Neurons directly induced from human skin cells and preparation method for neurons

A neuron cell and skin cell technology, applied in the field of biomedicine, can solve the problems of inability to obtain inducible neuron cells, inability to use inducible human neuron cells, lack of complete understanding of the characteristics of new cells, etc., and achieve full in vitro and in vivo characteristics. Evaluation data, good clinical application prospects, and the effect of improving motor function

Inactive Publication Date: 2012-11-28
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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Problems solved by technology

[0005] However, the above-mentioned technical methods still have the following defects: 1) so far, human skin cells cannot be induced by using the combination of transcription factors that induce mice; 2) in animal experiments, human inducible neurons can not be obtained Through transgenic technology, the reporter gene that marks the change of cell type is integrated into the mouse genome to make a transgenic mouse. After the skin cells are transformed into nerve cells, the required nerve cells can be selected by fluorescence or antibiotic resistance screening. , but because transgenic technology cannot be directly applied to humans, this cell screening method cannot be applied to the preparation of inducible human neuron cells; 3) It is generally believed that to fully evaluate the characteristics and functions of a new class of cells, a complete and in vivo experimental data, but the aforementioned studies only carried out in vitro experiments when evaluating mouse induced neuron cells, and did not transplant the obtained functional cells into the body for analysis and identification, thus lacking a complete understanding of the characteristics of this new type of cells

Method used

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  • Neurons directly induced from human skin cells and preparation method for neurons
  • Neurons directly induced from human skin cells and preparation method for neurons
  • Neurons directly induced from human skin cells and preparation method for neurons

Examples

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Embodiment 1

[0073] Example 1: Isolation and culture of adult skin cells

[0074] Scalp tissue was harvested from exfoliated scalps of patients undergoing neurosurgery and adult skin cells were isolated. The specific isolation and culture methods are as follows: the scalp tissue removed during the operation was immediately placed in ice-precooled PBS, and washed repeatedly with PBS to remove blood clots; the two-step digestion method of collagenase + trypsin was used to obtain a single cell suspension, which was placed in a place containing 2 mmol / L glutamine, 10% FBS, 50 U / ml penicillin / 50 U / ml streptomycin in DMEM cell culture medium (both purchased from GIBCO); adjust the cell density to 1×l0 5 / m1, placed at 37℃, 5%CO 2 Centrifuge the cells after primary culture for 5 days, digest them with 0.25% trypsin, stop the digestion reaction with the DMEM cell culture medium, and dilute the cell suspension at 5× l0 5 / m1 density subculture in culture flasks, at 37 ° C, 5% CO 2 Under the co...

Embodiment 2

[0076] Example 2: Viral vector construction and packaging

[0077] Using human total cDNA as a template, amplify three genes including Sox2, Ascl1 and Myt1l; integrate the genes into a lentiviral basic expression vector (Addgene Company) according to conventional molecular cloning methods; use 293T cells (Invitrogen Company) to package and amplify in large quantities Reconstructed viral vectors already carrying transcription factors. All gene cloning products were verified by PCR and gene sequencing.

Embodiment 3

[0078] Example 3: Viral vectors infect human skin cells

[0079] The day before transfection, skin cells were cultured in 8 × 10 5 Inoculate at a density of / m1; mix equal amounts of viral expression vectors containing Sox2, Ascl1 and Myt1l genes, add them to a culture dish, and culture overnight; 24 hours after transfection, replace the containing cells with fresh nerve cell culture medium Virus culture solution; detect the expression of foreign genes after 72 hours of culture. Morphological changes of the transfected cells were observed daily, and cells transformed into neuron-like morphological features were collected.

[0080] The main component of the nerve cell culture medium is DMEM / F12 basal culture medium (GIBCO Company), which is supplemented with 1×GlutaMax (Invitrogen Company), 2% B27 serum-free medium additive factor (Invitrogen Company), 1% N2 nerve cell growth Additive (Invitrogen), 100 ng / ml basic fibroblast growth factor (basic fibroblast growth factor, bFGF...

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Abstract

The invention belongs to the field of biomedicine and relates to neurons directly induced from human skin cells and a preparation method for the neurons. By the method, induced human neurons which can perform stable passage are directly prepared by importing complementary deoxyribonucleic acid (cDNA) containing reprogramming transcription factors, namely Sox2, Ascl1 and Myt1l into isolated adult skin cells, screening cells which express a human neuron marker and cloning the cells which express the human neuron marker. The invention is characterized in that (1) the neurons express human neuron-specific marker protein in vitro; and (2) the neurons can survive in vivo and exert functions. The human neurons with the functions are obtained without the process of multipotential stem cells, so that the technology is relatively simple and effective; a heterologous feeder layer is avoided, so that the neutrons have a good clinical application prospect; and the report related to the tumorigenesis is not known about the adopted transcription factors, so the neurons have relatively high biosafety.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for inducing neuron cells, in particular to a method for directly inducing neuron cells from human skin cells. Background technique [0002] Nervous system diseases such as brain injury, spinal cord injury, stroke, Parkinson's disease, neuronal amyotrophic lateral sclerosis, Alzheimer's disease, etc. have great social harm and difficult treatment, and have long been difficult problems in the medical field. Studies have shown that using human neuron cells to study the mechanism of nerve injury, screen new drugs that interfere with nerve function, and use them for transplantation to repair damaged nerve function is an effective and promising method; however, the source of functional nerve cells is limited. Commonly used embryonic stem cells (embryonic stem cells, ES cells) differentiated to obtain human neurons (Wichterle H, Lieberam I, Porter JA, et al. Directed differentiation of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0793C12N15/867C12N15/12
Inventor 朱剑虹王璞沙红英徐成仕吴惺
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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