the lineage of interest under suitable external conditions or in vivo microenvironment after obtaining the genome-wide histone methylation modification profile.
- ChlP-seq or ChlP-PCR was used to analyze the histone methylation status of stem cells in a lineage differentiation process and related transcription factors and real-time quantitation of these genes. PCR can be used to identify the specific differentiation stage of stem cells. (Note: Changes in histone methylation are first and gene expression changes, when the histone methylation status of a gene becomes more activated.
- Xenopus embryos IL Control of the onset of transcription. Cell. 1982, 30: 687-696.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN1005MUN2013 IN2013MN01005A (zh) | 2010-12-09 | 2011-12-02 | |
US13/992,619 US9523074B2 (en) | 2010-12-09 | 2011-12-02 | Sub-totipotent stem cell product and apparent hereditary modifying label thereof |
EP11847131.7A EP2636732B1 (en) | 2010-12-09 | 2011-12-02 | Sub-totipotent stem cell product and apparent hereditary modifying label thereof |
CN201180057318.8A CN103459592B (zh) | 2010-12-09 | 2011-12-02 | 亚全能干细胞产品及其表观遗传修饰标签 |
CA2820395A CA2820395C (en) | 2010-12-09 | 2011-12-02 | Sub-totipotent stem cell product and epigenetic modification label thereof |
AU2011341213A AU2011341213B2 (en) | 2010-12-09 | 2011-12-02 | Sub-totipotent stem cell product and apparent hereditary modifying label thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNPCT/CN2010/079608 | 2010-12-09 | ||
PCT/CN2010/079608 WO2012075636A1 (zh) | 2010-12-09 | 2010-12-09 | 预测干细胞分化潜能的表观遗传修饰标签 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2012075912A1 WO2012075912A1 (zh) | 2012-06-14 |
WO2012075912A9 true WO2012075912A9 (zh) | 2013-09-06 |
Family
ID=46206539
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2010/079608 WO2012075636A1 (zh) | 2010-12-09 | 2010-12-09 | 预测干细胞分化潜能的表观遗传修饰标签 |
PCT/CN2011/083380 WO2012075912A1 (zh) | 2010-12-09 | 2011-12-02 | 亚全能干细胞产品及其表观遗传修饰标签 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2010/079608 WO2012075636A1 (zh) | 2010-12-09 | 2010-12-09 | 预测干细胞分化潜能的表观遗传修饰标签 |
Country Status (6)
Country | Link |
---|---|
US (1) | US9523074B2 (zh) |
EP (1) | EP2636732B1 (zh) |
AU (1) | AU2011341213B2 (zh) |
CA (1) | CA2820395C (zh) |
IN (1) | IN2013MN01005A (zh) |
WO (2) | WO2012075636A1 (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103667190A (zh) * | 2012-09-07 | 2014-03-26 | 上海吉凯基因化学技术有限公司 | 一种诱导形成神经元的方法和组合物 |
CN103865873B (zh) * | 2012-12-12 | 2017-04-05 | 中国医学科学院基础医学研究所 | 亚全能干细胞分泌的外来体及其应用 |
CN110499283A (zh) * | 2018-05-17 | 2019-11-26 | 西安组织工程与再生医学研究所 | Wnt信号通路激活剂在制备改善低碱性磷酸酯酶症干细胞成骨分化能力异常产品中的应用 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7056738B2 (en) * | 2001-03-23 | 2006-06-06 | Tulane University | Early stage multipotential stem cells in colonies of bone marrow stromal cells |
JP2004248505A (ja) * | 2001-09-21 | 2004-09-09 | Norio Nakatsuji | 移植抗原の一部または全てを欠除したes細胞由来の未分化な体細胞融合細胞およびその製造 |
CN1778905B (zh) * | 2004-11-22 | 2010-05-05 | 赵春华 | 一种脂肪间充质干细胞的分离培养方法及其应用 |
GB0601538D0 (en) * | 2006-01-26 | 2006-03-08 | Univ Birmingham | Epigenetic analysis |
WO2009030092A1 (en) * | 2007-09-05 | 2009-03-12 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof |
US8524498B2 (en) * | 2009-05-29 | 2013-09-03 | The General Hospital Corporation | Methods and compositions for homologous recombination in human cells |
-
2010
- 2010-12-09 WO PCT/CN2010/079608 patent/WO2012075636A1/zh active Application Filing
-
2011
- 2011-12-02 EP EP11847131.7A patent/EP2636732B1/en active Active
- 2011-12-02 WO PCT/CN2011/083380 patent/WO2012075912A1/zh active Application Filing
- 2011-12-02 IN IN1005MUN2013 patent/IN2013MN01005A/en unknown
- 2011-12-02 US US13/992,619 patent/US9523074B2/en active Active
