WO2017219232A1 - Méthode de reprogrammation cellulaire basée sur un système tridimensionnel - Google Patents

Méthode de reprogrammation cellulaire basée sur un système tridimensionnel Download PDF

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WO2017219232A1
WO2017219232A1 PCT/CN2016/086536 CN2016086536W WO2017219232A1 WO 2017219232 A1 WO2017219232 A1 WO 2017219232A1 CN 2016086536 W CN2016086536 W CN 2016086536W WO 2017219232 A1 WO2017219232 A1 WO 2017219232A1
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scaffold
dimensional
polysaccharide
solution
calcium phosphate
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PCT/CN2016/086536
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English (en)
Chinese (zh)
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徐希明
邓纹纹
余青桐
余江南
曹霞
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江苏大学
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Priority to PCT/CN2016/086536 priority Critical patent/WO2017219232A1/fr
Priority to CN201680086898.6A priority patent/CN110312788A/zh
Publication of WO2017219232A1 publication Critical patent/WO2017219232A1/fr

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  • the invention relates to the field of biotechnology and tissue engineering, relates to a gene delivery system, and in particular to the establishment of a cell reprogramming method based on a three-dimensional system.
  • Biomimetic Collagen nanofibrous materials for bone tissue engineering [J].Advanced Engineering Materials,2010,12(9):B451-B466;Han,J.,Chen,L.,Luo,G.,Dai,B.,Wang,X. , Dai, J. Three-dimensional culture may promote cell reprogramming [J]. Organogenesis, 2013, 9(2): 118-120.].
  • the extracellular matrix typically includes collagen and laminin interlaced with the heparan sulfate proteoglycan.
  • laminin and heparan sulfate proteoglycans are distributed on the cell surface at the stage of two cells; while expression of type IV collagen and fibronectin can be detected in the cell cluster stage of blastocysts.
  • ECMs have multiple functions for cell growth, differentiation, and maintenance of tissue morphology. Cells interact with ECM via surface receptors (such as integrins), acting as a mechanism for mechanical riveting and cell membrane signaling.
  • surface receptors such as integrins
  • the invention establishes a novel and efficient non-viral three-dimensional gene delivery system, and is first applied to the induction of iPSCs in a three-dimensional environment.
  • the invention relates to a cationized Pleurotus eryngii polysaccharide, a calcium phosphate nanoparticle and a three-dimensional collagen scaffold, and the cationized Pleurotus eryngii polysaccharide modified calcium phosphate nanoparticle by a reverse microemulsion method, further improving the gene carrying of the non-viral vector Capacity; the polysaccharide-calcium phosphate-loaded gene nanoparticles were fused with a three-dimensional collagen scaffold to form a three-dimensional induction system of iPSCs.
  • the invention applies the three-dimensional system to the reprogramming research of human umbilical cord stem cells, evaluates the cell transfection efficiency and the cell reprogramming effect, and sets the two-dimensional gene delivery system under the same conditions as a positive control, and the experimental results show that the three-dimensional system indicates the three-dimensional system.
  • the gene delivery system is significantly superior to the two-dimensional gene delivery system in gene transfection efficiency; in addition, human-derived iPSCs induced by three-dimensional system appear iPSCs cell spheres on the 4th day after cell inoculation, and then gradually form embryoid body cells. ball.
  • HE staining, laser confocal microscopy, immunohistochemistry, karyotype analysis, and intratumoral tumors were identified, indicating that human iPSCs induced by three-dimensional system have the potential of whole germ layer differentiation similar to embryonic stem cells.
  • the present invention employs a chemical modification method to provide a highly efficient and non-toxic three-dimensional gene delivery system for reprogramming human umbilical cord stem cells (HUMSCs) into human induced pluripotent stem cells (hiPSCs).
  • HUMSCs human umbilical cord stem cells
  • hiPSCs human induced pluripotent stem cells
  • a cell reprogramming method based on a three-dimensional system specifically comprising the following steps:
  • Step 4 Expression of the Yamanaka factor in a three-dimensional system:
  • Step 5 Induction and amplification of iPSCs in a three-dimensional system:
  • Collagen scaffolds prepared from natural I collagen provide a safe and stable three-dimensional culture environment for HUMSCs.
  • Polysaccharide-calcium phosphate-loaded gene nanoparticles have good gene carrying capacity and are evenly distributed in three-dimensional scaffolds for HUMSCs to iPSCs.
  • Targeted reprogramming provides ample source of foreign genes and is capable of long-term release.
