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Method for constructing brain-like tissue using umbilical cord mesenchymal stem cells

A technology for stem cells and brain tissue, which is applied in the field of building brain-like tissue, and can solve problems such as the lack of methods for differentiation into neurons and glial cells, the lack of identification methods for neuron functions, and the lack of brain-like functions.

Active Publication Date: 2022-05-03
GENESIS STEMCELL REGENERATIVE MEDICINE ENG CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are still many difficulties to be solved in the application of human mesenchymal stem cells in clinical transformation. Although there are already methods for differentiating human embryonic stem cells (ESC, ES cells) into neurons, the use of human umbilical cord mesenchyme The method for stem cells to differentiate into neurons and glial cells has not yet been discovered; and the method for further constructing neurons differentiated from mesenchymal stem cells into brain-like neural networks in vitro is still blank
Moreover, the neuronal functions of mesenchymal stem cell differentiation and the constructed brain-like functions have not yet been characterized
Due to the current difficulty in obtaining ES cells and unresolvable ethical issues, the use of neurons differentiated from ES cells to construct brain-like tissues has great limitations in clinical application.

Method used

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  • Method for constructing brain-like tissue using umbilical cord mesenchymal stem cells
  • Method for constructing brain-like tissue using umbilical cord mesenchymal stem cells
  • Method for constructing brain-like tissue using umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Gene Editing of Human Umbilical Cord Mesenchymal Stem Cells

[0072] Human umbilical cord mesenchymal stem cells were gene-edited using the CRISPR / Cas9 system to construct a cell line expressing the light-sensitive channel (ChR2), which is called a gene-edited cell line. The method is as follows:

[0073] The ChR2(H134R)-EYFP cDNA from the plasmid vector pAAV-hSyn-hChR2(H134R)-EYFP (BioVector NTCC Type Culture Collection) was amplified by PCR and the ChR2(H134R)-EYFP fragment was inserted into AAVS1-pur-CAG -EGFP donor plasmid (when constructing this plasmid, the hrGFP gene in the AAVS1-pur-CAG-hrGFP plasmid (plasmid #52344, Addgene) was replaced by the EGFP gene, and the woodchuck hepatitis post-transcriptional regulatory element (woodchuckhepatitis post-transcriptional Regulatory element, WPRE) and human growth hormone (human growth hormone, hGH) Poly A inserted into the 3'-end of the EGFP gene) instead of EGFP, AAVS1-pur-CAG-ChR2 (H134R)-EYFP donor plasmid ...

Embodiment 2

[0075] Example 2 Culture of embryoid body-like neurospheres

[0076] The gene-edited human umbilical cord stem cell line obtained in Example 1 and the normal human umbilical cord mesenchymal stem cell line were mixed and subcultured at a ratio of 2:1 at the stem cell stage, and the steps were as follows:

[0077] 2.1 The mesenchymal stem cell line was grown on MEFs in a 6-well plate for 5-7 days, and 2 ml of human umbilical cord mesenchymal stem cell complete medium (NEM) was used to change the medium every day, and 2 μM bFGF factor was added every time the medium was changed.

[0078] 2.2 On the 5th-7th day when it can be subcultured, digest the clone with disapase for 2 minutes, and suspend culture in stem cell medium (NEM) to the 4th day (recalculate the number of days from the digestion, add 2 μM SB431542 (Stemgent) and 2 μM DMH1 factor (Torcris) without adding bFGF factor); from the 5th day, the stem cell culture medium was replaced with the neural induction medium (NIM) ...

Embodiment 3

[0079] Example 3 Differentiation of Brain Organoids

[0080] 3.1 Transfer the embryonic body (EB)-like neurospheres formed in Example 2 to a 6-well plate to adhere to the wall. Change the medium 2ml every 2 days, add SB431542 and CHIR factor 2μM until the 16th day.

[0081] 3.2 After culturing until the 16th day, the neural rosette neural tube-like structure neural precursor cells formed by adherent culture were blown up and cultured in suspension. After 7 days of suspension culture (Day 23), neurospheres with neural rosette neural tube-like structures were obtained.

[0082] 3.3 Embed neural rosette neural tube-like structure neurospheres in matrigel gel and culture in induction medium (NIM) until the 30th day. This process adopts neural induction medium (NIM) culture + insulin, N2 (LifeTechnology), B27 (Life Technology).

[0083] 3.4 Transfer the matrigel-coated neural rosette neural tube-like neurospheres to a low-adsorption 6-well plate and culture them on a shaker for ...

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Abstract

The invention discloses a method for constructing brain-like tissue using umbilical cord mesenchymal stem cells, which comprises the following steps: 1) performing stem cell culture on a human umbilical cord mesenchymal stem cell line to obtain subcultured cells; 2) subcultured cells Inducing neural differentiation to obtain differentiated neuron precursor cells, which are embryoid body-like neurospheres; 3) Inducing and culturing embryoid body-like neurospheres to obtain neural rosette neural tube-like neurospheres; 4) neural The rosette neural tube-like structure neurospheres were differentiated and cultured to obtain human brain organoids, which contain a mixture of various types of neurons. The present invention differentiates human mesenchymal stem cells into various neurons for the first time and constructs brain-like tissue, which can be used as a drug screening platform and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing brain-like tissue by using umbilical cord mesenchymal stem cells, in particular to a method for using human umbilical cord mesenchymal stem cells to induce differentiation into various functionally mature neurons in vitro and constructing brain-like tissues method of organization. Background technique [0002] Human umbilical cord mesenchymal stem cells are a kind of pluripotent cells with self-renewal ability, which not only maintain an undifferentiated state, but also have unlimited proliferation potential. Under certain conditions, it can differentiate into various functional cells, such as various types of nerve cells and glial cells. It is precisely because of this characteristic that mesenchymal stem cells have the natural advantages of studying early neural development and disease mechanisms, transplantation replacement therapy, gene therapy, drug scre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0793C12Q1/02
CPCC12N5/0697C12N5/0619G01N33/5008C12N2506/1369
Inventor 董毅殷鉴强刘定生
Owner GENESIS STEMCELL REGENERATIVE MEDICINE ENG CO LTD
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