- 2011-12-02 AU AU2011341213A patent/AU2011341213B2/en active Active
- 2011-12-02 CA CA2820395A patent/CA2820395C/en active Active
Also Published As
Publication number | Publication date |
---|---|
AU2011341213B2 (en) | 2016-08-18 |
EP2636732B1 (en) | 2018-05-30 |
IN2013MN01005A (zh) | 2015-09-25 |
US20140287930A1 (en) | 2014-09-25 |
EP2636732A4 (en) | 2014-05-07 |
WO2012075912A1 (zh) | 2012-06-14 |
AU2011341213A1 (en) | 2013-07-11 |
US9523074B2 (en) | 2016-12-20 |
EP2636732A1 (en) | 2013-09-11 |
CA2820395C (en) | 2017-03-07 |
WO2012075636A1 (zh) | 2012-06-14 |
CA2820395A1 (en) | 2012-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Shin et al. | Molecular signature of adult bone marrow-purified very small embryonic-like stem cells supports their developmental epiblast/germ line origin | |
JP6494515B2 (ja) | 幹細胞を1又は2以上の細胞系列に分化させる方法 | |
De Kock et al. | Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential | |
Marofi et al. | Gene expression of TWIST1 and ZBTB16 is regulated by methylation modifications during the osteoblastic differentiation of mesenchymal stem cells | |
Pisal et al. | Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth | |
Kouidhi et al. | Characterization of human knee and chin adipose‐derived stromal cells | |
US20230159887A1 (en) | Methods for Generating Thymic Cells in Vitro | |
Gao et al. | Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells | |
Scalise et al. | In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes | |
CN103459592B (zh) | 亚全能干细胞产品及其表观遗传修饰标签 | |
WO2012075912A9 (zh) | 亚全能干细胞产品及其表观遗传修饰标签 | |
Han et al. | Global transcriptome profiling of genes that are differentially regulated during differentiation of mouse embryonic neural stem cells into astrocytes | |
US20130309209A1 (en) | Formation of hematopoietic progenitor cells from mesenchymal stem cells | |
Denis et al. | Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways | |
Liedtke et al. | Neonatal mesenchymal-like cells adapt to surrounding cells | |
WO2018037091A1 (en) | Methods for the identification and isolation of hematopoietic stem and progenitor cells | |
Xiao et al. | Tuning FOXD3 expression dose-dependently balances human embryonic stem cells between pluripotency and meso-endoderm fates | |
Liu et al. | Nodal mutant e X traembryonic EN doderm (XEN) stem cells upregulate markers for the anterior visceral endoderm and impact the timing of cardiac differentiation in mouse embryoid bodies | |
Zhao et al. | Porcine skin-derived progenitor (SKP) spheres and neurospheres: distinct “stemness” identified by microarray analysis | |
Li et al. | Astrocyte‐like cells differentiated from a novel population of CD45‐positive cells in adult human peripheral blood | |
Kiyokawa et al. | Airway tissue stem cells reutilize the embryonic proliferation regulator, Tgfß-Id2 axis, for tissue regeneration | |
EP1961810A1 (en) | Method for obtaining intestinal stem-precursor cell | |
Lee et al. | The Transcription Factor CP2-like 1 Is Expressed in Very Small Embryonic-like Stem Cells and Other Adult Stem Cells: Implications for Cancer Stem Cells | |
Magro-Lopez et al. | Optimizing Nodal, Wnt and BMP signaling pathways for robust and efficient differentiation of human induced pluripotent stem cells to intermediate mesoderm cells | |
Creamer | The role of CDX4 during patterning of definitive hemogenic mesoderm |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11847131 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2820395 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011847131 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2011341213 Country of ref document: AU Date of ref document: 20111202 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13992619 Country of ref document: US |