  • the expression of exogenous genes in the three-dimensional system was significantly higher than that of the two-dimensional system by RT-PCR.
  • the results of iPSCs showed that the human iPSCs formed in the three-dimensional system have the ability to differentiate into the whole germ layer of embryonic stem cells. Stable amplification in vitro, passage to more than 20 generations, and can maintain a normal karyotype. This method provides a new three-dimensional environment for the induction of iPSCs, and provides a theoretical basis for the application of iPSCs from cell level to tissue level.
  • Figure 1 is a flow diagram of cell reprogramming in a three dimensional system.
  • Figure 2 is a scanning electron micrograph of a three-dimensional gene-loaded nanoparticle-collagen scaffold.
  • Figure 3 shows the morphological changes of HUMSCs in a three-dimensional gene-loaded nanoparticle-collagen scaffold under an optical microscope.
  • Figure 4 is a three-dimensional gene-loaded nanoparticle-collagen scaffold and HE staining of iPSCs therein.
  • Figure 5 shows the immunohistochemistry of iPSCs.
  • Figure 6 is a diagram of cell karyotype
  • Figure 7 shows the pluripotency of iPSCs by immunofluorescence staining.
  • Figure 8 shows the degree of methylation of the Oct4 and Nanog promoters, wherein hESCs are human embryonic stem cells.
  • Figure 9 shows the differentiation of the three germ layers of iPSCs cell spheres in vivo, where: g represents the stroma structure, which is the characteristic of endoderm, b and c represent the bone tissue and cartilage tissue, respectively, which is the characteristic of mesoderm differentiation, and n represents the neural tube. Germ marking.
  • cationic polysaccharide D-Sp LB medium (homemade); type I-collagen, type IV collagenase, agarose, ethidium bromide, L-glutamine, MINI26-1KT, biotinylated IgG (American sigma); trypsin, Tris base, ampicillin, streptomycin, DAB display kit (Biyuntian Biotechnology Research Institute); basic fibroblast growth factor (bFGF) (PeproTech, USA); plasmid extraction kit (Promega, USA); calcium chloride (National Pharmaceutical Group Chemical Reagent Co., Ltd.); MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide), PicoGreen dsDNA quantification Kit (Invitrogen, USA); basal medium DMEM, basal medium DMEM/F12, knockout DME, fetal bovine serum (FBS), knockout serum replacement (GIBCO, USA); Oligo
  • DH5 ⁇ glycerol bacteria Najing Jitian Biological
  • human umbilical cord mesenchymal stem cells Jiangsu Beike Biotechnology Co., Ltd.
  • mouse embryonic fibroblasts Shanghai Chinese Academy of Sciences stem cell bank
  • PEI Fat Diabetes/Severe Combined Immunodeficiency Mice (Center for Comparative Medicine, Yangzhou University)
  • plasmid non-viral plasmid OCT4, SOX2, KLF4 and CMYC (Guangzhou Fueneng Gene Co., Ltd.)
  • OCT4 upstream primer TGGAGGTGAT GGGTTAGG 18
  • OCT4 downstream primer CATCAAACTA CCCTATCATA ACC 23
  • NANOG upstream primer GTGAATGAAA GAGGAAAATG GAG 23
  • NANOG downstream primer AATAACCCAC CCCTATAATC C 21
  • PBS solution take NaCl 8.00g, KCl 0.20g, Na 2 HPO 4 .12H2O 3.5g, KH 2 PO 4 0.2g, add 800mL double distilled water, adjust the pH to 7.4, dilute to 1000mL, dispense, Autoclaved, stored at 4 ° C.
  • Knockout DMEM medium was sequentially added with 20% serum substitute (Knockout SR), 2 mmol/L L-glutamine, 0.1 mmol/L ⁇ -mercaptoethanol, 1% non-essential amino acid, 100 U/ mL qing-streptomycin solution, 4 ng / mL human recombinant basic fibroblast growth factor, mix, placed in a refrigerator at 4 ° C for use.
  • Kerckout DMEM medium was sequentially added with 20% serum substitute (Knockout SR), 2 mmol/L L-glutamine, 0.1 mmol/L ⁇ -mercaptoethanol, 1% non-essential amino acid, 100 U/ mL qing-streptomycin solution, 4 ng / mL human recombinant basic fibroblast growth factor, mix, placed in a refrigerator at 4 ° C for use.
  • Example 1 Preparation of a three-dimensional collagen scaffold:
  • Igepal CO-520 was dissolved in cyclohexane to prepare an Igepal CO-520 29% Igepal CO-520/cyclohexane mixture;
  • microemulsion A Take a clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then 650 ⁇ L 0.01M calcium chloride solution and 0.8mg cationic polysaccharide D-Sp, magnetically stirred Under the conditions, together with the addition to the net conical flask, continue to stir for 2min, forming microemulsion A;
  • microemulsion B Take another clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then add 650 ⁇ L of 0.06M disodium hydrogen phosphate and plasmid mixed solution 10 ⁇ L (including Oct4, Sox2) Klf4, c-Myc each 2.5 ⁇ g) together into the net conical flask, continue to stir for 2min, forming microemulsion B;
  • microemulsion A was added dropwise to the microemulsion B while stirring, until the whole system was transparent and clear, and the polysaccharide-calcium phosphate hybrid nanoparticle microemulsion was obtained;
  • each well containing the inoculated cell scaffold was added DMEM medium containing 10% FBS, and continued to culture for 4 h;
