Neuronal precursor cells derived from adipose-derived stem cells and preparation method and application of neuronal precursor cells

A technology of adipose stem cells and precursor cells, applied in biochemical equipment and methods, animal cells, nervous system cells, etc., can solve the problems of poor health and low purity of neuron precursor cells, and achieve good health, The effect of high activity and purity

Pending Publication Date: 2020-06-02
上海泉眼生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problems of poor health and low purity of the neuron precursor cells induced by the existing fat stem cells, the object of the present invention is to provide a kind of neuron precursor cells derived from fat stem cells, a preparation method and its use in the treatment of central nervous system. Use in Nervous System Injuries

Method used

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  • Neuronal precursor cells derived from adipose-derived stem cells and preparation method and application of neuronal precursor cells
  • Neuronal precursor cells derived from adipose-derived stem cells and preparation method and application of neuronal precursor cells
  • Neuronal precursor cells derived from adipose-derived stem cells and preparation method and application of neuronal precursor cells

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Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Isolation and Characterization of ADSCs

[0058] Fresh human adipose tissue was obtained from abdominal liposuction, washed several times with sterile phosphate buffered saline (100 U / ml penicillin, 100 μg streptomycin), cut into small pieces, and then treated with 0.075% (phosphoric acid) at 37°C. Salt buffer) of type I collagenase (purchased from sigma company) was incubated for 30 minutes with shaking, centrifuged (1500rpm / min) to remove the floating fat in the upper layer; the remaining suspension was filtered through 100m nylon membrane (purchased from Falcon company) Filter; then neutralize the filtrate with 10% FBS prepared in DMEM / F12, and centrifuge (1500 rpm / min) for 10 minutes; discard the supernatant, and collect the pelleted cells, in a medium containing 10% FBS and penicillin / streptomycin (100 U / ml penicillin, 100 μg streptomycin) in DMED / F12 and incubated at 37°C; after 24 hours, the plate was washed with PBS to remove suspended cells. After 5...

Embodiment 2

[0062] Example 2 Induction of ADSCs into neuronal cells

[0063] Before induction, the cells were first plated 24 hours in advance, and the cells were seeded into 24-well plates, then the medium was removed, rinsed twice with PBS, and then the induction solution was added. , after 24-48 hours of culture, fixation followed by immunocytochemical staining to examine the identity of neuronal cells.

[0064] Observe the morphological changes from day 0 (D0) to day 6 (D6) of induction respectively, such as figure 2 (A). Compare the protein expression of ADSCs without induction and 24 hours after induction, such as figure 2 (B) and figure 2 (C).

[0065] figure 2 This shows the neural induction effect of the induction solution on adipose stem cells. The neural structure is more complex and the neurons are more mature after the neurotrophic factor is added in the C picture than that without the addition in the B picture.

Embodiment 3

[0066] Example 3 Induction of ADSCs into neuronal precursor cells

[0067] It has been demonstrated that ADSCs can be induced to differentiate into neuronal cells. The present invention improves the inducer. The adipose stem cells are added to the culture medium containing the specific inducer for 24 hours, and then 10 ng / mL neurotrophic factor is added to the culture medium. Immunocytochemical staining, semi-quantitative RT-PCR and quantitative PCR were then used to examine the identity of neuronal precursor cells.

[0068] Follow the standard operating instructions for Qiagen RNeasy Mini Kit (#74104) from ~10 6 Total RNA was extracted from individual ADSCs, embryonic stem cells, and neuronal precursor cells. The extracted RNA is valid only when the OD260 / 280 value is higher than 1.8. The total RNA extracted above was reverse transcribed using Thermo Scientific RevertAid First Strand cDNA Synthesis kit (#K1621). RT-PCR consisted of quenching the reaction by adding at 70°C...

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Abstract

The invention discloses neuron precursor cells derived from adipose-derived stem cells, and a preparation method and application of the neuron precursor cells in treating central nervous system injury. The method for differentiating the adipose-derived stem cells into the neuronal precursor cells comprises the following steps: S1, adding the adipose-derived stem cells into a neuronal culture medium containing an inducer, and carrying out induction culture for 24 hours; and S2, adding neurotrophic factors into the neuron culture medium in the S1, and performing culturing for 24-72 hours. A neuron precursor cell population derived from adipose-derived stem cells is obtained by inducing and differentiating the adipose-derived stem cells through the specific inducer, and the neuronal precursorcells are good in health condition and high in activity and purity.

Description

technical field [0001] The invention relates to the technical field of stem cells and biomedicine, in particular to neuron precursor cells derived from adipose stem cells, a preparation method and their application in the treatment of central nervous system damage. Background technique [0002] Embryonic stem cells (ESCs) provide a sufficient cell source for the treatment of various degenerative diseases due to their unique self-renewal and differentiation totipotency. ESCs are virtually immortal in vitro and can differentiate into any lineage, but their clinical application is highly controversial due to their tumorigenic potential and difficulty in obtaining high purity of any single lineage without karyotypic abnormalities, as well as ethical concerns. [0003] Induced pluripotent stem cell (iPSC) technology can greatly overcome the ethical issues posed by ESCs, however, issues that hinder the application of ESCs still exist for iPSCs. Traditional protocols for iPSC indu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793A61K35/30A61P25/00
CPCA61K35/30A61P25/00C12N5/0619C12N2500/30C12N2501/13C12N2501/33
Inventor 高山峨
Owner 上海泉眼生物科技有限公司
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