Cell transdifferentiation method

A transdifferentiation and cell technology, applied in the field of cell biology, can solve the problems of decreased cell number and proliferation and differentiation potential, high cell virus infection rate, and low separation efficiency.

Inactive Publication Date: 2017-01-04
深圳市伊思科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, experimental and clinical studies mainly focus on bone marrow-derived MSCs, but bone marrow-derived MSCs have their own limitations: for example, with the increase of donor age, the number of cells and the proliferation and differentiation potential of bone marrow-derived MSCs are greatly reduced; The high rate of cel

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Preparation of lentivirus Lentivirus-ETV2.

[0069] The packaging plasma pCMV-dR8.2dvpr, envelope plasma pCMV-VSV-G, and pCCL-ETV2 were respectively amplified in Stbl3 competent cells (TransStbl3Chemically Competent Cell, Cat.No.CD521, TRANS), and the endotoxin-free plasmid was used to Plasmids were extracted using an extraction kit (Cat. No. DP118-02, TIANGEN) to obtain a total amount of 1 mg each.

[0070] 293T cells (Cat.No.CRL-3216, ATCC) with a cell number less than 10 were used, cultured in a complete medium, in a 100mm cell culture dish, and plasmid transfection was ready after the cells reached 80% growth density.

[0071] Add the three plasmids pCMV-VSV-G, pCMV-dR8.2 and pCCL-ETV2 into 1 mL of normal temperature serum-free medium Opti-MEM according to the mass ratio of 1:2.5:2.5 (or molar ratio 1:1:1) , mix gently to prepare a lentiviral expression vector solution (the concentration of the transfected plasmid in each 100mm cell culture dish is 1 μg / ...

Embodiment 2

[0082] Embodiment 2, fluorescence counting method is measured the titer of Lentivirus-ETV2

[0083] The day before the measurement, plate the 293T cells required for titer determination, add 5000 cells to each well of a 96-well cell culture plate, and the volume is 100 μL.

[0084] Before infection, perform a 10-fold serial dilution of the lentivirus sample to be detected. The operation method is: prepare 8 sterile 1.5mL centrifuge tubes, add 90 μL of DMEM complete medium to the centrifuge tubes, take 11 μL of the virus sample to be tested and add it to the first tube, mix well, take 11 μL and add it to the second tube. In each tube, continue the same operation until the last tube.

[0085] Select the desired cell wells and aspirate 90 μL of medium. Add 90 μL of the diluted virus solution. Place in the incubator, being careful not to blow up the cells. During the culture process, the medium was replaced appropriately to maintain the normal growth of the cells.

[0086] 37...

Embodiment 3

[0090] Example 3, Lentivirus-ETV2 infection target cell line

[0091] The target cell line used in this example is human myoblasts (HSKMM, Cat. No. 3520, Lot. No. 2152, ScienCell).

[0092] One day before infection, human myoblasts were plated in a 12-well cell culture plate at a density of 2×10 4 cells / well.

[0093] On the first day of infection, re-digest the cells in similarly treated cell culture wells or dishes, and count the number of cells.

[0094] Heat the complete medium (DMEM medium + 10% FBS) in a 37°C water bath.

[0095] According to the final concentration of 8 μg / μL, polybrene (Hexadimethrine bromide, Cat. No. H9268, Sigma) was added into the complete medium at 37°C, followed by adding lentivirus to obtain the infection medium.

[0096] Remove the medium in the 12-well cell culture plate, and add 400 μL of infection medium according to the ratio of the number of lentivirus to the target cell line at 2TU / cell. Among them, the small-volume infection system h...

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Abstract

The invention discloses a cell transdifferentiation method. The cell transdifferentiation method comprises the following steps that a lentiviral expression vector is adopted to prepare a lentivirus, the sequence of the lentiviral expression vector includes a transcription factor etv2; the lentivirus is adopted to infect a target cell line, culture is performed for 5-7 days, and then transdifferentiation of the target cell line is completed. The cell transdifferentiation method adopts the lentivirus as the carrier, the transcription factor etv2 is transfected to the target cell line, then culture is performed for 5-7 days to complete the transdifferentiation of the target cell line. FACS identification of the transdifferentiated target cell line shows that about 70% of cell expression endothelial cell molecular markers CD144 are found. It is found that genes relevant to blood vessels in transdifferentiated cells are improved at different degrees through QPCR identification of the transdifferentiated target cell line. The cell transdifferentiation method can achieve target cell linetransdifferentiation, so that cells of different sources are transdifferentiated into cellsrelevant to blood vessels, and accordingly the method can be used for cell therapy.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a cell transdifferentiation method. Background technique [0002] The primary problem of cell therapy is the source of cells. There are many types of pluripotent stem cells currently used in cardiovascular diseases, such as skeletal muscle myoblasts, bone marrow mesenchymal stem cells, endothelial progenitor cells, liver stem cells, hematopoietic stem cells, etc. In 1996, human embryonic stem cells (hESC) were successfully isolated and cultured in vitro by researchers. Since then, Amit et al. have confirmed through long-term culture and induction differentiation experiments that hESC not only has the pluripotency to differentiate into three germ layers, inner, middle and outer, but also has the self-replication ability of continuous division, and its division ability and totipotency are much higher than those of hESC. Partially differentiated pluripotent stem cells bring great hope ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
Inventor 林硕李松秦伟
Owner 深圳市伊思科生物科技有限公司
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