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Method for in vitro abduction and cultivation of multi-potentiality stem cell

A technology of pluripotent stem cells and feeder cells, applied in the field of biomedicine, can solve the problems of biosafety, infection, complex and changeable genome of pluripotent stem cells, etc., and achieve the effect of good biosafety

Inactive Publication Date: 2009-04-08
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the pluripotent stem cells obtained in the above studies all have the following defects: ① The process of inducing and culturing human pluripotent stem cells needs to be co-cultured with mouse embryonic fibroblasts (MEFs), which may cause pathogens from mice to infect humans iPS cells, which limit their clinical application
② Although the gene transfection method using virus as a carrier has high efficiency, there are biological safety problems
However, this method still has the above two defects. In addition, the introduction of six foreign genes makes the genome of induced pluripotent stem cells complex and changeable.

Method used

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  • Method for in vitro abduction and cultivation of multi-potentiality stem cell
  • Method for in vitro abduction and cultivation of multi-potentiality stem cell
  • Method for in vitro abduction and cultivation of multi-potentiality stem cell

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Experimental program
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Embodiment 1

[0028] According to the prior art, total RNA is extracted from human cell samples, reverse-transcribed into cDNA, and Oct3 / 4 and Nanog genes are amplified by PCR technology. The purified Oct3 / 4 was connected to the Nanog gene and the plasmid vector, and transformed into Escherichia coli for amplification.

[0029] Human skin fibroblasts were isolated and cultured. Under the condition of feeder-free cells, the purified plasmids containing 10 μg Oct3 / 4 and 10 μg Nanog transcription factor genes were transfected into human with transfection reagent lipofectamine2000 (Invitrogen Company). Adult cells were cultured in a cell induction medium, which was based on DMEM and added human basic fibroblast growth factor (fibroblast growth factor-2, bFGF) with a final concentration of 10 μg / L, Activin A (Activin A) with a final concentration of 10 μg / L, transforming growth factor β (transforming growth factor β, TGFβ) with a final concentration of 10 μg / L, epidermal growth factor (epidermal...

Embodiment 2

[0036] Human adult cells were isolated and cultured. Under the condition of no heterogeneous feeder layer cells (no feeder layer or using autologous cells consistent with the source of adult cells as feeder layer cells), the commercially available, containing 20 μg Oct3 / 4 and 20 μg Nanog A transcription factor protein was transferred into human adult cells and cultured in induction medium. The induction culture medium was based on DMEM, added with a final concentration of 40 μg / L human basic fibroblast growth factor (fibroblast growth factor-2, bFGF), 40 μg / L Activin A (Activin A), 35 μg / L L transforming growth factor β (transforming growth factor β, TGFβ), 35 μg / L epidermal growth factor (epidermal growth factor, EGF) and other cytokines and DNA methyltransferase inhibitors such as 20nmol / L trichostatin A , TSA) (the above are all final concentrations).

[0037] After the cells are cultured in the induction medium for a period of time, they are replaced with embryonic stem c...

Embodiment 3

[0042] Human (or monkey) adult cells are isolated and cultured, and Oct3 / 4 and Nanog transcription factor genes or proteins are transfected into human (or monkey) adult cells. Induce pluripotent stem cells under feeder-free conditions (or use autologous cells consistent with the source of human adult cells as feeder cells). The cell induction culture medium is based on DMEM, and the final concentration is 6 μg / L alkaline Fibroblast growth factor (fibroblast growth factor-2, bFGF), 6 μg / L activin A (ActivinA), 6 μg / L transforming growth factor β (transforming growth factor β, TGFβ), 6 μg / L epidermal growth factor (epidermal growth factor , EGF) and other cytokines and DNA methyltransferase inhibitors such as: 0.5mmol / L valproic acid (valproic acid, VPA) or 10nmol / L trichostatin (trichostatin A, TSA), the above are the final concentration.

[0043] On day 3-5 after transfection, the cells were replaced with embryonic stem cell culture medium to continue culturing. The medium w...

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Abstract

The invention discloses a method for inducing and culturing in vitro multi-potential stem cells, comprising the following steps: two transcription factors, i.e., Oct3 / 4 and Nanog, are transferred into audult cells, cultivated in cell induction culture solution under the condition of non-heterologous feeder cells, to induce and clone multi-potential stem cells; and the induced multi-potential stem cells are cloned in embryo stem cell culture solution, cultured and amplified under the condition of heterologous feeder cells, and the multipotentiality thereof is maintained. The multi-potential stem cells generated by the invention can avoid transplant rejection; the multi-potential stem cells induced by non-heterologous feeder cells has better clinical application value; the technology of adopting Oct3 / 4 and Nanog transcription factor genes or direct transfer of relevant protein is concise and has higher biological safety.

Description

technical field [0001] The invention belongs to the field of biomedicine. It specifically relates to a method for inducing and cultivating pluripotent stem cells in vitro. Background technique [0002] Embryonic stem cells (embryonic stem cells, ESCs) are pluripotent cells derived from the cell mass in the blastocyst stage of the embryo, with unlimited proliferation and multilineage differentiation potential (Evans MJ, 1981Martin, 1981). Human embryonic stem cells (hESCs) are considered to be promising donor cells for cell transplantation to treat juvenile diabetes, Parkinson's disease and heart failure (Thomson JA, 1998), but due to tissue rejection and the use of human embryos, Its application is limited. [0003] Recently, multiple research groups introduced four transcription factor genes into human adult cells through viral vectors, and then co-cultured with mouse embryonic fibroblasts (MEFs) to obtain reprogrammed human pluripotent stem cells in vitro (Lowry et al ....

Claims

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Application Information

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IPC IPC(8): C12N5/10
Inventor 葛坚陈梦飞
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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