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Technology for promoting direct trans-differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells

A technology of positive cells and hematopoietic stem cells, applied in the field of bioengineering, can solve the problems of low efficiency of MSCs, and achieve the effect of far-reaching medical application value

Inactive Publication Date: 2017-08-08
溯源生命科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of low efficiency of inducing MSCs from the above-mentioned umbilical cord blood, the object of the present invention is to provide a genetic engineering technique for inducing the differentiation of umbilical cord blood CD34 positive cells into MSCs, and the clinical treatment performed using the iMSCs obtained by this method

Method used

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  • Technology for promoting direct trans-differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells
  • Technology for promoting direct trans-differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells
  • Technology for promoting direct trans-differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This embodiment provides a method for obtaining MSCs by using lentivirus to infect CD34-positive hematopoietic stem cells derived from umbilical cord blood, which includes the following steps:

[0023] 1. Enrichment and storage of CD34 positive cells:

[0024] 1) The umbilical cord blood and hydroxyethyl starch 5:1 were thoroughly mixed, and left at room temperature for 40 minutes to precipitate red blood cells.

[0025] 2) Dispense 15ml of lymphocyte separation solution into a 50ml centrifuge tube and place at room temperature to equilibrate.

[0026] 3) Aspirate the supernatant in 1), and slowly add it to the lymphocyte separation medium that has been equilibrated to room temperature, taking care not to disturb the liquid level of the separation medium, the total volume can reach 50ml. The centrifuge was set to de-brake and centrifuged at 400G for 25 minutes.

[0027] 4) Aspirate the buffy coat layer between the two liquid phases, add 5 times the volume of PBS and mix...

Embodiment 2

[0036] This embodiment provides a method for using a non-integrating vector to transdifferentiate umbilical cord blood-derived CD34-positive hematopoietic stem cells to obtain iMSCs, which includes the following steps:

[0037] 1. Construction of non-integrating vector

[0038] 1) In order to better meet the standards of clinical cell therapy, we developed a transdifferentiation method for MSCs using non-integrating vectors. We first tested an episomal vector based on EBNA1. One week after nucleofection of cord blood CD34-positive cells, we obtained MSC-like cells.

[0039] 2) From Invitrogen (Carlsbad, CA). The hygromycin resistance gene element and the CMV promoter were removed by two restriction enzyme sites, NruI and BamHI, and at least one of the nucleotide sequence combinations of the six genes OCT4, NANOG, MYCL1, SNAI1, SNAI2, and TWIST1 An episomal vector cut from lentiviral vector and inserted into pCEP4. figure 2 It is a schematic diagram of the non-integrated e...

Embodiment 3

[0047] Example 3 Long-term culture of iMSCs and identification of genome stability

[0048] In this example, the iMSCs obtained in Example 2 were cultured for a long period of time, and the genome stability was identified according to the following steps:

[0049] 1. We cultured bone marrow iMSCs for more than 40 passages under MSCs culture conditions, and the population doubling time was 18-36 hours. Specific methods of cell culture and subculture:

[0050] 1) 24-well plates were pretreated with fibronectin (RetroNectin) (CH-296; Takara Bio, Inc., Shiga, Japan) according to the product instructions. After standing at room temperature for 2 hours or overnight at 4, wash with PBS to remove residual fibronectin and it can be used.

[0051] 2) MSCs were cultured in culture plates pretreated with RetroNectin. Every 2-3 days, iMSCs were treated with Accutase or trypsin for five minutes to digest the iMSCs into single cells, and passaged 4-6 times.

[0052] 2. After long-term in ...

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Abstract

The invention relates to a technology for promoting direct trans-differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells, and belongs to the technical field of bioengineering. With the application of the genetic engineering technology provided by the invention, efficient direct trans-differentiation of the umbilical cord blood CD34 positive cells from hematopoietic stem cells can be promoted, so that iMSCs can be obtained; the hematopoietic stem cells, which are co-cultured with a certain composition for enough time, can be transformed into MSCs through trans-differentiation; the composition includes at least one transcription factor selected from OCT4, NANOG, MYCL1, SNAI1, SNAI2 and TWIST1; and the composition can also include one or more cell factors disclosed by the invention and other compounds. With the application of the technical method provided by the invention, transient expression of the related transcription factors in the umbilical cord blood CD34 positive cells can be achieved by virtue of a virus infection method, and in the combination with an MSCs inducing medium, 1*10<9> MSCs can be obtained from 1*10<6> umbilical cord blood CD34 positive cells through the trans-differentiation, and the iMSCs cells, obtained from the trans-differentiation, are represented in the form of the MSCs and can express surface markers of the MSCs cells, such as CD29, CD44, CD73 and CD90.

Description

technical field [0001] The invention relates to a direct transdifferentiation technique for inducing the differentiation of umbilical cord blood CD34 positive cells into mesenchymal stem cells, and belongs to the technical field of bioengineering. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a kind of pluripotent stem cells originating from mesoderm, which can be isolated from various human tissues, such as bone marrow and adipose tissue. One of the characteristics of MSCs is their ability to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, muscle, bone marrow mesenchyme and adipose tissue, etc. This biological characteristic makes MSCs cells in clinical therapy and regenerative medicine has broad application prospects. [0003] MSCs can be conveniently obtained from a variety of adult tissues, including adipose tissue and bone marrow tissue, and methods for their isolation and culture are well established in the fie...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/078
CPCC12N5/0665C12N2501/105C12N2501/11C12N2501/115C12N2501/135C12N2501/15C12N2501/155C12N2501/165C12N2501/415C12N2501/599C12N2501/60C12N2501/603C12N2501/605C12N2506/11
Inventor 袁卫平张孝兵程涛庞雅坤
Owner 溯源生命科技股份有限公司
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