  • paraffin wax After the paraffin wax is completely immersed in the stent, it is embedded: first prepare the container (such as folding a small carton), pour the melted paraffin, quickly pick up the saturated paraffin tissue block, and cool it into a block. Yes;
  • the embedded wax block is fixed on a microtome and cut into thin slices, generally 5 to 8 ⁇ m thick.
  • the cut slices tend to wrinkle, and should be placed in heated water to be flattened, then attached to a glass slide, and dried in a 45 ° C incubator.
  • Sections were subjected to conventional dewaxing and hydration, and a part of the sections were used for alkaline phosphatase (AP) staining;
  • AP alkaline phosphatase
  • colchicine treatment add 24 ⁇ L to the cell culture flask 3 h before terminating the cell culture Colchicine, the final concentration of 0.8 ⁇ g / mL, and then returned to the incubator for 72h;
  • hypotonic treatment add 1mL 0.075mol / L KCl hypotonic solution to the centrifuge tube, gently pipet with a straw, so that the cells are evenly suspended in the hypotonic solution, add 6mL 0.075mol / L KCl hypotonic solution, Placed in a constant temperature water bath at 37 ° C, allowed to stand for 20 min, so that the cells swell and chromosomes dispersed;
  • Dropping tablets Add about 1 mL of the appropriate amount of fixative solution to the sediment, gently pipe the suspension cells with a pipette, and pipette the cell suspension. Drip 2 to 3 drops of water at a certain height and pre-soak clean. On the slide, immediately blow it away in one direction with the mouth, dry it several times on the alcohol, dry it, and mark each piece;
  • Dyeing Place the carrier piece with the cell suspension in the dyeing tank, so that there is a certain gap between the piece and the piece. Dip the diluted Giemsa dyeing solution into the dyeing tank, dye it for 20 minutes, take it out and rinse it with tap water. dry;
  • Genomic DNA extraction and quality inspection genomic DNA extraction and OD were performed using a special gDNA extraction kit, three cell samples (human umbilical cord mesenchymal stem cells, iPSCs induced by the experiment, and human embryonic stem cell line HN4). Concentration and agarose gel electrophoresis were determined.
  • Methylation treatment of qualified DNA 500 ng to 2 ⁇ g of gDNA was subjected to methylation treatment using a specialized methylation kit in strict accordance with the instructions to obtain 10 ⁇ L of methylated DNA.
  • Specific methylation procedure Add 20 ⁇ L of gDNA (500 ng to 2 ⁇ g) to 130 ⁇ L of CT solution (a reagent in the kit), mix gently, and operate according to the following procedure: 98 ° C, 10 min; 64 ° C, 2.5 h; 4 ° C, hold.
  • PCR amplification and recovery and purification PCR amplification was carried out using methylated DNA as a template.
  • the first round of PCR system (total volume 25 ⁇ L):
  • the first round of PCR amplification procedures 95 ° C for 3 min; 95 ° C for 30 sec, 53 ° C for 30 sec, 72 ° C for 30 sec, for 40 cycles; followed by 72 ° C for 5 min, the end.
  • the second round of PCR amplification procedures 95 ° C for 3 min; 95 ° C for 30 sec, 53 ° C for 30 sec, 72 ° C for 30 sec, 40 cycles; followed by 72 ° C for 5 min, the end.
  • the PCR product was recovered and purified: the second round of the PCR product was subjected to gel recovery and purification, and the concentration was determined by OD.
  • TA cloning The PCR purified product was cloned into T vector by T vector kit, transformed with DH5 ⁇ competent cells, plated, and cultured at 37 ° C overnight (see the T vector kit for specific procedures)
  • Sequencing analysis of the sequencing results First, the sequence alignment software was used for sequencing analysis, and then methylation analysis software was used to analyze the methylation analysis of the correct sequencing results, and the information of the methylation number of the CpG island was counted. As can be seen from Fig. 8, demethylation was evident in the OCT4 and NANOG promoter regions of iPSCs, and the degree of methylation was similar to that of the positive control human embryonic stem cells, but significantly different from the original human umbilical cord mesenchymal stem cells. This result further demonstrates the successful reprogramming of human umbilical cord mesenchymal stem cells in a three-dimensional gene-loaded nanoparticle-collagen scaffold.
  • iPSCs were digested and centrifuged, a cell suspension of 5 ⁇ 10 6 /mL was prepared and inoculated into the abdomen of a 5-week-old male NOD-SCID nude mouse, and then tumor growth was observed. After 5 to 8 weeks, the mice were sacrificed and the tumors were removed for HE staining analysis.
  • iPSCs cell spheres can successfully form teratomas in immunodeficient NOD-SCID nude mice, and teratomas show significant trigeminal differentiation, respectively, with endoderm glandular tissue, mesoderm Tissues such as bone and cartilage tissues and neural tubes of the ectoderm are representative. This result fully demonstrates that the three-dimensional gene-loaded nanoparticle-collagen scaffold can be successfully applied to cell reprogramming.

