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Methods for producing nerve cells from stem cells, nerve cells and uses thereof

A nerve cell and stem cell technology, applied in the field of cell transplantation, can solve the problem that nerve cells are not suitable for clinical application

Inactive Publication Date: 2014-06-25
AFFILIATED HOSPITAL OF NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that trophoblast cells derived from mice need to be co-cultured with human embryonic stem cells, and the contamination of animal-derived protein and possible animal-derived substances makes the nerve cells obtained by this method unsuitable for clinical application.
The limitation of using this method to generate cells for clinical use is that for any patient, the cells derived from embryonic stem cells are allogeneic cells, so immune rejection and corresponding immune response syndrome may occur after cell transplantation

Method used

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  • Methods for producing nerve cells from stem cells, nerve cells and uses thereof
  • Methods for producing nerve cells from stem cells, nerve cells and uses thereof
  • Methods for producing nerve cells from stem cells, nerve cells and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 : Isolation of human placental mesenchymal stem cells

[0079] The placenta used in this experiment was the term placenta of healthy puerpera, which was obtained from the Obstetrics Department of the Affiliated Hospital of Ningxia Medical University. The donors voluntarily donated without compensation and signed the informed consent. Approximately 20 g of fresh human placental tissue was dissected from the postpartum placenta under sterile conditions. The tissue was preserved in a 50 ml centrifuge tube containing 20 ml of DMEM (Invitrogen, product number 11885084, product number 11885084 ) culture medium. Placental tissue in the above protection solution was transferred to a cGMP laboratory within 30 minutes. Prior to processing, the tissue was washed three times in PBS (Invitrogen, product number 14040133) containing 1% penicillin / streptomycin.

[0080] Dissect the chorionic disc from the placental tissue using sterile surgical scissors, wash the chorion...

Embodiment 2

[0081] Example 2: Embryonic brain proteins induce mesenchymal stem cells to differentiate into neural cells

[0082] The steps of inducing mesenchymal stem cells to differentiate into nerve cells include:

[0083] 1. The human placental mesenchymal stem cells (MSCs) obtained in Example 1 were cultured in a standard medium (DMEM+10% FBS) to a density of 80%.

[0084] 2. The cells were digested with 200u / ml collagenase IV (Invitrogen) at 37°C for 3-5 minutes to obtain single cells.

[0085] 3. Transfer the cells into collagen-IV (Invitrogen)-coated culture flasks, add standard DMEM medium and culture for 24 hours.

[0086] 4. Replace the medium with fresh medium (DMEM+20% FBS) and culture for 16 hours.

[0087] 5. Continue culturing the cells in the initial differentiation medium for 16 hours, wherein the initial differentiation medium is a standard DMEM / F12 medium containing one-fold concentration of N2 mixed cofactor (Invitrogen), 2ug / ml heparin ( Heparin, Sigma), 1% Mini...

Embodiment 3

[0092] Example 3: Determination of neuron-specific proteins

[0093] The assay steps include:

[0094] 1, cell climbing sheet: the nerve cell (test group) that obtains from embodiment 3 is divided into 3 * 10 5 Densities were transferred to culture dishes with coverslips and cultured in neuronal culture medium (same as in Example 3, Step 6). In addition, uninduced human placental mesenchymal stem cells were cultured under the same conditions as a control group. The test group and the control group received exactly the same treatment in all the following steps.

[0095] 2. After 24 hours, wash the cells on the glass slide with cells (neuroid cells and control cells respectively) with PBS for 3 times, 5 minutes each time.

[0096] 3. Add 30% H to the slide 2 o 2 (1 part) and pure methanol (50 parts) for 30 minutes, and then washed twice with distilled water.

[0097] 4. Add a blocking solution containing 5% bovine serum albumin (BSA) dropwise on the cells of the glass sl...

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Abstract

The present invention belongs to the field of cell transplantation. Particularly, the present invention provides a method of producing neurons for treating injuries with the loss of neuron function from stem cells, wherein the stem cell is a human derived mesenchymal stem cell, preferably human derived placental mesenchymal stem cell, bone marrow mesenchymal stem cell, adipose mesenchymal stem cell and liver mesenchymal stem cell. The present invention also provides the uses of said method and the nerve cells produced by said method in the preparation of medicines for treating injuries with the loss of neuron function and in the treatment of injuries with the loss of neuron function. The present invention further provides a method of treating injuries with the loss of neuron function.

Description

technical field [0001] The invention belongs to the field of cell transplantation. In particular, the present invention relates to a method for generating nerve cells that can be used to treat neurologically impaired injuries, and the use of the method and the generated nerve cells in treating neurologically impaired injuries. The invention also relates to methods of treating neurologically disabling injuries. Background technique [0002] Parkinson's disease is a degenerative disorder of the central nervous system, usually caused by degeneration or lesions of dopamine-producing neurons in the midbrain. Clinicopathological manifestations of dopamine (Dopamine) deficiency. In theory, dopamine supplementation could alleviate or treat Parkinson's symptoms. However, since dopamine cannot pass through the human blood-brain barrier, the currently widely used therapeutic drug is the precursor of dopamine, L-DOPA. However, most of L-DOPA will be metabolized in the body before re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/079C12N5/02A61K35/12A61K35/28A61K35/50A61P25/00A61P25/16A61P25/28C12R1/91
CPCC12N2501/13C12N5/0668C12N2501/115C12N2501/105C12N2501/41A61K35/12C12N2501/91A61K35/30C12N2501/11C12N2506/025C12N2502/08C12N2506/1392C12N2501/385C12N5/0619A61K35/28A61P25/00A61P25/16A61P25/28
Inventor 杨银学魏军李玉奎王立斌马立君刘婷马晓娜范恒朱永朝叶萍金毅然
Owner AFFILIATED HOSPITAL OF NINGXIA MEDICAL UNIV
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