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Use of Activin A in human embryo stem cell trophoblast-free cell culture

A technology of human embryonic stem cells and trophoblast cells, applied in the field of biomedicine, can solve the problem that the in vitro culture conditions of human embryonic stem cells are not optimal, etc.

Active Publication Date: 2007-08-01
浙江煦顼技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conclusion, the in vitro culture conditions of human embryonic stem cells are not yet optimal

Method used

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  • Use of Activin A in human embryo stem cell trophoblast-free cell culture
  • Use of Activin A in human embryo stem cell trophoblast-free cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 , Culture of human embryonic stem cells

[0029] 1.1. Culture of human embryonic stem cells

[0030]The Non-CM formulation used in this example is as follows: 80% by weight of DMEM / F12 culture fluid, 20% by weight of serum substitute, 1mML-glutamic acid, 0.1mM β-mercaptoethanol, 1% by weight of non-CM Essential amino acids (Invitrogen, catalog number: 11140050).

[0031] The culture method of H1 human embryonic stem cells is as follows: the culture bottle is treated with matrigel glue, and a sterile medium is filled. Inoculate H1 human embryonic stem cells in culture medium, place them in a carbon dioxide incubator, and store them at 37°C in 5% volume ratio of CO 2 Cultured for 6 days.

[0032] The experiments were divided into 4 groups:

[0033] Group 1: CM culture, Non-CM culture, and FST culture with concentrations of 1ng / ml, 10ng / ml and 100ng / ml in CM;

[0034] Group 2: CM culture, Non-CM culture, and CM containing 300ng / ml FST added Activin A at a c...

Embodiment 2

[0047] Example 2 , quantitative RT-PCR analysis

[0048] According to the method of Noaksson, K. et al. (Noaksson, K., et al. Stem Cells. 2005, 23, 1460-7), RNA isolation and quantitative RT-PCR analysis were performed on the cells cultured for 6 days in Example 1.

[0049] Total RNA was prepared by RNAeasy kit (Qiagen) and used as a template for RT-PCR. Real-time quantitative PCR was performed on MyiQ real-time quantitative PCR detection system (Bio-Rad) using SYBR GreenI-based PCR Master Mix (Bio-Rad).

[0050] PCR primers are:

[0051] Upstream of Oct4: 5'-GAGAAGGATGTGGTCCGAGTGTG-3';

[0052] Downstream: 5'-CAGAGGAAAGGACACTGGTCCC-3';

[0053] Nanog upstream: 5'-TGAACCTCAGCTACAAACAGGTG-3';

[0054] Downstream: 5'-AACTGCATGCAGGACTGCAGAG-3'.

[0055] The PCR conditions are all: 94° C. for 30 seconds, 66° C. for 30 seconds, and 72° C. for 30 seconds for 35 cycles.

[0056] Each experiment was repeated at least 3 times. The experimental value for each ge...

Embodiment 3

[0058] Example 3 , the formation of teratoma

[0059] According to the method of step 1.1, culture H1 human embryonic stem cells with Non-CM containing 5ng / ml Activin A, passage once a week, treat with 1mg / ml Dispase protease at 37°C for 30 minutes during passage, and transfer to a new culture flask.

[0060] After 10 generations of culture, approximately 5×10 6 Cells were injected intramuscularly into NOD / SCID mice (Taconic). After 4-6 weeks, the tumor was removed, stained with hematoxylin-eosin and immunohistochemical staining according to the method in the literature (Fan G., et al.FEBS Lett.2000, 467(1):7-11) , and the result is shown in Figure 3.

[0061] The staining results in Figure 3 showed that these teratomas contained tissues derived from ectoderm, mesoderm, and endoderm. Among them, A is nerve tissue, B is bone tissue, C is liver tissue, A, B, and C are stained with HE; D is anti-pan-keratin antibody staining, showing the existence of ectodermal tissue epider...

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Abstract

The invention discloses the application of Activin A for cell culture in human blast stem cell panoistic layer, belonging to biomedical technology field. It is necessary and adequate for Activin A to maintain self- refresh and multi-function for human blast stem cell, it can induce expression of transcription factor Oct4 and Nanog. The stem cell still possess differentiation potence in mouse body for teratoma after 10- generation culture with panoistic cell culture medium containing 5 ng / ml Activin A, and it still maintains normal karyotype after 150- day culture and more than 20- generation.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular, the invention relates to the application of transforming growth factor Activin A for culture of human embryonic stem cells without trophoblast. Background technique [0002] Human embryonic stem cells are cells obtained from the inner cell mass of early human embryos under in vitro culture conditions and have the potential to develop into any type of cell in the body and have unlimited proliferation potential (Thomson JA., Itskovitz-Eldor J., Shapiro SS., et al. Science. Nov 6 1998;282(5391):1145-1147). Human embryonic stem cells have the ability to expand infinitely under specific culture conditions in vitro, maintain a normal karyotype and differentiate into any type of cell in the body, which provides an unlimited source of cells for the treatment of human diseases and damaged tissues. These diseases that may be treated with human embryonic stem cells include: diabetes, Alz...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 肖磊
Owner 浙江煦顼技术有限公司
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