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A method for inducing and culturing urinary-derived pluripotent stem cells by small molecules in vitro

A pluripotent stem cell and small molecule technology, applied in artificially induced pluripotent cells, non-embryonic pluripotent stem cells, animal cells, etc., can solve the problem of scarcity of stem cells, and achieve broad clinical application prospects, broad application prospects, enhanced The effect of blood flow restoration

Active Publication Date: 2017-01-18
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Being able to use a patient's own stem cells for therapy has the advantage of not inducing an immune response or rejection, however, tissue-specific stem cells are difficult to isolate from organs and tissues because they are rare

Method used

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  • A method for inducing and culturing urinary-derived pluripotent stem cells by small molecules in vitro
  • A method for inducing and culturing urinary-derived pluripotent stem cells by small molecules in vitro
  • A method for inducing and culturing urinary-derived pluripotent stem cells by small molecules in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Induction and culture of urine-derived pluripotent stem cells (hUSCs) by small molecules in vitro

[0040] The schematic diagram of the experimental steps for isolating and culturing hUSCs in vitro is as follows: figure 1 shown.

[0041] (1) Separation of urine-derived cells

[0042] Centrifuge 50-100mL of the obtained human urine sample at a speed of 500g for 5min, wash the pellet twice with PBS (phosphate buffered saline, pH7.2-7.4), discard the supernatant, and resuspend the cells with culture medium. It is: keratinocyte serum-free medium (KSFM) medium + 15% FBS (purchased from Gibco-Invitrogen Company), and the urine-derived cells are isolated;

[0043] (2) Primary culture of urine-derived cells

[0044] The obtained urine-derived cells were inoculated in a 24-well plate containing culture medium at a density of 500 cells / well, and the culture medium composition was: keratinocyte serum-free medium (KSFM) medium+10% FBS+penicillin (100U / ml )+Streptomyci...

Embodiment 2

[0049] Example 2 Small Molecule Induction and Culture of Urine-derived Pluripotent Stem Cells (hUSCs) in Vitro

[0050] A two-step induction method was adopted. In the first step, 5-aza-2'-deoxycytidine (5-AZA) was added to make the final concentration 2 μmol / L, and induced for 2 days; in the second step, 3- Deazaneplanocin A (DZNeP) and vitamin C (vitaminC) were induced, and the final concentrations reached 3 μmol / L and 40 μg / ml, respectively,

[0051] All the other steps are the same as in Example 1.

Embodiment 3

[0052] Example 3 Small Molecule Induction and Culture of Urine-derived Pluripotent Stem Cells (hUSCs) in Vitro

[0053] A two-step induction method was adopted. In the first step, 5-aza-2'-deoxycytidine (5-AZA) was added to make the final concentration 8 μmol / L, and induced for 2 days; in the second step, 3- Deazaneplanocin A (DZNeP) and vitamin C (vitaminC) were induced, and the final concentrations reached 4 μmol / L and 10 μg / ml, respectively,

[0054] All the other steps are the same as in Example 1.

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Abstract

The invention discloses a method for culture of urine-derived pluripotent stem cells by virtue of in vitro small molecule induction. The method comprises the following steps: obtaining urine-derived cells from a human urine sample; carrying out in vitro small molecule induction and culturing on the obtained primary cells to obtain P1 generation hUSCs clones growing with adherence; picking up the hUSCs clones in good growing state to inoculate; and continuously carrying out continuous cell culture to obtain hUSCs with the cells which are elongated and good in state. The hUSCs prepared by the method disclosed by the invention, by virtue of induction, can be directionally differentiated to osteoblasts, adipose cells, skeletal muscle cells, nerve cells, fibroblasts or smooth muscle cells. The hUSCs are transplanted into a diabetic lower extremity ischemia model, and research results show that the hUSCs can remarkably enhance blood flow recovery and angiogenesis in an ischemia part. The hUSCs prepared by the method can be applied to treating and repairing diseased tissues and organs of diabetes and diabetic complications, cardiovascular, cerebrovascular and renovascular diseases, Alzheimer's disease, osteoporosis, arthritis and the like as well as early-warning tumors of the urinary system and screening cell drugs.

Description

technical field [0001] The invention relates to a method for preparing pluripotent stem cells, in particular to a method for inducing and culturing human Urine-Derived Stem Cells (Human Urine-Derived Stem Cells, hUSCs) by small molecules in vitro. The invention belongs to the technical field of cell biology, Background technique [0002] The loss or dysfunction of tissues and organs is one of the major hazards to human health and the leading cause of human disease and death. According to a data in the United States, millions of Americans suffer from loss or dysfunction of various tissues and organs every year, requiring 8 million operations for repair each year, and the annual hospitalization days are between 40 and 90 million. , with an annual cost of more than $40 billion. In recent years, a series of major breakthroughs in the field of stem cells have ignited the hope of human beings to find tissue and organ regeneration and repair. [0003] Stem cells are a class of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074C12N5/077C12N5/079C12N5/071A61K35/32A61K35/35A61K35/34A61K35/33A61K35/30A61P3/10A61P9/14A61P17/02A61P9/00A61P25/28A61P19/10A61P19/02
Inventor 曾强陈海旭杨超付小兵相杭
Owner GENERAL HOSPITAL OF PLA
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