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Application of growth factor PDGF-C in maintaining pluripotency of stem cells and culture method and application for promoting pluripotency of stem cells

A PDGF-C, PDGF-CC technology, applied in germ cells, animal cells, embryonic cells, etc., can solve the problems that restrict the development of assisted reproduction technology of cloned animals to produce embryonic stem cells

Pending Publication Date: 2022-07-29
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many studies have found that in vitro culture conditions have an important impact on embryonic development, including a variety of growth factors, amino acid components, etc., but the important core components and regulatory mechanisms are still not fully understood, which seriously restricts the development of cloned animals. Production, acquisition of embryonic stem cells, and development of assisted reproductive technologies

Method used

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  • Application of growth factor PDGF-C in maintaining pluripotency of stem cells and culture method and application for promoting pluripotency of stem cells
  • Application of growth factor PDGF-C in maintaining pluripotency of stem cells and culture method and application for promoting pluripotency of stem cells
  • Application of growth factor PDGF-C in maintaining pluripotency of stem cells and culture method and application for promoting pluripotency of stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Required experimental materials

[0048] 1.1 Cells

[0049] Since mouse ESCs share many properties with human ESCs, such as Nanog, Oct4 and Sox2 are core pluripotency transcription factors 8-10 that maintain the stemness of mouse / human ESCs, they are often used as the primary research tools for embryonic stem cells and object. Mouse embryonic stem cell line E14 was provided by Baylor College of Medicine; mouse embryonic fibroblasts (MEF) were purchased from Guangzhou Ximei (Zhaomei) Biotechnology Co., Ltd.; A172-loxP mouse embryonic stem cell line was provided by Dr. Courtesy of Thomas P. Zwaka.

[0050] 1.2 Plasmids

[0051] pLoxP-3Flag-EGFP: kindly provided by Dr. Thomas P. Zwaka of Baylor College of Medicine.

[0052] pSalk-Cre: a gift from Dr. Thomas P. Zwaka, Baylor College of Medicine, USA.

[0053] 1.3 Antibodies and proteins

[0054] The antibodies and proteins used in this example are shown in Table 1.

[0055] Table 1 Antibody and protein suppliers a...

Embodiment 2

[0201] 1. Using PDGF-C to culture mouse embryonic stem cells

[0202] (1) Mouse embryonic stem cell E14 (the mouse embryonic stem cell used in the experiment was provided by Baylor College of Medicine, USA); PDGF-CC protein (purchased from Peprotech Company, Item No. 100-00cc-100).

[0203] (2) Recovery and culture of mouse embryonic stem cells

[0204] 1) Prepare mouse embryonic stem cell complete medium: Knockout DMEM 500mL+15%FBS+0.1mM MEM-NEAA+1%P / S+2mM L-Glutamine+0.1mM 2-Me+1000U / mL LIF, store at 4℃ .

[0205] 2) Preparation of 0.1% gelatin: Dissolve 0.1 g gelatin powder in 100 mL ddH 2 O, mix evenly, sterilize at high temperature, and store at room temperature.

[0206] 3) Prepare 10×PBS: potassium dihydrogen phosphate (KH 2 PO 4 ) 2.7g+ disodium hydrogen phosphate (Na 2 HPO 4 ) 14.2g + sodium chloride (NaCl) 80g + potassium chloride (KCl) 2.0g, add double distilled water to make up to 1L, mix well, and store at room temperature after high temperature sterilizati...

Embodiment 3

[0226] The culture method for protecting the early embryonic development of mice using PDGF-C includes the following steps:

[0227] 1) C57BL / 6 mice (6-8 weeks old) were purchased from JiCui Yaokang Company;

[0228] 2) Both HCG and PMSG were purchased from Calbiochem Company. Preparation: PMSG and hCG powder are prepared into 1IU / ul stock solution with pure water according to the instructions, and stored at -20 degrees after sub-packaging (30, 60 or 90ul volume sub-packaging). Before use, thaw at room temperature for about 5 minutes, and dilute 20-fold with 0.9% NaCl.

[0229] 3) Culture medium: culture medium for flushing embryos: M2 medium (Sigma-Aldrich, M7167) KSOM; basic medium for in vitro culture of embryos: KSOM medium (Sigma-Aldrich, MR-121-D). PDGF-CC protein (purchased from Peprotech Company, item number 100-00cc-100). Hyaluronidase (Sigma-Aldrich, H-4272)

[0230] 4) Make a glass Pasteur pipette for taking embryos: bake with an alcohol lamp outside the flame, ...

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Abstract

The invention provides a culture method for promoting pluripotency of stem cells by using PDGF-C. The culture method comprises the following steps: (1) directly adding PDGF-CC recombinant protein into a stem cell culture medium; 2) a plasmid or a virus for expressing PDGF-C; and 3) adding doxycycline into the stem cells of the induced overexpression PDGF-C to induce the expression of the PDGF-C. The PDGF-C can be combined with a PDGFR alpha receptor to activate a PDGFR pathway and promote the expression of pluripotent factors in stem cells, so that the effect of maintaining the pluripotency of the stem cells is achieved.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to the application of the growth factor PDGF-C in maintaining the pluripotency of stem cells and the culturing method and application of promoting the pluripotency of stem cells. Background technique [0002] Stem cells (SC) are a kind of undifferentiated cells with multi-directional differentiation potential and self-replication ability, which can differentiate into a specific tissue in vivo. Due to these two characteristics of stem cells, they have important value in clinical application, especially in the field of regenerative medicine, and become an important material for regeneration and repair of various tissues and organs. According to the classification of genetic origin, stem cells can be divided into embryonic stem cells (ESCs) and adult stem cells (ASCs)1. Both are hot areas of current stem cell research, but ASCs lack the potential to differentiate into...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2501/135C12N2501/235
Inventor 李旭日卢蔚斯
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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