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Method for differentiating and preserving non-integrated feeder-layer-free human urine-derived induced pluripotent stem cells into neural stem cells

A technology of neural stem cells and pluripotent stem cells, applied in artificially induced pluripotent cells, non-embryonic pluripotent stem cells, urinary tract/kidney cells, etc. source of great effect

Pending Publication Date: 2021-03-09
长沙科雅生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] An object of the present invention is to provide a non-integrated non-feeder-free human urine-derived induced pluripotent stem cell differentiation method to neural stem cells, to solve the existing integrated induction method clinical safety problems and the difficulty of obtaining neural stem cells, and at the same time Hope to establish a feeder-free, serum-free, non-integrated iPSCs cell bank, and store a large number of iPSCs-derived NSCs for autologous or allogeneic transplantation

Method used

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  • Method for differentiating and preserving non-integrated feeder-layer-free human urine-derived induced pluripotent stem cells into neural stem cells
  • Method for differentiating and preserving non-integrated feeder-layer-free human urine-derived induced pluripotent stem cells into neural stem cells
  • Method for differentiating and preserving non-integrated feeder-layer-free human urine-derived induced pluripotent stem cells into neural stem cells

Examples

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Effect test

Embodiment 1

[0023] This embodiment discloses a method for preparing non-integrated human urine-derived induced pluripotent stem cells without a feeder layer, which includes the following steps:

[0024] (1) Collect urine cells

[0025] Collect about 300ml of midstream urine, and add it equally into 50ml centrifuge tubes containing 2ml of penicillin / streptomycin double antibody;

[0026] Centrifuge at 400g for 10min; quickly pour off the supernatant to avoid pouring out the precipitate, leave about 5ml in each tube, and mix it into a centrifuge tube; add about 30ml of 5X penicillin / streptomycin PBS, mix gently; centrifuge at 400g for 10min, pour off the upper Clear, about 1ml of liquid remains.

[0027] (2) Cultivate and expand urine cells

[0028]Prepare a 12-well culture plate, coat it with 0.1% Gelatin for more than 30 minutes, and discard the liquid in the well before use; add the remaining liquid in step (1) to the coated 12-well culture plate, add 2ml REGM medium, and then add 2ul...

Embodiment 2

[0038] This example mainly illustrates the method for inducing neural stem cells from human urine-derived pluripotent stem cells obtained above, which specifically includes the following steps: take the induced pluripotent stem cells obtained above, plate them in a six-well plate, add 10 μM 27632, and wait until the cell density When the cell density reaches more than 90%, spread Matrigel-coated 12-well plate overnight; after the cell density reaches more than 95%, change the N2B27+2i induction medium, change the medium every day, and culture until the cells have no iPSCs characteristics; wash once with DMEM / F12, add N2B27 to maintain For the culture medium, scrape the cell mass, inoculate it into a 6-well plate coated with Matrigel in advance, maintain the culture medium with N2B27, and change the medium every day; when a rosette-like structure appears, add bFGF (final concentration 2 μg / ml) to the medium ), remove bFGF two days later, continue to culture for 2-3 days, suck of...

Embodiment 3

[0042] This example discloses a storage method for NSCs (iNSCs) obtained from iPSCs, which specifically includes the following steps: iNSCs are digested into single-cell suspension culture, and Neurospheres of different sizes are formed after 4-6 days. Collect these Neurospheres into 15ml centrifuge tubes, add 10ml of culture medium, wrap them in tinfoil, and store them at room temperature.

[0043] Place Neurospheres at room temperature for 7 days ( Figure 5 A), the experimental results show that Neurospheres can still maintain a good shape and still have the ability to differentiate like neurons; placed at room temperature for 9 days, as a control, iNSCs cultured in monolayers were also placed in 6-well plates, Wrap it in tinfoil and leave it at room temperature for 9 days. The results showed that the survival rate of Neurospheres was above 80%, but the survival rate began to decline after seven days, and the monolayer cells had shrunk rapidly and apoptotic ( Figure 5 B)...

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Abstract

The invention discloses a method for differentiating and preserving non-integrated feeder-layer-free human urine-derived induced pluripotent stem cells into neural stem cells, and belongs to the technical field of neuroscience and cell biology. The method comprises the following steps of: selecting and collecting urine-derived cells, culturing and amplifying, inducing pluripotent stem cells iPSCsfor induction culture, inducing the pluripotent stem cells to induce differentiation of neural stem cells NSCs, and preserving. According to the method disclosed by the invention, urine is selected asan iPSCs cell source, and the method has the advantages of being easily available, simple, low in cost, non-invasive and the like. The iPSCs cells are induced into NSCs by adopting an episome induction method, the whole link is free of animal-derived cells and other components, feeder layers are avoided, the method is non-integrated and virus infection-free, the availability and safety of the iPSCs-derived neural stem cells are greatly improved, and a novel method is provided for clinical autologous or allogeneic transplantation. Meanwhile, the cultured NSCs are stored in a form of nerve balls and can be preserved for a week at an ambient temperature, and a novel method is provided for long-term preservation and long-distance transportation.

Description

technical field [0001] The invention belongs to the field of neuroscience and cell biology technology, and specifically relates to a method for differentiating non-integrated human urine-derived induced pluripotent stem cells into neural stem cells without a feeder layer, and also relates to a preservation method for the neural stem cells. Background technique [0002] Uncertainties and inefficiencies in the derivation of induced pluripotent stem cells (iPSCs) have led to a plethora of induction methods. The initial generation of iPSCs depends on the transient expression of key transcription factors that maintain stem cell characteristics. There are many methods for inducing iPSCs, mainly including viral methods and non-viral methods. From the earliest use of retrovirus and lentiviral vectors to inducible lentiviral vectors, plasmids, episomes, transposons, direct mRNA transfection and protein addition, and the use of microRNA alone, these systems have obtained The stable g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/074
CPCC12N5/0623C12N5/0696C12N2501/115C12N2506/25C12N2506/45C12N2533/54C12N2533/90
Inventor 李志远李帅王利群唐焱邓思浩
Owner 长沙科雅生物科技有限公司
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