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Reprogramming method for efficient inducing of T cells into multipotent stem cells

A technology for pluripotent stem cells and reprogramming, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve the problem of iPSC preparation technology failing to achieve large-scale preparation, and achieve the effect of high immune cell differentiation efficiency

Active Publication Date: 2018-10-12
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] These findings indicate that the use of iPSC technology to manipulate T cells may be very useful for the development of more effective immunotherapy in the future, but the current iPSC preparation technology for clinical applications has not achieved large-scale production

Method used

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  • Reprogramming method for efficient inducing of T cells into multipotent stem cells
  • Reprogramming method for efficient inducing of T cells into multipotent stem cells
  • Reprogramming method for efficient inducing of T cells into multipotent stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1: Design and Construction of Episomal Vector Expression System

[0071] Such as figure 2 As shown, three kinds of episomal vectors were constructed in this example, all of which were amplified by direct polymerase chain reaction (PCR) from some or all of the open reading frames (ORFs) in the pluripotency determinant gene (using the The first and last 20-22 bases are used as primers), and the above ORF is inserted into the commercially available mammalian expression vector pCEP4 or related backbones containing OriP / EBNA1 to generate episomal vectors, all three episomal vectors contain at least An internal ribosome entry size (IRES), where the first vector is pEP4-E-O2S-E-N2K ( figure 2 A), which in turn comprises the first promoter, POU5F1, IRES2, SOX2, the second promoter, NANOG, IRES2 and KLF4; the second vector is pEP4-E-O2S-E-T2K ( figure 2 B), which sequentially comprises the third promoter, POU5F1, IRES2, SOX2, the fourth promoter, SV40LT, IRES2 and K...

Embodiment 2

[0072] Example 2: Primary isolation, culture and identification of T cell mononuclear cells

[0073] 1. Primary isolation and culture of T cell monocytes

[0074] Collect 6ml of blood sample, transfer it to a lymphocyte separation tube, centrifuge, take the mononuclear cell layer, wash it twice with DPBS centrifugation, take a sample and count it, and use flow cytometry to detect the expression of the surface molecules CD3+ and CD4+CD8+, Detect its positive rate and average fluorescence intensity index respectively (experimental result such as image 3 Shown in A), take 6×10 according to the counting result 6 Cells were seeded in 3 wells of 6-well plate coated with activator, added T cell serum-free medium 2ml / well, placed at 37°C, 5% CO 2 Cultivation in an incubator constitutes the expansion culture system of the present invention, and each well is supplemented with 1-2 ml of fresh T cell serum-free medium on the first day of expansion.

[0075] The T cell serum-free mediu...

Embodiment 3

[0080] Example 3: Episomal vector-induced reprogramming

[0081] 1. Method

[0082] a. Take 0.5~4×10 T cells activated and cultured for 2 days in Example 2 6, using the pEP4-E-O2S-E-N2K, pEP4-E-O2S-E-T2K and pCEP4-LM-2L episomal vectors constructed in Example 1 to electrotransfect target cells, and then inoculated in hiPSC induction medium and Matrigel or vitronectin or other cell matrix-coated six-well plates, and the transfection contents of each plasmid DNA were pEP4-E-O2S-E-N2K: pEP4-E-O2S-E-T2K: pCEP4-LM -2L=1:1:1.

[0083] b. After 48 hours, half of the medium was replaced with fresh hiPSC induction medium, and the culture was continued until 10 days, and the medium was changed every other day, that is, reprogramming was performed on a feeder-free system.

[0084] The specific components of the pluripotent stem cell induction medium are:

[0085] On the basis of each liter of T cell serum-free expansion medium in Example 2, one or more of the following small molecule...

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Abstract

The invention discloses a reprogramming method for efficient inducing of T cells into multipotent stem cells. The method comprises the following steps: S1, extracting monocyte from a blood sample, andadding the monocyte to an amplification culture system containing an activating agent for implementing selective T cell activating culture; S2, introducing an episomal vector which contains at leastone potential determinant into the T cells which are obtained in the step S1; S3, cultivating the T cells which are obtained in the step S2 and contain the episomal vector via a multipotent stem cellinducing medium, and performing inducing in a system free from a feeding layer, so that preprogrammed intermediate cells are obtained; and S4, after complete inducing, replacing the multipotent stem cell inducing medium in the step S3 with a multipotent stem cell medium for maintaining culture, so that cells that the expression of the potential determinant disappears and expression of endogenous multipotent genes, namely POU5F1, NANOG, TRA-1-60 and TRA-1-81, is activated are obtained, wherein the cells are induced multipotent stem cells. The method provided by the invention has the beneficialeffect that the T-cell derived induced multipotent stem cells can be simply and conveniently prepared in a large scale.

Description

technical field [0001] The invention belongs to the field of cells, and in particular relates to a reprogramming method for efficiently inducing T cells into pluripotent stem cells. Background technique [0002] In general, stem cells are undifferentiated cells capable of giving rise to a succession of mature, functional cells. Embryonic stem cells (ESCs), for example, are derived from embryos and are pluripotent, thus having the ability to develop into any organ or tissue type in the adult. Hematopoietic stem cells can give rise to any of the different types of terminally differentiated blood cells. Induced pluripotent stem cells (induced pluripotent stem cells), usually referred to as iPS cells or iPSCs, are a type of pluripotent stem cells artificially derived from non-pluripotent cells (usually adult cells). iPSCs were first generated from mouse cells in 2006 (Takahashi et al., 2006) and from human cells in 2007 (Takahashi et al., 2007; Yu et al., 2007). The study of ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N5/0696C12N15/85C12N2506/115C12N2510/00C12N2501/608C12N2501/604C12N2501/606C12N2501/605C12N2501/602C12N2501/60
Inventor 俞君英周桃陈涛涛董成友张颖
Owner 安徽中盛溯源生物科技有限公司
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