Abstract

La présente invention concerne une méthode de reprogrammation cellulaire basée sur un système tridimensionnel. Un polysaccharide cationique de type D de Pleurotus eryngii, du phosphate de calcium et quatre plasmides codant pour les facteurs de Yamanaka sont utilisés comme matériaux de base. Des nanoparticules hybrides de polysaccharide et de phosphate de calcium sont préparées au moyen de la méthode de micro-émulsion inverse et sont adsorbées sur la surface interne d'une matrice de collagène vide afin d'obtenir une matrice tridimensionnelle de collagène et de nanoparticules comprenant les gènes. Des cellules souches mésenchymateuses de cordon ombilical humain sont ajoutées à la matrice tridimensionnelle de collagène et de nanoparticules comprenant les gènes pour être reprogrammées, afin de former des sphères de cellules souches pluripotentes induites.
PCT/CN2016/086536 2016-06-21 2016-06-21 Méthode de reprogrammation cellulaire basée sur un système tridimensionnel WO2017219232A1 (fr)

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CN201680086898.6A CN110312788A (zh) 2016-06-21 2016-06-21 一种基于三维体系的细胞重编程方法

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154352A (zh) * 2010-12-30 2011-08-17 江苏大学 阳离子化多糖纳米粒基因传递系统及其制法
CN103097517A (zh) * 2010-06-11 2013-05-08 塞拉帝思股份公司 用于提高多能干细胞分化成肝细胞的3维支架
WO2015069469A1 (fr) * 2013-11-05 2015-05-14 Clontech Laboratories, Inc. Compositions de transfection sèches et procédés de fabrication et d'utilisation de celles-ci

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101829367B (zh) * 2009-12-30 2013-05-08 江苏大学 一种基因传递系统的三维纳米支架及其制备与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103097517A (zh) * 2010-06-11 2013-05-08 塞拉帝思股份公司 用于提高多能干细胞分化成肝细胞的3维支架
CN102154352A (zh) * 2010-12-30 2011-08-17 江苏大学 阳离子化多糖纳米粒基因传递系统及其制法
WO2015069469A1 (fr) * 2013-11-05 2015-05-14 Clontech Laboratories, Inc. Compositions de transfection sèches et procédés de fabrication et d'utilisation de celles-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAO, XIA ET AL.: "Incorporating pTGF- 0 1/calcium phosphate nanoparticles with fibronectin into 3-dimensional collagen/chitosan scaffolds: efficient, sustained gene delivery to stem cells for chondrogenic differentiation", EUROPEAN CELLS AND MATERIALS, vol. 23, 31 December 2012 (2012-12-31), pages 81 - 93, XP055448987, ISSN: 1473-2262 *
CAO, XIA ET AL.: "Non-viral co-delivery of the four yamanaka factors for generation of human induced pluripotent stem cells via calcium phosphate nanocomposite particles", ADVANCED FUNCTIONAL MATERIALS, vol. 23, no. 43, 20 November 2013 (2013-11-20), pages 5403 - 5411, XP055448989, ISSN: 1616-301X *
PARK HYUN-JI ET AL.: "Nonviral delivery for reprogramming to pluripotency and differentiation", ARCH. PHARM. RES., vol. 37, no. 1, 13 November 2013 (2013-11-13), pages 107 - 119, XP035312379, ISSN: 1976-3786 *

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